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aneurysms at the origin of the posterior inferior cerebellar artery ( pica ) remain a challenge for neurosurgeons because the close proximity to the brainstem and lower cranial nerves poses a high risk for surgical clipping.1)14)15 ) aside from such anatomic unchangeability of adjacent structures , complex anatomic features of pica origin aneurysms themselves also add high risks for both surgical and endovascular treatments.16)19 ) however , in the past decade , there have been substantial advances in device technologies and endovascular techniques . as a result , the number of patients who are not candidates for endovascular treatment is decreasing . in particular , the " self expanding stent technology " and " supercompliant balloon technology " have revolutionized the methods and results of endovascular treatment.14)22 ) combining a self expanding stent with a " cross - over technique " , which uses an inter - arterial communication as an access route to the other vascular part,9)12 ) the authors herein report on a bilateral approach for stent - assisted coiling of two complex pica origin aneurysms . a 71-year - old woman who had no past medical history presented to our emergency room with a sudden thunderclap headache , nausea , and vomiting . on neurological examination , the score on the glasgow coma scale was 11 ( e3v3m5 ) and isocoric pupil response was observed . initial computed tomography ( ct ) and ct angiography showed fischer grade 4 subarachnoid hemorrhage ( sah ) and a ruptured left pica aneurysm ( fig . cerebral angiography and 3d image reconstruction showed a left pica origin aneurysm measuring 7 mm in length with a dome to neck ratio of 0.7 and pica incorporation into the sac ( fig . 1c and 1d ) . as a treatment option , in view of anticipated surgical difficulty and patient 's preference , our neurovascular team decided on endovascular treatment . the use of a multi - catheter or balloon remodeling technique is likely to have a high risk of pica occlusion during coil embolization of this complex aneurysm ; therefore , coiling with placement of a stent in the vertebral artery ( va)-aneurysm - pica path was planned . however , an antegrade approach to the pica appeared to be very difficult and too risky , so a retrograde approach via the contralateral va was decided . for selection of the left pica arising from the aneurysm , a 6f guiding catheter was placed in the right va , an excelsior sl-10 microcatheter ( striker , fremont , ca ) was introduced into the right va via the guiding catheter to cross over the vertebrovertebral junction to the left va , and the microcatheter over a synchro-14 guidewire ( striker , fremont , ca ) was able to select the left pica smoothly ( fig . after being placed in the left pica , the excelsior microcatheter was exchanged for a rebar-18 microcatheter ( ev3 inc , irvine , ca ) that was compatible with a stent delivery system . another 6f guiding catheter was introduced into the left va and an excelsior sl-10 microcatheter was placed in that aneurysm through the guiding catheter for coil delivery . the right vertebral artery was non - dominant and smaller than the left vertebral artery . however , the size of the right vertebral artery was sufficient to accept the rebar-18 microcatheter . after deployment of a solitaireab stent ( ev3 inc , irvine , ca ) for protection of the pica ( fig . 1f ) , a total of eight detachable coils were packed in the aneurysm . as only a 20-mm - long stent ( 4 20 ) was available at that time , it covered the left pica , aneurysm neck , left va , and a short segment of the right va , in order . the patient received dual anti - platelet therapy for 30 days , and continued life - long aspirin . at the four - month follow - up evaluation , a previously healthy 43-year - old man visited our emergency room complaining of a severe headache for six hours . except for nuchal rigidity , brain ct showed right premedullary sah and minimal intraventricular hemorrhage mainly in the fourth ventricle , three - dimensional ct angiography showed a ruptured right pica origin aneurysm , and cerebral angiography was successively performed for visualization of anatomic details of the aneurysm . it had a maximal size of 11 mm and a low dome - to - neck ratio of 0.9 , and gave rise to the pica , which was incorporated into the sac ( fig . aiming at both aneurysm occlusion and pica preservation , coiling with placement of a stent in the va - aneurysm - pica path was considered the best method . all procedural steps were similar to those of case 1 , except for the use of two micro - catheters for coil delivery and use of a 4 22 mm enterprise stent . after placement of the first two coils , eliminating contrast filling of the aneurysm dome , systemic heparinization ( 3,000 iu ) was administered intravenously . however , thrombosis occurred in the aneurysm neck during placement of the last coil , which resulted in near occlusion of the pica flow . tirofiban was given as an intraarterial bolus of 0.5 mg over five minutes , resulting in recanalization of the occluded pica . 2b ) , no progressive thrombosis was observed and the pica flow was intact , so that the patient was awakened . on neurological examination , no neurological deficit was identified immediately or at one month after the procedure . the patient received dual anti - platelet therapy for 30 days , but was lost to follow - up afterwards . a 71-year - old woman who had no past medical history presented to our emergency room with a sudden thunderclap headache , nausea , and vomiting . on neurological examination , the score on the glasgow coma scale was 11 ( e3v3m5 ) and isocoric pupil response was observed . initial computed tomography ( ct ) and ct angiography showed fischer grade 4 subarachnoid hemorrhage ( sah ) and a ruptured left pica aneurysm ( fig . cerebral angiography and 3d image reconstruction showed a left pica origin aneurysm measuring 7 mm in length with a dome to neck ratio of 0.7 and pica incorporation into the sac ( fig . 1c and 1d ) . as a treatment option , in view of anticipated surgical difficulty and patient 's preference , our neurovascular team decided on endovascular treatment . the use of a multi - catheter or balloon remodeling technique is likely to have a high risk of pica occlusion during coil embolization of this complex aneurysm ; therefore , coiling with placement of a stent in the vertebral artery ( va)-aneurysm - pica path was planned . however , an antegrade approach to the pica appeared to be very difficult and too risky , so a retrograde approach via the contralateral va was decided . for selection of the left pica arising from the aneurysm , a 6f guiding catheter was placed in the right va , an excelsior sl-10 microcatheter ( striker , fremont , ca ) was introduced into the right va via the guiding catheter to cross over the vertebrovertebral junction to the left va , and the microcatheter over a synchro-14 guidewire ( striker , fremont , ca ) was able to select the left pica smoothly ( fig . after being placed in the left pica , the excelsior microcatheter was exchanged for a rebar-18 microcatheter ( ev3 inc , irvine , ca ) that was compatible with a stent delivery system . another 6f guiding catheter was introduced into the left va and an excelsior sl-10 microcatheter was placed in that aneurysm through the guiding catheter for coil delivery . the right vertebral artery was non - dominant and smaller than the left vertebral artery . however , the size of the right vertebral artery was sufficient to accept the rebar-18 microcatheter . after deployment of a solitaireab stent ( ev3 inc , irvine , ca ) for protection of the pica ( fig . 1f ) , a total of eight detachable coils were packed in the aneurysm . as only a 20-mm - long stent ( 4 20 ) was available at that time , it covered the left pica , aneurysm neck , left va , and a short segment of the right va , in order . the patient received dual anti - platelet therapy for 30 days , and continued life - long aspirin . at the four - month follow - up evaluation , a previously healthy 43-year - old man visited our emergency room complaining of a severe headache for six hours . except for nuchal rigidity , brain ct showed right premedullary sah and minimal intraventricular hemorrhage mainly in the fourth ventricle , three - dimensional ct angiography showed a ruptured right pica origin aneurysm , and cerebral angiography was successively performed for visualization of anatomic details of the aneurysm . it had a maximal size of 11 mm and a low dome - to - neck ratio of 0.9 , and gave rise to the pica , which was incorporated into the sac ( fig . aiming at both aneurysm occlusion and pica preservation , coiling with placement of a stent in the va - aneurysm - pica path was considered the best method . all procedural steps were similar to those of case 1 , except for the use of two micro - catheters for coil delivery and use of a 4 22 mm enterprise stent . after placement of the first two coils , eliminating contrast filling of the aneurysm dome , systemic heparinization ( 3,000 iu ) was administered intravenously . however , thrombosis occurred in the aneurysm neck during placement of the last coil , which resulted in near occlusion of the pica flow . tirofiban was given as an intraarterial bolus of 0.5 mg over five minutes , resulting in recanalization of the occluded pica . 2b ) , no progressive thrombosis was observed and the pica flow was intact , so that the patient was awakened . on neurological examination , no neurological deficit was identified immediately or at one month after the procedure . the patient received dual anti - platelet therapy for 30 days , but was lost to follow - up afterwards . pica origin aneurysms frequently have unfavorable anatomic findings , including a wide neck and pica incorporation into the sac.1)10)14 ) endovascular treatment of such aneurysms is associated with risk for pica occlusion or procedural failure . although special techniques have recently been addressed,6)8 ) two goals of aneurysm occlusion and pica preservation appear to be mostly secured by stent - assisted coiling , where a self expanding stent successively covers the pica , aneurysm neck , and va . however , in the case of pica incorporation , selecting the pica antegrade through the ipsilateral va is extremely difficult and risky . anatomy - wise , if the contralateral va were non - interrupted and large enough to accept a catheter , a contralateral and retrograde approach to the pica would be easier than the ipsilateral antegrade one . a few case studies regarding the " cross - over technique " using the communicating artery or vertebrovertebral junction as an access route have been reported in the literature.5)9 ) moret et al.14 ) suggested that the anatomic benefits afforded by this technique outweigh the potential risks associated with catheterization of another major cerebral artery . we believe that , in two cases in this study , without stent - assisted coiling using the " cross - over technique " , pica occlusion would have been inevitable . however , long - term follow - up results of this procedure are not available . in particular , the size of the pica is so variable that the issue of long - term stent patency remains unanswered . after far - lateral suboccipital exposure , pica origin aneurysms can be approached between the 11 cranial nerve inferiorly and the ninth and tenth cranial nerves superiorly . therefore , the 10 and 11 cranial nerves are at risk of injury during the operation , which can result in lower cranial nerve dysfunction.3)7)10)15 ) in addition , complex aneurysmal geometry may make it difficult for neurosurgeons to complete aneurysm clipping without occlusion of the pica . occlusion of the pica close to its origin may lead to serious morbidity and mortality , depending on the distribution of the medullary perforators and the presence of collateral supply from other cerebellar arteries . aneurysm clipping following occipital artery - pica bypass surgery may be a solution to this serious complication,2)19)20 ) however , it is too early to generalize the staged treatments . due to recent instrumental and technical developments leading to good results , endovascular treatment is considered the primary treatment for posterior circulation aneurysms . however , complex pica aneurysms , having a wide neck and pica incorporation , remain a challenge for both surgical and endovascular treatments . in such cases , a bilateral approach for

aneurysms of the posterior inferior cerebellar artery ( pica ) are rarely encountered . in particular , due to frequent anatomic complexity and the presence of nearby critical structures , pica origin aneurysms are difficult to treat . however , recent reports of anecdotal cases using advanced endovascular instruments and skills have made the results of endovascular treatment rather outstanding . pica preservation is the key to a successful endovascular treatment , based on the premise that a pica origin aneurysm is well occluded . to secure pica flow , stenting into the pica would be the best method , however , it is nearly impossible technically via the ipsilateral vertebral artery ( va ) if the pica arose at an acute angle from the sac . in such a case , a bilateral approach for stent - assisted coiling can be a creative method for achievement of two goals of both aneurysm occlusion and pica preservation : ipsilateral approach for coil delivery and contralateral cross - over approach for stent delivery via a retrograde smooth path into the pica .

this in vitro study was conducted in the chemistry and biochemistry institute , university of veterinary medicine , vienna , austria between june and august 2013 in accordance with the guidelines of the european union council ( 86/609/eu ) for the use of laboratory animals . the work does not require approval from the ethics committee as it used mouse embryos under the fifteenth day of gestation . primary mesencephalic cell cultures were prepared from c57/b16 embryos according to radad et al.10 to summarize , embryonic mouse mesencephala were dissected on the fourteenth day of gestation and cut into small pieces in a drop of dulbecco s phosphate - buffered saline ( dpbs ) ( invitrogen , darmstadt , germany ) , 2 ml of 0.2% trypsin solution ( invitrogen , darmstadt , germany ) and 2 ml of 0.02% dnase i solution ( roche , berlin , germany ) were added and the tissue was subsequently incubated in a water bath at 37c for 7 minutes ( min ) . then , 2 ml of trypsin inhibitor ( 0.125mg / ml ) ( invitrogen , darmstadt , germany ) were added , the tissue was centrifuged at 100 g for 4 min and the supernatant was aspirated . the tissue pellet was triturated 2 - 3 times with a fire - polished pasteur pipette , each time 0.02% dnase i ( invitrogen , darmstadt , germany ) was included in the medium . dissociated cells were plated at a density of 257,000 cells / cm in dulbecco s modified eagle s medium ( dmem ) ( sigma aldrich , hamburg , germany ) supplemented with 4 mm glutamine , 10 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( hepes ) buffer , 30 mm glucose , 100 iu / ml penicillin , 0.1 mg / ml streptomycin , and 10% heat - inactivated fetal calf serum ( sigma aldrich , hamburg , germany ) . the medium was exchanged on the first day in vitro ( div ) and on the third div . on the fifth div , half of the medium was replaced by serum - free dmem containing 0.02 ml b-27/ml ( invitrogen , darmstadt , germany ) dmem . serum - free supplemented dmem was used for feeding from the sixth div , and subsequently replaced every second day . a stock solution of tq ( sigma aldrich , hamburg , germany ) ( 10 mm ) four sets of cultures were treated as follows : the first set of cultures was treated with dmso and kept as untreated controls . the second set of cultures was treated with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days to investigate the effect of tq on the survival of dopaminergic neurons . the third set of cultures was treated with 10 m of mpp on the tenth div for 48 hours ( h ) . the fourth set of cultures was concomitantly treated with tq ( 0.01 , 0.1 , 1 , and 10 ) , and 10 m of mpp on the tenth div for 48 h. dopaminergic neurons were identified immunocytochemically by staining tyrosine hydroxylase . cultures were rinsed carefully with phosphate buffered saline ( pbs , ph 7.2 ) at the end of each treatment and fixed in 4% paraformaldehyde for 45 min at 4c . after washing with pbs , cells were permeabilized with 0.4% triton x-100 for 30 min at room temperature . cultures were washed 3 times with pbs and incubated with 5% horse serum ( vectastain abc elite kit , biozol diagnostica vertrieb gmbh , eching , germany ) for 90 min to block nonspecific binding sites . to determine the number of thir in cultures , cells were sequentially incubated with anti - th primary antibody overnight at 4c , biotinylated secondary antibody ( vectastain ) , and avidin - biotin - horseradish peroxidase complex ( vectastain ) for 90 min at room temperature and washed with pbs between stages . the reaction product was developed in a solution of diaminobenzidine ( 1.4 mm ) in pbs containing 3.3 mm hydrogen peroxide and stained cells were counted with a nikon inverted microscope in 10 randomly selected fields per well at 10x magnification . cellular injury was quantitatively assessed by measuring the activity of lactate dehydrogenase ( ldh ) released from damaged cells into the culture medium . the reaction was initiated by mixing 0.2 ml of cell - free supernatant ( diluted 1:1 with aqua dest . ) with potassium phosphate buffer containing - nicotinamide adenine dinucleotide ( nadh ) and sodium pyruvate ( 0.18 and 0.62 mm in potassium phosphate buffer ) in a final volume of 0.5 ml in 1 ml cuvettes . the decrease of nadh was spectrophotometrically ( novaspec ii , ge healthcare europe gmbh , freiburg , germany ) monitored . the ldh activity was calculated from the slope of the decrease in optical density at 334 nm over a 3 min - time period . the ldh release is proportional to the number of damaged or destroyed cells.11,12 lysotracker deep red ( life technologies , invitrogen , grand island , ny , usa ) is a red fluorescence dye used for labeling acidic organelles in live cells including autophagolysosomes . cultures were treated with 1 m of tq ( a concentration that significantly protected dopaminergic neurons in mpp - treated cultures ) on the eighth div and co - administered with mpp ( 10 m ) on the tenth div for 2 days . on the twelfth div , culture medium was aspirated and cultured cells were incubated with a new medium containing 100 nm lysotracker deep red fluorescence dye ( life technologies , invitrogen , grand island , ny , usa ) for 15 - 30 min at 37c . after washing with dpbs , cultured cells were photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 580/590 , g-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) . 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) is a lipophilic cationic dye that selectively enters into mitochondria . in healthy cells with high mitochondrial membrane potential ( m ) the dye remains in the monomeric from with green fluorescence in case of apoptotic or damaged cells . the jc-1 red : green ratio is used to estimate changes in m.13 the jc-1 was dissolved in dmso and further diluted in dmem ( 10 g / ml final concentration ) . after removal of the culture medium , cells were loaded with jc-1 for 15 min at 37c , rinsed twice with pbs , and photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 520 dm/520 ba , b-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) . fluorescence intensity of the red : green ratio was determined semi quantitively by using adobe photoshop software . cells were fixed with 4% paraformaldehyde for 45 min at 4c . after washing with pbs ( ph 7.2 ) , the dapi solution ( 2 m final concentration ) was added to the cultures at room temperature for 5 min in the dark . after washing with dpbs , 3 photos were taken randomly from each well with a coolpix 990 digital camera connected to an inverted microscope with epifluorescence attachment using an ultraviolet filter ( nikon , otawara , japan ) . nuclei with condensed and fragmented chromatin were counted when the photos were analyzed with adobe photoshop software . data was obtained from 12 wells ( from 2 repeats ) for each treatment condition . comparisons were made using anova and post - hoc duncan s test using the statistical analysis system program 1998 ( sas institute inc . , this in vitro study was conducted in the chemistry and biochemistry institute , university of veterinary medicine , vienna , austria between june and august 2013 in accordance with the guidelines of the european union council ( 86/609/eu ) for the use of laboratory animals . the work does not require approval from the ethics committee as it used mouse embryos under the fifteenth day of gestation . primary mesencephalic cell cultures were prepared from c57/b16 embryos according to radad et al.10 to summarize , embryonic mouse mesencephala were dissected on the fourteenth day of gestation and cut into small pieces in a drop of dulbecco s phosphate - buffered saline ( dpbs ) ( invitrogen , darmstadt , germany ) , 2 ml of 0.2% trypsin solution ( invitrogen , darmstadt , germany ) and 2 ml of 0.02% dnase i solution ( roche , berlin , germany ) were added and the tissue was subsequently incubated in a water bath at 37c for 7 minutes ( min ) . then , 2 ml of trypsin inhibitor ( 0.125mg / ml ) ( invitrogen , darmstadt , germany ) were added , the tissue was centrifuged at 100 g for 4 min and the supernatant was aspirated . the tissue pellet was triturated 2 - 3 times with a fire - polished pasteur pipette , each time 0.02% dnase i ( invitrogen , darmstadt , germany ) was included in the medium . dissociated cells were plated at a density of 257,000 cells / cm in dulbecco s modified eagle s medium ( dmem ) ( sigma aldrich , hamburg , germany ) supplemented with 4 mm glutamine , 10 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( hepes ) buffer , 30 mm glucose , 100 iu / ml penicillin , 0.1 mg / ml streptomycin , and 10% heat - inactivated fetal calf serum ( sigma aldrich , hamburg , germany ) . the medium was exchanged on the first day in vitro ( div ) and on the third div . on the fifth div , half of the medium was replaced by serum - free dmem containing 0.02 ml b-27/ml ( invitrogen , darmstadt , germany ) dmem . serum - free supplemented dmem was used for feeding from the sixth div , and subsequently replaced every second day . a stock solution of tq ( sigma aldrich , hamburg , germany ) ( 10 mm ) was prepared in dimethyl sulfoxide ( dmso ) . four sets of cultures were treated as follows : the first set of cultures was treated with dmso and kept as untreated controls . the second set of cultures was treated with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days to investigate the effect of tq on the survival of dopaminergic neurons . the third set of cultures was treated with 10 m of mpp on the tenth div for 48 hours ( h ) . the fourth set of cultures was concomitantly treated with tq ( 0.01 , 0.1 , 1 , and 10 ) , and 10 m of mpp on the tenth div for 48 h. cultures were rinsed carefully with phosphate buffered saline ( pbs , ph 7.2 ) at the end of each treatment and fixed in 4% paraformaldehyde for 45 min at 4c . after washing with pbs , cells were permeabilized with 0.4% triton x-100 for 30 min at room temperature . cultures were washed 3 times with pbs and incubated with 5% horse serum ( vectastain abc elite kit , biozol diagnostica vertrieb gmbh , eching , germany ) for 90 min to block nonspecific binding sites . to determine the number of thir in cultures , cells were sequentially incubated with anti - th primary antibody overnight at 4c , biotinylated secondary antibody ( vectastain ) , and avidin - biotin - horseradish peroxidase complex ( vectastain ) for 90 min at room temperature and washed with pbs between stages . the reaction product was developed in a solution of diaminobenzidine ( 1.4 mm ) in pbs containing 3.3 mm hydrogen peroxide and stained cells were counted with a nikon inverted microscope in 10 randomly selected fields per well at 10x magnification . cellular injury was quantitatively assessed by measuring the activity of lactate dehydrogenase ( ldh ) released from damaged cells into the culture medium . the reaction was initiated by mixing 0.2 ml of cell - free supernatant ( diluted 1:1 with aqua dest . ) with potassium phosphate buffer containing - nicotinamide adenine dinucleotide ( nadh ) and sodium pyruvate ( 0.18 and 0.62 mm in potassium phosphate buffer ) in a final volume of 0.5 ml in 1 ml cuvettes . the decrease of nadh was spectrophotometrically ( novaspec ii , ge healthcare europe gmbh , freiburg , germany ) monitored . the ldh activity was calculated from the slope of the decrease in optical density at 334 nm over a 3 min - time period . lysotracker deep red ( life technologies , invitrogen , grand island , ny , usa ) is a red fluorescence dye used for labeling acidic organelles in live cells including autophagolysosomes . cultures were treated with 1 m of tq ( a concentration that significantly protected dopaminergic neurons in mpp - treated cultures ) on the eighth div and co - administered with mpp ( 10 m ) on the tenth div for 2 days . on the twelfth div , culture medium was aspirated and cultured cells were incubated with a new medium containing 100 nm lysotracker deep red fluorescence dye ( life technologies , invitrogen , grand island , ny , usa ) for 15 - 30 min at 37c . after washing with dpbs , cultured cells were photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 580/590 , g-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) . 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) is a lipophilic cationic dye that selectively enters into mitochondria . in healthy cells with high mitochondrial membrane potential ( m ) the dye remains in the monomeric from with green fluorescence in case of apoptotic or damaged cells . the jc-1 red : green ratio is used to estimate changes in m.13 the jc-1 was dissolved in dmso and further diluted in dmem ( 10 g / ml final concentration ) . after removal of the culture medium , cells were loaded with jc-1 for 15 min at 37c , rinsed twice with pbs , and photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 520 dm/520 ba , b-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) . fluorescence intensity of the red : green ratio was determined semi quantitively by using adobe photoshop software . cells were fixed with 4% paraformaldehyde for 45 min at 4c . after washing with pbs ( ph 7.2 ) , the dapi solution ( 2 m final concentration ) was added to the cultures at room temperature for 5 min in the dark . after washing with dpbs , 3 photos were taken randomly from each well with a coolpix 990 digital camera connected to an inverted microscope with epifluorescence attachment using an ultraviolet filter ( nikon , otawara , japan ) . nuclei with condensed and fragmented chromatin were counted when the photos were analyzed with adobe photoshop software . data was obtained from 12 wells ( from 2 repeats ) for each treatment condition . comparisons were made using anova and post - hoc duncan s test using the statistical analysis system program 1998 ( sas institute inc . , treatment of cultures with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days produced no significant effects on either the survival rate or the morphology of thir neurons ( data not shown ) . treatment of cultures with mpp ( 10 m on the eighth div for 48 h ) decreased the number of dopaminergic neurons by around 40% compared with untreated control cultures ( figure 1a ) . surviving neurons after mpp treatment showed fewer , shortened , and thickened neurites ( figure 1b ) . co - treatment of cultures with tq ( on the eighth div for 4 days ) and mpp ( 10 m on the tenth div for 48 h ) prevented dopaminergic cell loss by around 25% at 0.1 and 1 m ( figure 1a ) , and improved the morphology of surviving neurons compared to mpp - treated cultures ( figure 1b ) . anti - th immunohistochemical staining of cultured cells showing : a ) survival of dopaminergic neurons in primary mesencephalic cell cultures . 100% corresponds to the total number of thir neurons after 12 div in untreated controls . values represent the meansem of 3 independent experiments with 4 wells in each treatment . in each well , 10 randomly selected fields were counted for th immunocytochemistry ( # p=0.001 , * p=0.008 , + p=0.009 ) . the mpp - treated cultures showed thir neurons with few , shortened and thickened neuritis ( arrows ) . treatment with tq improves the morphology of thir neurons compared to mpp - treated cultures . th - tryosine hydrolase , thir - tyrosine hydroxylase immunoreactive , div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq attenuated mpp - induced ldh increase in primary mesencephalic cell culture . the mpp ( 10 m from the tenth to twelfth div ) increased ldh release in the culture medium by 145% compared with untreated cultures ( figure 2 ) . the tq significantly decreased ldh release in the culture medium by around 70% at 0.1 and 0.1 m concentrations compared with mpp - treated cultures ( figure 2 ) . div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq increased lysotracker deep red fluorescence and the red : green fluorescence ratio of jc-1 , and decreased mpp - induced apoptotic cell death in primary mesencephalic cell culture . lysotracker deep red fluorescent intensity increased 3 folds ( 682% ) in the cultures co - treated with tq and mpp compared with the cultures treated with mpp alone ( 222% ) ( figure 3a ) . in parallel , cultures co - treated with mpp and tq showed higher red fluorescence than the cultures treated with mpp alone ( figure 3b ) . lysotracker deep red fluorescence staining of cultured cells showing : a ) lysotracker deep red fluorescence intensity in primary mesencephalic cell cultures . 100% corresponds to the density of lysotracker deep red in primary mesencephalic cell cultures after 12 div . fluorescence intensity was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) . ( # p=0.05 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased lysotracker deep red fluorescence intensity compared with mpp - treated cultures . div - day in vitro , sem - standard error of mean , tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium treatment of cultures with mpp ( 10 m on the tenth div for 48 h ) caused dissipation of m . cultures treated with mpp showed a significant decrease in red : green fluorescence ratio of jc-1 by around 17% compared with untreated controls ( figure 4a ) . however , co - treatment of mpp - treated cultures with 1 m tq from the eighth - twelfth div significantly increased m as it increased the red : green fluorescence ratio of jc-1 by around 24% compared with mpp - treated cultures ( figure 4a ) . as shown in figure 4b , mpp - treated cultures co - administered with tq displayed much higher red fluorescence than the cultures treated with mpp alone . 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) fluorescence staining of cultured cells showing : a ) red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures . 100% corresponds to the red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures after 12 div . red : green fluorescence ratio of jc-1 was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) . ( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased red fluorescence compared to mpp+-treated cultures which exhibits marked green fluorescence . tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium staining of cultured cells with the nuclear fluorescence dye , dapi revealed that mpp ( 10 m on the tenth div for 48 h ) increased the number of nuclei showing apoptotic features by 139% compared with untreated cultures ( figure 5a ) . against mpp , tq was shown to decrease the number of apoptotic nuclei by around 100% compared with mpp - treated cultures ( figure 5a ) . 4,6-diamidino-2-phenylindole fluorescence staining of cultured cells showing : a ) number of nuclei showing apoptotic features with condensed and fragmented chromatin in primary mesencephalic cell cultures . 100% corresponds to the number of apoptotic nuclei in untreated control cultures after 12 div . ( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq decreased the number of apoptotic nuclei compared to mpp - treated cultures . tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium summary of the neuroprotective effect of tq against mpp treatment in primary mesencephalic cell culture . treatment of cultures with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days produced no significant effects on either the survival rate or the morphology of thir neurons ( data not shown ) . treatment of cultures with mpp ( 10 m on the eighth div for 48 h ) decreased the number of dopaminergic neurons by around 40% compared with untreated control cultures ( figure 1a ) . surviving neurons after mpp treatment showed fewer , shortened , and thickened neurites ( figure 1b ) . co - treatment of cultures with tq ( on the eighth div for 4 days ) and mpp ( 10 m on the tenth div for 48 h ) prevented dopaminergic cell loss by around 25% at 0.1 and 1 m ( figure 1a ) , and improved the morphology of surviving neurons compared to mpp - treated cultures ( figure 1b ) . anti - th immunohistochemical staining of cultured cells showing : a ) survival of dopaminergic neurons in primary mesencephalic cell cultures . 100% corresponds to the total number of thir neurons after 12 div in untreated controls . values represent the meansem of 3 independent experiments with 4 wells in each treatment . in each well , 10 randomly selected fields were counted for th immunocytochemistry ( # p=0.001 , * p=0.008 , + p=0.009 ) . the mpp - treated cultures showed thir neurons with few , shortened and thickened neuritis ( arrows ) . treatment with tq improves the morphology of thir neurons compared to mpp - treated cultures . th - tryosine hydrolase , thir - tyrosine hydroxylase immunoreactive , div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq attenuated mpp - induced ldh increase in primary mesencephalic cell culture . the mpp ( 10 m from the tenth to twelfth div ) increased ldh release in the culture medium by 145% compared with untreated cultures ( figure 2 ) . the tq significantly decreased ldh release in the culture medium by around 70% at 0.1 and 0.1 m concentrations compared with mpp - treated cultures ( figure 2 ) . div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq increased lysotracker deep red fluorescence and the red : green fluorescence ratio of jc-1 , and decreased mpp - induced apoptotic cell death in primary mesencephalic cell culture . lysotracker deep red fluorescent intensity increased 3 folds ( 682% ) in the cultures co - treated with tq and mpp compared with the cultures treated with mpp alone ( 222% ) ( figure 3a ) . in parallel , cultures co - treated with mpp and tq showed higher red fluorescence than the cultures treated with mpp alone ( figure 3b ) . lysotracker deep red fluorescence staining of cultured cells showing : a ) lysotracker deep red fluorescence intensity in primary mesencephalic cell cultures . 100% corresponds to the density of lysotracker deep red in primary mesencephalic cell cultures after 12 div . fluorescence intensity was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) . ( # p=0.05 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased lysotracker deep red fluorescence intensity compared with mpp - treated cultures . div - day in vitro , sem - standard error of mean , tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium treatment of cultures with mpp ( 10 m on the tenth div for 48 h ) caused dissipation of m . cultures treated with mpp showed a significant decrease in red : green fluorescence ratio of jc-1 by around 17% compared with untreated controls ( figure 4a ) . however , co - treatment of mpp - treated cultures with 1 m tq from the eighth - twelfth div significantly increased m as it increased the red : green fluorescence ratio of jc-1 by around 24% compared with mpp - treated cultures ( figure 4a ) . as shown in figure 4b , mpp - treated cultures co - administered with tq displayed much higher red fluorescence than the cultures treated with mpp alone . 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) fluorescence staining of cultured cells showing : a ) red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures . 100% corresponds to the red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures after 12 div . red : green fluorescence ratio of jc-1 was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) . ( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased red fluorescence compared to mpp+-treated cultures which exhibits marked green fluorescence . tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium staining of cultured cells with the nuclear fluorescence dye , dapi revealed that mpp ( 10 m on the tenth div for 48 h ) increased the number of nuclei showing apoptotic features by 139% compared with untreated cultures ( figure 5a ) . against mpp , tq was shown to decrease the number of apoptotic nuclei by around 100% compared with mpp - treated cultures ( figure 5a ) . 4,6-diamidino-2-phenylindole fluorescence staining of cultured cells showing : a ) number of nuclei showing apoptotic features with condensed and fragmented chromatin in primary mesencephalic cell cultures . 100% corresponds to the number of apoptotic nuclei in untreated control cultures after 12 div . ( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq decreased the number of apoptotic nuclei compared to mpp - treated cultures . tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium summary of the neuroprotective effect of tq against mpp treatment in primary mesencephalic cell culture . in the present study , tq was investigated to ascertain whether it protected mesencephalic dopaminergic neurons against mpp - induced cell death through activation of enzymatic degradation , preservation of mitochondrial function , and inhibition of apoptotic cell death . clearly , mpp was found to significantly decrease the survival of dopaminergic neurons and increase the release of ldh into the culture medium . the mpp toxicity involves its selective uptake by dopaminergic neurons through the dopamine transporter and inhibition of mitochondrial complex i activity with subsequent mitochondrial depolarization.14 in parallel , the use of jc-1 fluorescence dye in our current study showed that mpp significantly decreased the m of cultured cells as indicated by the decreasing red : green fluorescence ratio of jc-1 . similar mpp - induced reduction of m was reported in other in vitro disease models.15,16 mitochondrial damage has long been implicated in the death of nigrostriatal dopaminergic neurons in both pd patients and experimental models.17,18 staining of primary dopaminergic cultures with blue - fluorescent dapi nucleic acid stain showed that a significant number of the cells displayed features of apoptosis , most notably chromatin condensation , and fragmentation . previously , tang et al19 and xu et al16 demonstrated that mpp caused apoptotic cell death in pc12 and sh - sy5y cells . the mpp - induced apoptosis was reported to occur as the result of disruption of mitochondrial transmembrane potential and opening of the permeability transition pore.20 similar to our previous report,9 co - treatment of primary mesencephalic cell cultures with tq and mpp was found to protect dopaminergic neurons and decreased the release of ldh into the culture medium . since that time , no evidence in the literature has shown how tq protected dopaminergic neurons in the primary mesencephalic cell culture . staining of cultures with lysotracker deep red showed that tq significantly increased the red fluorescence of the dye compared with mpp - treated cultures , indicating enhancement of the formation of many autophagolysosomes , the sites of lysosomal degradation , by tq . this is supported by the findings of he and klionsky21 who correlated the fluorescent signals of lysotracker deep red to the upregulation of autophagy in zebrafish . increased red fluorescence of lysotracker deep red is attributed to the formation of many autophagosomes and autophagolysosomes that retain much dye as the result of increasing their acidification . using jc-1 fluorescent dye showed that tq significantly enhanced m as it increased the red : green fluorescence ratio of jc-1 compared with mpp - treated cultures . the tq was similarly found to protect rat cortical neurons against ethanol- and a1 - 42-induced neurotoxicity through inhibition of mitochondrial membrane depolarization.22,23 counting of apoptotic nuclei using blue - fluorescent dapi nucleic acid stain indicated that tq decreased mpp - induced apoptotic cell death in primary mesencephalic cell cultures . in accordance , ullah et al22 reported that tq inhibited apoptotic cell death in ethanol - treated rat cortical neurons and attributed this effect of tq to the preservation of mitochondrial integrity . zhang et al24 reported that mitochondrial clearance protected cultured cortical neurons against ischemia - reperfusion - induced cell damage . in conclusion , correlating such results would therefore suggest that tq might activate a lysosomal degradative process in dopaminergic neurons , where clearance of damaged mitochondria results in reduced mitochondria - mediated apoptotic cell death . this might raise the possibility of using tq as a potentially therapeutic intervention in pd patients . whenever a manuscript contains material ( tables , figures , etc . ) which is protected by copyright ( previously published ) , it is the obligation of the author to obtain written permission from the holder of the copyright ( usually the publisher ) to reproduce the material in neurosciences . please submit copies of the material from the source in which it was first published .

objectives : to investigate potential mechanisms mediating the neuroprotective effect of thymoquinone ( tq ) on dopaminergic neurons.methods:this study was conducted in the chemistry and biochemistry institute , university of veterinary medicine , vienna , austria between june and august 2013 . primary cultures were prepared from embryonic mouse mesencephala ( ofi / spf ) at gestation day 14 . four sets of cultures were kept untreated , treated with tq on the eighth day in vitro ( div ) for 4 days , treated with 1-methyl-4-phenylpyridinium ( mpp+ ) on the tenth div for 48 hours and co - treated with thymoquinone and mpp+ . on the twelfth div , cultures were subjected to immunohistochemistry against tyrosine hydroxylase and fluorescent staining using lysotracker deep red , 5,5,6,6-tetrachloro-1,1,3,3-tetraethyl benzimidazolylcarbocyanine ( jc-1 ) and 4,6-diamidino-2-phenylindole stains.results:the mpp+ decreased the number of dopaminergic neurons by 40% , and increased the release of lactate dehydrogenase ( ldh ) into the culture medium . the tq significantly rescued dopaminergic neurons and decreased the release of ldh at the concentrations of 0.1 and 1 m . the tq significantly shifted the red fluorescent intensity of the lysotracker deep red , increased the mitochondrial membrane potential as it increased the red : green florescent ratio of jc-1 , and decreased mpp+-induced apoptotic cell death.conclusion:the tq protects dopaminergic neurons in primary mesencephalic culture by enhancing lysosomal degradation that clears damaged mitochondria and inhibits mitochondria - mediated apoptotic cell death .

bed rest , heart failure , depletion of intravascular volume , and aging often induce hypotension when the body or trunk is upright.15 generally , a tachycardic response occurs due to vagal withdrawal according to the downward blood shift that occurs under orthostatic stress in healthy humans,6 although orthostatic hypotension presents as an excessive drop in cardiac output ( q ) and lower tachycardic response in frail and elderly patients as a consequence of inadequate vascular contraction or autonomic dysfunction.7,8 numerous studies have suggested that advancing age is associated with decreases in vagal nerve activity.912 age - related decreases in vagal nerve activity have been suggested as contributing factors for orthostatic hypotension in elderly individuals.7,13 orthostatic hypotension has been reported in more than 60% of frail older patients in an acute geriatric ward.5 previous studies have observed orthostatic hypotension in 54% of seated older inpatients resting on a bed for more than 12 h.14 in light of these facts , methods of preventing hypovolemia and hypotension during orthostatic stress need to be investigated . pointed out that studies of orthostatic hypotension in frail patients , such as those with decompensated heart failure , are scarce and preventive interventions remain insufficient.2 clinically , frail patients are placed in fowler s position or a semiseated position on the bed . these positions are often clinically used without distinction between acute- or chronic - phase illness . the patient is placed in a reclined position with the backrest between 30 and 60 and with flexed or straight knees in fowler s position.15,16 breathing , eating , and other routine daily activities are considered to be facilitated in fowler s position.15,1720 some studies have suggested a relationship between trunk angle and hemodynamics during fowler s position.2124 cicolini et al . reported that blood pressure in fowler s position was intermediate between the seated and supine positions in both young healthy individuals and hypertensive patients.23,24 those studies indicated that the effects of orthostatic stress on hemodynamics in fowler s position may be substantial in frail patients . how posture can prevent the hypovolemia and hypotension caused by orthostatic stress during fowler s position is important and should be addressed . our previous study indicated that fowler s position with an upright upper trunk is effective for maintaining circulatory volume with lower tachycardia via vagal withdrawal compared with having the whole trunk upright in younger subjects.25 however , whether an upright upper trunk in fowler s position allows easy maintenance of circulatory volume in cases with autonomic hypofunction associated with aging remains unclear . we postulated that stroke volume ( sv ) would be higher and heart rate ( hr ) would be lower with the upper trunk upright than with the whole trunk upright in fowler s position in both older and younger subjects . in addition , we presumed that the lower hr would be caused by reduced sympathetic activation in older individuals but by reduced vagal withdrawal in younger individuals . the present study was performed as a cross - sectional study to assess our hypothesis with a fundamental study of the effects of the two fowler s positions on hemodynamics and hr variability parameters in healthy older and younger subjects . we assessed hemodynamics and hr variability in 11 male younger subjects ( mean age standard error of the mean [ sem ] : 20.50.2 years ; range : 2022 years ; weight : 60.61.8 kg ; height : 170.21.8 cm ) and 11 male community - dwelling older subjects ( mean age sem : 69.51.2 years ; range : 6479 years ; weight : 62.02.1 kg ; height : 165.41.8 cm ) . all subjects were interviewed regarding medical history , and blood pressure was checked after a period of rest . final decisions on subject selections were made by consultations between a medical doctor and researchers . potential subjects were excluded when they had a history of cardiovascular disease , respiratory disease or other chronic medical disease , or were not normotensive ( systolic bold pressure below 140 mmhg and diastolic blood pressure below 90 mmhg ) . all subjects in the final analysis were healthy and had no history of chronic or acute cardiovascular or respiratory disease or other chronic diseases . subjects were instructed to refrain from beverages containing caffeine or alcohol for 24 h before starting experiments and to refrain from eating and drinking after 2,200 h on the day prior to the experiments that started in the morning or to consume a light breakfast before experiments that started in the afternoon . all experiments were implemented between 1,100 and 1,400 h , because blood pressure fluctuates according to a circadian pattern stabilizing at approximately noon . the ethics commission of the international university of health and welfare approved the study , and all participants provided voluntary written consent to participate after being fully informed about the procedure , risks , and protocol . we collected physiological data with the patient in the supine position and two fowler s positions . prior to experiments , subjects rested in a thermoneutral room at 28c for 15 min and were set up for electrocardiography ( ecg ) , noninvasive arterial pressure monitoring , and impedance cardiography ( icg ) . measurements in the supine position were made with sufficient time between measurements in the two fowler s positions . between measurements in each fowler s position , subjects rested for more than 3 min . data were recorded for 5 min in each of the supine position and the two fowler s positions . subjects were placed in the fowler s positions on a bed at 30 of lower trunk inclination and 60 of upper trunk inclination ( ut60 ) and 60 of whole trunk inclination ( wt60 ) ( figure 1 ) . we defined the upper and lower segments at ut60 around the spinous process of the 10th thoracic vertebra . the height of the bottom of the upper and lower trunk was adjusted according to individual trunk size . all experiments were performed in our laboratory ( odawara , kanagawa , japan ) under controlled thermoneutral temperature . hr was estimated from type ii ecg ( ecg 100c ; biopac systems , goleta , ca , usa ) in all experiments . icg ( nico 100c ; biopac systems ) was performed using eight spot electrodes ( vitrode m ; nihon kohden , tokyo , japan ) attached to the neck and lower thorax of each subject for icg based on a previous study.26 sv was estimated from transthoracic bioimpedance using bernstein s method that has been validated as offering high reliability comparable to the technique of thermodilution.26 icg allows continuous noninvasive evaluation of sv . noninvasive continuous arterial pressure was measured in the radial artery by tonometry using an arterial pressure monitor ( bp-608ev ; omron colin , tokyo , japan ) . mean arterial pressure ( map ) was estimated as one third pulse pressure plus diastolic pressure . ecg , thoracic bioimpedance , and continuous arterial pressure signals were recorded on a personal computer using the mp150 data acquisition system ( biopac systems ) at a sampling rate of 1 khz . we performed frequency domain and time domain analysis of ecg for the assessment of vagal function . we calculated logarithm - transformed respiratory sinus arrhythmia ( ln rsa ) from the power spectrum of the rr intervals ( 0.150.4 hz ) . we thus calculated the square root of the mean of the sum of the squares of differences between adjacent rr intervals ( rmssd ) , number of pairs of adjacent rr intervals differing by more than 50 ms ( nn50 ) and the proportion derived by dividing nn50 by the total number of rr intervals ( pnn50 ) for 5 min . all parameters of hr variability reflect vagal modulation.27,28 changes in hemodynamic variables from supine and hr variability parameters in the three positions in subjects from the two age groups were compared by two - way mixed - design analysis of variance ( anova ) . when three positions two age group interactions were significant , one - way repeated - measures anova of the three positions in each subject and one - way anova of subjects from the two age groups in each position were used to evaluate simple main effects . subsequent post hoc analysis to determine differences between the three positions in each age group were evaluated using a paired t - test with shaffer s multiple comparison procedure in each age group.29 the level of statistical significance was set at p<0.05 . data were statistically analyzed using r for windows version 3.3.0 statistical software ( www.r-project.org ) and the car 2.14 package . we assessed hemodynamics and hr variability in 11 male younger subjects ( mean age standard error of the mean [ sem ] : 20.50.2 years ; range : 2022 years ; weight : 60.61.8 kg ; height : 170.21.8 cm ) and 11 male community - dwelling older subjects ( mean age sem : 69.51.2 years ; range : 6479 years ; weight : 62.02.1 kg ; height : 165.41.8 cm ) . all subjects were interviewed regarding medical history , and blood pressure was checked after a period of rest . final decisions on subject selections were made by consultations between a medical doctor and researchers . potential subjects were excluded when they had a history of cardiovascular disease , respiratory disease or other chronic medical disease , or were not normotensive ( systolic bold pressure below 140 mmhg and diastolic blood pressure below 90 mmhg ) . all subjects in the final analysis were healthy and had no history of chronic or acute cardiovascular or respiratory disease or other chronic diseases . subjects were instructed to refrain from beverages containing caffeine or alcohol for 24 h before starting experiments and to refrain from eating and drinking after 2,200 h on the day prior to the experiments that started in the morning or to consume a light breakfast before experiments that started in the afternoon . all experiments were implemented between 1,100 and 1,400 h , because blood pressure fluctuates according to a circadian pattern stabilizing at approximately noon . the ethics commission of the international university of health and welfare approved the study , and all participants provided voluntary written consent to participate after being fully informed about the procedure , risks , and protocol . we collected physiological data with the patient in the supine position and two fowler s positions . prior to experiments , subjects rested in a thermoneutral room at 28c for 15 min and were set up for electrocardiography ( ecg ) , noninvasive arterial pressure monitoring , and impedance cardiography ( icg ) . measurements in the supine position were made with sufficient time between measurements in the two fowler s positions . between measurements in each fowler s position , subjects rested for more than 3 min . data were recorded for 5 min in each of the supine position and the two fowler s positions . subjects were placed in the fowler s positions on a bed at 30 of lower trunk inclination and 60 of upper trunk inclination ( ut60 ) and 60 of whole trunk inclination ( wt60 ) ( figure 1 ) . we defined the upper and lower segments at ut60 around the spinous process of the 10th thoracic vertebra . the height of the bottom of the upper and lower trunk was adjusted according to individual trunk size . all experiments were performed in our laboratory ( odawara , kanagawa , japan ) under controlled thermoneutral temperature . hr was estimated from type ii ecg ( ecg 100c ; biopac systems , goleta , ca , usa ) in all experiments . icg ( nico 100c ; biopac systems ) was performed using eight spot electrodes ( vitrode m ; nihon kohden , tokyo , japan ) attached to the neck and lower thorax of each subject for icg based on a previous study.26 sv was estimated from transthoracic bioimpedance using bernstein s method that has been validated as offering high reliability comparable to the technique of thermodilution.26 icg allows continuous noninvasive evaluation of sv . noninvasive continuous arterial pressure was measured in the radial artery by tonometry using an arterial pressure monitor ( bp-608ev ; omron colin , tokyo , japan ) . mean arterial pressure ( map ) was estimated as one third pulse pressure plus diastolic pressure . ecg , thoracic bioimpedance , and continuous arterial pressure signals were recorded on a personal computer using the mp150 data acquisition system ( biopac systems ) at a sampling rate of 1 khz . we performed frequency domain and time domain analysis of ecg for the assessment of vagal function . we calculated logarithm - transformed respiratory sinus arrhythmia ( ln rsa ) from the power spectrum of the rr intervals ( 0.150.4 hz ) . we thus calculated the square root of the mean of the sum of the squares of differences between adjacent rr intervals ( rmssd ) , number of pairs of adjacent rr intervals differing by more than 50 ms ( nn50 ) and the proportion derived by dividing nn50 by the total number of rr intervals ( pnn50 ) for 5 min . changes in hemodynamic variables from supine and hr variability parameters in the three positions in subjects from the two age groups were compared by two - way mixed - design analysis of variance ( anova ) . when three positions two age group interactions were significant , one - way repeated - measures anova of the three positions in each subject and one - way anova of subjects from the two age groups in each position were used to evaluate simple main effects . subsequent post hoc analysis to determine differences between the three positions in each age group were evaluated using a paired t - test with shaffer s multiple comparison procedure in each age group.29 the level of statistical significance was set at p<0.05 . data were statistically analyzed using r for windows version 3.3.0 statistical software ( www.r-project.org ) and the car 2.14 package . figure 2 shows the results of changes in hemodynamic variables from supine at all positions . change in hr ( hr ) increased and change in sv ( sv ) decreased with the change from supine to an upright trunk in younger and older subjects . two - way mixed - design anova of hr and sv revealed significant main effects of the three positions ( hr , f = 31.73 ; sv , f = 15.73 ; p<0.05 ) , but no significant main effects of the two age groups ( p>0.05 ) and no significant three positions two age groups interactions ( p>0.05 ) . multiple comparisons for each age group showed that hr ( ut60 and wt60 in younger , 2.91.2 beats / min and 6.61.4 beats / min , respectively ; ut60 and wt60 in older , 2.80.7 beats / min and 5.00.9 beats / min , respectively ) was significantly lower at ut60 than at wt60 and at supine than at ut60 and wt60 . in addition , sv ( ut60 and wt60 in younger , 4.42.8 ml and 13.72.9 ml , respectively ; ut60 and wt60 in older , 0.33.3 ml and 7.43.1 ml , respectively ) was significantly higher at ut60 and supine than at wt60 in older and younger subjects ( p<0.05 ) . anova for changes in q ( q : ut60 and wt60 in younger , 0.10.2 l and 0.30.2 l , respectively ; ut60 and wt60 in older , 0.20.2 l and 0.00.2 l , respectively ) revealed a significant main effect of the three positions ( q , f = 3.55 , p<0.05 ) but no significant main effect of the two age groups ( p>0.05 ) and no significant interaction of three positions two age groups ( p>0.05 ) . multiple comparisons among each age group showed no significant differences between the three positions ( p>0.05 ) . anova of changes in map ( map : ut60 and wt60 in younger , 1.21.9 mmhg and 4.24.0 mmhg , respectively ; ut60 and wt60 in older , 0.52.8 mmhg and 2.72.6 mmhg , respectively ) revealed no significant main effects for the three positions ( p>0.05 ) or two age groups ( p>0.05 ) and no significant interaction of the three positions two age groups ( p>0.05 ) . although all parameters decreased with the change from supine to upright trunk in younger subjects , significant differences were not seen between all positions in older subjects . these parameters in older subjects were lower compared with younger subjects for each position ( table 2 ) . two - way mixed - design anova of ln rsa , rmssd , nn50 , and pnn50 revealed significant main effects of the three positions ( p<0.05 ) and two age groups ( p<0.05 ) but significant three positions two age groups interactions ( p<0.05 ) . post hoc analysis revealed significant simple main effects of the three positions in younger subjects ( ln rsa in younger , f = 8.67 ; rmssd in younger , f = 6.71 ; nn50 in younger , f = 5.95 ; pnn50 in younger , f = 7.12 ; p<0.05 ) but no significant simple main effect of the three positions in older subjects ( p>0.05 ) and significant simple main effects of the two age groups among each position ( ln rsa of supine , f = 34.6 ; rmssd of supine , f = 25.18 ; nn50 of supine , f = 27.45 ; pnn50 of supine , f = 29.77 ; ln rsa of ut60 , f = 24.72 ; rmssd of ut60 , f = 19.87 ; nn50 of ut60 , f = 20.43 ; pnn50 of ut60 , f = 19.46 ; ln rsa of wt60 , f = 14.36 ; rmssd of wt60 , f = 4.95 ; nn50 of wt60 , f = 6.59 ; pnn50 of wt60 , f = 6.12 ; p<0.05 ) . multiple comparisons among the three positions showed that all hr variability parameters were significantly higher at ut60 and supine position than at wt60 in younger subjects ( p<0.05 ) . figure 2 shows the results of changes in hemodynamic variables from supine at all positions . change in hr ( hr ) increased and change in sv ( sv ) decreased with the change from supine to an upright trunk in younger and older subjects . two - way mixed - design anova of hr and sv revealed significant main effects of the three positions ( hr , f = 31.73 ; sv , f = 15.73 ; p<0.05 ) , but no significant main effects of the two age groups ( p>0.05 ) and no significant three positions two age groups interactions ( p>0.05 ) . multiple comparisons for each age group showed that hr ( ut60 and wt60 in younger , 2.91.2 beats / min and 6.61.4 beats / min , respectively ; ut60 and wt60 in older , 2.80.7 beats / min and 5.00.9 beats / min , respectively ) was significantly lower at ut60 than at wt60 and at supine than at ut60 and wt60 . in addition , sv ( ut60 and wt60 in younger , 4.42.8 ml and 13.72.9 ml , respectively ; ut60 and wt60 in older , 0.33.3 ml and 7.43.1 ml , respectively ) was significantly higher at ut60 and supine than at wt60 in older and younger subjects ( p<0.05 ) . anova for changes in q ( q : ut60 and wt60 in younger , 0.10.2 l and 0.30.2 l , respectively ; ut60 and wt60 in older , 0.20.2 l and 0.00.2 l , respectively ) revealed a significant main effect of the three positions ( q , f = 3.55 , p<0.05 ) but no significant main effect of the two age groups ( p>0.05 ) and no significant interaction of three positions two age groups ( p>0.05 ) . multiple comparisons among each age group showed no significant differences between the three positions ( p>0.05 ) . anova of changes in map ( map : ut60 and wt60 in younger , 1.21.9 mmhg and 4.24.0 mmhg , respectively ; ut60 and wt60 in older , 0.52.8 mmhg and 2.72.6 mmhg , respectively ) revealed no significant main effects for the three positions ( p>0.05 ) or two age groups ( p>0.05 ) and no significant interaction of the three positions two age groups ( p>0.05 ) . although all parameters decreased with the change from supine to upright trunk in younger subjects , significant differences were not seen between all positions in older subjects . these parameters in older subjects were lower compared with younger subjects for each position ( table 2 ) . two - way mixed - design anova of ln rsa , rmssd , nn50 , and pnn50 revealed significant main effects of the three positions ( p<0.05 ) and two age groups ( p<0.05 ) but significant three positions two age groups interactions ( p<0.05 ) . post hoc analysis revealed significant simple main effects of the three positions in younger subjects ( ln rsa in younger , f = 8.67 ; rmssd in younger , f = 6.71 ; nn50 in younger , f = 5.95 ; pnn50 in younger , f = 7.12 ; p<0.05 ) but no significant simple main effect of the three positions in older subjects ( p>0.05 ) and significant simple main effects of the two age groups among each position ( ln rsa of supine , f = 34.6 ; rmssd of supine , f = 25.18 ; nn50 of supine , f = 27.45 ; pnn50 of supine , f = 29.77 ; ln rsa of ut60 , f = 24.72 ; rmssd of ut60 , f = 19.87 ; nn50 of ut60 , f = 20.43 ; pnn50 of ut60 , f = 19.46 ; ln rsa of wt60 , f = 14.36 ; rmssd of wt60 , f = 4.95 ; nn50 of wt60 , f = 6.59 ; pnn50 of wt60 , f = 6.12 ; p<0.05 ) . multiple comparisons among the three positions showed that all hr variability parameters were significantly higher at ut60 and supine position than at wt60 in younger subjects ( p<0.05 ) . the major findings of the present study were as follows : 1 ) the reductions in sv and tachycardic response were smaller with the upper trunk upright compared with the whole trunk upright , in healthy young and older individuals , and 2 ) the reduced tachycardic response with the upright upper trunk would be caused by decreased vagal withdrawal in younger subjects and reduced sympathetic activation in older subjects . orthostatic stress reportedly occurs with the shift in blood volume to the abdomen and legs in healthy individuals.30,31 at ut60 , subdivision of the trunk into upper and lower segments was defined by the spinous process of the 10th thoracic vertebra , under the presumption that since the heart is located close to the center of rotation of the bottom of the dorsal thorax , even if the upper trunk is upright , the level of the heart is lower with ut60 than with wt60 . in addition , the abdomen and pelvic segments at ut60 were not upright compared with wt60 . orthostatic stress might therefore be smaller at ut60 than at wt60 , suggesting differences in the distribution of the blood volume shift to lower parts of the body at ut60 compared to wt60 in both subject groups . moreover , these results were observed to be similar to the findings of our previous study in healthy young subjects.25 some previous studies have reported the effects of advancing age on sv under orthostatic stress induced by lower body negative pressure ( lbnp ) . these previous studies revealed that the reduction of sv was significantly lower among older individuals than among younger individuals under higher lbnp , although changes in sv did not differ between age groups under lower level lbnp.32,33 tsutsui et al . indicated that hypovolemia was less among older subjects than among the younger during higher level lbnp because leg compliance in the older subjects was reduced according to decreased capacitance function with age.33 with the present findings , significant differences in sv between age groups with the two fowler s positions were not observed . these results appear similar to previous findings during lower level lbnp but appear to differ from previous findings for higher - level lbnp.33 in fowler s position , since only the trunk and head were upright and the lower extremities were similar to the level in the supine position , orthostatic stress would be limited . differences in leg compliance with aging were thus indicated to have no effect on differences in sv between younger and older subjects , because the shift in blood volume to the lower extremities was regarded as too small . in this study , sv was less than approximately 14 ml during the two fowler s positions in both age groups . positive hr occurred with the change from supine to upright trunk and hr was lower at ut60 than at wt60 in both age groups . these tachycardic responses were suggested to be the result of the negative sv being lower at ut60 than at wt60 . however , since all hr variability parameters in older subjects differed from those of younger subjects in the three positions , the mechanisms underlying the tachycardic responses reflected in the present results were suggested to differ between younger and older subjects . generally , tachycardic response less than 100 beats / min is attributed to vagal withdrawal.34 we therefore concluded that the tachycardic responses in the present results were caused by vagal withdrawal in younger subjects . in addition , vagal withdrawal would be lower at ut60 than at wt60 because ln rsa , rmssd , nn50 , and pnn50 were higher at ut60 compared with wt60 in younger subjects . however , no significant differences were seen among all positions and ln rsa , rmssd , nn50 , and pnn50 were lower in older subjects than in younger subjects in each position . vagal function is well known to reduce with age.912 these results suggested that vagal function was reduced in older subjects and tachycardic response was caused by mechanisms other than vagal withdrawal . nevertheless , the tachycardic response was significantly different between the two fowler s positions in older subjects . some previous studies have indicated that sympathetic nerve function and sympathetic baroreflex function are maintained with age.10,32,3537 the tachycardic response of older subjects with an upright trunk might thus be suggested to result from sympathetic activation in the present study , rather than vagal withdrawal . from the above , compared with wt60 , ut60 might inhibit decreases in sv and inhibit tachycardic response by reducing vagal withdrawal in younger subjects and by reducing sympathetic activation in older subjects . although sv was higher at ut60 than at wt60 in both age groups , q and map did not differ between the two fowler s positions in either age group or between the two age groups in either fowler s position . these results suggest that the tachycardic response and vasomotor contraction response maintained q and map to compensate for reduced sv under orthostatic stress regardless of age . such findings may be attributed to the regulation of tachycardic response and vasomotor contraction via vagal withdrawal in younger individuals and via sympathetic activation in older individuals . our findings indicate that an upright upper trunk during fowler s position allowed maintenance of sv and inhibited tachycardic response compared to an upright whole trunk regardless of age , although the autonomic mechanisms underlying tachycardic responses differed between younger and older adults . an upright upper trunk in fowler s position might help to reduce orthostatic stress and facilitate routine activities and conversation in frail patients . some studies have suggested that leg compression bandaging could prevent orthostatic hypotension in older patients.2,38 although that method might indeed be effective , that approach requires a more time - consuming procedure and the patient may feel a sense of uncomfortable restriction from the bandaging . our proposed method offers the advantages that the procedure to tilt the upper trunk is very simple and patients do not require compression bandaging or use of stockings to maintain circulatory volume when the patient wants to raise the body on the bed . the present results suggest that reduced tachycardic response with an upright upper trunk may be regulated by reduced sympathetic activation compared with having the whole trunk upright during fowler s position in older subjects . however , the present study did not directly assess sympathetic nerve activity in the two fowler s positions or while supine . to corroborate our findings , confirmation by direct measurement of sympathetic nerve activity or venous plasma norepinephrine concentration in fowler s positions in younger and older subjects may be necessary . in consideration of using a fowler s position such as ut60 at clinical sites , validation in patients with orthostatic intolerance is needed because the influences of an upright upper trunk on hemodynamics and cardiovascular regulation in such patients have yet to be completely clarified . we demonstrated that an upright upper trunk during fowler s position allowed maintenance of sv and inhibited tachycardic responses compared to having the whole trunk upright in both younger and older subjects . such inhibition of tachycardic responses might be caused by reduced vagal withdrawal in younger subjects and by reduced sympathetic activation in older subjects . an upright upper trunk during fowler s position might be useful to maintain circulatory volume for reduced orthostatic stress and to facilitate daily activities in frail patients at clinical sites .

backgrounddownward shifts in blood volume with changing position generally cause tachycardic responses . age - related decreases in vagal nerve activity could contribute to orthostatic hypotension in older individuals . fowler s position is a reclined position with the back between 30 and 60 , used to facilitate breathing , eating , and other routine daily activities in frail and elderly patients.objectivethis study examined whether stroke volume ( sv ) was higher and heart rate ( hr ) lower in fowler s position with an upright upper trunk than in fowler s position with the whole trunk upright in both older and younger subjects , based on the assumption that lower hr would result from reduced sympathetic activation in older individuals.methodswe assessed hemodynamics and hr variability from electrocardiography , noninvasive arterial pressure and impedance cardiography in 11 younger male subjects ( age range , 2022 years ) and 11 older male subjects ( age range , 6479 years ) , using three positions : supine , or fowler s positions with either 30 of lower trunk inclination and 60 of upper trunk inclination ( ut60 ) or 60 of whole trunk inclination ( wt60 ) . comparisons were then made between age groups and between positions.resultsreductions in sv and tachycardic response were smaller with ut60 than with wt60 , in both younger and older subjects . in addition , reduced tachycardic response with upright upper trunk appeared attributable to decreased vagal withdrawal in younger subjects and to reduced sympathetic activation in older subjects.conclusionour findings indicate that an upright upper trunk during fowler s position allowed maintenance of sv and inhibited tachycardic response compared to an upright whole trunk regardless of age , although the autonomic mechanisms underlying tachycardic responses differed between younger and older adults . an upright upper trunk in fowler s position might help to reduce orthostatic stress and facilitate routine activities and conversation in frail patients .

frontalis sling surgery is the treatment option for myopathic blepharoptosis with poor levator muscle action . nontuberculous mycobacterial infections in the periocular region are rare and are usually caused by organisms belonging to runyon group iv including mycobacterium chelonae and mycobacterium fortuitum . chang et al . reported six cases of nontuberculous mycobacterial infection and found an association with nasolacrimal duct obstruction , implantation of foreign body , history of recent surgery , and immunosuppression . mauriello found implantation of a foreign body in 5 out 13 patients of nontuberculous mycobacterial infections of the periocular region . treatment of nontuberculous mycobacterial infections can be difficult because of their multi - drug resistance . treatment usually involves surgical debridement with removal of infected foreign body and a prolonged course of antibiotics like amikacin , clarithromycin , ciprofloxacin , and doxycycline . we report a rare case of nontuberculous mycobacterial infection of the silicone rod after frontalis sling surgery . a 65-year - old apparently healthy male presented with complaints of pain and swelling of both eyelids of 2 months duration . he had undergone cataract and frontalis sling surgery for myopathic blepharoptosis in both eyes 2 years prior to presentation . the best corrected visual acuity for distance was 20/400 in right eye and 20/25 in the left eye . examination of face showed multiple , erythematous nodules involving both upper eyelids and the brow . all nodules were localized to the scars from the previous ptosis surgery with a few showing signs of suppuration ( fig . the posterior segment showed hypertensive retinopathy in both eyes and a scar at the macula in the right eye . systemic workup was unremarkable and included a complete blood picture , chest x - ray and serological tests for hiv . the patient was clinically diagnosed to have chronic progressive external ophthalmoplegia status post bilateral frontalis sling and cataract surgery with multiple abscesses in forehead and upper eyelids . the patient underwent an incision and drainage of the abscesses in the brow and the purulent material was sent for microbiological examination . the smear showed the presence of acid fast bacilli on 20 % acid fast staining ( fig . 1b ) and culture was significant for staphylococcus sp . and m. fortuitum ( fig . the patient received intravenous amikacin ( 1 g / day , single dose ) and ciprofloxacin ointment locally . poor response to treatment necessitated exploration with explantation of the silicone rod sling in the right eye 10 days later . a repeat microbiological evaluation showed acid fast bacilli and significant growth of m. fortuitum on culture . as the isolate showed a similar antibiotic sensitivity pattern the patient was continued on intravenous amikacin ( 1 g / day ) . however , 11 days later , the patient suffered from another episode of pain and swelling in the left eye . a repeat exploration of the brow with drainage and silicone rod explantation was performed in the left eye . the patient showed a slow but definite improvement and received intravenous amikacin for 6 weeks . five months after commencement of therapy the patient was asymptomatic and the left upper eyelid position was maintained ( fig . 1a an apparently healthy 62-year - old man presented with painful , erythymatous nodules of bilateral brow and upper lids two years after frontalis sling surgery for severe myopathic ptosis . b microscopic picture of the purulent material showing long , slender acid fast bacilli along with polymorphonuclear cells ( ziehl neelsen stain , total magnification 1,000 ) . c sheep blood chocolate agar inoculated with purulent material along with silicone tube ( left eye brow after 11 days of treatment ) shows confluent growth of cream colored , opaque , medium size colonies of staphylococcus aureus and tiny , semi - opaque colonies of mycobacterium fortuitum ( incubation : 4 days , 5 % co2 , 37 c ) . d five months after sensitivity - based antibiotic treatment and explantation of bilateral silicone rods , the patient showed resolution of symptoms . the left upper eyelid maintained an elevated position after silicone rod explantation a an apparently healthy 62-year - old man presented with painful , erythymatous nodules of bilateral brow and upper lids two years after frontalis sling surgery for severe myopathic ptosis . b microscopic picture of the purulent material showing long , slender acid fast bacilli along with polymorphonuclear cells ( ziehl neelsen stain , total magnification 1,000 ) . c sheep blood chocolate agar inoculated with purulent material along with silicone tube ( left eye brow after 11 days of treatment ) shows confluent growth of cream colored , opaque , medium size colonies of staphylococcus aureus and tiny , semi - opaque colonies of mycobacterium fortuitum ( incubation : 4 days , 5 % co2 , 37 c ) . d five months after sensitivity - based antibiotic treatment and explantation of bilateral silicone rods , the patient showed resolution of symptoms . several authors have reported about nontuberculous mycobacterial infections after periocular surgery [ 3 , 4 ] . the median time interval between prior surgery and onset of infection in these reports was 6 weeks ( range 0.511 months ) [ 3 , 4 ] . in our patient , multiple erythematous nodules with suppuration along the tract of the silicone rod more than 2 years after surgery led to the suspicion and eventual diagnosis of nontuberculous mycobacteria . some aspects in our patient need to be highlighted . first is that a bilateral infection by nontuberculous mycobacteria involving the silicone rod after frontalis sling surgery has not been reported earlier . the lodgement of nontuberculous mycobacteria in the brow region in our case may be related to the persistent irritation from the silicone rod and its predeliction for fat [ 4 , 5 ] . sequestration of nontuberculous mycobacteria with fat allows its growth without detection by normal immunosurveillance [ 4 , 5 ] . in our patient , sequestration of nontuberculous mycobacteria in the brow fat may have been responsible for delayed and bilateral infection involving the silicone slings . further , fitzgerald et al . found nontuberculous mycobacteria in 82 % of fat globules in their series of 71 cases . complete resolution in our case occurred only after silicone slings were removed from both eyes . finally , removal of the silicone sling did not result in a recurrence of blepharoptosis . this phenomenon is postulated to be due to scarring and fibrosis along the tract of the silicone rod . nontuberculous mycobacterial may be responsible for bilateral delayed infection after frontalis surgery with silicone slings in an immunocompetent adult . surgical removal of infected slings and prolonged course of antibiotics are required for complete resolution . the authors do not have any financial interest or any conflicting relationship in any of the issues or products referred to in the manuscript . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution and reproduction in any medium , provided the original author(s ) and source are credited .

purposethe purpose of this study is to report a case of nontuberculous mycobacterial infection after frontalis sling surgery.methoda 65-year - old man presented with bilateral painful , erythematous lesions in the brow and upper eyelids . he had a history of frontalis sling surgery for myopathic ptosis 2 years back and all lesions were found localized to the tract of the silicone rod used in the previous frontalis sling surgery.resultincision and drainage of the lesions with microbiological analysis revealed significant growth of coagulase negative staphylococcus and mycobacterium fortuitum . sensitivity - based antibiotic treatment with intravenous amikacin was started , but poor response necessitated eventual explantation of both silicone rods for relief of symptoms . culture of the explanted rods revealed similar results of m. fortuitum infection . five months after the acute presentation , the patient is asymptomatic.conclusionnontuberculous mycobacterial infection may be a delayed onset complication in frontalis sling surgery using silicone rods .

according to the who , over 3 000 000 children under the age of five die each year due to causes related to environmental risk factors . identification of recognized environmental health risk factors is considered to be one of the most important objectives of health policy , as it relates to the everyday work of medical health - care professionals including family doctors , pediatricians , and nurses . determination of mentioned risk factors will allow implementation of proper preventive measures , and a system of providing parents and teachers with information about health risks will , in turn , decrease the risks which may improve the effectiveness of health care . the who green page questionnaire was designed as a potential tool to determine and monitor children s environmental conditions in all places where they live and develop . it can be a valuable source of supplemental information acquired in the course of children s and parents appointments with family physicians , and it may also be a great support for doctors in determining children s environmental risks at home and school . the who green page questionnaire has not yet been analyzed in terms of its practical utility with respect to medical diagnostics . the expected goal of this research was to assess the possibility of implementation of the who green page as a tool to supplement basic medical interviews with potential environmental health risk factors for children and determination of real risk factors currently existing in home and school environments . the who green page questionnaire was implemented with parents of children from urban , suburban , and rural environments who visited a family practice doctor . due to exclusion of questionnaires missing the majority of data ( more than 50% of the answers were missing ) , the responses of 159 patients were analyzed . when we analyzed the questionnaires with less than 50% of the questions unanswered , the denominator for percentage calculation was lower than that for the 159 parents . wilk test was carried out . for measurable ( quantitative ) variables , arithmetic means , and standard deviations were calculated , while for qualitative variables , the frequency ( percentage ) was determined . the analysis of qualitative variables was based on contingency tables and the test . to compare quantitative variables in two non - related and related groups , wallis test was conducted for means of variables that did not meet the criteria for variance analysis . in total 159 parents took part in the study , including 87 parents of girls ( 58% of the examined ) and 63 parents of boys ( 42% of the examined ) . the average age of the children was 11.2 years ( sd 6.2 , median 11 ) . the children mostly lived with both parents ( 91.3% , 136 ) , while 6.7% ( 10 ) of them lived with their mothers only . the majority of the examined cases came from urban areas ( 56.2% , 87 ) , while 38.3% ( 59 ) and 5.2% ( 8) were from rural and suburban areas respectively ( p<0.05 ) . overpopulation at home was noted only among 4.4% ( 7 ) of the respondents . domestic animals were present around the homes of 74.5% ( 117 ) of the respondents , and the distribution of answers was statistically significant ( p<0.05 ) , as it relates to the child s place of living . contact with domestic animals was predominant in rural areas ( animals were present in 91.53% of cases ) as opposed to urban and suburban areas ( 63.95% and 57.14% respectively ; p<0.05 ) . it was noted that 24.3% ( 34 ) of caregivers expressed concern about their children s environment ( vs 75.7% , 106 ) , which did not maintain statistically significant dependency with the children s living environment ( p>0.05 ) . knowledge and awareness of particular existing environmental risks were noted in 23.7% ( 32 ) of the subjects . most of the respondents ( 96.2% , 153 ) were aware of the presence of disease - transmitting factors : 42.5% of the subjects knew disease - transmitting factors . the distribution of the answers to this question was dependent on the children s living environment it was distinctively different in rural areas ( disease - transmitting factors were identified by 64.3% of the examined ) as opposed to urban and suburban areas ( correct answers were given by 31.4% and 33.3% the examined respectively ) , p<0.05 . in addition 7.0% ( 11 ) of the surveyed stated that their children had sustained injuries in connection with road traffic prior to the questionnaire study , and one child had sustained injuries in connection with fire . the distribution of the answers to those questions was not dependent on the children s living environment ( p>0.05 ) . existing exposure to chemical substances ( pesticides and detergents ) was confirmed by 5.2% ( 8) of the respondents , and 6.4% ( 10 ) of the respondents reported that their children had been poisoned before the questionnaire as a result of contact with chemical substances . furthermore , 9.6% ( 15 ) maintained that there existed a threat from poisonous animals . the distribution of the answers to the questions about contact with chemical substances and threat from poisonous animals was not dependent on the children s living environment ( p>0.05 ) . following analysis of the respondents answers , it was determined that the examined children lived in a densely built - up area in 79.6% ( 125 ) of the cases and in a low - risk geographical zone in 76.3% ( 119 ) of the cases . the food they ate was of appropriate quality according to 94.9% ( 149 ) of the respondents , the indoor air quality was considered to be good by 81.5% ( 119 ) of the respondents , and the outdoor air quality was considered to be average by 51.0% ( 80 ) of the respondents . the land they lived on was seen as appropriate by 72.9% ( 113 ) of the respondents , and 92.3% ( 143 ) of the examined thought that sewage was disposed of in an appropriate way . appropriate disposal of waste was confirmed by 87.9% ( 138 ) of the respondents , and the noise level was considered low by 68.2% ( 107 ) of the examined . exposure to chemical substances was low according to 70.7% ( 111 ) of the respondents . road traffic was seen as low by 44.6% ( 70 ) of the examined ( table 1table 1abc of environmental conditions homeschoolabcabcbuilt - up environment79.6% ( 125)20.4% ( 32)0.0% ( 0)60.8% ( 90)38.5% ( 57)0.7% ( 1)geographical zone76.3% ( 119)23.1% ( 36)0.6% ( 1)71.3% ( 102)28.0% ( 40)0.7% ( 1)food94.9% ( 149)4.5% ( 7)0.6% ( 1)83.5% ( 111)15.8% ( 21)0.8% ( 1)indoor air quality81.5% ( 119)17.1% ( 25)1.4% ( 2)64.1% ( 84)34.4% ( 45)1.5% ( 2)outdoor air quality40.8% ( 64)51.0% ( 80)8.3% ( 13)35.0% ( 50)55.9% ( 80)9.1% ( 13)water - drinkable86.5% ( 128)13.5% ( 20)0.0% ( 0)82.1% ( 110)17.9% ( 24)0.0% ( 0)land / soil72.9% ( 113)25.8% ( 40)1.3% ( 2)63.2% ( 91)35.4% ( 51)1.4% ( 2)sewage disposal92.3% ( 143)7.7% ( 12)0.0% ( 0)88.0% ( 125)11.3% ( 16)0.7% ( 1)waste disposal87.9% ( 138)11.5% ( 18)0.6% ( 1)79.2% ( 114)20.1% ( 29)0.7% ( 1)noise68.2% ( 107)28.7% ( 45)3.2% ( 5)28.6% ( 42)51.7% ( 76)19.7% ( 29)exposure to chemical substances70.7% ( 111)28.7% ( 45)0.6% ( 1)65.3% ( 96)32.7% ( 48)2.0% ( 3)road traffic44.6% ( 70)43.9% ( 69)11.5% ( 18)25.2% ( 37)56.5% ( 83)18.4% ( 27)environmental risk factors : a , proper ; b , average ; c , potential . differences between answers are statistically significant ( p<0.05 ) . ) . environmental risk factors : a , proper ; b , average ; c , potential . ( 90 ) of cases , the children schools were in very well - built areas , and the schools were situated in low - risk geographical zones in 71.3% ( 102 ) of cases . the food offered at school was of proper quality according to 94.4% ( 149 ) of the examined . indoor air quality at schools was good according to 64.1% ( 84 ) of the respondents , and outdoor air quality was considered to be average by 55.9% ( 80 ) . the land where their schools stand was considered appropriate by 63.2% ( 91 ) of the examined , and 88.0% ( 125 ) of the examined thought that sewage was disposed of in an appropriate way . appropriate waste disposal was confirmed by 79.2% ( 114 ) of the respondents , and the noise level was considered average by 51.7% ( 76 ) of the examined . exposure to chemical substances was low according to 65.3% ( 96 ) of the respondents . road traffic was seen as moderate by 56.5% ( 83 ) of the examined ( table 1 ) . the majorities of the children involved in the environmental assessment were of school age , lived with both parents , and came from urban areas . the caregivers expressed minor concerns about the children s living and study environments as well as low knowledge and awareness of existing environmental risks . a very low percentage of parents stated that their children sustained injuries in connection with road traffic , and in one case , a child sustained injuries in connection with fire ; these were not statistically significantly dependent on where they lived , exposure to chemical substances , or threat from poisonous animals . both the places they lived and studied were located in low - risk geographical zones . in the opinions of the majority of the respondents , the food , indoor and outdoor air , tap water , land , sewage and waste , noise level , exposure to chemical substances , and road traffic were appropriate quality in both places . this picture is positive and does not fully correspond with reality from the hygienic point of view and after performing an environmental interview , which emphasizes people s low awareness of environmental risks . the questionnaire may prove to be a useful tool in determining the kind of potential environmental risk factors that exist in children s living and study places . the who green page supplements the information of the basic medical interview by being a part the patient s medical history .

background : the objective of this study was to assess the possibility of implementation of the who green page as a tool to supplement basic medical interviews with environmental health risk factors for children.methods : the who green page questionnaire was tested on parents of children who visited family practice doctors.results : a total of 159 parents took part in the study . it was noted that 24.3% of caregivers expressed concern about their children s environment without naming the risk factors . it was also found that 23.7% of the parents demonstrated knowledge and awareness of existing real environmental risks , and 7.0% of them stated that their children had sustained injuries in connection with road traffic prior to the questionnaire study.conclusions : the who green page will provide additional information to the basic medical interview and , if regularly updated , will allow for monitoring of changing environmental conditions of children .

toxoplasmosis is an infection caused by the intracellular protozoon t. gondii , for which the primary host is cats . sexual reproduction of this parasite takes place in the intestinal environment of cats , and the oocysts produced are eliminated together with the feces . the oocysts remain viable in the external environment for up to one year . within this setting , t. gondii contaminates secondary hosts through these hosts ' contact with sand , foods , or plants that have been contaminated by the oocysts . after ingestion of the oocysts , the oocyst wall becomes thinned by the digestive juices , thereby leading to release of sporozoites into the intestinal lumen . the sporozoites rapidly invade the intestinal epithelium , though the vascular endothelium . at this stage of the evolution , they are transformed into tachyzoites , which are the invasive form of the parasite . through tachyzoite dissemination , the parasite will lodge in a wide variety of tissues in the secondary host , in which large numbers of cysts that are rich in bradyzoites are produced . the secondary hosts are commonly humans , rodents , birds , crustaceans , domestic animals , and practically all other warm - blooded animals , which explains why consumption of raw or undercooked meat can contaminate other hosts with bradyzoites from tissue cysts . ingestion of sporozoites occurs mainly through eating poorly washed fruits or vegetables , doing gardening , coming into contact with children who were playing on the ground or on sand , and so on . data in the literature consistently draw attention to the importance of proper guidance regarding the commonest means of becoming infected with t. gondii , with the aim of preventing this event within the pregnancy cycle and the possible harm done to the fetus [ 2 , 3 ] . a study conducted in the economic research service in the united states concluded that approximately half of the cases of toxoplasmosis occurred due to consumption of raw or undercooked meat and that the annual cost of treating such cases in that country was approximately u$ 7.7 billion . this high amount was due mainly to the congenital complication resulting from vertical transmission of the parasite to the fetus . although toxoplasmosis is extremely benign among immunocompetent women , there is a risk of vertical transmission of the parasite when this infection occurs during pregnancy . the risk increases with advancing gestational age in the following manner : first trimester 1025% , second trimester 3050% , and third trimester 6090% . this seems to occur as a function of the increase in the mass of the placenta , thus increasing the tissue available for tachyzoites to invade . on the other hand , the increase in the volume of the placenta also contributes towards increasing the quantity of cysts rich in bradyzoites , which implies a greater risk of fetal infection in cases of reactivation of the pathological condition . investigation of igg and igm levels should be incorporated into the prenatal routine for the commonest infections and for teratogenic infections , including rubella , cytomegalovirus , and toxoplasmosis . diagnoses of acute or progressive toxoplasmosis infection are based on detection of its specific antibodies , with the aim of making an early diagnosis of maternal seroconversion and instituting appropriate treatment . this is extremely important for preventing vertical transmission of t. gondii and its deleterious effects on the fetus [ 69 ] . findings of toxoplasmosis - specific igm indicate occurrences of acute infection , although these antibodies may be present in the maternal serum from one week to 18 months after the date of invasion by the parasitic tachyzoites . specific igg appears in the maternal serum between one and two months after the initial infection . in this light , it is clear that it is absolutely impossible for clinicians to define the starting time of the acute infection based only on the presence of igg and igm . moreover , the specificity and sensitivity of igm are not 100% , but in the ranges of 93.3 to 100% and 77.5 to 99.1% , respectively , according to evaluations on the six elisa kits most commonly used in the united states , in a study commissioned by the food and drugs administration ( fda ) . the igg avidity test is the laboratory resource currently accepted for the diagnosis of the approximate time when the initial infection occurred . this measures the affinity of igg for binding to the antigens of t. gondii , and it tends to increase with the length of time that has elapsed since the initial infection [ 1013 ] . a mexican study on 100 pregnant women showed that there was no correlation between igm levels and the igg avidity test results . thus , it was demonstrated in that study that the igg avidity test is useful for inferring the infection phase and assisting in managing toxoplasmosis during pregnancy . in this manner , avidity test results of up to 30% allow it to be said that the initial infection occurred not more than four months earlier . on the other hand , results of more than 60% indicate that the initial infection occurred at least six months earlier . results of between 30 and 60% are inconclusive regarding the exact time of the initial infection . however , in practice , all the antibodies discussed above only allow conclusions consisting of suppositions and probabilities . a prospective study assayed igg avidity for toxoplasmosis among 146 pregnant women who presented with igm positive for t. gondii . the patients underwent a multiplex nested polymerase chain reaction ( pcr ) for dna of t. gondii in the amniotic fluid , maternal blood , and blood of the umbilical cord . fifty ( 34.9% ) of the pregnant women presented with low igg avidity ( less than 30% ) . the pcr performed on the amniotic fluid or performed at birth was positive in the cases of nine patients with low igg avidity . among these nine patients , only three presented with congenital toxoplasmosis . none of the pregnant women with high or threshold avidity presented positive pcr results in the amniotic fluid or congenital toxoplasmosis . there were no diagnoses of congenital toxoplasmosis among the patients with negative pcr results in the amniotic fluid . thus , it was concluded from that study that using igg avidity in association with pcr on the amniotic fluid was important for diagnosing congenital toxoplasmosis . a prospective cohort study evaluated women with toxoplasmosis infection that had been detected in the prenatal screening at three centers , by means of real - time pcr on the amniotic fluid . pcr analysis was performed on 261 of the 377 patients who were included in the study . the sensitivity and negative predictive values were , respectively , 92.2% ( 95% confidence interval , ci : 8198% ) and 98.1% ( 95% ci : 9599.5% ) . there was no significant association with the trimester of pregnancy in which the infection occurred . thus , real - time pcr was considered to be an important tool for predicting fetal infection due to t. gondii and for making more appropriate treatment decisions . in a study conducted in france , at the university hospital of toulouse , 352 amniocentesis procedures were performed on pregnant women who were infected with t. gondii . the sensitivity and specificity of the diagnosis of congenital toxoplasmosis by means of pcr on the amniotic fluid were 91% and 99.5% , respectively . pcr was also performed on the placenta , which showed specificity of 52% and sensitivity of 99% . the specific igg and igm tests on the cord blood presented specificity of 91% and 92% and sensitivity of 53% and 64% , respectively . it was concluded in that study that pcr on the amniotic fluid has higher sensitivity and specificity for diagnosing congenital toxoplasmosis , thus enabling a more certain diagnosis of fetal infection . a study conducted at the university of the suez canal , in egypt , on 358 pregnant women with toxoplasmosis , showed sensitivity of 92.9% and specificity of 94.4% for pcr on the amniotic fluid , with a positive predictive value of 76.5% and a negative predictive value of 98.5% for the diagnosis of congenital toxoplasmosis . based on these studies , it has been accepted that the gold - standard examination for identifying fetal infection is pcr , which has high sensitivity and specificity when performed on amniotic fluid samples obtained from the sixteenth week of pregnancy onwards . in this manner , diagnosing the presence of dna particles from t. gondii in this medium can be done with great certainty . since colonization of the fetal kidneys occurs around two to three weeks after invasion by the tachyzoites of t. gondii and production of the amniotic fluid occurs basically by means of fetal diuresis , it can be concluded that a positive pcr indicates the presence of infection in the fetus , which has been eliminating t. gondii dna into the amniotic environment , in its urine . attention has also been drawn to the possibility of reactivation of latent toxoplasmosis in immunosuppressed patients . maternal immunosuppression and contamination by t. gondii may also be related to higher risk of fetal disruption disorders . likewise , if it is considered that the pregnancy cycle naturally confers immunosuppression on pregnant women , it would not , in our view , be unreasonable to consider that reactivation of latent toxoplasmosis might occur in some pregnant women . the world health organization and the centers for disease control and prevention recommend pyrimethamine , sulfadiazine , and folinic acid as the standard of care for people with congenital toxoplasmosis . these medications were proven to be effective in a randomized prospective study called the national collaborative chicago based congenital toxoplasmosis study ( nccbts ) . this study found that treatment with the three aforementioned medications significantly decreased adverse signs and symptoms associated with congenital toxoplasmosis , including ocular and central nervous system symptoms and sensorineural hearing loss . for patients with sensitivity to sulfadiazine , when a diagnosis of acute maternal toxoplasmosis infection is made , spiramycin should immediately be administered to the mother , at a dose of 2 to 3 g per day . this medication does not go beyond the placental barrier , but it diminishes the risk of vertical transmission by up to 60% . likewise , if pcr on the amniotic fluid is positive , institution of bacteriostatic therapy that crosses the placental barrier is essential , with the aim of diminishing the fetal malformations that result from acute toxoplasmosis . the following drugs can then be administered : sulfadiazine , at a dose of 3.0 g per day , and pyrimethamine , at a dose of 50 mg per day . however , these medications have high teratogenic potential , since they noticeably diminish fetal serum folate synthesis . it is known that insufficient concentration of serum folates noticeably diminishes synthesis of the enzyme tetrahydrofolate reductase . this enzyme acts within the cell medium to transform homocysteine , which is a cytotoxic and teratogenic agent , into methionine , which is an essential amino acid . high tissue concentration of homocysteine is therefore one of the causes of higher incidence of structural abnormalities in fetuses . for this reason , the administration of sulfadiazine and pyrimethamine should not be continuous and should be alternated with spiramycin administration every three weeks . in addition , a precursor of serum folate synthesis should be administered : folinic acid , at a dose of 10 to 20 mg , two to three times a week , while the patient is receiving sulfadiazina and pyrimethamine . administration of these three drugs decreases the risk that the fetus might develop disruption abnormalities due to t. gondii infection , by 70% . this adverse effect may be avoided with the simultaneous administration of folic acid during treatment [ 29 , 30 ] . despite these preventative measures , weekly monitoring of cell counts and platelet counts should be done to assess the level of marrow suppression and adjust these medications as necessary . recently , the society of obstetricians and gynecologists of canada proposed a clinical practice guideline to the toxoplasmosis in pregnancy . this guideline reinforces the conclusion that the combination of pyrimethamine , sulfadiazine , and folinic acid should be offered as treatment for women in whom fetal infection has been confirmed or is highly suspected , usually by a positive amniotic fluid pcr ( level i - b of evidence ) . women who are immunosuppressed or hiv - positive should be offered screening because of the risk of reactivation and toxoplasmosis encephalitis ( level i - a of evidence ) . the necessity of the treatment of t. gondii in human immunodeficiency - virus infected pregnant women is proved by several case reports [ 3234 ] . in study realized in brazil that presents high incidence of toxoplasmosis in pregnancy , porto and duarte analyzed the correlation between toxoplasmosis in pregnant women and incidence of congenital toxoplasmosis . among the pregnant women classified as confirmed cases of toxoplasmosis in pregnancy ( n = 19 ) , the congenital toxoplasmosis risk was six times greater than that in the probable / possible group . no case of congenital toxoplasmosis was identified in the group of pregnant women classified as unlikely to have toxoplasmosis in pregnancy . the children with no prenatal treatment ( 46.2% n = 242/524 ) presented a risk almost of congenital toxoplasmosis three times greater than the treated children ( odds ratio or 2.77 ; 95% ci 1.544.97 ; p = 0.001 ) . complete prenatal treatment was identified as a protecting factor for congenital toxoplasmosis ( or 0.35 ; 95% ci 0.190.65 ; p = 0.001 ) . in study realized in germany with 685 pregnant women and 33 children with positive serological tests for t. gondii infection , the overall transmission rate of t. gondii from the infected mother to her child was 4.8% . the prenatal treatment with spiramycin until sixteenth week was followed by at least 4 weeks of combination therapy with pyrimethamine , sulfadiazine , and folinic acid independent of the infection stage of the fetus . these results are consistent with other studies in which only a small study population was included [ 37 , 38 ] . other alternative proposed treatment to toxoplasmosis in pregnancy is the spiramycin / cotrimoxazole association . this association was used in 76 pregnant women with positive serological tests for t. gondii . the treatment consisted of spiramycin 3,480 g 4 times a day , cotrimoxazole 960 mg ( sulfamethoxazole 800 mg plus trhmetroprim 160 mg ) twice a day , and folinic acid 4 mg / day . the intrauterine transmission rate was only 2.6% ( two babies ) and none of them showed signs or symptoms of congenital infection at birth or during followup . the treatment did not need to be stopped in any mother because of adverse drug effects . in a case report , tamaru et al . the ultrasound realized at 23 weeks of pregnancy evidenced fetal ascites , cardiac effusion , cardiomegaly , enlarged lateral ventricles , and thickened placenta . sulfadoxine ( 500 mg / day ) and pyrimethamine ( 25 mg / day ) were administered from 25 to 27 weeks . cyclic administration of azithromycin ( 500 mg / day for 3 days followed by an interval of no medication for 4 days ) was subsequently started at 28 weeks and continued for 3 weeks followed by an interval of no medication for a week until delivery . then cyclic administration of acetylspiramycin ( 1.2 g / day for 3 weeks followed by an interval of no medication for 2 weeks ) was combined with azithromycin for 31 weeks . fetal ascites , cardiomegaly , enlarged lateral ventricles , and thickened placenta persisted until delivery ; cardiac effusion of the fetus completely disappeared at 29 weeks , which the authors considered to be the partial effect of administration of azithromycin started at 28 weeks . figure 1 shows a diagram with the sequence of serological screening of toxoplasmosis during pregnancy and its treatment . from a critical analysis on the data presented , we can conclude that it is essential to have results available from the igg avidity test and from pcr on the amniotic fluid , so that treatment of pregnant women affected by t. gondii can be adequately managed . without these test results , no conclusions regarding the presence or absence of vertical transmission of t. gondii to the fetus can be reached and doubts will remain regarding whether medications that cross the placental barrier , to treat the fetus , should be introduced . this uncertainty therefore could condemn a fetus that might have become contaminated across the placenta to develop a variety of congenital abnormalities , with serious postnatal repercussions for the family and for society as a whole , given that the ensuing costs to the public healthcare system are far from small .

a brief report on the nature and epidemiology of t. gondii infection is firstly presented . the importance of the specific igg avidity test and polymerase chain reaction ( pcr ) for toxoplasmosis is discussed , along with their significance and importance as auxiliary methods for determining the most likely time for the initial infection by this coccidian and for defining the therapeutic strategy . lastly , practical comments are made in relation to the classical therapeutic regimens , with special attention to the indications for fetal treatment , when this is necessary .

increasing prevalence of morbid obesity has augmented the demand for bariatric surgery , which is so far the most effective and durable therapy for the treatment of morbidly obese patients . deep vein thrombosis ( dvt ) and pulmonary embolism ( pe ) are the major risk factors of mortality after bariatric surgery [ 24 ] ; venous thrombosis of mesenteric vessels may also contribute to significant morbidity . although the risk of postoperative dvt following bariatric surgery does not exceed 2% in most studies , there are currently no strategies to identify individuals who have the highest risk for dvt . diagnosis of dvt in morbidly obese patients is a challenging clinical task because a considerable proportion of cases remains asymptomatic . to date , there are different prophylactic strategies available for prevention of dvt ; however , optimal personalized prophylactic regimes in individual patients are yet not defined . anticoagulation with low molecular weight heparin , anti - factor xa inhibitors , and unfractionated heparin are widely used in bariatric surgery for prevention of dvt and pe , but appropriate dosing of anticoagulation is still controversial . furthermore , the risk of dvt among morbidly obese patients undergoing bariatric surgery appears to persist despite the use of aggressive prophylactic anticoagulation therapy . identification of individuals with increased risk for dvt is also crucial for the balance between dvt prevention and the risk of bleeding , which is another major complication of bariatric surgery . recently , many different and complex changes in plasma coagulation factors have been described in patients with obesity . obesity is linked to elevated expression of the prothrombotic molecules plasminogen activator inhibitor-1 and tissue factor ( tf ) and increased platelet activation . a significant increase in median levels of circulating microparticles was observed in obese patients compared to controls , including annexin v - mp , endothelial - derived , leukocyte - derived , and tissue factor bearing mp compared to controls . the same study also showed that obese patients had a significantly higher median peak thrombin and increased median endogenous thrombin potential compared to controls . the same group of researchers has shown that after sleeve gastrectomy , the decrease of bmi in morbidly obese subjects was matched with a decrease of circulating microparticles of endothelial , platelet , and leukocyte origin . thromboelastography ( teg ) is used to monitor viscoelastic properties of blood clots , from formation to lysis . over the last decade , teg has gained significant importance in the management of high - risk bleeding populations , liver transplantation , and certain other clinical conditions . although teg is mostly used to monitor hypocoagulability states , recent evidence shows the importance of teg in evaluation of hypercoagulability in the perioperative setting . thromboelastography has recently gained wide scientific attention in obesity and hypercoagulation trends observed in these individuals . a clear relationship between hypercoagulability detected by whole - blood thromboelastometry and aggregometry and several studies addressed the role of hypercoagulability monitoring in the perioperative period of bariatric surgery . a recent systematic review about hypercoagulable state and teg indicated that further studies are needed to determine the ultimate role of this method for prediction of hypercoagulable states and postoperative thrombosis . the aim of this study was to determine hypercoagulability states in obese patients undergoing bariatric surgery and to assess the alterations of coagulation parameters in the perioperative setting using teg . we selected the cutoff values of teg parameters for defining hypercoagulability based on the studies discussed above . we also evaluated the potential of clinical and standard laboratory tests to predict hypercoagulable states in patients referred to bariatric surgery . sixty consecutive patients undergoing laparoscopic bariatric surgery ( gastric bypass or gastric plication ) were recruited at the department of surgery , lithuanian university of health sciences hospital from october 2014 to june 2015 . all the patients underwent general anesthesia in normothermal conditions and were given standardized thromboembolic prophylaxis with 500 ml 6% dextran 70 ( fresenius , poland ) after induction of anesthesia and fraxiparine 0.3 ml sc ( glaxosmithkline , poland ) starting in the morning of pod1 until discharge from the hospital . we excluded patients on anticoagulation therapy , as well as those with chronic kidney / liver failure , coagulation disorders ( liver cirrhosis , known hematological , or hereditary coagulation disorder ) , previous history of dvt , or using oral contraception . the patients were followed for clinically evident thrombotic complications for 1 year after the study by arranging clinical appointments 1 month and 1 year after the surgery . the study was approved by the regional kaunas ethics committee ( be-2 - 10 ) . thromboelastography was performed using the teg 5000 thromboelastograph hemostasis analyzer system ( braintree , usa ) . thromboelastographic alterations were assessed at 4 time points : at baseline prior to induction of anesthesia , immediately after the end of the surgery , and on the morning of postoperative days 1 ( pod1 ) and 2 ( pod2 ) . the following parameters of thromboelastography were assessed : r - time ( reaction time , min . ; normal range [ nr ] : 510 ) ; k - time ( clotting time , min ; nr : 13 ) ; -angle ( degrees ; nr : 5372 ) ; ma parameter ( maximum amplitude , millimeters ; nr : 5070 ) ; and g ( clot strength , dynes per square centimeter ; nr : 4.610.9 ) . briefly , mixing 1 ml of citrated blood with kaolin by an inversion technique was performed by the physician conducting teg . then , 20 l of 0.2 m calcium chloride was added to the plain teg cup , followed by 340 l of the blood / kaolin mix . data ( age , bmi , coagulation parameters , and teg values ) are presented as mean values and standard deviation ( sd ) . qualitative variables were compared using the chi - square or fisher s exact test , as appropriate . spearman correlation analysis was performed to determine the correlation between clinical or standard laboratory test values ( crp , esr , d - dimers , and fibrinogen ) and teg parameters . receiver operating characteristic ( roc ) curve analysis was performed to determine optimal fibrinogen levels for prediction of hypercoagulable states g 11 dynes / cm and ma 68 mm . statistical analysis was performed using the spss 20.0 software package ( chicago , il ) . sixty consecutive patients undergoing laparoscopic bariatric surgery ( gastric bypass or gastric plication ) were recruited at the department of surgery , lithuanian university of health sciences hospital from october 2014 to june 2015 . all the patients underwent general anesthesia in normothermal conditions and were given standardized thromboembolic prophylaxis with 500 ml 6% dextran 70 ( fresenius , poland ) after induction of anesthesia and fraxiparine 0.3 ml sc ( glaxosmithkline , poland ) starting in the morning of pod1 until discharge from the hospital . we excluded patients on anticoagulation therapy , as well as those with chronic kidney / liver failure , coagulation disorders ( liver cirrhosis , known hematological , or hereditary coagulation disorder ) , previous history of dvt , or using oral contraception . the patients were followed for clinically evident thrombotic complications for 1 year after the study by arranging clinical appointments 1 month and 1 year after the surgery . the study was approved by the regional kaunas ethics committee ( be-2 - 10 ) . thromboelastography was performed using the teg 5000 thromboelastograph hemostasis analyzer system ( braintree , usa ) . thromboelastographic alterations were assessed at 4 time points : at baseline prior to induction of anesthesia , immediately after the end of the surgery , and on the morning of postoperative days 1 ( pod1 ) and 2 ( pod2 ) . the following parameters of thromboelastography were assessed : r - time ( reaction time , min . ; normal range [ nr ] : 510 ) ; k - time ( clotting time , min ; nr : 13 ) ; -angle ( degrees ; nr : 5372 ) ; ma parameter ( maximum amplitude , millimeters ; nr : 5070 ) ; and g ( clot strength , dynes per square centimeter ; nr : 4.610.9 ) . briefly , mixing 1 ml of citrated blood with kaolin by an inversion technique was performed by the physician conducting teg . then , 20 l of 0.2 m calcium chloride was added to the plain teg cup , followed by 340 l of the blood / kaolin mix . data normality was assessed using kolmogorov - smirnov test . data ( age , bmi , coagulation parameters , and teg values ) qualitative variables were compared using the chi - square or fisher s exact test , as appropriate . spearman correlation analysis was performed to determine the correlation between clinical or standard laboratory test values ( crp , esr , d - dimers , and fibrinogen ) and teg parameters . receiver operating characteristic ( roc ) curve analysis was performed to determine optimal fibrinogen levels for prediction of hypercoagulable states g 11 dynes / cm and ma 68 mm . statistical analysis was performed using the spss 20.0 software package ( chicago , il ) . the study included 60 consecutive patients ( 18 males and 42 females ) referred to bariatric surgery , with a mean age of 39.111.9 years ( age range 1862 ) and a mean bmi of 47.58.5 years ( bmi range 34.384.9 ) . gastric bypass surgery was performed in 38 ( 63.3% ) patients and gastric plication in 22 ( 36.7% ) patients . twenty - one patients ( 35% ) were active smokers at the time of the surgery , and 39 patients ( 65% ) had previous diagnosis of arterial hypertension . we followed all our patients for clinically evident thrombotic complications for 1 year after the study and none of them had thrombotic events during the follow - up period ( data not shown ) . forty - six patients ( 76.7% ) had g parameter < 11 and 14 patients ( 23.3% ) exhibited hypercoagulability prior to surgery , as defined by g value 11 ( table 1 ) . comparison of clinical parameters and standard laboratory tests between the 2 groups ( g < 11 vs. g 11 ) did not reveal significant differences in bmi and crp levels ( table 1 ) . the age of patients in the g < 11 group was lower ( 37.8 ) than in the g 11 group ( 43.6 ) , but the difference was not significant ( p=0.117 ) . the only parameter that was significantly higher in the g 11 group compared to the g < 11 group was fibrinogen levels , which were 4.23 and 3.8 g / l , respectively ( p=0.021 ; table 1 ) . among all study participants , 17 patients ( 28.3% ) had ma 68 mm and 43 patients ( 71.7% ) had ma < 68 mm . patients with ma 68 were significantly older ( 44.2 years ) than in the group of patients with ma < 68 mm ( 37.1 years ; p=0.046 ; table 1 ) . fibrinogen levels were also higher in the hypercoagulable group ( ma 68 mm ) than in the remaining subjects with ma < 68 mm ( table 1 , p=0.025 ) mean values of teg parameters in the perioperative period of study participants are summarized in table 2 . there was an increase in r - time from preoperative levels ( 6.74 s ) to pod1 ( 6.96 s ) , but it reached significance only at pod2 ( 7.43 s , p=0.022 ; table 2 ) . k - time and a - angle remained at levels similar to the initial teg results recorded on pod1 and pod2 ( table 2 ) . there was a trend towards hypercoagulability and higher values of ma and g compared with initial teg values and results obtained on pod2 ; however , the differences were not significant ( table 2 ) . dynamics of fibrinogen and d - dimer during the perioperative period are also presented in table 2 . there was a significant increase in d - dimer levels in the postoperative period after bariatric surgery . average fibrinogen levels also increased significantly from basal values prior to surgery , from 3.90 to 4.16 we also aimed to assess the changes in hypercoagulability status from baseline to pod2 . at baseline , 46 patients ( 76.7% ) had g parameter < 11 ( table 3 ) , but on pod2 13.0% of this group had g 11 . out 14 patients ( 23.3% ) showing hypercoagulability prior to surgery , as defined by g value 11 , 12 patients remained hypercoagulable on pod2 ( table 3 ) . prior to surgery , 17 patients ( 28.3% ) had ma 68 mm and 14 of them remained hypercoagulable on pod2 ( table 3 ) . forty - three patients ( 71.7% ) had ma < 68 mm at baseline , but 7 of them became hypercoagulable by pod2 . to assess the relationship between clinical variables and standard laboratory parameters with baseline teg values , we performed correlation analysis ( table 4 ) . similarly , markers of inflammation , crp , and erythrocyte sedimentation rate ( esr ) also did not correlate with coagulation parameters recorded by teg ( table 4 ) . preoperative fibrinogen levels were associated with k - time ( r=0.313 , p=0.018 ) , -angle ( r=0.333 , p=0.011 ) , ma ( r=0.431 , p=0.001 ) , and g ( r=0.387 , p=0.003 ; table 4 ) . interestingly , we also observed a weak but significant correlation between age and r - time ( r=0.280 , p=0.03 ) , k - time ( r=0.259 , p=0.046 ) , and -angle ( r=0.260 , p=0.045 , table 4 ) . to evaluate the potential of plasma fibrinogen levels to predict hypercoagulability measured by teg , we performed roc curve analysis . figure 1 shows that plasma fibrinogen level could predict clot strength ( g ) 11 dynes / cm with area under the curve ( auc)=0.680 ( 95% confidence interval ( ci ) : 0.530.80 , p=0.044 ) . roc curve analysis of plasma fibrinogen levels to predict ma 68 mm produced an auc of 0.679 ( 95% ci : 0.530.83 , p=0.041 , figure 2 ) . g / l , with sensitivity of 80.0% and specificity of 64.3% ( figure 2 ) . the study included 60 consecutive patients ( 18 males and 42 females ) referred to bariatric surgery , with a mean age of 39.111.9 years ( age range 1862 ) and a mean bmi of 47.58.5 years ( bmi range 34.384.9 ) . gastric bypass surgery was performed in 38 ( 63.3% ) patients and gastric plication in 22 ( 36.7% ) patients . twenty - one patients ( 35% ) were active smokers at the time of the surgery , and 39 patients ( 65% ) had previous diagnosis of arterial hypertension . we followed all our patients for clinically evident thrombotic complications for 1 year after the study and none of them had thrombotic events during the follow - up period ( data not shown ) . forty - six patients ( 76.7% ) had g parameter < 11 and 14 patients ( 23.3% ) exhibited hypercoagulability prior to surgery , as defined by g value 11 ( table 1 ) . comparison of clinical parameters and standard laboratory tests between the 2 groups ( g < 11 vs. g 11 ) did not reveal significant differences in bmi and crp levels ( table 1 ) . the age of patients in the g < 11 group was lower ( 37.8 ) than in the g 11 group ( 43.6 ) , but the difference was not significant ( p=0.117 ) . the only parameter that was significantly higher in the g 11 group compared to the g < 11 group was fibrinogen levels , which were 4.23 and 3.8 g / l , respectively ( p=0.021 ; table 1 ) . among all study participants , 17 patients ( 28.3% ) had ma 68 mm and 43 patients ( 71.7% ) had ma < 68 mm . patients with ma 68 were significantly older ( 44.2 years ) than in the group of patients with ma < 68 mm ( 37.1 years ; p=0.046 ; table 1 ) . fibrinogen levels were also higher in the hypercoagulable group ( ma 68 mm ) than in the remaining subjects with ma < 68 mm ( table 1 , p=0.025 ) mean values of teg parameters in the perioperative period of study participants are summarized in table 2 . there was an increase in r - time from preoperative levels ( 6.74 s ) to pod1 ( 6.96 s ) , but it reached significance only at pod2 ( 7.43 s , p=0.022 ; table 2 ) . k - time and a - angle remained at levels similar to the initial teg results recorded on pod1 and pod2 ( table 2 ) . there was a trend towards hypercoagulability and higher values of ma and g compared with initial teg values and results obtained on pod2 ; however , the differences were not significant ( table 2 ) . dynamics of fibrinogen and d - dimer during the perioperative period are also presented in table 2 . there was a significant increase in d - dimer levels in the postoperative period after bariatric surgery . average fibrinogen levels also increased significantly from basal values prior to surgery , from 3.90 to 4.16 we also aimed to assess the changes in hypercoagulability status from baseline to pod2 . at baseline , 46 patients ( 76.7% ) had g parameter < 11 ( table 3 ) , but on pod2 13.0% of this group had g 11 . out 14 patients ( 23.3% ) showing hypercoagulability prior to surgery , as defined by g value 11 , 12 patients remained hypercoagulable on pod2 ( table 3 ) . prior to surgery , 17 patients ( 28.3% ) had ma 68 mm and 14 of them remained hypercoagulable on pod2 ( table 3 ) . forty - three patients ( 71.7% ) had ma < 68 mm at baseline , but 7 of them became hypercoagulable by pod2 . to assess the relationship between clinical variables and standard laboratory parameters with baseline teg values , we performed correlation analysis ( table 4 ) . similarly , markers of inflammation , crp , and erythrocyte sedimentation rate ( esr ) also did not correlate with coagulation parameters recorded by teg ( table 4 ) . preoperative fibrinogen levels were associated with k - time ( r=0.313 , p=0.018 ) , -angle ( r=0.333 , p=0.011 ) , ma ( r=0.431 , p=0.001 ) , and g ( r=0.387 , p=0.003 ; table 4 ) . interestingly , we also observed a weak but significant correlation between age and r - time ( r=0.280 , p=0.03 ) , k - time ( r=0.259 , p=0.046 ) , and -angle ( r=0.260 , p=0.045 , table 4 ) . to evaluate the potential of plasma fibrinogen levels to predict hypercoagulability measured by teg , we performed roc curve analysis . figure 1 shows that plasma fibrinogen level could predict clot strength ( g ) 11 dynes / cm with area under the curve ( auc)=0.680 ( 95% confidence interval ( ci ) : 0.530.80 , p=0.044 ) . the best cutoff point for fibrinogen to predict g 11 was 3.97 roc curve analysis of plasma fibrinogen levels to predict ma 68 mm produced an auc of 0.679 ( 95% ci : 0.530.83 , p=0.041 , figure 2 ) . g / l , with sensitivity of 80.0% and specificity of 64.3% ( figure 2 ) . much research has been targeted at establishment of optimal prophylactic anticoagulation regimens in bariatric surgery ; however , a consensus over this strategy has not been achieved . although the incidence of thromboembolic complications in patients undergoing bariatric surgery is not very high , the mortality rates associated with pe in this subgroup of patients is striking . routine coagulation tests , including aptt , inr , and platelet count , are not able to predict hypercoagulative status in the perioperative period . therefore , alternative testing and monitoring of coagulation status for identification of individuals at increased risk of vte is needed . in the present study we aimed to evaluate coagulation status in patients undergoing bariatric surgery and to assess dynamics of coagulation parameters in the perioperative setting using teg . the major finding of our study is that many patients referred to bariatric surgery show a trend towards hypercoagulability on teg . we used teg cutoff values of g 11 dynes / cm and ma 68 mm to define hypercoagulability based on previous studies that have shown these thresholds to be linked with thrombotic events . our results also show that fibrinogen level is the only indicator of hypercoagulative state among the routinely used laboratory parameters ; however , used alone , it is not able accurately predict hypercoagulative status . we believe that the results of our study add new insights into the monitoring of coagulation status in bariatric surgery . an increasing number of studies show the importance of teg for monitoring of coagulation status in the perioperative period . the major finding of the present study is that many patients referred to bariatric surgery show hypercoagulability as determined by teg in the perioperative setting . based on results of previous studies , we used g 11 dynes / cm to define a hypercoagulable state found that strongest predictor of vte in surgical patients was elevated g value ; for every 1 dyne / cm increase in g , the odds of a vte increased by 25% . a significant proportion ( 23% ) of our patients were in hypercoagulable state prior to surgery , which supports previous data obtained by rotem analysis . it is important to point out that 18 patients ( 30% ) also had g 11 at pod2 . we routinely stop low molecular weight heparins ( lmwh ) on discharge and this group of patients might benefit from extended antithrombotic prophylaxis , but this remains to be assessed in further studies . interestingly , we followed all our patients for clinically evident thrombotic complications for 1 year after the study and none of them had thrombotic events during the follow - up period . the other teg parameter that may be used to identify hypercoagulable states and to predict dvt in the postoperative period is ma . recent studies have also shown that ma parameter 68 mm is linked with increased risk of thrombotic complications and postoperative myocardial infarction . it is worth pointing out that in our study 17 out 60 patients had ma 68 mm prior to surgery , but this proportion did not increase dramatically at pod2 ( 28.3% and 35.0% , respectively ) ; however , forfori et al . showed a substantial ( from 20% to 50% ) increase in the percentage of patients with ma 68 mm by pod3 . a potential reason why we did not observe a significant increase in the proportion of patients with ma 68 on pod2 could be the shorter overall surgery time ( 91.420.0 min ) as compared to the forfori et al . another reason might be related to the different regimens used for thrombosis prophylaxis . in our study , patients received dextran solution during induction of anesthesia . dextran is known for its anti - platelet activity realized through reduction of platelet adhesion to von willebrand factor and impairment of platelet activation by thrombin . we also observed a significant increase in r - time from preoperative levels to pod2 . these results indicate that a prophylactic regimen of 0.3 ml fraxiparine qd started on pod1 seems to reduce activation of the coagulation system in the postoperative period , similar to the prophylaxis regimen based solely on fraxiparine . were the first to show that morbidly obese patients have accelerated fibrin formation and higher ma values than lean patients referred to elective surgery , as determined by teg . in our study , the patients with clot strength g 11 dynes / cm had higher average plasma fibrinogen concentration than patients in the g < 11 group , and these findings support previously published results . we observed significant correlations between fibrinogen concentration and k - time , -angle , ma , and g values as determined by teg . to evaluate the potential of plasma fibrinogen levels to predict hypercoagulability measured by teg plasma fibrinogen level predicted clot strength ( g ) 11 dynes / cm with an auc=0.680 and the optimal cutoff point for fibrinogen to predict g 11 was 3.97 similarly , roc curve analysis of plasma fibrinogen levels to predict ma 68 mm resulted in auc of 0.679 with an optimal cutoff point for fibrinogen at 3.85 similar cutoff values of fibrinogen for prediction of hypercoagulability were observed in a spanish study . our results show that fibrinogen level is the only indicator of hypercoagulative state among routinely used laboratory parameters ; however , moderate auc values obtained in the study should be reassessed in larger studies . it is worth pointing out that some studies have shown that hyperfibrinogenemia is a potential factor in heparin resistance , and these patients might need enhanced vte prophylaxis regimens . obese patients have a higher prevalence of inherited and acquired thrombophilias compared to the general population . diabetes , which is closely linked to obesity , is also related to increased incidence of recurrent dvt . a recent study has reported that the inflammation biomarker crp was associated with hypercoagulable states evaluated by rotem in a group of patients referred to bariatric surgery . in the present study we did not observe a significant correlation between teg parameters and crp or esr levels . several previous studies have shown that bmi increases the risk of vte . in the present study , bmi did not correlate with teg values , which was also observed previously by a research group . a recent study showed that central obesity , but now overall weight , is linked with increased thrombin generation and is a better predictor of complications . firstly , it was not aimed at determining the optimal anticoagulation regimes for bariatric surgery , and that remains a challenging task in further studies . we have selected the teg cutoff values based on some previous studies that have shown these thresholds to be linked with the risk of thrombotic events and used them in earlier studies : g 11 dynes / cm and ma parameter ma 68 mm ; however , we agree that the prediction of prothrombotic states based on teg results only is difficult and further research in the field is required . we also did not use doppler ultrasound to monitor subclinical vein thrombosis ; therefore , we might have missed some cases of obscure vein thrombosis in the follow - up . to date , it is not clear whether patients showing hypercoagulation parameters on teg should be managed differently , and the optimal strategies for dvt prevention in this group have yet to be established in well - designed randomized clinical trials . interestingly , some studies have suggested the need for extended anticoagulation regimens after bariatric surgery because some dvt occurs later after surgery . we speculate that patients showing hypercoagulability on pod2 by teg might benefit from receiving a prolonged prophylactic anticoagulation , but this needs to be assessed in further studies . potentially , a combination of teg results and other coagulation markers could provide more accurate prediction models , and this needs to be established in the future . bariatric surgery is clearly associated with significant weight loss and will retain a major role in treatment of morbid obesity . furthermore , the balance between the risk of bleeding and risk of thrombotic complications remains very important , posing further challenges in the field . a considerable proportion of patients referred to bariatric surgery show a trend towards hypercoagulability as determined by teg . this study shows the potential of hypercoagulation monitoring by teg in the perioperative setting of bariatric surgery .

backgroundobesity is known as a major risk factor for postoperative vein thrombosis . thromboelastography ( teg ) is used to monitor viscoelastic features of blood clots . the aim of this study was to determine hypercoagulable states in patients undergoing bariatric surgery and to assess dynamics of coagulation parameters in the perioperative setting using teg.material/methodswe included 60 consecutive patients undergoing bariatric surgery . teg alterations were assessed at 4 time points : at baseline , after the surgery , and on postoperative day 1 ( pod1 ) and 2 ( pod2 ) . hypercoagulable state was defined when patients showed clot strength ( g ) of 11 dynes / cm2 or maximum amplitude ( ma ) 68 mm.resultsfourteen patients ( 23.3% ) out of 60 showed hypercoagulability prior to surgery on teg . fibrinogen levels were significantly higher in the g 11 group compared to the g < 11 group , at 4.2 and 3.8 g / l , respectively ( p=0.02 ) . seventeen patients ( 28.3% ) had ma 68 mm at baseline . fibrinogen levels increased significantly from 3.90 at baseline to 4.16 g / l in pod2 ( p<0.001 ) . there was an increase in mean reaction time from baseline ( 6.74 s ) to pod2 ( 7.43 s , p=0.022 ) . we found a correlation between baseline fibrinogen levels and ma ( r=0.431 , p=0.001 ) or g ( r=0.387 , p=0.003 ) . roc curve analysis showed that fibrinogen levels can predict clot strength ( g ) 11 dynes / cm2 with auc=0.680 ( p=0.044).conclusionsa considerable proportion of patients referred to bariatric surgery show a trend towards hypercoagulability on teg . this study shows the potential of hypercoagulation monitoring by teg in the perioperative setting of bariatric surgery .

it is a multisystem disorder affecting the gastrointestinal , respiratory , hepatobiliary , and reproductive systems , as well as the sweat glands ( 1 - 6 ) . cf is caused by a mutation in the cystictransmembrane conductance regulator ( cftr ) gene , leading to defective ion transport of the cftr protein ( 7 - 10 ) . although numerous mutations in the cftr gene have been reported as a primary cause in the clinical manifestations and outcome of cf ( 9 ) , recent research has focused on the role of other factors such as gender ( 11 , 12 ) , race ( 1 ) , age ( 11 ) , geographic area ( 2 ) , infection with pseudomonas aeruginosa ( 11 , 13 ) , religion ( 8) , culture ( 1 , 14 ) , and nutritional status ( 13 - 15 ) . recently , improvement and progress in outcomes have been observed through better understanding of the disease , finding the effective factors in the start and progression of the disease , and ultimately in better management of the disease ( 16 ) . these factors have led to earlier diagnosis and more timely treatment , which could affect the clinical course and change the outcome for a patient . therefore , the link among researchers and clinicians will be stronger due to extensive research on the disease and improved introduction of the disease ( 17 , 18 ) . in addition , the research results could have an effective role in changing of the culture and laws of a society . retrospective research , in spite of some disadvantages , plays an important role in depicting the disease ( 16 , 19 ) . this research has been conducted after searching the electronic databases and performing a literature review , to ensure that no similar study had been undertaken in the region . this study aimed to assess the association between outcomes and demographic status in azeri turkish patients with cystic fibrosis . this was a cross sectional study conducted at the educational and treatment children s hospital of the university of medical sciences and the medical genetic laboratory , tabriz , iran , from march 2001 to september 2014 . the educational and treatment children s hospital is a governmental , specialized , and referral hospital with 150 beds and six wards in northwestern iran and dr . m. rafeey is the top , and the most professional , physician in this field and region . this study was performed on azeri turks , who are members of one of the largest ethnic groups in iran ( 20 ) . all the medical records of the patients were reviewed using the census method , and further information was gathered from patients , or their parents , through telephone interviews . the diagnosis of cf was based on typical clinical features and confirmed by sweat chloride level > 60 meq / l , according to the method of gibson and cooke , or detection of mutations in the cftr gene known to cause cf ( 21 ) . the medical records of 442 patients who met the inclusion criteria were separately reviewed by two authors . patients with incomplete records and who were not diagnosed as having cf were excluded with the reviewer s agreement . the kappa agreement rate was higher than 85% , as inclusion criteria to the next phase . in the end , 331 cases were included in this study , which was conducted over a 13-year period from 2001 to 2014 . data included patient outcome , gender , age , residence , ranking in birth order of the family s children , family history , consanguineous marriage , degree of consanguineous marriage , and weight at birth . ranking in birth order of the family s children was divided into two groups , including one child as the first group , and two children or more as the second group . a positive family history for cf was considered as having had a first , second , or third - degree relative affected with cf ( 22 ) . a consanguineous marriage was defined as a union between two persons who are related as first cousins ( 3rd degree ) , first cousins once removed or double second cousins ( 4 degree ) , and second cousins ( 5 degree ) . the first degree and the second degree , first cousins were classified as the first group and the others as the second group . demographic data were analyzed using spss 18 for means and standard deviations for normally distributed continuous variables , and median and interquartile ranges were obtained for non - normally distributed continuous variables . the comparison between variables was analyzed with a chi - square or fisher s exact test , and independent samples t test . logistic regression analyses were carried out to identify variables independently associated with gender . in all analyses , odds ratio ( or ) with a 95% confidence interval , and p < 0.05 indicated statistical significance . ethical aspects were considered and approval for the study was gained by the ethics committee of the university ( no = 5/4/1775 ) . participants were assured of confidentiality , and consent was obtained from the patients or their parents . the diagnosis of cf was based on typical clinical features and confirmed by sweat chloride level > 60 meq / l , according to the method of gibson and cooke , or detection of mutations in the cftr gene known to cause cf ( 21 ) . the medical records of 442 patients who met the inclusion criteria were separately reviewed by two authors . patients with incomplete records and who were not diagnosed as having cf were excluded with the reviewer s agreement . the kappa agreement rate was higher than 85% , as inclusion criteria to the next phase . in the end , 331 cases were included in this study , which was conducted over a 13-year period from 2001 to 2014 . data included patient outcome , gender , age , residence , ranking in birth order of the family s children , family history , consanguineous marriage , degree of consanguineous marriage , and weight at birth . ranking in birth order of the family s children was divided into two groups , including one child as the first group , and two children or more as the second group . a positive family history for cf was considered as having had a first , second , or third - degree relative affected with cf ( 22 ) . a consanguineous marriage was defined as a union between two persons who are related as first cousins ( 3rd degree ) , first cousins once removed or double second cousins ( 4 degree ) , and second cousins ( 5 degree ) . the first degree and the second degree , first cousins were classified as the first group and the others as the second group . demographic data were analyzed using spss 18 for means and standard deviations for normally distributed continuous variables , and median and interquartile ranges were obtained for non - normally distributed continuous variables . the comparison between variables was analyzed with a chi - square or fisher s exact test , and independent samples t test . logistic regression analyses were carried out to identify variables independently associated with gender . in all analyses , odds ratio ( or ) with a 95% confidence interval , and p < 0.05 indicated statistical significance . ethical aspects were considered and approval for the study was gained by the ethics committee of the university ( no = 5/4/1775 ) . participants were assured of confidentiality , and consent was obtained from the patients or their parents . demographic data of this study is shown in table 1.there was a significant difference between frequencies of deceased and living patients ( p < 0.001 ) . in the application of the kolmogorov - smirnov test , age was assumed as non - normal distribution that uses the sample median and the iqr ( minimum - maximum ) , consequently , median age for living patients was 0.4 years ( 7 days30 years ) and median age at death was 4.8 years ( 3 months 43 years ) . abbreviations : d , days ; iqr , minimum - maximum ; m , months ; y , years . all data were mentioned as frequency ( % ) . for deceased cf - patients , median age at death and living cf - patients , median age at time of the study have been considered , because of non - normal of age and weight at birth , median and iqr were applied . eighty - five patients expired during the 13 years of the study with a mean of six deaths per year and at a ratio of 1:3 . during the time of the study , 77 ( 90.6% ) of the 85 deceased cf patients had died under the age of four years . among the 246 living cf patients , 131 ( 53.3% ) were less than four years of age . ( % ) . at first , we calculated the association between outcome and gender . at the time of the study , 51 ( 26.7% ) of the deceased patients were males and 34(24.3% ) were females . the test did not show any significant difference between outcome and gender ( or = 1.13 [ 95% ci , 0.68 - 1.87 ] , p = 0.6 ) . in the next step , there was a significant difference between the outcome and the geographic location ( or = 0.55 [ 95% ci , 0.32 - 0.95 ] , p = 0.03 ) . there was no significant difference between outcome and ranking among the family s children , while 38 ( 23.9% ) were first - born children , and 47 ( 27.3% ) were second or later born children ( or = 0.83 [ 95% ci , 0.50 - 1.37 ] , p = 0.4 ) . in this study , we analyzed family history status among these patients , and the risk of mortality was approximately two times higher in patients with a positive family history than in those with a negative family history ( or = 1.91 [ 95% ci , 1.09 - 3.36 ] , p = 0.02 ) . there was a statistically significant difference between the outcome and consanguineous marriage ( or = 1.94 [ 95% ci , 1.14 - 3.29 ] , p = 0.01 ) , but no significant difference in degree of positive consanguineous marriage ( or = 0.68 [ 95% ci , 0.34 - 1.37 ] , p = 0.2 ) . the risk of mortality was 1.94 times higher for patients in consanguineous marriages than for those in non - consanguineous marriages . finally , weight at birth was computed and compared in cf patients , both deceased and living . there was no significant difference between the outcome and weight at birth ( or = 1 [ 95% ci , 1 - 1.001 ] , p = 0.7 ) ( tables 1 and 3 ) . there was no statistically significant difference when logistic regression analysis was considered with the or for all of the variables ( p > 0.05 ) . associations between gender , ranking in birth order of the family s children , degree of consanguineous marriage , and weight at birth were not significant in terms of outcome , and therefore , omitted from the final model . when residential area , family history , and consanguineous marriage were entered into the model , the difference associated with outcome and or was somewhat reduced . results of logistic regression analysis illustrated the adjusted or for the final model and are shown in table 3 . at first , we calculated the association between outcome and gender . at the time of the study , 51 ( 26.7% ) of the deceased patients were males and 34(24.3% ) were females . the test did not show any significant difference between outcome and gender ( or = 1.13 [ 95% ci , 0.68 - 1.87 ] , p = 0.6 ) . in the next step , there was a significant difference between the outcome and the geographic location ( or = 0.55 [ 95% ci , 0.32 - 0.95 ] , p = 0.03 ) . there was no significant difference between outcome and ranking among the family s children , while 38 ( 23.9% ) were first - born children , and 47 ( 27.3% ) were second or later born children ( or = 0.83 [ 95% ci , 0.50 - 1.37 ] , p = 0.4 ) . in this study , we analyzed family history status among these patients , and the risk of mortality was approximately two times higher in patients with a positive family history than in those with a negative family history ( or = 1.91 [ 95% ci , 1.09 - 3.36 ] , p = 0.02 ) . there was a statistically significant difference between the outcome and consanguineous marriage ( or = 1.94 [ 95% ci , 1.14 - 3.29 ] , p = 0.01 ) , but no significant difference in degree of positive consanguineous marriage ( or = 0.68 [ 95% ci , 0.34 - 1.37 ] , p = 0.2 ) . the risk of mortality was 1.94 times higher for patients in consanguineous marriages than for those in non - consanguineous marriages . finally , weight at birth was computed and compared in cf patients , both deceased and living . there was no significant difference between the outcome and weight at birth ( or = 1 [ 95% ci , 1 - 1.001 ] , p = 0.7 ) ( tables 1 and 3 ) . there was no statistically significant difference when logistic regression analysis was considered with the or for all of the variables ( p > 0.05 ) . associations between gender , ranking in birth order of the family s children , degree of consanguineous marriage , and weight at birth were not significant in terms of outcome , and therefore , omitted from the final model . when residential area , family history , and consanguineous marriage were entered into the model , the difference associated with outcome and or was somewhat reduced . results of logistic regression analysis illustrated the adjusted or for the final model and are shown in table 3 . the association between non - genetic variables and mortality in individuals with cf has been established ( 2 , 6 ) . despite improved management and increased awareness , outcome remains an important issue for cf patients , because non - genetic parameters , especially demographic factors , have been ignored by researchers and clinicians . the current study was performed to highlight the associations between demographic parameters and outcomes in cf patients . in this study , the ratio of deceased patients to living patients was 1:3 , with a significant difference . eighty - five patients expired during the period of 13 years , with a mean of six deaths per year . in a study performed by dodge et al . in the uk from 1996 to 2003 , there were 1,066 deaths , with a mean of 133 deaths per year ( 15 ) . therefore , based on these findings the mortality rate is lower in our study than in the uk , perhaps because this study has been performed on an ethnic group in iran with a low incidence of cf . in addition , it is likely that a number of deaths were not recorded or were not reported by the family in our study . in a previous study in sweden , lannefors et al . reported data from 1971 - 1999 showing that while the incidence of cf was 1/5600 live - births , the mean and median age of living cf patients were 18 12 and 16 years , respectively . in this study , regarding deceased patients , 45% were older than 18 years and 10% were older than 35 years . in deceased patients , the median age at death was 26 years ( range 0 - 72 ) ( 2 ) . in another study , also , this statistic had been observed in median age at death . delayed diagnosis and inappropriate health care were risk factors for a younger median age at death in non - european countries ( 24 ) . in our study , living cf patients had lower mean , median , and were of an older age than that reported in developed countries . also , in deceased cf patients the mean and median age at death were lower than in developed countries . we hypothesize that this difference may be due to the lack of proper equipment , inadequate diagnostic processes , and absence of treatment by specialists and expert staff , as well as poor nutritional status in our region compared to developed countries ( 13 , 24 ) . in our study , there was no significant difference between outcome and gender ; however , the mortality rate was somewhat higher in males than females . a number of studies have indicated that males have a better survival rate than females , while lannefors et al . in another study , accumulated data related to the mortality rate between genders reflects a higher mortality rate in cf females compared to males ( 6 ) . cultural issues and intrinsic ethnic differences ( 1 ) , as well as religion ( 8 , 25 ) and geographical elements , ( 9 ) for example , females are more susceptible to cf because of their body functions ; consequently , they more often report problems than males . expression of problems can be observed more in females than males in our region because of cultural issues ( 26 ) . reasons for this difference may be unclear , so it is necessary to perform additional investigations in a large population from neighboring countries to elucidate this issue . in the current study , there was a significant difference between outcome and location of residence . the mortality rate of cf was 50% higher in rural patients than urban patients , in spite of less air pollution ( 10 ) , lower stress , sufficient exercise , and organic nutritional intake in rural areas ( 13 , 27 ) . the availability of appropriate facilities to diagnosis and treat the disease is an important factor in patient survival . growth and lung health of children is influenced by proper treatment , and decreased pulmonary function is associated with morbidity and shortened survival for cf patients ( 28 ) . continuous care , suitable food , and daily physiotherapy are the main elements in survival of cf ( 27 ) . the relationship between improved nutritional support and increased survival in cf has been established , and retention of balanced and stable nutritional status is the best method of treatment for cf ( 13 , 27 ) . preventive treatments , such as adequate exercise , have been introduced at early ages for cf patients , and it appears to be an important factor in health maintenance in patients with cf ( 27 ) . in addition , a healthy lifestyle ( 27 ) , related to appropriate family functioning ( 29 ) , elevated levels of social class , education , and higher income ( 19 , 30 ) are determinates of survival for cf patients . patients living in urban areas are usually more likely to benefit from these conditions . in this study , there was no significant difference between outcome and ranking in birth order of the family s children , although the mortality rate was slightly higher for the second or subsequent children than for the first - born . management of patients with cf is one of the greatest challenges for families , and the family plays an important role in the treatment of cf ( 30 ) . cf patients need constant care such as physiotherapy , various medications , proper dietary intake , exercise , and adequate support ( 21 , 31 ) . on the other hand , high socioeconomic status with adequate income , suitable occupation , and employment status were identified as factors related to a good prognosis in affected individuals ( 30 ) . in crowded families , patients with a positive family history , such as a diagnostic category ( 32 , 33 ) , had a higher mortality rate than those with a negative family history . there was a significant difference , with the risk of mortality approximately two times , or 91% , higher with a positive family history than a negative family history , in the current study . ormond et al . published findings of a study stating that individuals affected by cf are segregated into four categories based on severity of disease . patients with a family history of cf showed a broader range of superficial severity with an unknown reason ( 34).understanding the reasons these patients have poorer prognoses than others will need additional research . considering consanguineous marriage as a predicating factor of outcome , children born to related parents were 94% more at risk for death than children of non - consanguineous marriage , especially in consanguineous marriage of the first - cousin type ( 3rd - degree ) . social conditions , religion , economic situations , and traditional beliefs are essential considerations in the preference for consanguineous marriage . in muslim populations , first - cousin unions ( 3rd - degree ) between a man and his father s or mother s brother s daughter , and the opposite , were detected frequently . the incidence of consanguinity has been reported to be 56% in middle east with saudi arabia having the highest prevalence at 89% in families with cf ( 35 ) . most studies have shown that early mortality is higher in the progeny of consanguineous unions than in non - consanguineous marriage ( 36 ) . ultimately , weight at birth was reviewed in deceased patients and living patients and no significant difference between them was found . the mean of weight at birth in the living cf population was slightly higher than the deceased cf population . cf is an autosomal recessive disorder affecting several organs , such as the digestive system , which is manifested in fat malabsorption , steatorrhea , and poor growth ( 2 , 21 ) . patients who intake adequate dietary requirements and gain proper weight have better pulmonary function ( 1 , 13 ) . an increased rate of mortality was associated with malnutrition , growth retardation , and declining lung function ( 1 , 13 , 37 ) . it is thought that in consanguineous marriage this was observed and that low weight at birth in cf patients causes shortened survival . the presence of ranking among the family s children , degree of consanguineous marriage , and weight at birth do not rule in the mortality of cf , although location of residence , family history , and consanguineous marriage are predictors of increased mortality in cf patients . the primary weakness of this study was that it was a retrospective study and may have limited accuracy , but it does lead the way to prospective research . another weak point was the investigation was limited to a specialist group , which decreases the ability to generalize the study results . data were limited to the children s hospital of the university of medical sciences and medical genetic laboratory in tabriz , and it was possible that some cf patients were not referred to these centers . the strength of this study was the adequate sample size in this population , and that it was conducted over a long period of time . data that was approved by two reviewers were collected from educational , referral , and therapeutic centers , and were designed to evaluate the effects of non - genetic factors on outcomes . this study was conducted in a population with a unique religion , ethnic origin , and culture , which could be a basis for the performance of comparative studies . this study indicates an association between outcome and the number of demographic factors in cf patients and emphasized the role of these factors in outcomes . clinicians need to be aware and educate patients and their families about treatments such as the correct use of medicines ( oral nutritional supplements , antibiotics , and nebulizers ) , physiotherapy , and proper nutrition , which are essential for a positive prognosis , especially in patients living in rural areas . it should be required that the government and relevant organizations focus on informing the public of the dangers of consanguinity . couples with consanguineous marriages , especially with a positive family history of the disease , require more frequent and thorough evaluations before pregnancy . other studies were performed for evaluating the association , if any , between outcome and other demographic factors . in this study , the ratio of deceased patients to living patients was 1:3 , with a significant difference . eighty - five patients expired during the period of 13 years , with a mean of six deaths per year . in a study performed by dodge et al . in the uk from 1996 to 2003 , there were 1,066 deaths , with a mean of 133 deaths per year ( 15 ) . therefore , based on these findings the mortality rate is lower in our study than in the uk , perhaps because this study has been performed on an ethnic group in iran with a low incidence of cf . in addition , it is likely that a number of deaths were not recorded or were not reported by the family in our study . in a previous study in sweden , lannefors et al . reported data from 1971 - 1999 showing that while the incidence of cf was 1/5600 live - births , the mean and median age of living cf patients were 18 12 and 16 years , respectively . in this study , regarding deceased patients , 45% were older than 18 years and 10% were older than 35 years . in deceased patients , the median age at death was 26 years ( range 0 - 72 ) ( 2 ) . in another study , also , this statistic had been observed in median age at death . delayed diagnosis and inappropriate health care european countries ( 24 ) . in our study , living cf patients had lower mean , median , and were of an older age than that reported in developed countries . also , in deceased cf patients the mean and median age at death were lower than in developed countries . we hypothesize that this difference may be due to the lack of proper equipment , inadequate diagnostic processes , and absence of treatment by specialists and expert staff , as well as poor nutritional status in our region compared to developed countries ( 13 , 24 ) . in our study , there was no significant difference between outcome and gender ; however , the mortality rate was somewhat higher in males than females . a number of studies have indicated that males have a better survival rate than females , while lannefors et al . 15 ) showed 45% and 49% of males with cf expired , respectively . in another study , accumulated data related to the mortality rate between genders reflects a higher mortality rate in cf females compared to males ( 6 ) . cultural issues and intrinsic ethnic differences ( 1 ) , as well as religion ( 8 , 25 ) and geographical elements , ( 9 ) may be reasons for the differences . for example , females are more susceptible to cf because of their body functions ; consequently , they more often report problems than males . expression of problems can be observed more in females than males in our region because of cultural issues ( 26 ) . reasons for this difference may be unclear , so it is necessary to perform additional investigations in a large population from neighboring countries to elucidate this issue . in the current study the mortality rate of cf was 50% higher in rural patients than urban patients , in spite of less air pollution ( 10 ) , lower stress , sufficient exercise , and organic nutritional intake in rural areas ( 13 , 27 ) . the availability of appropriate facilities to diagnosis and treat the disease is an important factor in patient survival . growth and lung health of children is influenced by proper treatment , and decreased pulmonary function is associated with morbidity and shortened survival for cf patients ( 28 ) . continuous care , suitable food , and daily physiotherapy are the main elements in survival of cf ( 27 ) . the relationship between improved nutritional support and increased survival in cf has been established , and retention of balanced and stable nutritional status is the best method of treatment for cf ( 13 , 27 ) . preventive treatments , such as adequate exercise , have been introduced at early ages for cf patients , and it appears to be an important factor in health maintenance in patients with cf ( 27 ) . in addition , a healthy lifestyle ( 27 ) , related to appropriate family functioning ( 29 ) , elevated levels of social class , education , and higher income ( 19 , 30 ) are determinates of survival for cf patients . patients living in urban areas are usually more likely to benefit from these conditions . in this study , there was no significant difference between outcome and ranking in birth order of the family s children , although the mortality rate was slightly higher for the second or subsequent children than for the first - born . management of patients with cf is one of the greatest challenges for families , and the family plays an important role in the treatment of cf ( 30 ) . cf patients need constant care such as physiotherapy , various medications , proper dietary intake , exercise , and adequate support ( 21 , 31 ) . on the other hand , high socioeconomic status with adequate income , suitable occupation , and employment status were identified as factors related to a good prognosis in affected individuals ( 30 ) . in crowded families , patients with a positive family history , such as a diagnostic category ( 32 , 33 ) , had a higher mortality rate than those with a negative family history . there was a significant difference , with the risk of mortality approximately two times , or 91% , higher with a positive family history than a negative family history , in the current study . ormond et al . published findings of a study stating that individuals affected by cf are segregated into four categories based on severity of disease . patients with a family history of cf showed a broader range of superficial severity with an unknown reason ( 34).understanding the reasons these patients have poorer prognoses than others will need additional research . considering consanguineous marriage as a predicating factor of outcome , children born to related parents were 94% more at risk for death than children of non - consanguineous marriage , especially in consanguineous marriage of the first - cousin type ( 3rd - degree ) . social conditions , religion , economic situations , and traditional beliefs are essential considerations in the preference for consanguineous marriage . in muslim populations , first - cousin unions ( 3rd - degree ) between a man and his father s or mother s brother s daughter , and the opposite , were detected frequently . the incidence of consanguinity has been reported to be 56% in middle east with saudi arabia having the highest prevalence at 89% in families with cf ( 35 ) . most studies have shown that early mortality is higher in the progeny of consanguineous unions than in non - consanguineous marriage ( 36 ) . ultimately , weight at birth was reviewed in deceased patients and living patients and no significant difference between them was found . the mean of weight at birth in the living cf population was slightly higher than the deceased cf population . cf is an autosomal recessive disorder affecting several organs , such as the digestive system , which is manifested in fat malabsorption , steatorrhea , and poor growth ( 2 , 21 ) . patients who intake adequate dietary requirements and gain proper weight have better pulmonary function ( 1 , 13 ) . an increased rate of mortality was associated with malnutrition , growth retardation , and declining lung function ( 1 , 13 , 37 ) . it is thought that in consanguineous marriage this was observed and that low weight at birth in cf patients causes shortened survival . the presence of ranking among the family s children , degree of consanguineous marriage , and weight at birth do not rule in the mortality of cf , although location of residence , family history , and consanguineous marriage are predictors of increased mortality in cf patients . the primary weakness of this study was that it was a retrospective study and may have limited accuracy , but it does lead the way to prospective research . another weak point was the investigation was limited to a specialist group , which decreases the ability to generalize the study results . data were limited to the children s hospital of the university of medical sciences and medical genetic laboratory in tabriz , and it was possible that some cf patients were not referred to these centers . the strength of this study was the adequate sample size in this population , and that it was conducted over a long period of time . data that was approved by two reviewers were collected from educational , referral , and therapeutic centers , and were designed to evaluate the effects of non - genetic factors on outcomes . this study was conducted in a population with a unique religion , ethnic origin , and culture , which could be a basis for the performance of comparative studies . this study indicates an association between outcome and the number of demographic factors in cf patients and emphasized the role of these factors in outcomes . clinicians need to be aware and educate patients and their families about treatments such as the correct use of medicines ( oral nutritional supplements , antibiotics , and nebulizers ) , physiotherapy , and proper nutrition , which are essential for a positive prognosis , especially in patients living in rural areas . it should be required that the government and relevant organizations focus on informing the public of the dangers of consanguinity . couples with consanguineous marriages , especially with a positive family history of the disease , require more frequent and thorough evaluations before pregnancy . other studies were performed for evaluating the association , if any , between outcome and other demographic factors .

backgroundoutcomesforcystic fibrosis patients are improving rapidly . the demographic factors are notable variables inoutcomes , which can be evaluated and modified.objectivesthis study was designed to investigate the association between outcome and demographic factors in patients with cystic fibrosis.patients and methodsthis was a cross - sectional study and data were gathered for 331 patients using the census method , from march 2001 to september 2014 in iran . data was analyzed using logistic regression analysis , chi - square test , and independent sample t test using spss 18 . odds ratio with confidence intervals of 95% and p < 0.05 were considered significant.resultsthere were 85 ( 25.7% ) deceased patients and 246 ( 74.3% ) living patients at the time of the study . of the 246 living cf patients , 202 ( 82.2% ) were less than nine years of age , and 77 ( 90.6% ) out of the 85 deceased cf patients had died younger than four years of age . there was a significant difference between outcome and location of residence . the risk of mortality was 50% less in urban patients than in rural patients ( p = 0.03 ) . the risk of mortality was approximately two times higher in patients with a positive family history than in those with a negative family history ( p = 0.02 ) . the proportion of mortality was approximately two times , or 94% , higher for those in a consanguineous marriage than for those in a non - consanguineous marriage ( p = 0.01).conclusionsthe results demonstrated that the mortality rate was higher in cf patients with a positive family history , a consanguineous marriage , and residence in a rural area . therefore , demographic factors play an important role in the outcome of cystic fibrosis . unfortunately , these parameters , which can be managed easily and with low cost , have been overlooked .

approximately 1 - 5% of peripheral blood t cells express the t - cell receptor instead of the conventional t - cell receptor . the versus t - cell lineage commitment during intrathymic t - cell development seems to be controlled by the signal strength provided to the t - cell receptor . in healthy donors , most blood t cells carry a specific t - cell receptor composed of v9 and v2 elements . in addition to effector functions shared with t cells , the v9v2 t cells can acquire professional antigen - presenting capacity characteristic of dendritic cells . in contrast to t cells , v9v2 t cells do not see processed antigenic peptides presented by major histocompatibility complex molecules , but rather recognize small phosphorylated non - peptide molecules ( phosphoantigens ) produced by many microorganisms but also by transformed eukaryotic cells . while microbial phosphoantigens are active at pico- to nanomolar concentrations , micromolar concentrations of the eukaryotic phosphoantigen isopentenyl pyrophosphate ( ipp ) are required for t - cell activation . such high concentrations are not achieved in the mevalonate pathway of isoprenoid synthesis used in non - transformed cells . interestingly , human v9v2 t cells can kill a broad variety of epithelial tumor and leukemia / lymphoma cells . the sensitivity of tumor cells to t - cell - mediated killing is increased upon treatment of tumor cells with aminobisphosphonates ( n - bps ) , drugs that are used in clinical practice for the treatment of osteoporosis and bone metastasis in cancer patients . n - bps inhibit the ipp - processing enzyme farnesyl diphosphate synthase ( fpps ) , thereby leading to an accumulation of ipp , which is then sensed by the t cells . t cells are poor producers of interleukin-2 ( il-2 ) , which is required for expansion of t cells . therefore , attempts to activate tumor - reactive t cells endogenously by treating patients with n - bps must take into consideration an appropriate supply of il-2 . alternative strategies consider the adoptive transfer of in vitro expanded tumor - reactive t cells [ 10 - 14 ] . the critical role of fpps in the control of intracellular ipp levels , and thus of the sensitivity of tumor cells toward t - cell killing , has been recently demonstrated using short hairpin rna - mediated knock - down of fpps . knock - down of fpps caused tumor cells , which otherwise were not recognized by t cells , to be susceptible to t - cell killing . therefore , v9v2 t cells recognize and kill tumor cells on the basis of the unbalanced isoprenoid metabolic pathway in transformed cells , a pathway that is stable in non - malignant cells . the discovery that n - bps activate t cells by inhibiting fpps , thereby leading to accumulation of ipp , has paved the way for proof - of - principle studies to activate t cells in patients with advanced cancer . in a phase i clinical trial , dieli and colleagues treated patients with hormone - refractory prostate cancer with a standard application of the n - bp zoledronate ( 4 mg intravenous infusion every 21 days ) either with or without additional low - dose ( 6 10 iu ) subcutaneous application of il-2 . various parameters , including subset analysis of t cells , and serum levels of prostate - specific antigen and cytokines , were monitored over time . although the two cohorts comprised only a few patients , statistically significant effects of zoledronate plus il-2 on the mobilization and effector cell maturation of t cells were recorded . very importantly , the two cohorts showed distinct clinical outcomes , with clinical responses seen in six of nine patients treated with zoledronate plus il-2 but only in one of nine patients treated with zoledronate alone . interestingly , a correlation between favorable outcome at 12 months and t - cell numbers or functional status ( or both ) was observed . similarly , wilhelm and colleagues had previously shown that the combined application of n - bp plus low - dose il-2 can induce objective tumor responses in patients with lymphoid malignancies . together , these studies support the view that application of n - bps plus il-2 is safe , induces in vivo activation / maturation of t cells , and may have beneficial effects in advanced cancer ( figure 1a ) . ( a ) n - bps inhibit farnesyl diphosphate synthase ( fpps ) , thus preventing processing of isopentenyl pyrophosphate ( ipp ) to farnesyl diphosphate ( fpp ) . the combined application of n - bp plus il-2 leads to in vivo activation of t cells . ( b ) alternatively , t cells can be activated in vitro with n - bp or synthetic phosphoantigens in the presence of antigen - presenting cells ( apc ) and can be subsequently expanded to large cell numbers by an exogenous supply of il-2 for subsequent adoptive transfer into cancer patients . the cell preparation can be performed under gmp ( good manufacturing practice ) conditions . in a case study reported by laggner et al . , regression of lung and bone metastases was observed in a patient with advanced stage melanoma upon systemic treatment with zoledronate and localized radiotherapy . although t - cell subsets were analyzed , it is difficult to ascertain a substantial role of t - cell activation in the resolution of metastases in this single case , particularly since il-2 was omitted in the treatment of this patient . in addition to their t - cell activating properties , n - bps also exhibit direct anti - tumor activities by both inhibiting proliferation and inducing apoptosis in tumor cells . while zoledronate seems to be the most potent t - cell - activating substance among the n - bps licensed for clinical application , derivatives of zoledronate with further improved t - cell - stimulating capacity and enhanced direct anti - tumor activity are under development . such modified n - bps might also exert improved in vivo activation of t cells when given to patients together with il-2 . t - cell - based immunotherapeutic strategy is the adoptive transfer of in vitro expanded v9v2 t cells from tumor patients ( figure 1b ) . recently , efficient protocols for the large - scale in vitro expansion of v9v2 t cells based on stimulation with synthetic phosphoantigens or zoledronate have been established . first results indicate that the repetitive adoptive transfer of in vitro expanded t cells is well tolerated and may induce anti - tumor responses in patients with solid tumors , including renal cell carcinoma and myeloma . the protocol developed by dieli et al . for the in vivo activation of t cells based on zoledronate plus low - dose il-2 application is ready to be explored in larger clinical trials and in other tumor entities with poor prognosis , for example , pancreatic ductal adenocarcinoma where it might be combined with standard regimens such as gemcitabine . it is conceivable that this protocol can be further improved , for instance , by combination with tumor - targeting monoclonal antibodies . along this line , it has been shown that b - cell lymphoma or breast tumor cell killing by fc receptor - expressing t cells is enhanced in the presence of targeting antibodies rituximab ( cd20 ) or trastuzumab ( her2/neu ) , respectively . moreover , a t - cell - stimulating synthetic phosphoantigen was found to enhance the depletion of cd20 b cells by rituximab in a non - human primate model in vivo , pointing to the possible use of phosphoantigen plus anti - cd20 antibodies in the treatment of cd20 leukemias and lymphomas . furthermore , cytokines promoting homeostatic proliferation and survival of t cells , such as il-15 , or cytokines potentiating the cytolytic activity and pro - inflammatory response , such as il-21 , might be combined with il-2 or used instead of il-2 . this could be considered both for in vivo application together with n - bps and for optimization of in vitro expansion of t cells . in addition , future study protocols might include the combination of in vivo activation of t cells ( by n - bp or phosphoantigen plus il-2 ) followed by the adoptive transfer of in vitro expanded t cells , finally , it should be stressed that t - cell - based immunotherapy is not expected to replace established therapeutic protocols . rather , it might offer additional benefit to the patient , for instance , in combination with conventional chemotherapy .

t lymphocytes are a numerically small subset of t cells with potent cytotoxic activity against a variety of tumor cells . human t cells expressing the v9v2 t cell antigen receptor recognize endogenous pyrophosphate molecules that are overproduced in transformed cells . moreover , the intracellular accumulation of such pyrophosphates is strongly enhanced by aminobisphosphonates used in the treatment of osteoporosis and bone metastasis in certain cancer patients . a new concept of cancer immunotherapy is based on the endogenous activation of t cells with aminobisphosphonates plus low - dose interleukin-2 .

a cross - sectional and longitudinal study was conducted that included 22 medical clinics ( i.e. , general practitioners ) or general / university - affiliated hospitals from different areas in japan , using the same software ( codic ) to compile electronic medical records , as a working study group , the japan diabetes clinical data management ( jddm ) study ( 9 ) . a detailed description of the study has been published previously ( 9,10 ) . all consecutive patients with type 2 diabetes who visited each clinic / hospital between 2000 and 2009 and whose diabetes was diagnosed before 2009 were included ( n = 45,876 ) . in total , 4,798 drug - naive patients were recruited on their first visits between 2000 and 2009 from across japan . all patients met the japan diabetes association criteria for type 2 diabetes ( 11 ) . all case participants had their height , weight , hba1c , blood pressure ( bp ) , and lipids measured . bmi was calculated as weight ( kg ) divided by height squared ( m ) . waist circumference ( wc ) was assessed at the top of the iliac crest at the end of a normal expiration ( 12 ) . the jddm protocol , which is in accordance with the declaration of helsinki , received ethical approval from the institutional review boards of all of the participating institutions and was undertaken in accordance with the ethical guidelines for clinical studies of the japanese ministry of health , labor , and welfare . the morning after an overnight fast , venous blood was sampled for the baseline measurements of the hba1c level and plasma concentrations of glucose , ldl cholesterol , hdl cholesterol , triglycerides ( tgs ) , creatinine , and insulin . hba1c , expressed in national glycohemoglobin standardization program units , was measured by high - performance liquid chromatography . plasma total cholesterol , hdl cholesterol , and tgs were assessed with standard enzymatic spectrophotometric techniques . ( 13 ) , except when tgs exceeded 400 mg / dl ( in that case , data were treated as missing ) . albumin concentrations in random spot urine samples were determined by turbidimetric immunoassay , and creatinine levels were determined by the enzymatic method . the abbott imx insulin assay ( dainabot ; abbott laboratories , tokyo , japan ) is used to quantitatively measure insulin at bml , inc . this assay shows no cross - reactivity with proinsulin ( < 0.005% ) . within the assay , for every year , the coefficients of variation at mean values of 59.6 and 873.8 the following three mrfs were evaluated as defined by the revised national cholesterol education program criteria ( 14 ) and the world health organization criteria for asia ( 15 ) : 1 ) wc 80 cm ( men ) or 75 cm ( women ) ; 2 ) systolic bp 130 mmhg , diastolic bp 85 mmhg , or antihypertensive medications ; and 3 ) hdl cholesterol < 1.04 mmol / l , tgs 1.68 mmol / l , or lipid medications . sex , age , bmi , wc , mean bp , hba1c , fasting insulin , tgs , ldl cholesterol , hdl cholesterol , and microalbuminuria were compared by five consecutive 2-year periods from 2000 to 2009 using one - way anova and the bonferroni post hoc test . statistical analyses included the unpaired student t test , one - way anova , the test for categorical variables , univariate linear correlations , and ancova . multiple linear regression analysis was used to assess variables that were independently associated with variations in fasting insulin levels . both fasting insulin and bmi differences over this 10-year term were assessed using ancova to adjust for potential confounders ( sex , age , and fasting plasma glucose [ fpg ] ) . the morning after an overnight fast , venous blood was sampled for the baseline measurements of the hba1c level and plasma concentrations of glucose , ldl cholesterol , hdl cholesterol , triglycerides ( tgs ) , creatinine , and insulin . hba1c , expressed in national glycohemoglobin standardization program units , was measured by high - performance liquid chromatography . plasma total cholesterol , hdl cholesterol , and tgs were assessed with standard enzymatic spectrophotometric techniques . ( 13 ) , except when tgs exceeded 400 mg / dl ( in that case , data were treated as missing ) . albumin concentrations in random spot urine samples were determined by turbidimetric immunoassay , and creatinine levels were determined by the enzymatic method . the abbott imx insulin assay ( dainabot ; abbott laboratories , tokyo , japan ) is used to quantitatively measure insulin at bml , inc . this assay shows no cross - reactivity with proinsulin ( < 0.005% ) . within the assay , for every year , the coefficients of variation at mean values of 59.6 and 873.8 pmol / l were 4 and 2.5% , respectively . between assays , the coefficients of variation at mean values of 59.6 and 872.4 the following three mrfs were evaluated as defined by the revised national cholesterol education program criteria ( 14 ) and the world health organization criteria for asia ( 15 ) : 1 ) wc 80 cm ( men ) or 75 cm ( women ) ; 2 ) systolic bp 130 mmhg , diastolic bp 85 mmhg , or antihypertensive medications ; and 3 ) hdl cholesterol < 1.04 mmol / l , tgs 1.68 mmol / l , or lipid medications . sex , age , bmi , wc , mean bp , hba1c , fasting insulin , tgs , ldl cholesterol , hdl cholesterol , and microalbuminuria were compared by five consecutive 2-year periods from 2000 to 2009 using one - way anova and the bonferroni post hoc test . statistical analyses included the unpaired student t test , one - way anova , the test for categorical variables , univariate linear correlations , and ancova . multiple linear regression analysis was used to assess variables that were independently associated with variations in fasting insulin levels . both fasting insulin and bmi differences over this 10-year term were assessed using ancova to adjust for potential confounders ( sex , age , and fasting plasma glucose [ fpg ] ) . comparisons between groups were analyzed for five consecutive 2-year periods versus each variable value in 20002001 , when observation began . sex , age , mean bp , and the prevalence of mrfs ( % wc , hypertension , and dyslipidemia ) did not differ significantly among year groups . waist circumference ( cm ) and bmi were significantly different in the 20062007 and 20082009 groups . fpg , hba1c , fasting insulin , and tgs were significantly different in each group in 2002 and thereafter . compared with 20002001 , the bmi increased significantly in 2006 and later , but the difference was slight . blood pressure did not change during the 10-year period . for lipids , compared with 20002001 , tgs increased in 2002 and thereafter . as a visual representation of this study , profile plots were created showing the estimated marginal means for the fasting insulin levels / bmi values in each of the 2-year groups ( fig . further analysis using ancova adjusted for age and fpg showed differences among year groups , with an increase in bmi and a decrease in fasting insulin ( p < 0.001 ) . in each year group , bmi and fasting insulin values in females were higher than in males ( p < 0.001 ) . multiple linear regression analysis was performed to identify factors affecting fasting insulin levels ( table 2 ) . factors that significantly affected the fasting insulin value , in order of greater , were wc , bmi , year group , mean bp , hdl cholesterol , tgs , and microalbuminuria . the factors most affecting fasting insulin were wc and bmi , but even after correction for sex , age , and hba1c , the values decreased during the 10-year period . there was also a highly linear relationship between wc and bmi ( r = 0.87 , p < 0.001 ) . patients ' characteristics for each 2-year group estimated marginal mean for ancova results among year groups for fasting insulin ( a ) and bmi ( b ) . covariates in each model were evaluated based on age = 56.7 years and fpg = 9.1 pmol / l . in this 10-year study of drug - naive japanese diabetic patients evaluated at their first clinic / hospital visit , fasting insulin levels were found to decrease over time , with wc and bmi being the most important factors after corrections . from 2000 to 2009 , when the current study was conducted , a diabetes status survey in japan also estimated an increase in the number of diabetic patients , from 6.9 to 12.5 million ( 3 ) . it is clear that , in the last 10 years , the insulin secretion ability of japanese individuals has gradually decreased each year . the reason for this is the following changes that were observed in mrfs that play a role in insulin resistance . as mrfs , in the current study , the relationships with wc , bmi , bp , lipids , and microalbuminuria were examined . the factors most involved with the decrease in fasting insulin levels were wc and bmi . fasting insulin levels were affected by age , sex , and hba1c . even after adjustment for these , based on our study , the role of mrfs ( wc , bmi , bp , lipids , and microalbuminuria ) seems important in the pathogenesis of type 2 diabetes in japanese individuals . the asia - pacific region has been considered to be the major site of a rapidly emerging epidemic of diabetes ( 16 ) , and with its large populations , it is of prime importance for the epidemiology of diabetes . approximately 13.5% of the japanese population now has either type 2 diabetes or impaired glucose tolerance ( 17 ) . furthermore , it is thought that westernization of the lifestyle and an increased percentage of fat in the diet play a role in increasing insulin resistance ( 18 ) . in asian populations , the -cells may lose their ability to compensate for the decrease in insulin sensitivity seen with the development of central adiposity . loss of -cell function has been demonstrated to appear before the development of the obesity - induced decreased insulin sensitivity among subpopulations of japanese and japanese americans who develop type 2 diabetes ( 19,20 ) . the japanese have a higher prevalence of polymorphisms for at least three genes that code for proteins thought to play key roles in lipid and glucose metabolism : the 3-adrenergic receptor , the peroxisome proliferator activated receptor , and calpain-10 ( 18,21 ) . the interaction between changes in lifestyle and the thrifty genotype characteristic of many japanese people may play a significant role in the increasing prevalence of diabetes and associated cardiovascular risk in this population . although this type of genetic background has not changed , compared with 30 years ago , dietary habits have become westernized with a high - fat diet . surprisingly , the daily calorie intake has not changed over the last 50 years and remains at ~2,000 kcal . however , 50 years ago , when a traditional japanese diet was consumed , the percentage of fat in the calorie intake was ~7% , but now it is > 27% , a sudden , almost fourfold increase in only 50 years ( 3 ) . therefore , an imbalance with the characteristic japanese insulin secretion ability has developed . as a result , the current state of obesity and poor insulin effectiveness has become very significant . since japanese people with their lower insulin secretion ability can not completely compensate for this , the prevalence of diabetes is increasing dramatically . in addition , it has been shown that japanese americans have experienced a higher prevalence of type 2 diabetes than in japan . research conducted in seattle , wa , suggests that lifestyle factors associated with westernization play a role in exacerbating this susceptibility to diabetes ( 22 ) . bmi is a strong determinant of insulin resistance , and it is concordant with the evidence that the mean bmis of representative epidemiologic studies of japanese diabetic patients are from 23 to 25 kg / m lower than in the studies of other ethnic populations ( 23,24 ) . bmi and wc thresholds vary among ethnicities , and values are lower for asian populations . local and regional data have shown that the same level of bmi connotes a greater degree of obesity in asians compared with caucasians ( 25 ) , and that asians are prone to disorders such as diabetes , hypertension , and dyslipidemia at lower levels of bmi than caucasian populations ( 25 ) . the use of bmi as a measure of body proportion is a limitation because of its inability to provide information on body fat distribution and central adiposity . the current study also indicates that , for japanese , a slight increase in wc / bmi may be as informative with regard to diabetes risk due to decreasing fasting insulin levels . decreased -cell function and decreased insulin sensitivity are the two major risk factors for the development of type 2 diabetes . the ability of -cells to compensate for a decrease in insulin sensitivity enables these individuals to maintain normal glucose levels . the fundamental pathological sequence leading to type 2 diabetes is presumed to be the development of an obesity - induced decrease in insulin sensitivity followed by hyperglycemia when the -cells can no longer compensate ( 27 ) . however , it is still unknown whether -cell dysfunction ( 28 ) , decreased insulin sensitivity ( 29 ) , or a combination of both defects is the primary abnormality leading to type 2 diabetes ( 30 ) . although the risk factors for type 2 diabetes are similar among ethnically diverse populations , there are ethnic differences in insulin sensitivity and -cell function among groups at high risk for type 2 diabetes . since the study design was cross - sectional , causal relationships can not be explored . first , as our study included only type 2 diabetic patients in diabetes clinics / hospitals , our findings can not be generalized to other patients in a typical practice or community population . also , fasting insulin levels are limited measures of insulin sensitivity and -cell function . other environmental or lifestyle factors , including dietary fat or fiber , alcohol consumption , physical activity , smoking , and socioeconomic status might be related to the decreasing trend in fasting insulin over the 10-year period . nevertheless , we have shown that fasting insulin levels in subjects with newly diagnosed diabetes have been decreasing progressively from 2000 to 2009 , and this decrease has been associated with an increase in mrfs , particularly wc and bmi . in conclusion , the superimposition of low pancreatic -cell reserve upon a lifestyle background of metabolic parameters appears to result in hyperglycemia and diabetes in japan .

objectiveto investigate the relationship between fasting insulin levels and metabolic risk factors ( mrfs ) in type 2 diabetic patients at the first clinic / hospital visit in japan over the years 2000 to 2009.research design and methodsin total , 4,798 drug - naive japanese patients with type 2 diabetes were registered on their first clinic / hospital visits . conventional clinical factors and fasting insulin levels were observed at baseline within the japan diabetes clinical data management ( jddm ) study between consecutive 2-year groups . multiple linear regression analysis was performed using a model in which the dependent variable was fasting insulin values using various clinical explanatory variables.resultsfasting insulin levels were found to be decreasing from 2000 to 2009 . multiple linear regression analysis with the fasting insulin levels as the dependent variable showed that waist circumference ( wc ) , bmi , mean blood pressure , triglycerides , and hdl cholesterol were significant , with wc and bmi as the main factors . ancova after adjustment for age and fasting plasma glucose clearly shows the decreasing trend in fasting insulin levels and the increasing trend in bmi.conclusionsduring the 10-year observation period , the decreasing trend in fasting insulin was related to the slight increase in wc / bmi in type 2 diabetes . low pancreatic -cell reserve on top of a lifestyle background might be dependent on an increase in mrfs .

somatization is a condition in which the patient experiences unexplained medical symptoms , and is sometimes called functional somatic complaint.1,2 the condition is particularly hard to detect in the elderly and its effects should not be underestimated as it can become a serious medical condition.3 high comorbidity between medically unexplained physical symptoms and other psychiatric disorders has been found among elderly patients.4 one survey revealed that general practitioners believe somatization to be an important cause of physical complaints among the elderly,5 and some investigators found that it is difficult to differentiate between the functional somatic and psychiatric origins of somatic complaints , in particular if they are overlapping.6 somatic symptoms may be related to depression , as depressed elderly people commonly manifest their suffering through physical symptoms.7,8 in previous studies , we showed that depressed elderly people tend to express their frustrations more as physical symptoms than nondepressed individuals , and that this was more the case in elderly depressed than younger depressed people.9,10 in addition , other factors such as culture , education , social values , or even ageism make it difficult for elderly people to be open or assertive , and as a result , physical complaints would seem to be a coping mechanism that they use to deal with this inner conflict.11 somatic symptoms may present themselves differently due to the influence of ethnic factors . for example , south americans have been found to have the highest rates of somatization11,12 and the condition is also common in asian people , especially among those who are experiencing depression , regardless of their age.13 an ethnic difference has also been found in terms of the types of symptoms reported . for instance , one symptom described as a heavy head is significantly more common among asians than among americans , caucasians , and africans.11 it may be that culture and ethnic background frame how individuals express themselves , whether they benefit or look bad when coping with types of stress or conflict , and in certain situations . for example , in thai society , elderly people are expected to have higher moral standards than younger people , so it may be unacceptable to express feelings such as sexual desire , even with a doctor , even if they still have them . somatic symptoms , on some levels , may also be related to personality traits such as neuroticism . neuroticism is the actual inclination to see distressing thoughts easily , such as frustration , anxiety , depressive disorder , or susceptibility . it is also at times referred to as emotional instability , inverse emotional stability , or neuroticism has been found to be one of the strongest predictors of somatism,14 and alexithymia is another personality trait that is found to have an influence on the condition.15,16 based on these results , somatization is viewed psychoanalytically as a defense mechanism against internal conflict whereby an individual finds it difficult to express himself or herself directly.17,18 it is also influenced by age and culture , and as mentioned before , can be viewed as a repository for frustration . moreover , hypothetically , somatization should be prominent in individuals who display social inhibition , meaning it is difficult for them to effectively access help from others or express themselves directly.19 social inhibition is thought to reflect complex interpersonal behavior that is related to social withdrawal , regardless of whether it is related to anxiety , depression , or even apathy.2022 social inhibition , however , as measured by the interpersonal inventory of interpersonal problems , tends to be viewed as a trait rather than as a symptom.23 therefore , with regard to the role of social inhibition , in theory it is believed to act as a link or mediator between personality traits and somatization , and previous studies have found a correlation between it and somatization , although mostly accompanied by the presence of negative affectivity ( or neuroticism).19,2426 however , little is known about the relationship between somatization and social inhibition in the elderly , in whom somatization is a common condition . we were interested in studying the relationship between these variables , as well as personality traits in addition to neuroticism . this study , therefore , aimed to explore the effects of personality traits on somatization and social inhibition as well as some sociodemographic data , based on the hypothesis that personality traits have either a direct effect on somatization , or indirectly via social inhibition ( figure 1 ) . these have not previously been investigated . this study was part of a wider study conducted in 200927 which was approved by the ethics committee at the faculty of medicine , chiang mai university . the authors analyzed the data of 126 elderly participants who provided demographic data and who completed the inventory of interpersonal problems ( iip-64),27,28 the sixteen personal ity factor ( 16 pf ) , and symptom checklist ( scl)-9029,30 questionnaires . demographic data concerning the sex , age , income , and education of the participants were obtained . participants aged 60 years or more were recruited ( in thailand , people over 60 years old are defined as elderly ) . income was assessed based on the gross monthly household incomes of the participants ( < 1 unit to 9 units , 1 unit equals 5,000 baht or 167 us dollars ) . the iip-6428 is a self - reporting questionnaire which measures interpersonal difficulties across eight subscales as follows : domineering , vindictive , nonassertive , socially inhibited , overly accommodating , self - sacrificing , and intrusive / needy . the scale consists of 64 items which are rated on a 5-point scale , ranging from 0 ( not at all ) to 4 ( extremely ) , and the subscales can be modeled geometrically , as with a circumplex model . each subscale signifies an octant in this model . the thai version has demonstrated a good overall internal consistency of =0.95.27 the test retest reliability of the iip-64 subscale using intraclass correlation coefficient ( icc ) ranged from 0.68 to 0.76 and 0.81 for the total score , although only the subscale socially inhibited ( =0.79 ) was used for the analysis . the scl-9029,30 is a 90 item self - reporting questionnaire used to assess psychological problems and symptom distress . each item assesses symptom severity on a 5-likert scale , where 0 represents not at all and 4 represents extremely . there are nine symptoms included in the instrument : somatization , obsessive compulsive , interpersonal sensitivity , hostility , depression , anxiety , paranoid ideation , phobic anxiety , and psychoticism . the thai version of scl-90 was developed by chooprayoon30 and has been used on both a clinical and nonclinical population . in this study , only somatization was used . for the somatization subscale , a cronbach s alpha of 0.85 has been generated using this instrument.10 the 16 pf , as developed by cattell et al,31 is a tool used for assessing personality based on eight dimensions or 16 characteristics : warmth , intellect , emotional stability , dominance , liveliness , rule - consciousness , social boldness , sensitivity , vigilance , abstractedness , privateness , apprehensiveness , openness to change , self - reliance , perfectionism , and tension . the results are rated by giving a score of 1 or 2 , then comparing the results with a standard score . it has been shown to have adequate reliability : cronbach 0.80 over a 2-week period and 0.70 over a 2-month period.32 the thai version of 16 pf was developed by cheuaphakdi and phornphatkul,33 and has been widely used to assess personality and its relationship to clinical samples . the cronbach s alpha found when using the tool in this study ranged between 0.61 and 0.88.33 in this study , descriptive statistics were examined across all variables . path analysis using amos 18 ( ibm corporation , armonk , ny , usa ) was applied to establish the effects of the personality trait predictors as well as the outcomes ( somatization and social inhibition ) . a graphical illustration of path analysis is an easy , convenient , and effective way to present the complicated relationships among variables that exist as it allows one to see the influence of personality traits on somatization and social inhibition at the same time , and also the indirect effect of personality traits or social inhibition on somatization . in the first step , we analyzed the direct effects of the significant , correlated demographic variables , plus each personality trait variable , on somatization and social inhibition , after which we introduced mediator variables and estimated the direct and indirect effects . path analysis was undertaken using the maximum - likelihood estimation method with all single indicators allowed to be correlated . two fit indices commonly used in the confirmatory factor analysis literature were used to evaluate the model fit , these being the goodness of fit index ( gfi)34 and the comparative fit index ( cfi).35 the root mean square error of approximation ( rmsea ) and standardized root - mean - square residual ( srmr ) methods were also used as this is an evaluation statistic that is relatively unaffected by sample size and is suitable for assessing models of differing complexity.35,36 modification indices were also applied . the authors analyzed the data of 126 elderly participants who provided demographic data and who completed the inventory of interpersonal problems ( iip-64),27,28 the sixteen personal ity factor ( 16 pf ) , and symptom checklist ( scl)-9029,30 questionnaires . demographic data concerning the sex , age , income , and education of the participants were obtained . participants aged 60 years or more were recruited ( in thailand , people over 60 years old are defined as elderly ) . income was assessed based on the gross monthly household incomes of the participants ( < 1 unit to 9 units , 1 unit equals 5,000 baht or 167 us dollars ) . the iip-6428 is a self - reporting questionnaire which measures interpersonal difficulties across eight subscales as follows : domineering , vindictive , nonassertive , socially inhibited , overly accommodating , self - sacrificing , and intrusive / needy . the scale consists of 64 items which are rated on a 5-point scale , ranging from 0 ( not at all ) to 4 ( extremely ) , and the subscales can be modeled geometrically , as with a circumplex model . each subscale signifies an octant in this model . the thai version has demonstrated a good overall internal consistency of =0.95.27 the test retest reliability of the iip-64 subscale using intraclass correlation coefficient ( icc ) ranged from 0.68 to 0.76 and 0.81 for the total score , although only the subscale socially inhibited ( =0.79 ) the scl-9029,30 is a 90 item self - reporting questionnaire used to assess psychological problems and symptom distress . each item assesses symptom severity on a 5-likert scale , where 0 represents not at all and 4 represents extremely . there are nine symptoms included in the instrument : somatization , obsessive compulsive , interpersonal sensitivity , hostility , depression , anxiety , paranoid ideation , phobic anxiety , and psychoticism . the thai version of scl-90 was developed by chooprayoon30 and has been used on both a clinical and nonclinical population . in this study , only somatization was used . for the somatization subscale , a cronbach s alpha of 0.85 has been generated using this instrument.10 the 16 pf , as developed by cattell et al,31 is a tool used for assessing personality based on eight dimensions or 16 characteristics : warmth , intellect , emotional stability , dominance , liveliness , rule - consciousness , social boldness , sensitivity , vigilance , abstractedness , privateness , apprehensiveness , openness to change , self - reliance , perfectionism , and tension . the results are rated by giving a score of 1 or 2 , then comparing the results with a standard score . it has been shown to have adequate reliability : cronbach 0.80 over a 2-week period and 0.70 over a 2-month period.32 the thai version of 16 pf was developed by cheuaphakdi and phornphatkul,33 and has been widely used to assess personality and its relationship to clinical samples . the cronbach s alpha found when using the tool in this study ranged between 0.61 and 0.88.33 the continuous scores of each variable were used for analysis . path analysis using amos 18 ( ibm corporation , armonk , ny , usa ) was applied to establish the effects of the personality trait predictors as well as the outcomes ( somatization and social inhibition ) . a graphical illustration of path analysis is an easy , convenient , and effective way to present the complicated relationships among variables that exist as it allows one to see the influence of personality traits on somatization and social inhibition at the same time , and also the indirect effect of personality traits or social inhibition on somatization . in the first step , we analyzed the direct effects of the significant , correlated demographic variables , plus each personality trait variable , on somatization and social inhibition , after which we introduced mediator variables and estimated the direct and indirect effects . path analysis was undertaken using the maximum - likelihood estimation method with all single indicators allowed to be correlated . two fit indices commonly used in the confirmatory factor analysis literature were used to evaluate the model fit , these being the goodness of fit index ( gfi)34 and the comparative fit index ( cfi).35 the root mean square error of approximation ( rmsea ) and standardized root - mean - square residual ( srmr ) methods were also used as this is an evaluation statistic that is relatively unaffected by sample size and is suitable for assessing models of differing complexity.35,36 modification indices were also applied . the age of the participants ranged from 60 to 93 years ( mean = 71.7 ; standard deviation [ sd ] = 6.7 ) ; 50.8% of the group were male and 54.8% were married . the average number of years spent in education among the participants was 7.6 ( sd = 5.2 ) . out of the 16 personality factors , only seven personality traits significantly related to either social inhibition or somatization were selected : emotional stability , dominance , reasoning , vigilance , apprehension , self - reliance , and tension ( table 1 ) . vigilance was found to correlate with both social inhibition and somatization ( r=0.229 , p<0.001 and r=0.192 , p<0.05 , respectively ) . the other personality traits correlated in the range of 0.185 to 0.267 . personality traits revealing significant correlations with social inhibition and somatization were included in the hypothesized model . sex , reasoning , apprehension , and self - reliance were excluded from the model as they yielded low estimates in terms of regression weighting ( p0.05 ) . as a result , only emotional stability , dominance , and vigilance were retained . however , the direct effect of age on social inhibition , dominance on social inhibition , and social inhibition on somatization was not significant ( p>0.05 ) , so these three paths were deleted from the model . age was found to have a direct impact only on somatization ( =0.25 , z=3.20 , p=0.001 ) , and then indirectly on social inhibition via the education level as they were significantly correlated ( r=0.37 ) . a lower education level was shown to have a direct association with high levels of somatization ( =0.37 , z=4.80 , p<0.001 ) as well as social inhibition ( =0.19 , z=2.17 , p=0.03 ) . emotional stability had a direct effect on both somatization ( =0.19 , z=2.58 , p=0.01 ) and social inhibition ( =0.19 , z=2.27 , p=0.023 ) . dominance had an effect on somatization ( =0.18 , z=2.45 , p = 0.014 ) and a direct effect on vigilance ( =0.33 , z=3.86 , p<0.001 ) , and vigilance had an effect on social inhibition ( =0.19 , z=2.50 , p=0.024 ) . more importantly , modification indices did not suggest a link between social inhibition and somatization , as had been speculated beforehand.18,25 the r for somatization was 0.35 , whereas for social inhibition it was 0.10 . the model showed a very good fit to the data , with =3.55 , df=6 , p=0.738 , cfi = 1.00 , tli = 1.097 , nfi = 0.968 , gfi = 0.992 , rmsea = 0.00 , and srmr = 0.031 , and all paths were significant ( p<0.05 ) . to our knowledge , this is the first study investigating the link between personality traits and both somatization and social inhibition at the same time . above all , age and education were found to have an effect on both symptoms , with education found to have the strongest influence when compared to personality traits . this result may have been due to the level of intelligence of the individuals involved , as found in previous studies.3739 age was found to have both a direct and indirect impact on these symptoms when combined with personality traits . as mentioned earlier , here our model showed that it had an impact on both outcomes , meaning that elderly people in the study who had a low emotional stability score tended to have both symptoms . dominance , meanwhile , was found to have a direct effect on somatization and an indirect effect on social inhibition . it is important to note that without the presence of the dominance trait and other factors in the model , emotional stability had no significant effect on somatization . this may be explained by the fact that somatization is related to depression , that is , it may be influenced by depression , which in turn is related to neuroticism ( or inverse emotional stability).4046 neitzert et al compared the relationships between neuroticism , depression , and somatic symptoms in a healthy student sample , finding that neuroticism and depression are significantly and positively related to somatic symptoms reporting , even after controlling for the effects of neuroticism.47 however , our study focused exclusively on the importance of personality traits with regard to social inhibition and somatization when depression is not involved , which is why the effect of emotional stability was not dramatic . it is interesting to note that somatization is related to dominance , which is defined by the presence of characteristics such as assertiveness , forcefulness , aggressiveness , and competiveness . what can be understood from this relationship between dominance and somatization ? some assumptions we would like to make here are that since the dominance personality trait is forceful an assertive personality akin to a type a it has been found to be related to a narcissistic personality.48 it may therefore be speculated that sociocultural influences have a role to play , particularly among the thai elderly , with regard to the dominance personality trait . in thai society , older people tend to feel powerless and that they lack authority.49 they tend to submit and expect not be independent , and this undermines their pride and self - esteem . when compared to younger people with these personality types , it may be rather difficult for dominant elderly people to assert themselves , or to express their frustrations , meaning that somatic complaints may reflect an idiom of distress . dominance indirectly endorses social inhibition through the suspicion trait , while skepticism ( vigilance ) may make an older person socially withdraw rather than step forward for help in a time of need , as the elderly are expected by thai society to be knowledgeable and self - fulfilled . as for social inhibition , there is evidence that it has a relationship with emotional stability,23 in contrast to the findings of kingma et al who found that neuroticism had no effect on social inhibition.50 this can be attributed to the fact that there were differences in terms of the neuroticism - related outcomes measured and instruments used in these studies . it is interesting to note that the results here showed no direct effect of social inhibition on somatization , as had been hypothesized in the model , even though both social inhibition and somatization are significantly correlated . in fact , social inhibition did have a significant effect on somatization , but this was reduced to a nonsignificant level when other personality traits were included . this suggests that these personality traits outweigh the effect social inhibition has on somatization . to summarize , somatization symptoms , without depression , were related to ( instead of being influenced by ) social inhibition and both were influenced by age , education level , as well as inverse emotional stability , dominance , and vigilance personality traits . what outcomes here are of use when dealing with elderly people experiencing somatization and social inhibition , but without depression ? the model used here suggests we should consider an individual s level of education ( or intelligence ) , his or her age , and also look out for the personality traits of emotional stability ( or neuroticism ) , dominance , and vigilance . to help patients feel safe , therapists should reduce the level of fear and anxiety patients experience , while creating a way for them to express their self - esteem and pride . approaching the problems in this way should help them to cope with stress or frustrations better rather than avoiding their problems and using somatic complaints as a way to cope with stress or frustration . in addition , building trust should help improve their socialization levels , which in turn may prevent them experiencing somatic preoccupation . first , the sample size used in this study was rather small , and this will have had an impact on the statistical analyses . second , this study investigated a nonclinically diagnosed sample of somatization sufferers using the scl-90 self - reporting tool , whereas ideally , clinician rated structured interviews should have been used to assess the presence or otherwise of the condition . third , this study was cross - sectional in nature , therefore it was not possible to evaluate causality . our study identified personality traits and sociodemographic variables that have an effect on somatization and social inhibition . emotional stability , dominance , and vigilance , as well as age and level of education were found to have an effect on these symptoms . therefore , primary care workers should perhaps pay more attention to these factors when dealing with elderly with somatization . however , further studies with larger sample size and longitudinal design is encouraged to warrant the likelihood of the model . in addition , the authors encourage investigating how depression has its effect on these variables in any further study .

purposesomatization is a common symptom among the elderly , and even though personality disorders have been found to be associated with somatization , personality traits have not yet been explored with regard to this symptom . the aim of this study is to investigate the relationship between personality traits and somatization , and social inhibition.patients and methodsas part of a cross - sectional study of a community sample , 126 elderly thais aged 60 years or over completed self - reporting questionnaires related to somatization and personality traits . somatization was elicited from the somatization subscale when using the symptom checklist scl-90 instrument . personality traits were drawn from the 16 personality factor questionnaire and social inhibition was identified when using the inventory of interpersonal problems . in addition , path analysis was used to establish the influence of personality traits on somatization and social inhibition.resultsof the 126 participants , 51% were male , 55% were married , and 25% were retired . the average number of years in education was 7.6 ( standard deviation = 5.2 ) . emotional stability and dominance were found to have a direct effect on somatization , as were age and number of years in education , but not sex . also , 35% of the total variance could be explained by the model , with excellent fit statistics . dominance was found to have an indirect effect , via vigilance , on social inhibition , which was also influenced by number of years in education and emotional stability . social inhibition was not found to have any effect on somatization , although hypothetically it should.conclusionemotional stability , dominance , and vigilance , as well as age and the number of years in education , were found to have an effect on somatization . attention should be paid to these factors in the elderly with somatization .

plasma cell neoplasms are characterized by the neoplastic proliferation of a single clone of plasma cells , typically producing a monoclonal immunoglobulin . plasma cell neoplasms can present as a single lesion ( solitary plasmacytoma ) or as multiple lesions ( multiple myeloma ) . solitary plasmacytomas most frequently occur in the bone ( plasmacytoma of bone ) , but can also be found outside the bone in soft tissues ( extramedullary plasmacytoma).1 approximately 5% of all cases of plasma cell disorders are solitary plasmacytomas of the bone.2 however , the involvement of the pancreas is rare . we report a case of an extramedullary plasmacytoma of the pancreas diagnosed using ultrasonography - guided fine needle aspiration ( eus - fna ) with a review of the literature . a 58-year - old woman was transferred to our hospital for further evaluation of pelvic pain that was aggravated by walking and began approximately 2 months before admission . the initial laboratory tests showed a hemoglobin level of 10.6 g / dl , and blood urea nitrogen was 12.0 mg / dl . the level of total protein level was 9.7 g / dl and the albumin level was 3.3 g / dl . skeletal surveys detected a small osteolytic lesion without a sclerotic rim in the left parietal bone and a large bone destructive osteolytic lesion in the right inferior pelvic bone ( fig . further evaluation showed a serum free light chain lambda level of 3,475.3 mg / l and a 2-microglobulin level of 4.6 mg / l . contrast - enhanced abdominal computed tomography ( ct ) revealed a suspicious ill - defined marginated mass in the body of the pancreas ( fig . 2 ) . to further characterize the lesion , magnetic resonance imaging ( mri ) of the pancreas was performed . t2-weighted mri indicated that the pancreatic proximal body contained a mass of subtle high signal intensity ( fig . linear eus ( eu - me1 ultrasound system ; olympus , tokyo , japan ) ( gf - uct 240 ; olympus ) revealed a 1.21.0-cm sized , hypoechoic , heterogeneous , well - defined round mass in the pancreatic body ( fig . 4a ) ; fna was performed via a transgastric approach and five passages were made with a 22-gauge needle ( echotip ultra , echo-22 ; cook endoscopy , winston - salem , nc , usa ) ( fig . the cytopatholgy results showed a small cell neoplasm and the immunohistochemical profile was compatible with plasmacytoma ( fig . the patient began combination therapy consisting of bortezomib , mephalan , and prednisolone with local radiation therapy for the right pelvic bone lesion . multiple myeloma is a disease characterized by malignant proliferation of plasma cells typically involving medullary bones . extramedullary plasmacytomas represent 3% to 4% of all plasma cell neoplasms and have a male predominance , with a three to five times higher incidence in men than in women ; they usually occur in the sixth and seventh decades of life . approximately 80% to 90% of tumors develop in the head and neck area , although gastrointestinal involvement has been reported in 10% of cases . pancreatic involvement of myeloma is relatively rare , with an incidence rate of 2.3% based on autopsy studies . however , previous studies failed to describe the type of disease entity , and did not distinguish between multiple myeloma , myelomatosis , solitary bone myeloma , or extramedullary plasmacytoma.3 - 6 the radiological differentiation of extramedullary plasmacytoma of the pancreas from other pancreatic tumors such as poorly differentiated pancreatic neoplasm , lymphoma , and metastasis is difficult . most cases of plasma cell infiltration of the pancreas are microscopic , and well - formed masses are unusual . the latter may present as a focal mass or a diffuse enlargement of the pancreas ; typically , the patient presents with jaundice and abdominal pain related to the obstruction of the biliary tree because pancreatic plasmacytoma is often located in the head of the pancreas . in the present case , the mass was detected incidentally because it was located in the body of the pancreas . on ultrasound , pancreatic plasmacytoma appears as a multilobulated heterogenous or a homogenous hypoechoic mass . the ct features have been described as a focal multilobulated solid hypodense mass with homogeneous intravenous contrast enhancement or diffuse enlargement of the pancreas . mri features include pancreatic enlargement with a lobulated contour , lower signal intensity than that of the liver on t1-weighted images , and diffusely increased signal intensity on t2-weighted pulse sequences with heterogeneous enhancement.7,8 in our case , imaging findings were compatible with those of previous western reports . the diagnosis of an extramedullary plasmacytoma is based on evidence of a monoclonal plasma cell tumor outside the bone marrow . in the present case eus - fna has an excellent safety profile regarding the risk of pancreatitis , bleeding , and perforation . major complications have been reported in 2.5% of 355 patients who underwent eus - fna for a solid pancreatic mass.9 potential complications include acute pancreatitis , infection , and sedation - related complications ; no deaths have been reported.10 seeding of the needle track with tumor cells is rare . previous studies reported that the risk of seeding of pancreatic adenocarcinoma via eus - fna is 2.2% , which is low compared with that ( 16.3% ) of ct or transabdominal ultrasonography - guided percutaneous biopsy.11,12 moreover , seeding of a pancreatic extramedullary plasmacytoma during eus - fna is yet to be reported . in conclusion , we safely performed eus - fna in a patient with pancreatic plasmacytoma , and were able to provide an accurate visualization and histopathologic diagnosis . although there are approximately three case reports describing pancreatic involvement of multiple myeloma diagnosed by eus - fna in the english language literature,7,13,14 to the best of our knowledge , this is the first time it is reported in korea .

extramedullary plasmacytoma involves organs outside the bone marrow ; however , involvement of the pancreas is rare . we recently experienced a case of extramedullary plasmacytoma of the pancreas that was diagnosed by endoscopic ultrasonography - guided fine needle aspiration ( eus - fna ) . eus - fna , which has a high diagnostic accuracy and an excellent safety profile , is the modality of choice for establishing tissue diagnosis . we report a case of extramedullary plasmacytoma of the pancreas diagnosed using eus - fna .

restless legs syndrome ( rls ) is a sensorimotor disorder characterized by an urge to move the legs that are associated with unpleasant paresthesias . usually , the symptoms worsen when at rest and at night and are relieved by movement . various studies suggest that genetic component , iron - deficiency , disturbances in the dopaminergic neurotransmitter system and abnormality in spinal conduction pathways are associated with the disorder . transcranial magnetic stimulation ( tms ) , developed in 1985 , is a noninvasive technique with the ability to stimulate neurons in the cerebral cortex through the scalp safely and with minimal discomfort . repetitive tms ( rtms ) is a technique that delivers long trains of closely spaced pulses to specific brain areas in order to alter cortical activity and connectivity . previous studies have suggested that low - frequency rtms decreases the excitability of the cortex while high - frequency rtms increases it . recently , rtms has been widely applied in the treatment of patients with psychiatric disorders , epilepsy , migraine , chronic pain , and neurodegenerative disorders , including parkinson 's disease ( pd ) , although its mechanism is not yet well understood . numerous reports have demonstrated that rtms of the human primary motor cortex induces the release of dopamine in the putamen , which indicates that rtms probably modulates striatal dopaminergic neurotransmission . currently , an increasing number of studies have provided support for a link between rls and pd . some studies have found that the prevalence of rls is higher in patients with pd than in the general population . based on the common mechanism involving disturbances in the dopaminergic neurotransmitter system between rls and pd , we investigated whether rtms application to the cortex was beneficial in patients with rls . we included fourteen idiopathic rls patients treated at the sleep clinic of our hospital between 2011 and 2012 that were diagnosed according to the international criteria of the international restless legs syndrome study group set in 2003 . the exclusion criteria were as follows : ( i ) all secondary rls stemming from a vitamin deficiency , iron - deficiency anemia , pregnancy , diabetes mellitus , severe metabolic disorders , liver dysfunction , or renal disease ; ( ii ) peripheral neuropathy and radiculopathy ; ( iii ) a history of psychiatric disease ; ( iv ) neuropathic pain ; ( v ) leg cramps or epilepsy ; ( vi ) use of a cardiac pacemaker , vagal nerve stimulator , or any metal implants ; ( vii ) other severe medical diseases . patients were not placed on any new medications , including dopaminergic agonists , psycholeptics , or benzodiazepines ; if they were already taking them for at least 4 weeks prior to the initiation of the study , they continued the medicine throughout the study at the prescribed dosage . all patients provided written informed consent , and the study had the approval of hospital ethics committee . we administered rtms at 15 hz using a magstim system ( magstim super rapid stimulator , magstim company , whitland , dyfed , uk ) with a figure - eight coil . one rtms train consisted of 75 pulses delivered at 15 hz with an intertrain interval of 10 min . in one session , 600 pulses ( 8 rtms trains ) were delivered to each hemisphere . one session was performed per day for 5 continuous days and stopped for 2 days . the patients were seated in a comfortable chair , and the coil was positioned at the leg representation in the motor cortex of frontal lobe . the optimal stimulation position for the tibialis anterior muscle was located by stimulating the presumed motor cortex at every 1 cm in a 6-cm . the resting motor threshold ( rmt ) was defined as the minimal stimulus intensity that produced a motor evoked potential in the relaxed muscle with a peak - to - peak amplitude of > 50 mv on 50% of 10 trials . the international rls rating scale ( irls - rs ) , pittsburgh sleep quality index ( psqi ) , hamilton anxiety scale ( hama ) and hamilton depression scale ( hamd ) were used to evaluate the severity of rls , quality of sleep , and the severity of anxiety and depression , respectively . the assessments were taken at the baseline ( prior to stimulation ) , at end of 14 session , and at 1- and 2-month posttreatment by a trained clinical neurologist . one - way analysis of variance was used to compare the means of scale scores at different time points . a p < 0.05 was considered as statistically significant . we included fourteen idiopathic rls patients treated at the sleep clinic of our hospital between 2011 and 2012 that were diagnosed according to the international criteria of the international restless legs syndrome study group set in 2003 . the exclusion criteria were as follows : ( i ) all secondary rls stemming from a vitamin deficiency , iron - deficiency anemia , pregnancy , diabetes mellitus , severe metabolic disorders , liver dysfunction , or renal disease ; ( ii ) peripheral neuropathy and radiculopathy ; ( iii ) a history of psychiatric disease ; ( iv ) neuropathic pain ; ( v ) leg cramps or epilepsy ; ( vi ) use of a cardiac pacemaker , vagal nerve stimulator , or any metal implants ; ( vii ) other severe medical diseases . patients were not placed on any new medications , including dopaminergic agonists , psycholeptics , or benzodiazepines ; if they were already taking them for at least 4 weeks prior to the initiation of the study , they continued the medicine throughout the study at the prescribed dosage . all patients provided written informed consent , and the study had the approval of hospital ethics committee . we administered rtms at 15 hz using a magstim system ( magstim super rapid stimulator , magstim company , whitland , dyfed , uk ) with a figure - eight coil . one rtms train consisted of 75 pulses delivered at 15 hz with an intertrain interval of 10 min . in one session , 600 pulses ( 8 rtms trains ) one session was performed per day for 5 continuous days and stopped for 2 days . then , another 4 days of stimulation were given . in total , 14 sessions were performed for each patient in our study . the patients were seated in a comfortable chair , and the coil was positioned at the leg representation in the motor cortex of frontal lobe . the optimal stimulation position for the tibialis anterior muscle was located by stimulating the presumed motor cortex at every 1 cm in a 6-cm . the resting motor threshold ( rmt ) was defined as the minimal stimulus intensity that produced a motor evoked potential in the relaxed muscle with a peak - to - peak amplitude of > 50 mv on 50% of 10 trials . the international rls rating scale ( irls - rs ) , pittsburgh sleep quality index ( psqi ) , hamilton anxiety scale ( hama ) and hamilton depression scale ( hamd ) were used to evaluate the severity of rls , quality of sleep , and the severity of anxiety and depression , respectively . the assessments were taken at the baseline ( prior to stimulation ) , at end of 14 session , and at 1- and 2-month posttreatment by a trained clinical neurologist . one - way analysis of variance was used to compare the means of scale scores at different time points . a p < 0.05 was considered as statistically significant . among 14 patients , there were 4 males and 10 females with a mean age of 59.22 10.10 years and a range of 4673 years . the irls - rs scores at the four - time points assessed ( baseline , end of 14 session , 1- and 2-month posttreatment ) are summarized in table 1 . all of the irls - rs , psqi and hama scores showed continuous and significant improvement posttreatment compared to baseline . the irls - rs , psqi , hama , and hamd scores in the fourteen idiopathic rls patients irls - rs : international rls rating scale ; psqi : pittsburgh sleep quality index ; hama : hamilton anxiety scale ; hamd : hamilton depression scale . * different from baseline , p<0.05 . our results indicated a significant improvement in irls - rs scores after 14 sessions , from 23.86 5.88 to 11.21 7.23 . this proved the effect of rtms in treating rls and that the effect could last for some time ( at least 2 months , according to our study ) after the stimulation . khedr et al . found that repeated sessions of rtms could produce prolonged changes in enhanced dopamine function that may be responsible for long - lasting clinical effects . after rtms treatment , psqi scores decreased from 15.00 4.88 to 9.29 3.91 and persisted at least 2 months after treatment . the long - lasting change in psqi scores was consistent with the improvement in irls - rs scores . found rls patients had significantly higher levels of anxiety and depression . in our study , rls patients showed obvious improvements in anxiety , and further improvements occurred in the 2 months after treatment . in addition , the patients depression improved after treatment , although this change was not statistically significant . it is likely that rtms improved symptoms of rls directly rather than its associated symptoms , such as mood disorders , which in turn led to an improvement in symptoms of rls . many studies have indicated that rls is not associated with the use of antidepressants , and some studies have suggested that antidepressants might exacerbate rls symptoms . in addition , depression did not improve as significantly as the symptoms of rls in our study . thus , we believe that rtms alleviates rls directly rather than through the treatment of associated symptoms . found that high - frequency rtms over the supplementary motor area ( sma ) improved irls - rs scores significantly after 5 and 10 sessions of stimulation . this result is consistent with ours , although the configuration of stimulation parameters differed , and they did not evaluate sleep quality , anxiety or depression . in burcu 's study , the stimulus frequency was 5 hz , and the stimulation was centered at points 3 cm anterior to the leg motor area at the sagittal midline . their parameters were taken from a previous study of pd , in which 5 hz rtms was administered over the sma , resulting in a modest improvement of motor symptoms . a recent study verified connectivity between the sma and the primary motor cortex . because stimulating either the sma or the primary motor cortex can alleviate rls syndrome , we speculate a common effect of rtms on upstream sma and downstream primary motor cortex . the exact mechanism underlying the treatment of rls with rtms is complex and not clearly understood . previous studies have revealed a shortened cortical silent period in the anterior tibialis muscle in patients with rls , which indicated a disturbed supraspinal inhibition mediated by decreased excitation of cortical inhibitory interneurons , thus leading to the hyperexcitability of spinal pathways . another probable mechanism involves the release of endogenous dopamine in the striatum , based on single photon emission computed tomography studies . however , a study conducted in japan suggested that chronic rtms had a limited effect on the dopaminergic system . hence , there may be multiple mechanisms of action involved in modulating symptoms of rls . revealing the exact mechanism is interesting and valuable and should be the subject of future study . in addition , no adverse effects were observed during stimulation or after treatment , and all patients showed good compliance . thus , 15 hz rtms delivered over the leg representation area of motor cortex is a safe treatment for rls . medications traditionally used to treat rls provide dramatic immediate benefits but may augment rls symptoms over time . thus , rtms and medications each possess advantages and provide patients with a variety of treatment options . however , our study had a limited sample size and no control group , therefore , a large case - control study is necessary to provide more convincing evidence . in addition , an optimized rtms paradigm should be established . in the future , we plan to compare cortical excitement before and after rtms treatment using neurophysiological and imaging measurements , such as paired pulse tms and functional magnetic resonance imaging , to identify the mechanism underlying rls . in conclusion , our study proves the utility of rtms for the treatment of rls patients despite the study 's limited sample size . in the future , a large case - control study should be performed , and the rtms protocol should be optimized .

background : repetitive transcranial magnetic stimulation ( rtms ) is a noninvasive technique used to alter cortex excitability that has been proposed as an efficient method for treating brain hyperexcitability or hypoexcitability disorders . the aim of this study was to investigate whether high - frequency rtms could have any beneficial effects in restless legs syndrome ( rls).methods : fourteen patients with rls were given high - frequency rtms ( 15 hz , 100% motor threshold ) to the leg representation motor cortex area of the frontal lobe for 14 sessions over 18 days . patients were diagnosed according to the international criteria proposed by the international restless legs syndrome study group in 2003 . the international rls rating scale ( irls - rs ) , pittsburgh sleep quality index ( psqi ) , hamilton anxiety scale ( hama ) and hamilton depression scale were used to evaluate the severity of rls , sleep quality , anxiety and depression , respectively . the scale scores were evaluated at four - time points ( baseline , end of the 14th session , and at 1- and 2-month posttreatment ) . one - way analysis of variance was used to compare scale scores at different time points.results:there was significant improvement in the irls - rs ( from 23.86 5.88 to 11.21 7.23 , p < 0.05 ) , psqi ( from 15.00 4.88 to 9.29 3.91 , p < 0.05 ) , and hama ( from 17.93 7.11 to 10.36 7.13 , p < 0.05 ) scale scores at the end of 14th session , with ongoing effects lasting for at least 2 months.conclusions:high-frequency rtms can markedly alleviate the motor system symptoms , sleep disturbances , and anxiety in rls patients . these results suggest that rtms might be an option for treating rls .

the high incidence of fractures of the angle of the mandible is attributed to a thinner cross - sectional area relative to the neighboring segments of the mandible , the curvature of trajectories in the angle region , and the presence of third molars , particularly those that are impacted , which weakens the region2 . it is therefore not uncommon for an oral and maxillofacial surgeon to encounter fractures of the angle of the mandible in their day - to - day practice . the oral and maxillofacial surgeon 's preference for the approach to a fracture site depends on accessibility , ease of procedure , aesthetic demands by the patient , and surgical expertise . various approaches are used for the fixation of fractures of the angle region of the mandible . this technique had certain disadvantages such as an unaesthetic scar and the risk of facial nerve injury , although exposure and direct application of the plate was better with this approach3 . to counteract these disadvantages , this approach involves operating entirely through an incision made in the oral mucosa / gingiva and is frequently used by surgeons . the disadvantages included placement of the plate in an anatomically unfavorable position , thin soft tissue coverage leading to an increase in dehiscence and exposure of the plate , and breakage of the plate due to a greater degree of intraoperative plate bending , which required to adapt to the complex contours of the superior border of the mandible . other disadvantages include placement of plate closer to the dentition , allowing an easier and shorter path for bacterial pathogens to move from the periodontal sulcus to the fixation hardware and more prevalent loosening of the screw , as there is less bone density on the superior aspect of the mandible and the alveolus4 . the disadvantages of the transoral approach prompted surgeons to find an alternative method , namely the transbuccal approach . this approach involves an intraoral incision plus a small incision on the facial skin , which permits the use of a transbuccal trocar to allow instruments such as a drill or screwdriver to pass through . advantages include no external scarring , fixation of the plates on the thicker lateral cortical plate of the mandible in a sagittal plane , greater soft tissue coverage , less chance of plate fracture as weakening of plates by over - bending is avoided , lower infection rate due to less movement of pathogens from the third molar region , and direct visualization and confirmation of desired occlusion during fixation45 . since the miniplate fixation differs for the transoral and transbuccal approaches , we decided to compare the two approaches in the management of angle fractures of the mandible . a total of 60 patients reporting to goa dental college and hospital from march 2013 to december 2014 were included in this prospective study and were randomly divided into 2 equal groups based on the type of approach employed for fracture fixation ( group a , transoral approach ; group b , transbuccal approach ) . the study was ethically approved by the goa dental college and hospital 's review committee and written informed consent was obtained from all patients participating in the study . we included patients with an age between 15 to 60 years , unilateral / bilateral mandibular angle fractures or fractures associated with other facial bone fractures with radiographic preoperative displacement of the fracture segments ranging from 1 to 5 mm , patients with controlled systemic conditions , and those willing to follow - up . we excluded patients who refused to follow - up or had a medically compromised status and patients with infections or pathologic or comminuted fractures . the degree of anatomical displacement was studied with digital orthopantomogram ( opg ) and posteroanterior ( pa ) mandible radiographs . complete hematological investigations were performed and all patients were started on intravenous antibiotics that were continued for 5 days postoperatively in all patients . erich arch bars were applied to the maxillary and mandibular dentition a day prior to surgery . all patients were operated under general anesthesia with nasotracheal intubation following a standard surgical protocol by a single oral and maxillofacial surgeon . in group a , following local infiltration of the intraoral site with 2% lignocaine with 1:80,000 adrenaline , an incision was planned extending from the anterior border of the ascending ramus at the level of maxillary occlusal plane . the incision was then carried down just along the lateral portion of the anterior ramus and continued forward approximately 5 mm from the junction of the attached mucosa and vestibule to extend anteriorly to the level of the mandibular first molar . b ) fractured segments were stabilized and fixed with a 2.5 mm 4-hole titanium miniplate with a gap , and were secured with monocortical screws that were 2.5-mm in diameter and 6 to 8 mm in length . general anesthesia was reversed and the patient was extubated and shifted to the recovery room . in group b , in addition to the transoral incision , a small extraoral stab incision was given to permit the insertion of the transbuccal cannula.(fig . b ) the location of the extraoral stab incision was guided by the location of the fracture line and the position of the facial vessels . the trocar was advanced into the operative site with blunt dissection through the stab incision , perforating the periosteum in the area planned for plate fixation.(fig . c ) the cheek retractor was applied to stabilize the trocar assembly during movement towards and away from the fracture site . a drill bit that was 11.5 cm in length and 2.3 mm in diameter the procedure followed for fracture reduction was similar to that of the transoral approach , except that after fracture reduction , the trocar assembly was removed and the extraoral skin incision was sutured with 5.0 ethilon ( johnson & johnson , new brunswick , nj , usa ) suture.(fig . d - h ) all patients were hospitalized for 5 days and were placed on a liquid diet for 2 weeks , followed by a soft diet for another 4 weeks . intraoperatively , patients were evaluated for the ease of surgical access for fixation and the surgical time ( time from incision to closure ) . the ease of surgical access for fixation in either approach was evaluated by the operating surgeon and graded as 1 , good ; 2 , fair ; and 3 , poor , based on the visual analogue scale6 . radiographic evaluation of fracture reduction between the two groups was done by measuring the gap between the fractured segments of the mandible in postoperative opg radiographs . all radiographs were performed using the orthophos xg machine ( sirona dental systems , bensheim , germany ) with similar exposure parameters . on the radiographs measurements of the fracture gap were conducted on these 4 defined points with a digital caliper6.(fig . 3 , 4 ) postoperative complications such as scarring ( in group b ) , occlusal discrepancy , infection , nonunion , and malunion were evaluated at each regular follow - up period . evaluation of scarring in group b was done with photographs at the 6th month postoperatively . the scoring for the scar was as follows : 1 , hypertrophic scar ; 2 , invisible scar ; and 3 , barely visible scar7 . postoperative occlusion was evaluated using the following scoring system : 1 , pre trauma ; 2 , minor discrepancy ; and 3 , major discrepancy8 . the data was tabulated and subjected to statistical analysis ( spss version 13 ; spss inc . , the mean age in this study was 26.73 years ( range , 17 - 53 years ) , with a peak incidence in the second and third decades of life ( n=46 , 76.7% ) which showed male predominance ( n=58 , 96.7% ) . road traffic accidents accounted for the majority of the cases ( n=52 , 86.7% ) . isolated mandibular angle fracture was seen in 20 patients ( 33.3% ) , with a higher incidence of right sided fracture ( n=36 , 60.0% ) when compared to the left ( n=24 , 40.0% ) . the ease of surgical access for fixation revealed no statistical significance when compared between the two groups . ( table 1 ) the mean surgical time for each group was 37 minutes and did not vary between groups.(table 2 ) postoperative radiographic tracing for both groups was done on the opg . it was noted that the reduction in the gap in group b was uniform from points a to d , whereas in group a , there was gradual increase in the distance between the fractured segments.(table 3 ) there was no statistical difference at point a for both groups . however , points b ( p=0.030 ) , c ( p=0.016 ) , and d ( p=0.004 ) were statistically different between groups.(table 4 ) with regard to postoperative complications , scar evaluation in group b at 6 months revealed 1 patient ( 3.3% ) with a hypertrophic scar , 6 patients ( 20.0% ) with barely visible scars , and 23 patients ( 76.7% ) with invisible scars . infection was noted in 2 patients ( 6.7% ) in group b , compared to 6 patients ( 20.0% ) in group a at 3 months postoperatively . the cause of the infection could be traced to the infected plates that were removed under local anesthesia , and patients were prescribed a course of oral antibiotics for 5 days . no cases of malunion or non - union were noted in the two groups . with regard to postoperative occlusion , 28 patients in group b had a score of 1 ( pre - trauma occlusion ) , compared to 16 patients in group a ( p=0.027 , significant ) . twelve patients in group a had a score of 2 ( mild discrepancy ) , compared to 2 patients in group b ( p=0.016 , significant ) . two patients in group a had a score of 3 ( major discrepancy ) , compared to no patients in group b.(table 5 ) the occlusal discrepancy was noted only in the first week postsurgery in either group and was corrected using elastic traction in all patients . the mandibular angle is subjected to forces between the muscles of mastication and the supra - hyoid group of muscles , resulting in unstable rotation of distal and proximal fragments . the presence of an impacted third molar tooth in the line of fracture may result in the fracture being compounded intraorally , which may distract away from bone or interfere in ideal fracture reduction9 . although the management of mandibular angle fractures is still a topic of debate , the treatment is dictated by the principles of fixation and aesthetic demand by the patient . as treatments and equipment have evolved , miniplate fixation can now be carried out in an anatomically favorable position using a transbuccal approach . however , some surgeons do not prefer the transbuccal technique due to the theoretical risk of damage to the facial nerve and an unfavorable facial scar1011 . in this study of 60 patients , the incidence of mandibular angle fractures was seen in ages ranging from 17 to 53 years , with a mean age of 26.73 years . the peak incidence of fractures was seen in the second and third decades of life ( n=46 , 76.7% ) with a definite predilection in males ( n=58 ) . road traffic accidents was the most common etiological factor , ( n=52 , 86.7% ) followed by assault ( n=8 , 13.3% ) . the findings were in unison with a study conducted by kumar et al.12 , which reported the pattern of maxillofacial fractures in 2,731 patients . the highest incidence of fractures in this study was found in the second and third decades of life ( n=1,535 , 56% ) . road traffic accidents were the most frequent cause ( n=2,086 , 76% ) , followed by assault ( n=260 , 12% ) . another similar study13 looked at 214 patients and stated that the incidence of angle fractures was higher in the male population and was most common in the third decade of life . although we report that surgical access is facilitated with the transbuccal approach , we did not observe any statistically significant differences between the two approaches for this parameter . surgical time is defined as the time taken from incision and exposure of the fractured site to closure . this finding contradicted studies in the literature that have shown increased surgical time with the transbuccal approach when compared to the transoral approach1011 . radiographic evaluation of fracture reduction was performed by studying the gap using tracings done on the opg . however , points b ( p=0.030 ) , c ( p=0.016 ) , and d ( p=0.004 ) were statistically different between groups . the reduction obtained in group b was uniform from points a to d , whereas in group a , there was a gradual increase in the distance between the fractured segments . we believe that the favorable position of the miniplate in the transbuccal approach brings about better control of the tensile and compressive forces , resulting in more uniform reduction in the fracture gap from points b to d. this observation was in accordance with a study on three - dimensional models by kroon et al.14 and choi et al.15 , who observed bony gaps along the inferior fracture border and found that this fracture movement was a contributory factor for subsequent complications , including infection . a study by wan et al.4 states that in transbuccal approach , no patients developed facial nerve palsy , whereas 1 patient out of 227 ( 45% ) developed a hypertrophic scar from the 6-mm facial skin incision . another study by sugar et al.10 reported similar findings in a population of 84 patients . no incidence of unsatisfactory facial scarring and facial nerve palsy from the transbuccal approach was noted . this is in accordance with our study , which reported 1 case ( 3.3% ) of hypertrophic scarring and no incidence of facial nerve palsy in group b. three months after surgery , only two patients in group b had an infection , as compared to six patients in group a. this was due to the infected plate , which was retrieved under local anesthesia . a course of oral antibiotics for 5 days was subsequently prescribed and the healing was uneventful . a study by barry and kearns9 reported infection in 4 out of 50 patients in which the plate was retrieved at an out - patient department . another study by ellis and walker16 reported infection occurring within two weeks of surgery in 2 out of 81 patients ; this infection was treated initially with oral antibiotics , which resulted in normal fracture healing . the gold standard in management of mandibular fracture is to establish the pre - trauma occlusion with minimal postoperative complications . when postoperative occlusion was assessed , the transoral group had significantly more occlusal discrepancy than the transbuccal group . the discrepancy in occlusion was observed only in the first week postsurgery and was managed using light guiding elastics in all patients , with no re - surgical intervention required in any patient . malocclusion may be due to the presence of concomitant fractures which may contribute to instability at the mandibular angle fracture site10 . this is in concordance with our study , which showed concomitant fractures in 11 patients ( 73.3% ) in group a and 9 patients ( 60% ) in group b. the rate of postoperative malocclusion reported in the literature ranges from 0% to as high as 7.5% . sugar et al.10 presented a study showing a strong preference of surgeons for fixation using a transbuccal approach . the principal reasons given were ease of use , minimal requirement for plate bending , and facilitation of plate placement in the neutral mid - point area of the mandible . our experience with the transbuccal approach was somewhat similar . a meta - analysis by al - moraissi and ellis17 states that the use of one miniplate is superior to the use of two miniplates in the management of mandibular angle fractures , as the incidence of postoperative complications was considerably lower . this is concordant with the present study , which showed better results when a single miniplate was used either transorally or transbuccally . in conclusion , although both approaches have inherent advantages and disadvantages , the transbuccal approach was superior to the transoral approach with regard to radiographic reduction in the fracture gap , inconspicuous external scarring , and fewer postoperative complications . we did not find increased operating time or damage to the facial nerve , which was observed by other authors when the transbuccal approach was employed . we preferred the transbuccal approach over the transoral approach due to ease of use , minimal requirement for plate bending , and facilitation of plate placement in the neutral mid - point area of the mandible . a study employing a larger sample size and without any confounding variables is ongoing to define our results even more precisely .

objectiveswe compared the transbuccal and transoral approaches in the management of mandibular angle fractures.materials and methodssixty patients with mandibular angle fractures were randomly divided into two equal groups ( a , transoral approach ; group b , transbuccal approach ) who received fracture reduction using a single 2.5 mm 4 holed miniplate with a bar using either of the two approaches . intraoperatively , the surgical time and the ease of surgical assess for fixation were noted . patients were followed at 1 week , 3 months , and 6 months postoperatively and evaluated clinically for post - surgical complications like scarring , infection , postoperative occlusal discrepancy , malunion , and non - union . radiographically , the interpretation of fracture reduction was also performed by studying the fracture gap following reduction using orthopantomogram tracing . the data was tabulated and subjected to statistical analysis . a p - value less than 0.05 was considered significant.resultsno significant difference was seen between the two groups for variables like surgical time and ease of fixation . radiographic interpretation of fracture reduction revealed statistical significance for group b from points b to d as compared to group a. no cases of malunion / non - union were noted . a single case of hypertrophic scar formation was noted in group b at 6 months postsurgery . infection was noted in 2 patients in group b compared to 6 patients in group a. there was significantly more occlusal discrepancy in group a compared to group b at 1 week postoperatively , but no long standing discrepancy was noted in either group at the 6 months follow-up.conclusionthe transbuccal approach was superior to the transoral approach with regard to radiographic reduction of the fracture gap , inconspicuous external scarring , and fewer postoperative complications . we preferred the transbuccal approach due to ease of use , minimal requirement for plate bending , and facilitation of plate placement in the neutral mid - point area of the mandible .

motor and sensory functioning , cognitive functioning , autonomic functioning , as well as emotional and social functioning all depend on anatomic and physiological integrity of the neural networks . this integrity may be disturbed by numerous factors , varying from intrinsic brain disease to mood disorders and from metabolic disturbances and general disease to exogenous intoxications . intrinsic brain disease may be focal , multifocal or generalized , and examples from these categories are brain tumors , multiple sclerosis and encephalitis , respectively . but optimal functioning of the brain is also determined by an intact environment . cerebral functioning in a person who is generally ill as a result of infectious disease or disseminated cancer may be severely impaired . consciousness and cognition may be hampered by a disturbed liver function , by hypoglycemia or by acidosis . and exogenous intoxications by carbon monoxide or alcohol , but also by numerous sorts of medication , such as sedative drugs , antidepressants and anti - epileptic drugs , may have a strong impact on functioning of the brain . in patients with brain tumors , disorders of cerebral functioning are a major concern and it is important to realize that not only the tumor itself but also our broad array of therapeutic interventions and all kinds of metabolic , psychological and social factors contribute to the cerebral condition that determines how the patient is able to express his emotions , how he is able to think , how he functions in his work and in his social environment , and , finally , what his quality of life is . brain tumors almost invariably cause severe symptoms , such as focal neurological deficits , cognitive deficits , and focal or generalized epileptic seizures . apart from that , brain tumors may give rise to increased intracranial pressure , resulting in headache or impairment of consciousness . brain tumors have this significant impact on the brain , since they force the non - tumoral brain tissue not only to adapt to the presence of an expanding mass , but also to the invasion of healthy brain tissue by infiltrating tumor cells . it is a great challenge to clarify the underlying mechanisms accounting for the changes in the brain s functional status resulting from the presence of this foreign entity and leading to cognitive impairments , to an alteration of emotional functioning , and to epileptic seizures . when reviewing the current knowledge in neuro - oncology , there is a strong need for theory regarding the complex relations between the tumor on the one hand , and epilepsy and cognitive and emotional status on the other hand . the study of these relations is complicated by the fact that patients with primary brain tumors undergo a large number of therapeutic interventions , starting with surgery , which is almost invariably followed by irradiation , and in a number of cases by chemotherapy . apart from these anti - tumor treatment modalities , brain tumor patients may receive antiepileptic drugs , antidepressants and corticosteroids , all having their specific impact on cerebral functioning . cognition is a collective term for a number of functions that can be subdivided into different domains . the six most important domains are ( 1 ) information processing , ( 2 ) psychomotor functioning , ( 3 ) attention , ( 4 ) verbal memory , ( 5 ) working memory , and ( 6 ) executive functioning . in order to get properly informed on the cognitive abilities of an individual patient , a number of cognitive tests should be performed , measuring these separate functions . the more extensive the test battery , the more detailed the information on the cerebral functioning of the patient . a number of studies on cognitive functioning in glioma patients have been published . severe neuropsychological impairments have been found in up to 89% of patients with high - grade gliomas ( hgg ) [ 24 ] . a comparable percentage of patients with low - grade gliomas ( lgg ) display cognitive deficits [ 512 ] . the most striking collective finding of these studies is that cognitive deficits are not restricted to the area of the brain where the tumor is located and where it has caused local damage , but rather should be traced to a more global dysfunctioning of the brain : memory disturbances , loss of concentration , difficulties with planning and language , and psychomotor slowness . it appears that the rate of tumor growth is related to the degree of cognitive dysfunction : a fast - growing brain tumor causes more profound cognitive deficits than a slow - growing tumour [ 13 , 14 ] . this is in accordance with the observation that brain tumor patients show remarkable preservation of cognitive functioning when compared to patients with acute lesions of the same size . when comparing the devastating effect of these acute lesions with that of slowly growing tumors , it is clear that plasticity plays a major role in the resilience to cognitive deficits in brain tumor patients . epilepsy could be considered as one of the manifestations of brain dysfunction and is often the first manifestation of a brain tumor . between 10 and 15% of adult patients presenting with epileptic seizures are diagnosed with a brain tumor as the underlying cause of the seizures [ 16 , 17 ] . conversely , most patients suffering from brain tumors develop epileptic seizures at some point during the course of their disease . this is the case in 85% of lgg patients , whereas about 50% of hgg patients will experience epileptic seizures at some point of their disease . we do not know why some patients develop epileptic seizures whereas others , suffering from the same tumor type in the same location , do not . surgery , radiotherapy , anti - epileptic drugs , glucocorticoids and various chemotherapy regimens may have influence on cerebral functioning of brain tumor patients . but also the tumor itself , psychological distress , depression and fatigue have a certain impact , and usually a combination of all these factors eventually destines how the patient will function . this makes it difficult to sort out what the contribution of separate treatment modalities may be . the primary aim of surgery is to reduce tumor mass if the localization of the tumor allows this . talacchi and coworkers ( 2011 ) reported a series of 29 glioma patients in whom extensive neuropsychological examination was performed both pre- and postoperatively . they found postoperative impairment in comparison to preoperative functioning in 38% of patients and the reverse ( postoperative improvement in comparison to preoperative functioning ) in 24% of patients . this illustrates that surgical treatment of brain tumors may be of benefit to cognitive function , since it reduces intracranial pressure and compression of brain tissue , but may also cause damage whether transient or more permanent . another study in 23 patients harboring a lgg in the language areas stressed the importance of neuropsychological assessment before operation : the authors found transient worsening of verbal working memory immediately after surgery which recovered within three months . improvement of language function after initial worsening following tumor resection was also reported by sanai et al . . especially the first reports on cognitive damage in young children who had been treated with radiotherapy for brain tumors or acute leukemia were alarming . later , radiation induced neurotoxicity was also reported in adults who had been treated with radiotherapy for primary brain tumors or brain metastases [ 22 , 23 ] . our findings suggested that a mean of 6 years after diagnosis the tumor itself , rather than the radiotherapy had the most deleterious effect on cognitive functioning ; only high fraction dose radiotherapy ( > 2 gy ) resulted in significant added cognitive deterioration . in a follow - up study of the same cohort at a mean of 12 years after first diagnosis , however , it appeared that long - term survivors of lgg who did not have radiotherapy had stable cognitive status in contrast to patients who had received radiotherapy . it is important to realize that this attentional deficit developed independently of the localization of the tumor and that treatment in this patient group had been restricted to focal irradiation . this finding indicates that global cognitive dysfunctioning may result from local damage . in patients with hgg , it is more difficult to determine the long term role of radiotherapy because in these patients the duration of survival is much shorter than in lgg patients . similar to surgical treatment , radiotherapy in hgg often leads to tumor response and this may result in improvement of cognitive functioning . a rapid decline of cognitive functioning occurs in almost all cases of glioma when tumor progression or tumor recurrence occurs . epileptic seizures necessitate the prescription of anti - epileptic drugs , and especially the older or first - generation anti - epileptic drugs ( phenytoin , carbamazepine and valproic acid ) may have a negative influence on cognition . it is interesting and important to note that levetiracetam , a second generation drug , may have a positive influence , instead of a deteriorating influence on cognition . glucocorticoids are usually very effective in the treatment of vasogenic edema , but may also induce new problems with regard to cognitive and emotional functioning . nowadays , temozolomide is incorporated in the treatment of most newly diagnosed hgg s . the use of temozolomide is not associated with neurocognitive side effects and may even have a beneficial effect on seizure frequency , but it is very well possible that other drugs will become part of glioma combined treatment regimens in the future and some of these drugs may prove to have neurotoxic side effects . in conclusion , brain tumors may negatively affect cerebral functioning and various combinations of all therapeutic modalities , as summarized here , may have an additional negative , but in some situations also a positive impact . the study of brain functions and the way these functions are affected by tumor progression and by various treatment modalities is usually performed by means of measurement of function : neurological examination , a variety of neuropsychological function tests , performance status scales and quality of life questionnaires are examples of instruments to directly or indirectly assess brain function . all these functions , however , depend on the integrity of the cerebral network and direct measurement of network parameters would have obvious advantages . electroencephalography ( eeg ) and magnetoencephalography ( meg ) are potential instruments to study these parameters . if it would become possible to identify certain characteristics of the human cerebral network , more insight into the effects of local lesions , such as brain tumors , and into the effects of systemic treatments on the integrity and plasticity of the cerebral network could result . we will give a short overview of network theory and the possibilities to study neural networks in the following paragraphs . network or graph theory originates from both the fields of mathematics and sociology . combining these two has led to a number of methods of analyzing different types of networks by representing them in an abstract , theoretical figure called a graph. the challenge of the study of networks including the study of neural networks is to find the optimal method of describing and defining all kinds of biological and social networks . most optimal functioning networks have so - called small - world characteristics , referring to a locally clustered architecture in combination with long distance connections . this means that parts of the network that are virtually remote from each other can actually be linked through only a few steps . this co - existence of integration and segregation in complex networks has only recently been elucidated . random networks were first described in the 20th century and seemed promising to model complex networks [ 30 , 31 ] . however , these graphs did not meet up to the expectations of explaining the abovementioned small - world characteristics of networks . they proposed a very simple model of a one - dimensional network ( see fig . 1 ) . in the regular network , each node or vertex is only connected to its next , with likelihood p , connections or edges are chosen at random and connected to other nodes , also chosen randomly . with increasing p , more and more edges become randomly reconnected and finally , for p = 1 , the network is completely random . this comprehensible model allows investigation of all types of networks , ranging from completely regular to completely random.fig . 1schematic drawing of a random , a regular , and a small - world network . in the regular network therefore , it has both a high clustering coefficient ( c ) and a long path length ( l ) , while the random network ( right ) combines a low c and a low l. the intermediate of the two : the so - called small - world network ( middle ) can be achieved by relocating but a few long - distance connections from the regular network , which causes l to decrease drastically but preserves a high c. thus it combines the best of two worlds schematic drawing of a random , a regular , and a small - world network . in the regular network ( left ) , each node is only connected to its neighbors . therefore , it has both a high clustering coefficient ( c ) and a long path length ( l ) , while the random network ( right ) combines a low c and a low l. the intermediate of the two : the so - called small - world network ( middle ) can be achieved by relocating but a few long - distance connections from the regular network , which causes l to decrease drastically but preserves a high c. thus it combines the best of two worlds the intermediate between random and regular architecture proved to be crucial to the understanding of the small - world phenomenon . two measures appear to be important for the classification of a network on the continuum of regular to random . the first measure is the clustering coefficient ( c ) , which can be defined as the likelihood that neighbors of a vertex are also connected . this is defined as the average of the shortest distance between pairs of nodes counted in the number of edges . using these two measures , we can resume the theory as follows : regular networks are very clustered ( high c ) but it takes a lot of steps to get from one side of the graph to the other ( high l ) . in contrast , random networks lack this high clustering ( they have a low c ) and the path length is short ( low l ) . neither of the two have small - world properties . however , these small - world properties occur already when p is only slightly higher than 0 : now the path length l drops sharply , while the clustering coefficient ( c ) hardly changes . thus networks with only a few ( randomly ) rewired connections combine both high clustering and a small path length : this phenomenon is referred to as the small - world phenomenon . these measures c and l make it possible to define the index of small - worldness . the discovery of these tools to describe small - world networks initiated a widespread interest in all kinds of complex networks and gave rise to a wide range of theoretical and experimental studies . one of the most intriguing , and certainly the most complex network is the human brain . brain activity is commonly studied by making use of functional magnetic resonance imaging ( fmri ) , eeg or meg . eeg has been a routine instrument in the study of brain function for a number of decades . it measures electrical flow and is especially useful for the diagnosis of epilepsy and other pathological cerebral conditions , such as encephalitis . meg is a more sophisticated method to measure brain activity and has a higher spatial resolution . meg registers brain activity by measuring magnetic flow , and the scalp and the skull do not distort signal registration in contrast with eeg . within the signal measured by eeg and meg , different frequency bands can be distinguished : delta ( 0.54 hz ) , theta ( 48 hz ) , lower alpha ( 810 hz ) , upper alpha ( 1013 hz ) , beta ( 1330 hz ) , lower gamma ( 3055 hz ) , and upper gamma band ( 5580 hz ) . a fundamental conception for the study of brain dynamics is synchronization or functional connectivity . the basic assumption of functional connectivity is that statistical interdependencies between time series of brain activity at separate areas reflect functional interactions between these brain regions . this functional connectivity can be calculated on the basis of the amount of synchrony of brain activity measured in two different areas . the conception is that multiple local networks are maintained by long - distance patterns of functional connectivity and this results in higher and complex brain functions , such as planning , memory , and executive functioning [ 3538 ] . functional connectivity between brain areas may thus be used to construct graphs of the brain ( see fig . 2 for an example in an meg recording ) . the two prerequisites of local segregation , referring to local specialization in specific tasks , and integration , combining information from lower - level networks at a higher and more global level , are thought to be crucial for optimal brain functioning [ 3941 ] . the small - world network is a highly adequate model of organization in the brain , because it supports both segregated as well as integrated information processing . brain tumors may interfere with neuronal structures and the resulting disturbances of anatomical connectivity may lead to alterations of functional connectivity patterns [ 43 , 44].fig . synchronization of signals from different regions of the brain is a measure of connectivity of these regions part of a 151-channel meg recording . synchronization of signals from different regions of the brain is a measure of connectivity of these regions during the last decade , we have shown that most lgg and hgg patients perform significantly worse than healthy controls on almost all cognitive tests . in order to better understand why glioma patients develop those global cognitive deficits , we investigated the correlation between resting - state meg - based functional connectivity and cognitive functioning in patients with lgg . we therefore studied correlations between time series of 126 meg channels and we used the so - called synchronization likelihood ( sl ) as one of the measures of synchronization ( or functional connectivity ) . the sl varies between zero ( no synchronization at all ) and one ( total synchronization ) . we found that lgg patients showed increased sl in the delta , theta , and lower gamma frequency bands in comparison to healthy controls , while connectivity in the lower alpha band was decreased . in another study we also found a correlation between cognitive functioning and network architecture in lgg patients . these findings partly confirmed our previous studies , in which we also found changes in functional connectivity and network architecture in brain tumor patients with various histologies [ 47 , 48 ] . there were some contradictory details between the earlier publications and the results of the later papers , especially regarding differences between tumor patients and healthy controls in the pattern and the direction of changes in connectivity in the various frequency bands . possible explanations for these differences in both functional connectivity and network topology may relate to the different patient samples : a homogeneous group of lgg patients versus a more heterogeneous sample of brain tumor patients . despite these contradictory details , it is most likely that conceptions like functional connectivity and network topology are pivotal for cognitive functioning . both cognitive performance and functional connectivity are disturbed in lgg patients , and correlations exist between these two phenomena . brain tumor patients consistently show pathologically increased connectivity particularly in the lower frequency bands ( delta , theta ) , which is related to poorer cognitive functioning . we further investigated network characteristics in glioma patients with epilepsy by means of meg registrations . functional connectivity was calculated in six frequency bands , as were a number of network measures such as normalized clustering coefficient and path length . increased theta band connectivity appeared to be related to a higher total number of seizures . furthermore , higher number of seizures was related to a less optimal , more random brain network topology . these results indicate that ( pathologically ) increased theta band connectivity is related to a higher number of epileptic seizures in brain tumor patients , suggesting that theta band connectivity changes are a hallmark of tumor - related epilepsy . furthermore , a more random brain network topology is related to greater vulnerability to seizures . thus , functional connectivity and brain network architecture may prove to be important parameters of tumor - related epilepsy . brain tumors almost invariably cause severe symptoms , such as focal neurological deficits , cognitive deficits , and focal or generalized epileptic seizures . apart from that , brain tumors may give rise to increased intracranial pressure , resulting in headache or impairment of consciousness . brain tumors have this significant impact on the brain , since they force the non - tumoral brain tissue not only to adapt to the presence of an expanding mass , but also to the invasion of healthy brain tissue by infiltrating tumor cells . it is a great challenge to clarify the underlying mechanisms accounting for the changes in the brain s functional status resulting from the presence of this foreign entity and leading to cognitive impairments , to an alteration of emotional functioning , and to epileptic seizures . when reviewing the current knowledge in neuro - oncology , there is a strong need for theory regarding the complex relations between the tumor on the one hand , and epilepsy and cognitive and emotional status on the other hand . the study of these relations is complicated by the fact that patients with primary brain tumors undergo a large number of therapeutic interventions , starting with surgery , which is almost invariably followed by irradiation , and in a number of cases by chemotherapy . apart from these anti - tumor treatment modalities , brain tumor patients may receive antiepileptic drugs , antidepressants and corticosteroids , all having their specific impact on cerebral functioning . cognitive functioning may be impaired in many conditions that affect the brain . but cognitive functioning as such is an ill - defined entity . cognition is a collective term for a number of functions that can be subdivided into different domains . the six most important domains are ( 1 ) information processing , ( 2 ) psychomotor functioning , ( 3 ) attention , ( 4 ) verbal memory , ( 5 ) working memory , and ( 6 ) executive functioning . in order to get properly informed on the cognitive abilities of an individual patient , a number of cognitive tests should be performed , measuring these separate functions . the more extensive the test battery , the more detailed the information on the cerebral functioning of the patient . a number of studies on cognitive functioning in glioma patients have been published . severe neuropsychological impairments have been found in up to 89% of patients with high - grade gliomas ( hgg ) [ 24 ] . a comparable percentage of patients with low - grade gliomas ( lgg ) display cognitive deficits [ 512 ] . the most striking collective finding of these studies is that cognitive deficits are not restricted to the area of the brain where the tumor is located and where it has caused local damage , but rather should be traced to a more global dysfunctioning of the brain : memory disturbances , loss of concentration , difficulties with planning and language , and psychomotor slowness . it appears that the rate of tumor growth is related to the degree of cognitive dysfunction : a fast - growing brain tumor causes more profound cognitive deficits than a slow - growing tumour [ 13 , 14 ] . this is in accordance with the observation that brain tumor patients show remarkable preservation of cognitive functioning when compared to patients with acute lesions of the same size . when comparing the devastating effect of these acute lesions with that of slowly growing tumors , it is clear that plasticity plays a major role in the resilience to cognitive deficits in brain tumor patients . epilepsy could be considered as one of the manifestations of brain dysfunction and is often the first manifestation of a brain tumor . between 10 and 15% of adult patients presenting with epileptic seizures are diagnosed with a brain tumor as the underlying cause of the seizures [ 16 , 17 ] . conversely , most patients suffering from brain tumors develop epileptic seizures at some point during the course of their disease . this is the case in 85% of lgg patients , whereas about 50% of hgg patients will experience epileptic seizures at some point of their disease . we do not know why some patients develop epileptic seizures whereas others , suffering from the same tumor type in the same location , do not . surgery , radiotherapy , anti - epileptic drugs , glucocorticoids and various chemotherapy regimens may have influence on cerebral functioning of brain tumor patients . but also the tumor itself , psychological distress , depression and fatigue have a certain impact , and usually a combination of all these factors eventually destines how the patient will function . this makes it difficult to sort out what the contribution of separate treatment modalities may be . the primary aim of surgery is to reduce tumor mass if the localization of the tumor allows this . talacchi and coworkers ( 2011 ) reported a series of 29 glioma patients in whom extensive neuropsychological examination was performed both pre- and postoperatively . they found postoperative impairment in comparison to preoperative functioning in 38% of patients and the reverse ( postoperative improvement in comparison to preoperative functioning ) in 24% of patients . this illustrates that surgical treatment of brain tumors may be of benefit to cognitive function , since it reduces intracranial pressure and compression of brain tissue , but may also cause damage whether transient or more permanent . another study in 23 patients harboring a lgg in the language areas stressed the importance of neuropsychological assessment before operation : the authors found transient worsening of verbal working memory immediately after surgery which recovered within three months . improvement of language function after initial worsening following tumor resection was also reported by sanai et al . . especially the first reports on cognitive damage in young children who had been treated with radiotherapy for brain tumors or acute leukemia were alarming . later , radiation induced neurotoxicity was also reported in adults who had been treated with radiotherapy for primary brain tumors or brain metastases [ 22 , 23 ] . our findings suggested that a mean of 6 years after diagnosis the tumor itself , rather than the radiotherapy had the most deleterious effect on cognitive functioning ; only high fraction dose radiotherapy ( > 2 gy ) resulted in significant added cognitive deterioration . in a follow - up study of the same cohort at a mean of 12 years after first diagnosis , however , it appeared that long - term survivors of lgg who did not have radiotherapy had stable cognitive status in contrast to patients who had received radiotherapy . it is important to realize that this attentional deficit developed independently of the localization of the tumor and that treatment in this patient group had been restricted to focal irradiation . this finding indicates that global cognitive dysfunctioning may result from local damage . in patients with hgg , it is more difficult to determine the long term role of radiotherapy because in these patients the duration of survival is much shorter than in lgg patients . similar to surgical treatment , radiotherapy in hgg often leads to tumor response and this may result in improvement of cognitive functioning . a rapid decline of cognitive functioning occurs in almost all cases of glioma when tumor progression or tumor recurrence occurs . epileptic seizures necessitate the prescription of anti - epileptic drugs , and especially the older or first - generation anti - epileptic drugs ( phenytoin , carbamazepine and valproic acid ) may have a negative influence on cognition . it is interesting and important to note that levetiracetam , a second generation drug , may have a positive influence , instead of a deteriorating influence on cognition . glucocorticoids are usually very effective in the treatment of vasogenic edema , but may also induce new problems with regard to cognitive and emotional functioning . nowadays , temozolomide is incorporated in the treatment of most newly diagnosed hgg s . the use of temozolomide is not associated with neurocognitive side effects and may even have a beneficial effect on seizure frequency , but it is very well possible that other drugs will become part of glioma combined treatment regimens in the future and some of these drugs may prove to have neurotoxic side effects . in conclusion , brain tumors may negatively affect cerebral functioning and various combinations of all therapeutic modalities , as summarized here , may have an additional negative , but in some situations also a positive impact . the study of brain functions and the way these functions are affected by tumor progression and by various treatment modalities is usually performed by means of measurement of function : neurological examination , a variety of neuropsychological function tests , performance status scales and quality of life questionnaires are examples of instruments to directly or indirectly assess brain function . all these functions , however , depend on the integrity of the cerebral network and direct measurement of network parameters would have obvious advantages . electroencephalography ( eeg ) and magnetoencephalography ( meg ) are potential instruments to study these parameters . if it would become possible to identify certain characteristics of the human cerebral network , more insight into the effects of local lesions , such as brain tumors , and into the effects of systemic treatments on the integrity and plasticity of the cerebral network could result . we will give a short overview of network theory and the possibilities to study neural networks in the following paragraphs . network or graph theory originates from both the fields of mathematics and sociology . combining these two has led to a number of methods of analyzing different types of networks by representing them in an abstract , theoretical figure called a graph. the challenge of the study of networks including the study of neural networks is to find the optimal method of describing and defining all kinds of biological and social networks . networks usually combine two seemingly opposing concepts : integration and segregation . most optimal functioning networks have so - called small - world characteristics , referring to a locally clustered architecture in combination with long distance connections . this means that parts of the network that are virtually remote from each other can actually be linked through only a few steps . this co - existence of integration and segregation in complex networks has only recently been elucidated . random networks were first described in the 20th century and seemed promising to model complex networks [ 30 , 31 ] . however , these graphs did not meet up to the expectations of explaining the abovementioned small - world characteristics of networks . they proposed a very simple model of a one - dimensional network ( see fig . 1 ) . in the regular network , each node or vertex is only connected to its next , with likelihood p , connections or edges are chosen at random and connected to other nodes , also chosen randomly . with increasing p , more and more edges become randomly reconnected and finally , for p = 1 , the network is completely random . this comprehensible model allows investigation of all types of networks , ranging from completely regular to completely random.fig . 1schematic drawing of a random , a regular , and a small - world network . in the regular network ( left ) , each node is only connected to its neighbors . therefore , it has both a high clustering coefficient ( c ) and a long path length ( l ) , while the random network ( right ) combines a low c and a low l. the intermediate of the two : the so - called small - world network ( middle ) can be achieved by relocating but a few long - distance connections from the regular network , which causes l to decrease drastically but preserves a high c. thus it combines the best of two worlds schematic drawing of a random , a regular , and a small - world network . in the regular network ( left ) , each node is only connected to its neighbors . therefore , it has both a high clustering coefficient ( c ) and a long path length ( l ) , while the random network ( right ) combines a low c and a low l. the intermediate of the two : the so - called small - world network ( middle ) can be achieved by relocating but a few long - distance connections from the regular network , which causes l to decrease drastically but preserves a high c. thus it combines the best of two worlds the intermediate between random and regular architecture proved to be crucial to the understanding of the small - world phenomenon . two measures appear to be important for the classification of a network on the continuum of regular to random . the first measure is the clustering coefficient ( c ) , which can be defined as the likelihood that neighbors of a vertex are also connected . this is defined as the average of the shortest distance between pairs of nodes counted in the number of edges . using these two measures , we can resume the theory as follows : regular networks are very clustered ( high c ) but it takes a lot of steps to get from one side of the graph to the other ( high l ) . in contrast , random networks lack this high clustering ( they have a low c ) and the path length is short ( low l ) . neither of the two have small - world properties . however , these small - world properties occur already when p is only slightly higher than 0 : now the path length l drops sharply , while the clustering coefficient ( c ) hardly changes . thus networks with only a few ( randomly ) rewired connections combine both high clustering and a small path length : this phenomenon is referred to as the small - world phenomenon . these measures c and l make it possible to define the index of small - worldness . the discovery of these tools to describe small - world networks initiated a widespread interest in all kinds of complex networks and gave rise to a wide range of theoretical and experimental studies . one of the most intriguing , and certainly the most complex network is the human brain . brain activity is commonly studied by making use of functional magnetic resonance imaging ( fmri ) , eeg or meg . eeg has been a routine instrument in the study of brain function for a number of decades . it measures electrical flow and is especially useful for the diagnosis of epilepsy and other pathological cerebral conditions , such as encephalitis . meg is a more sophisticated method to measure brain activity and has a higher spatial resolution . meg registers brain activity by measuring magnetic flow , and the scalp and the skull do not distort signal registration in contrast with eeg . within the signal measured by eeg and meg , different frequency bands can be distinguished : delta ( 0.54 hz ) , theta ( 48 hz ) , lower alpha ( 810 hz ) , upper alpha ( 1013 hz ) , beta ( 1330 hz ) , lower gamma ( 3055 hz ) , and upper gamma band ( 5580 hz ) . a fundamental conception for the study of brain dynamics is synchronization or the basic assumption of functional connectivity is that statistical interdependencies between time series of brain activity at separate areas reflect functional interactions between these brain regions . this functional connectivity can be calculated on the basis of the amount of synchrony of brain activity measured in two different areas . the conception is that multiple local networks are maintained by long - distance patterns of functional connectivity and this results in higher and complex brain functions , such as planning , memory , and executive functioning [ 3538 ] . functional connectivity between brain areas may thus be used to construct graphs of the brain ( see fig . 2 for an example in an meg recording ) . the two prerequisites of local segregation , referring to local specialization in specific tasks , and integration , combining information from lower - level networks at a higher and more global level , are thought to be crucial for optimal brain functioning [ 3941 ] . the small - world network is a highly adequate model of organization in the brain , because it supports both segregated as well as integrated information processing . brain tumors may interfere with neuronal structures and the resulting disturbances of anatomical connectivity may lead to alterations of functional connectivity patterns [ 43 , 44].fig . synchronization of signals from different regions of the brain is a measure of connectivity of these regions part of a 151-channel meg recording . synchronization of signals from different regions of the brain is a measure of connectivity of these regions during the last decade , we have shown that most lgg and hgg patients perform significantly worse than healthy controls on almost all cognitive tests . in order to better understand why glioma patients develop those global cognitive deficits , we investigated the correlation between resting - state meg - based functional connectivity and cognitive functioning in patients with lgg . we therefore studied correlations between time series of 126 meg channels and we used the so - called synchronization likelihood ( sl ) as one of the measures of synchronization ( or functional connectivity ) . the sl varies between zero ( no synchronization at all ) and one ( total synchronization ) . we found that lgg patients showed increased sl in the delta , theta , and lower gamma frequency bands in comparison to healthy controls , while connectivity in the lower alpha band was decreased . in another study we also found a correlation between cognitive functioning and network architecture in lgg patients . these findings partly confirmed our previous studies , in which we also found changes in functional connectivity and network architecture in brain tumor patients with various histologies [ 47 , 48 ] . there were some contradictory details between the earlier publications and the results of the later papers , especially regarding differences between tumor patients and healthy controls in the pattern and the direction of changes in connectivity in the various frequency bands . possible explanations for these differences in both functional connectivity and network topology may relate to the different patient samples : a homogeneous group of lgg patients versus a more heterogeneous sample of brain tumor patients . despite these contradictory details , it is most likely that conceptions like functional connectivity and network topology are pivotal for cognitive functioning . both cognitive performance and functional connectivity are disturbed in lgg patients , and correlations exist between these two phenomena . brain tumor patients consistently show pathologically increased connectivity particularly in the lower frequency bands ( delta , theta ) , which is related to poorer cognitive functioning . we further investigated network characteristics in glioma patients with epilepsy by means of meg registrations . functional connectivity was calculated in six frequency bands , as were a number of network measures such as normalized clustering coefficient and path length . increased theta band connectivity appeared to be related to a higher total number of seizures . furthermore , higher number of seizures was related to a less optimal , more random brain network topology . these results indicate that ( pathologically ) increased theta band connectivity is related to a higher number of epileptic seizures in brain tumor patients , suggesting that theta band connectivity changes are a hallmark of tumor - related epilepsy . furthermore , a more random brain network topology is related to greater vulnerability to seizures . thus , functional connectivity and brain network architecture may prove to be important parameters of tumor - related epilepsy . these disturbances may consist of function loss ( especially loss of complex functions ) , and epileptic seizures . it has been shown that both types of cerebral function disruption are associated with changes in functional connectivity and network architecture . although we are only in the very beginning of unraveling the extremely complex network architecture of the brain , the findings in brain tumor patients may prove to be of great importance for our future strategies in the treatment of these patients . possibilities to successfully combine brain tumor surgery and epilepsy surgery will improve , and we will be able to longitudinally study the influence of various treatment strategies on the network . in short , we will be able to study the plasticity of the brain in a direct and non - invasive way .

brain functions , including cognitive functions , are frequently disturbed in brain tumor patients . these disturbances may result from the tumor itself , but also from the treatment directed against the tumor . surgery , radiotherapy and chemotherapy all may affect cerebral functioning , both in a positive as well as in a negative way . apart from the anti - tumor treatment , glioma patients often receive glucocorticoids and anti - epileptic drugs , which both also have influence on brain functioning . the effect of a brain tumor on cerebral functioning is often more global than should be expected on the basis of the local character of the disease , and this is thought to be a consequence of disturbance of the cerebral network as a whole . any network , whether it be a neural , a social or an electronic network , can be described in parameters assessing the topological characteristics of that particular network . repeated assessment of neural network characteristics in brain tumor patients during their disease course enables study of the dynamics of neural networks and provides more insight into the plasticity of the diseased brain . functional mri , electroencephalography and especially magnetoencephalography are used to measure brain function and the signals that are being registered with these techniques can be analyzed with respect to network characteristics such as synchronization and clustering . evidence accumulates that loss of optimal neural network architecture negatively impacts complex cerebral functioning and also decreases the threshold to develop epileptic seizures . future research should be focused on both plasticity of neural networks and the factors that have impact on that plasticity as well as the possible role of assessment of neural network characteristics in the determination of cerebral function during the disease course .

a medication error can be defined as a failure in the treatment process that leads to , or has the potential to lead to , harm to the patient. the use of the term failure signifies that the process has fallen below an attainable standard . as per the us institute of medicine 's report ( 1999 ) to err is human , every year 7000 preventable adverse drug reactions in usa were due to medication errors . to the best of our knowledge , this is the first report on the adverse event of intradermal hypersensitivity testing with cloxacillin resulting in pain and skin necrosis as a result of medication error . we describe a case report of cloxacillin - induced skin necrosis which occurred following intradermal skin testing for screening of hypersensitivity response . intradermal skin testing for diagnosing hypersensitivity response to cloxacillin was done for three patients who were admitted in the surgery ward of a tertiary care hospital . this included a 12-year - old boy diagnosed with cryptorchidism and admitted for inguinal exploration , a 28-year - old male admitted for bilateral breast mass reduction , and a 52-year - old male who was admitted for inguinal mass exploration . the test dose was given in the forearm of each patient , one after the other , using the same diluted preparation of cloxacillin . the nurse who prepared the test dose solution had apparently mixed 500 mg of cloxacillin sodium in 2 ml of distilled water , and had used 0.1 ml of the solution for intradermal injection in each of the three patients . however , the institute protocol for intradermal skin testing was only 0.5 mg per test dose . all three patients complained of intense pain , itching and rash over the site of injection within 30 min of receiving the test dose intradermal . this was followed by darkening of skin over the injected area which progressed to necrosis within 6 to 12 h and was associated with persistent pain [ figures 1 and 2 ] . after 12 h of receiving the test dose , pain subsided spontaneously in all three patients . skin necrosis at the site of intradermal test dose skin necrosis at the site of intradermal test dose adverse drug reactions are one of the important causes for significant morbidity and mortality among patients . among various causes for adverse drug reactions , medication errors include administration of a drug which is inappropriate for the condition or at an inappropriate dose . a study by kopp et al . , on medication errors in intensive care patients showed that around 17% of medication errors resulted in adverse drug reactions which were preventable . further , 83% of medication error had the potential to cause adverse drug reactions . in a study by bates , approximately 28% of adverse drug events were due to medication errors which were preventable . further , studies had also shown that the most common cause of medication error ( 36% ) was due to overdose followed by selection of wrong drug and wrong route of drug administration . this article highlights the seriousness of the reaction which could potentially occur with cloxacillin due to medication error . since the drug was given at 50 times the dose recommended for intradermal testing as per the institute protocol , the patient faced a serious risk of developing serious adverse events such as anaphylaxis . there had been reports of patients developing anaphylaxis even with the low dose used for penicillin hypersensitivity intradermal skin test . many of the medication errors occurred due to oversight or negligence during the process of prescribing , dispensing , storage and administration of drug . in a hospital setup , this could occur due to oversight by the nursing staff responsible for administering the drug . some of the common reasons could be overtime working , under trained staff , unsupervised nursing interns , complicated and unclear protocols , interpersonal communication gap between health care professionals and also poor availability of ideal resources . in this report , the drug was diluted in a wrong way by an unsupervised nursing intern . there is an important role for pharmacovigilance centers that can contribute to the detection and prevention of medication errors and increasing the safety of patients . these centers must alert health care professionals about the importance of reporting medication errors through bulletins and journals .

medication error is one of the important causes of preventable adverse drug reactions . it can occur in the form of administration of a wrong drug , in the wrong dose , to the wrong patient , in an unsuitable dosage form , for the wrong duration or by using an inappropriate route of administration . intradermal skin testing for cloxacillin hypersensitivity is done at low doses to check for drug allergy . in this report , three patients were given 50 times higher dose of cloxacillin than recommended for skin testing , resulting in pain and necrosis at the site of injection . the error occurred due to wrong dilution of the drug as done by a nursing intern . some reasons for this could be overtime working , under trained staff , unsupervised nursing interns , complicated and unclear protocols , interpersonal communication gap between health care professionals and also poor availability of ideal resources . pharmacovigilance centers must alert health care professionals about the significance of reporting medication errors through bulletins and journals .

. chronic lower respiratory tract disease , defined as asthma , emphysema , chronic bronchitis , bronchiectasis , and chronic obstructive pulmonary disease ( copd ) , is the third leading cause of death in people aged 65 years and older.1 according to 2010 census data , 13% of the us population , or 40.3 million people , are older than age 65 , which is higher than any previous census . additionally , the population is aging at an increasingly faster rate each year . between 2000 and 2010 , the population age 65 years and over increased by 15.1% compared to the total us population which only increased by 9.7%.2 with such a large and rapidly growing aging population it is critical to understand how the body changes with age and how this impacts the entire respiratory system . understanding the aging process in the lungs is necessary in order to provide optimal care to our aging population . the structure of the thoracic cavity , which houses and protects the lungs , is vital for optimal lung function . changes to the spine , muscles , and ribs over time with aging impact normal lung function . as people normally age , narrowing of the intervertebral disk spaces causes kyphosis or curvature of the spine.3 this curvature decreases the space between the ribs and creates a smaller chest cavity.4 while a small amount of anterior curvature or kyphosis of the thoracic spine is normal , an angle greater than 40 , which is the 95th percentile of normal , is defined as hyperkyphosis.5,6 after age 40 , the kyphosis angle begins to increase more rapidly in women than men , from a mean of 43 in women aged 5560 years , to a mean of 52 in women 7680 years of age.5 the prevalence and incidence of hyperkyphosis is reported in older adults varying from approximately 20%40% among both men and women.5,6 in a study of 55 nonsmoking women with variable degrees of thoracic kyphosis lombardi et al7 found that with increasing vertebral angle there was a significant decline in the fraction of exhaled volume in 1 second ( fev1 ) and vital capacity ( vc ) during spirometry testing.7 this effect was most significant once the kyphotic angle was over 55. culham et al hypothesized that this effect is not from a decrease in thoracic cavity size alone , but due to the rib space narrowing , which decreases the length of the intercostal muscles.8 the angle of the muscle fibers in relation to the ribs may also affect the efficiency and decrease the movement of the lower ribs during inspiration.8 these changes are structural and based on the origin and insertion of the muscles . in addition to structural changes , there are changes in intrinsic function of the muscles with age . overall muscle function in the body decreases by 2% annually as we age.9,10 aging is associated with reduced inspiratory and expiratory respiratory muscle strength.11 respiratory muscle decline can lead to an inability to ventilate in the face of increasing demands , such as that seen in respiratory disease . there is also evidence that at the cellular level , the muscles of elderly individuals have less mitochondrial adenosine triphosphate reserves to sustain a sudden increase in metabolic demand.12 if an elderly person becomes ill with pneumonia , and therefore has increased metabolic demands for oxygen in the setting of decreased respiratory muscle strength , decreased cellular energy reserve , and decreased overall muscle function , he or she may not be able to meet those demands . this leads to an increased risk of respiratory failure in older individuals.13 with aging there is a decreased ability to clear mucus from the lungs . two mechanisms primarily contribute to this decline : 1 ) reduced cough strength and 2 ) alterations in the body s ability to clear particles in the airways . coughing is a maneuver that requires generation of a high forced expiratory flow . during a cough maneuver , inspiratory muscles contract to allow the lungs to take in a large tidal volume necessary to augment a sustained high expiratory flow.14 next , the expiratory muscles contract to build high positive intrapleural and intraairway pressures for the development of peak expiratory flow rates.15 finally , when the glottis is opened , the cough occurs , and the mucus is expelled from the airway into the mouth . any decrease in the strength of the respiratory muscles will greatly impact an individual s ability to generate the force required for an effective cough.16 aging is associated with both inspiratory and expiratory respiratory muscle strength reduction.11 polkey et al showed a 13% decrease in transdiaphragmatic pressure gradients , a surrogate for diaphragm strength , in older subjects ( ages 6781 ) as compared to younger subjects ( ages 2140).17 tolep et al compared the maximum transdiaphragmatic pressure ( pdimax ) obtained during voluntary maximal inspiratory efforts in nine young ( 1928 years ) and ten elderly ( 6575 years ) subjects and found that the average pdimax of the elderly subjects ( 128 9 cm h2o ) was significantly lower than the average pdimax of the younger subjects ( 171 8 cm h2o).18 more specifically , there is age - related atrophy of muscle fibers , termed sarcopenia , which may also explain the reduced respiratory strength in the elderly . the decrease in muscle fiber strength can be as high as 20% by age 70.1821 there are complex changes involving the mitochondria , muscle fiber disorganization , age - related muscle fiber transitions , and metabolic shifts in the aging muscle that can also explain the reduction in muscle strength.22 the mucociliary elevator refers to the action of ciliated cells along the upper and lower airway to beat in synchrony , trapping and clearing mucus and foreign particles out of the lungs.23 the upper airway nasal mucociliary cells work to remove large particles before they enter the smaller airways , and the lower airway mucociliary cells remove fine particles from the airway over a longer period of time.24 there are alterations in both the clearance of large and small particles with aging . de oliveira - maul et al used the clearance of saccharin that was inhaled through the nares of healthy subjects to measure large airway nasal mucociliary function . they demonstrated that in people over age 40 , there was a delayed nasal mucociliary clearance time of saccharin compared to subjects under 40 years of age.25 using radiolabeled particles that can travel past the upper airway and enter into the smaller airways in healthy nonsmoking subjects , svartengren et al evaluated clearance of the labeled particles in different age groups ranging from age 1981 years . they found that age alone was negatively associated with airway clearance of radiolabeled particles at 1 , 2 , 7 , 14 , and 21 days.26 the association between age and decreased clearance by the mucociliary elevator may be due to beat frequency of the cilia . the studies of beat frequency in cilia are confounded by the presence of cigarette smoking , which has a large impact on beat frequency . age has not been a statistically significant predictor of decreased beat frequency.27 the impact of structure and functional changes created by the aging process is summed up in figure 1 . in addition to these age - related structural changes in the lung , advanced age contributes to systemic immune dysfunction . of particular interest is the basal activation of the innate immune system in aged individuals in the absence of an immunologic threat.28 this phenomenon , referred to as inflamm - aging , is marked by elevated levels of tissue and circulating proinflammatory cytokines in aged subjects.28 specifically , increased levels of interleukin ( il)-1 , il-6 , and tumor necrosis factor- ( tnf- ) have been observed in aging rodent and human studies.2932 theoretically , heightened levels of these cytokines in the absence of an immunologic threat or infectious target may be a contributory factor to reduced elasticity and destruction of the delicate lung parenchyma with advanced age . related to inflamm - aging is the blunted immune response , known as immunosenescence , following a pathogenic threat or tissue injury.28 multiple studies have established reduced levels of mediators such as tnf- , il-6 , interferon- , nitric oxide , monocyte chemoattractant protein-1 , and macrophage inflammatory protein-1 after different types of antigenic stimulation in aged animals.3338 this basal level of inflammation , for example , elevated levels of il-6 , has been suggested to contribute to this subsequent immunosenescence following an immune challenge.28,39 using a model of il-6 knockout mice , gomez et al demonstrated restoration of cytokine production of il-1 , il-12 , and tnf- following lipopolysaccharide ( lps ) challenge in aged il-6 knockout animals to levels comparable to young wild - type.40 these data suggest that the basal elevation of circulating il-6 observed in aged wild - type animals prior to stimulation may contribute to the inability to upregulate cytokine production in the presence of an infectious threat as represented by lps.40 in addition to these cytokine alterations with aging , more recent data demonstrate a role for micrornas in inflamm - aging and cellular senescence.41 specifically , microrna 146a has been associated with a senescent associated proinflammatory status in the setting of vascular remodeling.42 together , these data support the relationship between inflamm - aging and immunosenescence , suggesting that a disruption in the balance of pro- and anti - inflammatory mediators results in a baseline proinflammatory environment with advanced age that subsequently dampens an appropriate innate and adaptive immune response ( figure 2 ) . in addition to this reduced activation , there are data that support a shift in the temporal response to injury with aging , such that this initial immunosenescence over time results in a protracted immune response and chronic inflammation.43,44 these studies will be discussed in depth later in the context of pulmonary inflammation with aging ; however , it is important to consider that this imbalance of immune mediators , delayed immune activation , and protracted course of inflammation may result in increased morbidity and mortality in aging individuals following infection , environmental exposures , or systemic injury4548 the lung has immunologic defenses that are both complex and resourceful , utilizing both an innate and adaptive immune response to inhaled antigens . adaptive immunity ( acquired immunity ) is antigen - specific and is required to ward off encapsulated bacteria , viruses , and intracellular pathogens . this form of immunity relies on immunologic memory and lymphocyte production of antibodies to nonself threats . toll like receptors ( tlrs ) are key molecules in recognition and initiation of the innate immune response . in the context of aging , there are conflicting data regarding the impact of age on murine and human expression of tlrs or downstream signaling mediators.38,4951 while some of these murine studies on monocytes and macrophages report reduced expression of one or several tlrs,34,49 another report demonstrates alterations in downstream tlr signaling involving p36.51 reduction in p38 signaling is supported by studies in human monocytes from aged subjects , where dampened p38 signaling was associated with diminished phosphorylation of p38.37 additionally , these authors observed a reduction in tlr1 but no changes in tlr2 expression . while the data are divergent on how aging impacts tlr expression , the data do suggest that alteration in the tlr pathways plays a role in an age - related aberrant initiation of the innate immune response and may contribute to an inability in rapidly recognizing and eradicating a pathogen . in studies of cigarette smoke exposure , elevated expression and nuclear translocation of nuclear factor - k murine neutrophil chemokines , cxcl1 and cxcl2 , were observed in aged mice.52 this was accompanied by a protracted neutrophilia in the lung parenchyma.52 moreover , following exposure to environmental toxins such as diesel exhaust , the increased pulmonary neutrophilia in lung parenchyma led to congestion and delayed clearance in aged animals as compared to young mice.53 these data suggest that inhaled pollutants cause a prolonged , aberrant pulmonary immune response , which may translate into increased tissue damage , playing a part in environmental , age - related pathology like copd . pulmonary infection with francisella tularensis in aged rodents demonstrated delayed production of neutrophil chemokines in conjunction with an attenuated neutrophil recruitment at early times points,43 supporting the concept of an aberrant initial immune response with age . at later time points , inoculation of lps into the respiratory tract of aged mice was associated with subsequent heightened levels of chemokines cxcl1 and cxcl2 , il-1 , and lingering pulmonary neutrophilia at 72 hours in aged animals as compared to young.44 considering the delicate lung alveolar architecture and the highly hydrolytic enzymatic degranulation products of activated neutrophils , this may contribute to excessive tissue damage and reduced lung function over time . in addition to age - related perturbations in recruitment following inhalational injury or infectious insult , mclachlan et al examined cytotoxic activity of monocytes in older patients compared to younger patients in response to lps exposure , and found that older patients display less reactive oxygen species ( ros).54 cytotoxicity generated by the production of ros and reactive nitrogen intermediates ( rni ) is a key function of m1 , or proinflammatory macrophages , responsible for activating the type 1 helper t cells ( th1 ) pathway . alteration in macrophage polarization marked by reduced ros and rni is reported with advanced age.55 supporting the study by mclachlan et al , alveolar macrophages from aged rats had reduced basal and lps - activated levels of ros and rni.56 our lab and others have also demonstrated that aging is associated with lower levels of inducible nitric oxide synthase ( inos ) , an enzyme that regulates production of ros and rni under control of the interferon- receptor.57,58 in conjunction with these changes in macrophage phenotype and cytotoxicity , others found that neutrophils from older individuals ( 85 years ) produced less superoxide.54,59 these changes in reactivity have implications for compromising host defenses with age . in addition to changes in innate system functioning with age , there are changes seen in adaptive immunity with age . in order to activate b- and t cells , dendritic cells ( dcs ) must migrate from sites of tissue injury and infection to local lymph nodes . dc migration in response to chemokine ligand-21 , a key dc chemokine that is localized in lymph nodes and binds chemokine receptor type 7 and presents on dc cell membranes , was reduced in aged mice as compared to young.60 interestingly , following respiratory infection in the lungs of aged mice with either mouse hepatitis virus-1 , respiratory syncytial virus , influenza a virus , or severe acute respiratory syndrome coronavirus , elevated expression of prostaglandin d2 correlated with reduced homing of lung dcs to regional lymph nodes and t cell activation.61 functional antagonism of prostaglandin d2 resulted in upregulation of chemokine receptor type 7 , the critical receptor for dc migration , and improved dc homing to draining lymph nodes , subsequently improving t cell activation and survival after viral infection.61 there are many adaptive immune functions that are inefficient with age . the thymus is primarily responsible for producing nave t cells and is replaced by fatty tissue by age 60 years.62 this leads to an increase in memory t cells relative to nave t cells.63 both nave cd4 + and cd8 + t cells are reduced in aged animals and humans relative to their memory t cells counterparts.6466 in regard to cd8 + t cells , it has been suggested that repeated or latent cytomegalovirus infection may results in expansion of cd8 + memory cells , again diminishing the nave cd8 + t cell pool.67 moreover , the proliferative aptitude of cd4 + t cells from aged donors appears to be reduced following t cell receptor engagement with high - dose anti - cd3 antibody in comparison to young controls,68 suggesting a weaker novel pathogen - specific immune response . meyer et al looked at the ratio of cd4 + to cd8 + t cells in bronchoalveolar lavage fluid in young versus old normal volunteers and found an increase in cd4+/cd8 + ratio as a function of age , suggesting there are fewer nave cells available to be converted to memory cells in the face of a novel infection.69,70 recently , forkhead box n1 , a transcription factor known to play a role in embryonic thymus development , has been shown to play a critical role in preventing thymic involution and preservation of nave t cell subsets with aging.71 overexpression of forkhead box n1 was demonstrated to increase early thymic progenitors , decrease splenic cd4 + memory t cells , and increase splenic nave cd4 + and cd8 + t cells.71 these data suggest a possible potential target to increase the number of nave t cells in aged individuals , increasing the ability of the elderly to respond to novel antigens . the antibody - secreting capacity of b cells the prevalence of copd is two to three times higher in people over age 60.73,74 it is projected that from 1990 to 2020 , copd will move from the sixth to the third leading cause of death worldwide.75 the rotterdam study found that of healthy 55-year - olds without copd , one in six women and one in four men will develop copd later in life , with the risk for developing copd over the coming 40 years being 24% and 16% , respectively.76 cigarette smoking is the greatest risk factor for developing copd in genetically susceptible individuals . copd is characterized by airway and lung inflammation , mucociliary dysfunction , alveolar destruction , and airway fibrosis.77 the increased burden of copd seen in the elderly population may be due to age - associated changes in the structure and function of the lung , increasing the pathogenetic susceptibility to copd . these changes , described in elderly lifelong nonsmokers , are characterized by airspace dilatation resulting from loss of supporting tissue without alveolar wall destruction , similar to changes seen with copd.77 the global initiative for chronic obstructive lung disease ( gold ) criteria , accepted by the american thoracic society and the european respiratory society , is the standard for the diagnosis and classification of copd , and is assessed by measuring the ratio of fev1 to the forced vital capacity ( fvc).78 fev1 peaks between ages 2036 years , and then begins to decline as we age.79 the annual rate of decline after the age of 25 is 20 ml per year and further declines to a loss of 38 ml per year after age 65.79 fvc begins to decline later in life than fev1 and at a slower pace . because of the unparalleled rate of decline , use of the fev1/fvc ratio alone to diagnose copd will over represent a copd diagnosis when no such pathology may exist.80 to complicate this matter , ohar et al found that copd is underdiagnosed in the united states , arguing that this is due to underutilization of spirometry as a screening test for copd.81 therefore , it is recommended to practitioners that a combination of spirometry and symptoms typical of copd be utilized in the diagnosis of copd in the elderly . these include osteoporosis , mental illness , malnutrition , risk of cardiovascular disease , and skeletal muscle dysfunction.82 low body mass index is commonly seen in patients with copd and has been shown to be inversely related to mortality in copd.83,84 anxiety and depression are prevalent comorbidities and have also been shown to be related to negative outcomes in copd . it is estimated that 40% of individuals with copd have depression , compared to a prevalence of 15% in the general population.85 cognitive impairments are common and associated with copd in the elderly . it is estimated that anywhere between 42%70% of aged persons with copd have concomitant dementia or neurocognitive impairment.8688 this may be related to hypoxemia and hypercapnia associated with copd.89 it may also be due to the common occurrence of cardiovascular disease in elderly with copd.90 these conditions certainly impact the ability to tolerate and comply with prescribed copd therapies . treatment options are varied and include metered dose inhaler , dry powder inhaler , or nebulized formulations . there are many factors which may impact effective treatment use in the elderly copd population including arthritis , weakness , poor manual dexterity , cognitive impairments , and visual limitations.91 careful consideration regarding treatment recommendations must be made in the aging copd population . the size of the thoracic cavity decreases , limiting lung volumes and altering the muscles that aid in respiration . muscle function on a cellular level is less efficient and has decreased reserve . cough strength is reduced in the elderly population due to anatomic changes and muscle atrophy . clearance of particles from the lung through the mucociliary elevator is negatively impacted and associated with ciliary dysfunction . there are many complex changes in immunity with aging that increase susceptibility to infections , including a less robust immune response from both the innate and adaptive immune systems . finally , copd has the highest prevalence in the elderly and deserves special consideration in regard to treatment in this fragile population . additional research is needed to improve our understanding of the determinants of lung aging and the effects on lung immunity .

there are many age - associated changes in the respiratory and pulmonary immune system . these changes include decreases in the volume of the thoracic cavity , reduced lung volumes , and alterations in the muscles that aid respiration . muscle function on a cellular level in the aging population is less efficient . the elderly population has less pulmonary reserve , and cough strength is decreased in the elderly population due to anatomic changes and muscle atrophy . clearance of particles from the lung through the mucociliary elevator is decreased and associated with ciliary dysfunction . many complex changes in immunity with aging contribute to increased susceptibility to infections including a less robust immune response from both the innate and adaptive immune systems . considering all of these age - related changes to the lungs , pulmonary disease has significant consequences for the aging population . chronic lower respiratory tract disease is the third leading cause of death in people aged 65 years and older . with a large and growing aging population , it is critical to understand how the body changes with age and how this impacts the entire respiratory system . understanding the aging process in the lung is necessary in order to provide optimal care to our aging population . this review focuses on the nonpathologic aging process in the lung , including structural changes , changes in muscle function , and pulmonary immunologic function , with special consideration of obstructive lung disease in the elderly .

among menstrual disorders , menorrhagia , dysmenorrhea , and abnormal cycles are highly prevalent and are considered as disabling conditions among women at reproductive ages . dysmenorrhea is defined as painful cramps in lower abdomen during menstruation without any evidence of pelvic pathologic lesions . this condition occurs right before or during menstruation without any describable macroscopic pelvic pathology . in general , 50% of women suffer from dysmenorrhea and 10% experience this condition for 13 days a month . one study reported the prevalence of dysmenorrhea to be over 75% . in another study in sweden , more than 72% of 19-year - old women suffered from this condition ( 34% , 23% , and 15% of the participants had mild , moderate , and severe symptoms , respectively ) . dysmenorrhea can reduce efficiency and production due to daily stresses and socioeconomic losses . in the us , at least 600 million hours and 2 billion dollars are lost due to dysmenorrhea every year . in addition , dysmenorrhea affects young girls ' opinion about femininity and method of childbirth . for the treatment and control of dysmenorrhea , there are many ways : common drug interventions for reduction of pain include nonsteroidal anti - inflammatory drugs ( nsaids ) and oral contraceptive pills . despite their usefulness , their side effects include diarrhea , stomachache , nausea , renal complications , hepatic complications , blood problems , edema , sleepiness , indigestion , and fatigue . other treatment methods also include supplementary vegetarian diet , vitamins , minerals , nerve block , spinal manipulation , magneto - inertial fusion , percutaneous electrical nerve stimulation , acupressure , acupuncture , rest , muscle relaxation , reflexology , hand pressure , heat , aromatherapy , and physical therapy . since the acupressure is a new therapy , the side effects of drug therapies are not readily acceptable . hence , but the rationales for using complementary and alternative medicine have been widely used for many years , and they are accepted by many people and doctors . approximately 38.2 million adult people in america have used complementary medicine ( herbs and supplements ) in 2002 . moreover , more than half of them said that complementary medicine was important to their health and well - being , besides the points examined in this study . in reviewing the past 30 years , no article has been found . hence , the acupressure points in this research are entirely new . according to the gate control theory of pain , cutaneous stimulation through massaging , needling , and scratching can stimulate large fiber nerves that transfer nervous impulses to the spinal cord . in case this stimulation is continued , it can close pain gates , eventually reducing the feeling of pain . evidence has shown acupressure as a noninvasive , inexpensive , safe , and simple method for reduction of labor pain . in the study by wang et al . , ( 2010 ) acupressure was applied at sanyinjiao ( sp-6 ) acupoint for 20 min to assess its effect on menstrual pain and distress in young women suffering from dysmenorrhea . the results revealed a significant decrease in pain in acupressure and self - care training groups based on short - form mcgill pain questionnaire . in another study in iran , acupressure at sp-6 acupoint significantly reduced the intensity of dysmenorrhea in the intervention group compared to the control group 30 min and 1 , 2 , and 3 h after the intervention ( p < 0.05 ) . in the present study , our aim is to perform acupressure at guan yuan ( rn-4 ) and qu gu ( rn-2 ) acupoints located on the abdominal meridian between the navel and the symphysis pubis and compare its effect to that of using ibuprofen ( control group ) . rn-2 acupoint is also located on the midline , 5 cuns below the navel , 1 cun between the two grooves of the interphalangeal joints of the massager s middle finger . the rationale for using self - care education to increase awareness of one s own body and the adoption of appropriate health behavior and lifestyle modification such as diet and exercise will lead to the improvement of dysmenorrheal . considering the limited interventions in this regard and importance of complementary medicine , the present study aims to compare self - care behaviors training , acupressure at rn-4 and rn-2 acupoints , and ibuprofen consumption . the present randomized clinical trial was conducted on 120 nonmedical students ( three groups of 40 ) living in dormitories of shiraz university shiraz , iran . the sample size was determined based on the study by kashefi et al . and considering the loss rate of 10% . of course , the initial sample comprised 157 patients with primary dysmenorrhea , of which 37 patients were excluded from the trial due to lack of inclusion criteria [ figure 1 ] . consort diagram of sampling at first , five dormitories of shiraz university were selected randomly . then , one , two , and two dormitories were randomly considered as control , self - care behaviors training , and acupressure groups , respectively . the inclusion criteria of the study were being single , being below 25 years old , having primary dysmenorrhea with pain intensity of four and above based on visual analog scale ( vas ) , having regular menstrual cycles ( 2135 days ) , having experienced dysmenorrhea in the recent six menstrual cycles ( self - report ) , not having used oral contraceptive pills , and treatment interventions ( nsaids , antiprogestins , and supplements ) since 2 months before the study , not suffering from physical ( vertebral fracture , herniated disk , acute inflammation , deep vein thrombosis , and gynecological diseases ) and mental disorders , not consuming special medications such as benzodiazepines and antidepressants , and not having experienced stress due to parents ' divorce or loss of first - degree relatives during the recent 6 months . on the other hand , the exclusion criteria of the study were having symptoms such as burn , itch , and abnormal vaginal discharges , experiencing stressors ( parents ' divorce , loss of first - degree relatives , etc . ) during the study , and not being willing to participate in the study . all interventions and inclusion criteria carried out by the researcher , and research was done in student dormitories . before the intervention , intensity of pain was determined in all the three groups . it should also be noted that researcher s performance of acupressure was confirmed by a specialist . in doing so , pressure was repeatedly applied with the thumb and was controlled by a scale to equalize its amount in each application . first group , in the acupressure group , pressure was applied on the abdominal meridian using the thumb for 20 min ( 15 s pressure , 15 s rest ) over the 1 2 days of menstruation for two cycles . pain intensity was assessed h , 1 h , and 2 h after application of pressure . it should be mentioned that pain intensity was assessed by the researcher s assistant to avoid bias . the method of compression in acupressure groups was done after obtaining confirmation from a trained researcher ( i.e. , from a specialist in physical medicine and rehabilitation ) . after confirmation from the researcher , the intervention was conducted . in the second group , self - care behaviors were conducted in two parts : teaching theoretical base of using lecture , discussion , and experience sharing was performed through four sessions ( one - 6090 min session a week ) . the educational content included anatomy and physiology of the genital system , nutrition during the menstrual cycle ( based on books , articles , and a nutritionist s advice , emphasis was put on the resources that were effective in dysmenorrhea ) , the second part was practical training and includes isometric exercises during the menstrual cycle . it should be noted that isometric exercises were educated to researcher by a master of physical education , and researcher was trained to girls in the gymnasium dormitory . isometric exercise duration was 8 weeks , in the first 4 weeks was conducted by the researcher . in the second 4 weeks with an appropriate images of playing sports the girls were given regarding how exercise . and the girls without researcher continued ( a total of 8 weeks ) . to ensure the exercise was done in the dormitory to follow up , as well as phone calls , for continued exercise classes were encouraged after the 1 4 weeks . food recommendations and other educational content have been on the books and articles . in group three , the control group received the routine treatment for dysmenorrhea , i.e. , taking ibuprofen 400 mg three times a day for three cycles . of course , they were provided with the educational pamphlet at the end of the study . questionnaires were completed in the student dormitories and after the intervention ; to avoid sampling bias in research , pain assessment was conducted by a research assistant . in this study , intensity of pain was assessed using mcgill pain questionnaire developed by melzack , which has been introduced as a reliable instrument for the assessment of pain intensity . however , since this questionnaire takes a long time to be completed , its short form was designed and has been frequently used in persian studies . the short form of mcgill pain questionnaire consists of 15 sensory ( 11 questions ) and emotional ( 4 questions ) items , and the patients determine the quality of their pain by selecting one of the options of none , mild , average , and severe . the validity and reliability of this scale in the current research were based on the study performed by adelmanesh et al . that reported cronbach s alpha coefficients of 0.951 , 0.832 , and 0.840 for sensory , emotional , and total scores , respectively . wallis test was used to compare the three study groups . besides , paired t - test was employed to compare the means before and after the intervention . finally , qualitative variables were compared using chi - square test . besides , written informed consents for taking part in the study were obtained from all the participants . hence , thesis number : 92 - 6900 , irct : 2013071013940n1 and ethic number : 1392 - 12 - 11 - 6900 . the present randomized clinical trial was conducted on 120 nonmedical students ( three groups of 40 ) living in dormitories of shiraz university shiraz , iran . the sample size was determined based on the study by kashefi et al . and considering the loss rate of 10% . of course , the initial sample comprised 157 patients with primary dysmenorrhea , of which 37 patients were excluded from the trial due to lack of inclusion criteria [ figure 1 ] . consort diagram of sampling at first , five dormitories of shiraz university were selected randomly . then , one , two , and two dormitories were randomly considered as control , self - care behaviors training , and acupressure groups , respectively . the inclusion criteria of the study were being single , being below 25 years old , having primary dysmenorrhea with pain intensity of four and above based on visual analog scale ( vas ) , having regular menstrual cycles ( 2135 days ) , having experienced dysmenorrhea in the recent six menstrual cycles ( self - report ) , not having used oral contraceptive pills , and treatment interventions ( nsaids , antiprogestins , and supplements ) since 2 months before the study , not suffering from physical ( vertebral fracture , herniated disk , acute inflammation , deep vein thrombosis , and gynecological diseases ) and mental disorders , not consuming special medications such as benzodiazepines and antidepressants , and not having experienced stress due to parents ' divorce or loss of first - degree relatives during the recent 6 months . on the other hand , the exclusion criteria of the study were having symptoms such as burn , itch , and abnormal vaginal discharges , experiencing stressors ( parents ' divorce , loss of first - degree relatives , etc . ) during the study , and not being willing to participate in the study . all interventions and inclusion criteria carried out by the researcher , and research was done in student dormitories . before the intervention , intensity of pain was determined in all the three groups . it should also be noted that researcher s performance of acupressure was confirmed by a specialist . in doing so , pressure was repeatedly applied with the thumb and was controlled by a scale to equalize its amount in each application . first group , in the acupressure group , pressure was applied on the abdominal meridian using the thumb for 20 min ( 15 s pressure , 15 s rest ) over the 1 2 days of menstruation for two cycles . pain intensity was assessed h , 1 h , and 2 h after application of pressure . it should be mentioned that pain intensity was assessed by the researcher s assistant to avoid bias . the method of compression in acupressure groups was done after obtaining confirmation from a trained researcher ( i.e. , from a specialist in physical medicine and rehabilitation ) . after confirmation from the researcher , the intervention was conducted . in the second group , self - care behaviors were conducted in two parts : teaching theoretical base of using lecture , discussion , and experience sharing was performed through four sessions ( one - 6090 min session a week ) . the educational content included anatomy and physiology of the genital system , nutrition during the menstrual cycle ( based on books , articles , and a nutritionist s advice , emphasis was put on the resources that were effective in dysmenorrhea ) , the second part was practical training and includes isometric exercises during the menstrual cycle . it should be noted that isometric exercises were educated to researcher by a master of physical education , and researcher was trained to girls in the gymnasium dormitory . isometric exercise duration was 8 weeks , in the first 4 weeks was conducted by the researcher . in the second 4 weeks with an appropriate images of playing sports the girls were given regarding how exercise . and the girls without researcher continued ( a total of 8 weeks ) . to ensure the exercise was done in the dormitory to follow up , as well as phone calls , for continued exercise classes were encouraged after the 1 4 weeks . food recommendations and other educational content have been on the books and articles . in group three , the control group received the routine treatment for dysmenorrhea , i.e. , taking ibuprofen 400 mg three times a day for three cycles . of course , they were provided with the educational pamphlet at the end of the study . questionnaires were completed in the student dormitories and after the intervention ; to avoid sampling bias in research , pain assessment was conducted by a research assistant . in this study , intensity of pain was assessed using mcgill pain questionnaire developed by melzack , which has been introduced as a reliable instrument for the assessment of pain intensity . however , since this questionnaire takes a long time to be completed , its short form was designed and has been frequently used in persian studies . the short form of mcgill pain questionnaire consists of 15 sensory ( 11 questions ) and emotional ( 4 questions ) items , and the patients determine the quality of their pain by selecting one of the options of none , mild , average , and severe . the validity and reliability of this scale in the current research that reported cronbach s alpha coefficients of 0.951 , 0.832 , and 0.840 for sensory , emotional , and total scores , respectively . wallis test was used to compare the three study groups . besides , paired t - test was employed to compare the means before and after the intervention . finally , qualitative variables were compared using chi - square test . this study was approved by the ethics committee of shiraz university of medical sciences . besides , written informed consents for taking part in the study were obtained from all the participants . hence , thesis number : 92 - 6900 , irct : 2013071013940n1 and ethic number : 1392 - 12 - 11 - 6900 . the mean age of the participants was 20.02 1.44 , 20.12 1.50 , and 20.37 1.54 years in the acupressure , training , and control groups , respectively ( p = 0.569 ) . besides , the mean time of beginning of pain after menarche was 1.80 0.82 years in the acupressure group , 1.90 0.77 years in the training group , and 1.87 0.79 years in the control group ( p = 0.844 ) . according to the results of kruskal wallis test , the three groups were homogeneous regarding these two variables [ table 1 ] . comparison of the age of in three groups ( acupressure - education of self - care behaviors and control ) in the training group , the mean intensity of pain was 16.90 7.43 before the intervention and 10.56 5.71 after the intervention , and the results of paired t - test showed that the difference was statistically significant ( p < 0.001 ) . in the acupressure group also , the mean intensity of pain was 20.25 6.93 before the intervention , and the results showed a significant difference in this regard before and 1 and 2 months after the intervention ( p < 0.001 ) [ table 2 ] . comparison of the intensity of pain after and before the acupressure and educational groups based on the mcgill questionnaire in the control group , the mean intensity of pain was 31.77 6.36 before the intervention , 23.52 5.55 1 month after the intervention ( p < 0.001 ) , 9 5.99 2 months after the intervention ( p < 0.001 ) , and 14.57 6.40 3 months after that ( p comparison of the intensity of pain after and before the acupressure and educational and control groups based on the mcgill questionnaire based on mcgill pain questionnaire , the mean intensity of pain was 10.65 5.71 in the training group , 19 5.41 in the control group , and 14.40 6.87 in the acupressure group after the intervention . wallis test , showed that these differences were statistically significant , and both interventions were more effective compared to the control group [ table 4 ] . comparison of the difference mean scores severity of pain of in three groups ( acupressure - education of self - care behaviors and control of the mcgill questionnaire according to the present study results , discussion is on three main axes : in our study , the mean intensity of pain significantly decreased in the self - care training group . chang and chuang conducted a study to introduce the effective factors in dysmenorrhea . in that study , the participants with moderate dysmenorrhea received self - care behaviors training , and the results showed that training self - care behaviors at schools were one of the effective methods in reduction of dysmenorrhea . chen et al . also carried out a research on self - care behaviors using herbal medicines and complementary medicine in primary dysmenorrhea among taiwanese adolescents . the results of the study by chen et al . revealed the necessity to educate individuals regarding the effect of food diet and effective herbal medications on the treatment of dysmenorrhea . in that study , the participants mentioned the reduction of physical activities as a self - care behavior , but the researchers modified this opinion and trained individuals regarding aerobic exercises as a self - care behavior to decrease their pain . , who investigated the effect of heavy exercises on the treatment of dysmenorrhea using mcgill pain questionnaire . the results of that study demonstrated that exercising was effective in reduction of pain without any side effects . in contrast , shafaie - sehati et al . found no significant difference between athletic and nonathletic groups with respect to the frequency of dysmenorrhea . in the present study , self - care behaviors training resulted in a reduction of pain and exercising was among the bases of this training . exercising enters the body in the state of hypoxia where beta - endorphin levels increase . therefore , hypoxia and acidosis stimulate production of beta - endorphins , and the relationship between breathing and beta - endorphin production can be associated with the effect of acidosis on the two through different mechanisms . reduction of pain after self - care training can also be attributed to the control of prostaglandins . in fact , aerobic exercises empty the uterus from waste materials and prostaglandins , which are the main cause of pain , eventually reducing the duration of pain through the menstrual cycle . in another survey exercise after 6 weeks is created flexibility in the hamstring , back and hip muscles , and reduces pain . the current study at the second part findings showed a significant difference in the acupressure group s pain intensity before and after the intervention . considering the limited number of studies conducted on rn-4 and rn-2 acupoints , the results of performance of acupressure at other points have been discussed in this section . in the study by yeh et al . , pressure was applied for 1 min four times a day for 2 days , which is different from our study . the results showed a significant difference in each group s pain intensity before and after the intervention . in the present study , however , the study groups ' pain intensity was significantly different only based on mcgill pain questionnaire . other researches have also revealed the effectiveness of acupressure in treatment of dysmenorrhea and relaxation . in a review study about efficacy of self - acupressure showed several studies ( eight rcts and two quasi - rcts ) have a positive effect and effective on primary dysmenorrhea . in another study concluded that enough evidence not exists for the effectiveness of acupressure or acupuncture and rct larger studies are needed . the analgesic effect of acupressure can be due to release of endorphins and glucocorticoids that block pain gates . acupressure , in fact , refers to the touch technique for balancing human body s energy flow or qi . this method leads to the release of various neurotransmitters , which cut the nervous signals transferred by the nervous system . it also inhibits secretion of prostaglandins , reduces stimulation of cerebral cortex , and regulates secretion of endocrine hormones . based on mcgill pain questionnaire scores , the control group s pain intensity scores were significantly different before and after the intervention . similarly , the results of the studies by zafari and witt et al . indicated the effectiveness of ibuprofen . however , the results obtained by salmalian were on the contrary to those of the present study . in general , nsaids are the first - line treatment , but their side effects , including gastrointestinal problems , make patients choose alternative therapies . overall , the results of the current study showed a significant difference among the three groups regarding the intensity of pain , and that training and acupressure were more effective compared to consumption of ibuprofen . in this study , new and effective acupoints were employed , whereas the previous studies had not used such a combination of effective points . one of the limitations of our study was the difficulty in obtaining the required permissions from dormitories and performing the procedures privately . hence , probable physiological tolerance of pain in each people is different , so feel pain is different , as well as contrast to the reactions and feelings of loss pain after pain relief methods will be different , which is beyond the control of the researcher . another limitation was to assess the severity of pain after intervention . in order to solve the problem and avoid bias in sampling , finally , due to the different time periods , research conducted at the same time for all units is not possible . future studies are recommended to compare the effects of acupressure at other acupoints and common chemical and herbal drugs on primary dysmenorrhea in larger populations . suggestions for future research : ( 1 ) comparison the effect of other point of acupressure on severity of primary dysmenorrhea with the rn-2 , rn-4 points . ( 2 ) the application of educational models such as basnef models on knowledge and attitudes of people with primary dysmenorrhea . according to the current study results , the most effective method in reduction of dysmenorrhea was self - care behaviors training followed by acupressure at rn-4 and rn-2 acupoints and consumption of ibuprofen . considering the side effects of consuming ibuprofen drug , training and acupressure as nonpharmacological , easy , safe , and inexpensive methods can be incorporated into the curricula of girls ' high schools . considering the effectiveness of acupressure in reducing dysmenorrheal , obstetricians , gynecologists , and midwives have recommended acupressure not only in the treatment of dysmenorrhea but also in reducing labor pain and pain control . the effective nondrug method that is the responsibility of the midwifery , and in clinics and delivery room applications and this study is funded by the shiraz university of medical sciences , shiraz , iran . this study is funded by the shiraz university of medical sciences , shiraz , iran . all authors contributed to the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work .

background : dysmenorrhea is one of the common problems during reproductive ages , with prevalence rate of 6090% . this study aimed to compare the effects of acupressure at guan yuan ( rn-4 ) and qu gu ( rn-2 ) acupoints , self - care behaviors training , and ibuprofen on the intensity of primary dysmenorrhea based on mcgill pain questionnaire.materials and methods : in the randomized clinical trial , 120 females , aged between 18 and 25 years , with primary dysmenorrhea , randomly selected from five dormitories of shiraz university , shiraz , iran were screened and randomized into acupressure group , in that pressure was applied for 20 min over the 1st 2 days of menstruation for two cycles . in the second group , the training group took part in four educational sessions each lasting for 6090 min and control group received ibuprofen 400 mg . the intensity of pain before and after the intervention was measured using short - form mcgill pain questionnaire . the data were entered into the spss statistical software ( version 16 ) and analyzed using kruskal wallis test , paired t - test , and chi - square test.results:a significant difference was found in the mean intensity of pain before and after the intervention in all the three study groups . the mean score of pain intensity was 10.65 5.71 in the training group , 19 5.41 in the control group , and 14.40 6.87 in the acupressure group after the intervention . the results of kruskal wallis test revealed that both interventions were more effective compared to consumption of ibuprofen.conclusion:training and acupressure were more effective than ibuprofen in the reduction of dysmenorrhea . thus , they can be considered as trainable methods without side effects in adolescent girls .

the term nonconvulsive status epilepticus ( ncse ) is defined as a range of conditions in which electrographic seizure activity is prolonged and results in nonconvulsive clinical symptoms , . the clinical features of ncse may be subtle and diverse , and confirmation relies on the electroencephalogram ( eeg ) . several approaches to establishing criteria for ncse in adults have been published ( table 1 ) , , , and some clinical states may lie along an ictal interictal continuum . periodic discharges are often regarded on the interictal end of the spectrum , but when accompanied by clinical features including impaired vigilance and when signs resolve proximate to treatment , then this lends evidence to the ictal nature of periodic discharges along this spectrum . periodic lateralized epileptiform discharges ( pleds ) , in particular , have sparked controversy with regard to being part of the ictal vs. interictal spectrum . periodic lateralized epileptiform discharges can occur in the temporal vicinity of an overt seizure , thus reflecting an ictal interictal continuum phenomenon that can continue for weeks ( chronic pleds ) in conscious patients with retained alpha or basic rhythm . however , when they occur in a patient with altered level of consciousness , some authors regard it as ictal , , , , , , , , . since changes in mental status are common in epileptic encephalopathies and developmental delay such as in patients with cerebral palsy , it can be difficult or impossible to determine whether they are in status epilepticus , unless there is a discernible clinical improvement with treatment . we report the case of a patient with chronic epilepsy and cerebral palsy ( cp ) who presented with deterioration from an already impaired baseline . although the eeg revealed slow pleds which are not typical of ncse , the patient showed a good clinical and eeg response to benzodiazepines with pled regression . this 49-year - old man with cerebral palsy , mental retardation , and epilepsy residing in a long - term care facility for the prior 13 years had been awake , noncommunicative , but responsive to auditory and visual stimuli . he was treated with phenytoin and levetiracetam and had yearly breakthrough seizures over the prior 10 years . he had recently developed fever and dyspnea with a drop in oxygen saturation and was suspected of having aspiration pneumonia complicated by sepsis . he was given intravenous antibiotics , fluids , and supplemental oxygen and was transferred to our facility . following a mild generalized tonic clonic seizure , his examination remained persistently unchanged but he was now unresponsive to auditory , visual , and noxious stimuli , with a fluctuating level of alertness . plasma phenytoin level was low at 34.8 mol / l ( normal : 4080 mol / l ) . 1 ) showed intrusions of excess slow left - sided periodic lateralized epileptiform discharges ( pleds ) on a background of normal alpha pattern . administration of 1 mg of iv lorazepam resulted in resolution of the slow pleds ( fig . 2 ) and improvement in the patient 's neurological status , which now became responsive to auditory , noxious , and visual stimuli that occurred in a matter of 15 min after administration of lorazepam . by the next morning , his condition had greatly improved and he was near his baseline status . he received additional intravenous loading doses of phenytoin and levetiracetam on the same day , and his maintenance doses were adjusted . brain ct showed a large central meningioma as well as a small posterior fossa meningioma ( fig . review of his old brain ct films ( done seven years ago ) showed no such lesions . because of his poor quality of life and the risks involved , neurosurgery was not considered an option , and the patient was managed medically with antiepileptic medication . 4 ) , and the patient was discharged to a long - term care facility for further care . this patient with chronic epilepsy and cerebral palsy ( cp ) showed deterioration from a baseline that was already impaired , a finding described in ncse in those with developmental disability , . the eeg findings were not typical of ncse in that slow pleds lie in the gray zone along the ictal interictal continuum . cerebral palsy is a complex , heterogeneous lifelong condition with significant disability and long - term challenges that persist into adult life . epilepsy is reported in 35% to over 60% of patients with cp , and is more frequent in patients with more severe disability . sixteen percent of children with cp and epilepsy may develop nonconvulsive and/or convulsive status epilepticus . brain tumors , particularly primary ones , are associated more frequently with both convulsive and nonconvulsive status epilepticus , . electroencephalographic changes in mass lesions reflect irritability around the penumbra of the lesion , in this case , on the left . one can speculate that the focal structural lesion in the setting of chronic diffuse brain dysfunction , along with intercurrent infection and low aed levels , represented the multiple risk factors seen in the recurrence of seizures and appearance of ncse , . periodic lateralized epileptiform discharges ( fig . 1 ) consist of periodic spike - and - slow wave or sharp - and - slow wave complexes , typically with a frequency of 12 hz . in our patient , the frequency was ~ 0.5 hz . periodic lateralized epileptiform discharges occur in a variety of conditions such as strokes , tumors , and infections ; these together comprise around 50% of the underlying etiologies . slow growing extraaxial tumors such as meningiomas usually produce little eeg disturbance unless they compress and displace brain tissue or cause hydrocephalus such as in our case . periodic lateralized epileptiform discharges are reported to support a diagnosis of ncse if they meet certain conditions , in particular , good proximate clinical and eeg response to benzodiazepines with pled regression ( table 1 ) . when eeg and clinical regression are not clear , spect or pet scanning can guide the clinician in the possible ictal ( versus interictal ) nature of the periodic discharges , . there are diagnostic and management challenges in patients with long - term disability whose cognitive and neurological baselines are impaired and not always known to the clinical caregivers . additionally , a diagnosis of ncse may be difficult to make when this baseline is unknown and when the eeg findings represent a pattern that lies along the ictal interictal continuum . electroencephalogram can establish where along this continuum the patient resides , following which treatment that balances the risk benefit needs of the patient , and the needs of chronic caregivers , can then be provided and monitored so as to offer the patient the optimal chance for regaining lost function with minimal risk .

the presence of cerebral palsy and that of slow growing brain tumors are risk factors for convulsive and nonconvulsive status epilepticus . nonconvulsive status epilepticus ( ncse ) needs electroencephalographic ( eeg ) monitoring to be confirmed as it may be clinically subtle . furthermore , it may present with a variety of ictal eeg morphologies . we report a case of a patient with cerebral palsy and a large central meningioma . electroencephalogram showed a slow pattern of periodic lateralized epileptiform discharges ( pleds ) ( a pattern considered as being situated in the ictal interictal continuum ) on an alpha background . the patient was treated for ncse successfully with benzodiazepines followed by up - titration of his antiepileptic drug doses .

access to adequate and safe blood transfusion facilities is integral to any basic health care delivery infrastructure . blood transfusions are also inherently embedded with risks ranging in severity from minor to life threatening . judicious patient selection with pragmatic pretransfusion assessments of risk versus benefit to the potential recipient combined with stringent quality control is an effective mode of reducing transfusion related adverse events . in addition , continuous monitoring of transfusion related complications can promote patient care and safety . the goal of hemovigilance was to observe , identify , and prevent the occurrence or recurrence of transfusion related unwanted events so as to increase the safety , efficacy , and efficiency of the blood transfusion process , covering the entire blood transfusion chain of donors to recipients . on the basis of this core principle , the haemovigilance programme of india ( hvpi ) was launched by the indian pharmacopoeia commission in collaboration with the national institute of biologicals on december 10 , 2012 . the hvpi that comes under the pharmacovigilance programme of india ( pvpi ) , tracks adverse reactions related to blood transfusions and blood product administration in affiliated blood banks across india . our institute obtained approval for a component in the year 2011 and affiliation to the hvpi , in 2013 . this study was carried out with the objective of observing and analyzing the acute transfusion reactions ( atrs ) encountered in the blood bank of this remote north eastern teaching hospital . this workup would enable us to develop insight into atr patterns not yet documented from remote centers like ours and compare them with that of larger national and international transfusion centers . the study was conducted in the department of transfusion medicine , ( hvpi centre ) , and the department of pharmacology , ( pvpi centre ) of this north east indian medical institute and attached teaching hospital . approval for conducting the study was obtained from the research protocol evaluation committee and the institutional ethical committee . this was a retrospective observational study in which all atrs reported to the blood bank over a period of 20 months ( may 2013 to january 2015 ) were reviewed and analyzed . the hospital is following the standard operating procedures adapted from the cdsco technical manual for transfusion , starting with donor selection , phlebotomy , component processing , testing for infectious diseases , storage , cross matching , issue of component , and follow - up of the recipient . the paramedical staff involved in blood transfusion has been trained in transfusion practices , enabling them to identify and manage any encountered transfusion reaction at the earliest . prior to the transfusion , they are required to cross - check for any clerical errors , abo - rh group of the patient and the blood bag , types of blood products and blood unit number , inspect the blood bag for hemolysis , clot and leakage , and expiry date . in the event of a transfusion reaction , the transfusion reaction form is being filled with information pertaining to : the date and time of initiation and cessation of the transfusion , time of the reaction , patients pre- and post - transfusion vital signs , approximate volume of blood transfused , as well as clinical signs and symptoms . the reaction form along with the posttransfusion blood sample ( 2 ml in citrate vial and 2 ml in plain vial ) , urine sample , and the leftover blood product bag , and transfusion set are immediately sent to the blood bank . the blood bank performs a thorough evaluation of the suspected transfusion reaction and rechecks the blood requisition form , returned blood / component unit number , abo - rh grouping and screening for irregular antibodies . blood grouping of the patient is repeated and compared with the pretransfusion sample . the blood bag and posttransfusion blood sample is checked for hemolysis and compared with the pretransfusion sample . in case of a suspected hemolytic reaction , further investigations done include : qualitative and quantitative estimation of plasma hemoglobin % direct antiglobulin testhemoglobinuria : gross visual examination and urine hemoglobin by dipstickshematuria : microscopic examinationserum total and unconjugated bilirubinperipheral blood smears examination for the presence of schistocytes and spherocytes . qualitative and quantitative estimation of plasma hemoglobin % direct antiglobulin test hemoglobinuria : gross visual examination and urine hemoglobin by dipsticks hematuria : microscopic examination serum total and unconjugated bilirubin peripheral blood smears examination for the presence of schistocytes and spherocytes . compatibility testing is repeated on pre- and post - transfusion sample : diamed - id microtyping system . in case of a reaction such as transfusion - related acute lung injury ( trali ) and pulmonary embolism , chest x - ray report is cross checked . based on the clinical features mentioned in the transfusion reaction form , and the laboratory reports , the reactions are classified according to the standard criteria defined by the american association of blood banks . after completion of transfusion reaction workup and categorization of the transfusion reaction , data are submitted to the hvpi . the data collected for this study were analyzed for frequency , percentage , mean , and standard deviation . statistical software used was microsoft office excel 2007 and ibm spss statistics version 20 ( statistical package for the social sciences ibm corporation ) . the chi - square test was used where applicable , and p < 0.05 was considered significant . nc is the number of component / whole blood units transfused and nt is the total number of transfusion units [ table 1 ; column 3]risk of transfusion reactions with a particular component / whole blood : ntrc / nc 100 where ntrc is the total number of reactions encountered with a particular component / whole blood , and nc is the total units of component / whole blood transfused [ table 1 ; column 5]risk of a particular reaction with a particular component / whole blood : nptr / nc 100 where nc is the total number of components / whole blood units transfused [ table 2 ; column 2 , 3 , and 4]frequency of a particular reaction in relation to total number of transfusions : nptr / nt 100 where nptr is the total number of the particular transfusion reaction and nt is the total number of transfusion units [ table 2 ; column 5]frequency of particular reaction in relation to total number of transfusion reactions : nptr / ntr 100 where nptr is the total number of the particular transfusion reaction and ntr is the total number of transfusion reactions [ figure 1 ] . nc is the number of component / whole blood units transfused and nt is the total number of transfusion units [ table 1 ; column 3 ] risk of transfusion reactions with a particular component / whole blood : ntrc / nc 100 where ntrc is the total number of reactions encountered with a particular component / whole blood , and nc is the total units of component / whole blood transfused [ table 1 ; column 5 ] risk of a particular reaction with a particular component / whole blood : nptr / nc 100 where nptr is the total number of a particular transfusion reaction , and nc is the total number of components / whole blood units transfused [ table 2 ; column 2 , 3 , and 4 ] frequency of a particular reaction in relation to total number of transfusions : nptr / nt 100 where nptr is the total number of the particular transfusion reaction and nt is the total number of transfusion units [ table 2 ; column 5 ] frequency of particular reaction in relation to total number of transfusion reactions : nptr / ntr 100 where nptr is the total number of the particular transfusion reaction and ntr is the total number of transfusion reactions [ figure 1 ] . frequency of wb / component transfusions and associated atrs type of transfusion reaction with respect to associated wb / component transfusions relative frequency of acute transfusion reactions the study was conducted in the department of transfusion medicine , ( hvpi centre ) , and the department of pharmacology , ( pvpi centre ) of this north east indian medical institute and attached teaching hospital . approval for conducting the study was obtained from the research protocol evaluation committee and the institutional ethical committee . this was a retrospective observational study in which all atrs reported to the blood bank over a period of 20 months ( may 2013 to january 2015 ) were reviewed and analyzed . the hospital is following the standard operating procedures adapted from the cdsco technical manual for transfusion , starting with donor selection , phlebotomy , component processing , testing for infectious diseases , storage , cross matching , issue of component , and follow - up of the recipient . the paramedical staff involved in blood transfusion has been trained in transfusion practices , enabling them to identify and manage any encountered transfusion reaction at the earliest . prior to the transfusion , they are required to cross - check for any clerical errors , abo - rh group of the patient and the blood bag , types of blood products and blood unit number , inspect the blood bag for hemolysis , clot and leakage , and expiry date . in the event of a transfusion reaction , the transfusion reaction form is being filled with information pertaining to : the date and time of initiation and cessation of the transfusion , time of the reaction , patients pre- and post - transfusion vital signs , approximate volume of blood transfused , as well as clinical signs and symptoms . the reaction form along with the posttransfusion blood sample ( 2 ml in citrate vial and 2 ml in plain vial ) , urine sample , and the leftover blood product bag , and transfusion set are immediately sent to the blood bank . the blood bank performs a thorough evaluation of the suspected transfusion reaction and rechecks the blood requisition form , returned blood / component unit number , abo - rh grouping and screening for irregular antibodies . blood grouping of the patient is repeated and compared with the pretransfusion sample . the blood bag and posttransfusion blood sample is checked for hemolysis and compared with the pretransfusion sample . in case of a suspected hemolytic reaction , further investigations done include : qualitative and quantitative estimation of plasma hemoglobin % direct antiglobulin testhemoglobinuria : gross visual examination and urine hemoglobin by dipstickshematuria : microscopic examinationserum total and unconjugated bilirubinperipheral blood smears examination for the presence of schistocytes and spherocytes . qualitative and quantitative estimation of plasma hemoglobin % direct antiglobulin test hemoglobinuria : gross visual examination and urine hemoglobin by dipsticks hematuria : microscopic examination serum total and unconjugated bilirubin peripheral blood smears examination for the presence of schistocytes and spherocytes . compatibility testing is repeated on pre- and post - transfusion sample : diamed - id microtyping system . in case of a reaction such as transfusion - related acute lung injury ( trali ) and pulmonary embolism , chest x - ray report is cross checked . based on the clinical features mentioned in the transfusion reaction form , and the laboratory reports , the reactions are classified according to the standard criteria defined by the american association of blood banks . after completion of transfusion reaction workup and categorization of the transfusion reaction , data are submitted to the hvpi . the data collected for this study were analyzed for frequency , percentage , mean , and standard deviation . statistical software used was microsoft office excel 2007 and ibm spss statistics version 20 ( statistical package for the social sciences ibm corporation ) . the chi - square test was used where applicable , and p < 0.05 was considered significant . nc is the number of component / whole blood units transfused and nt is the total number of transfusion units [ table 1 ; column 3]risk of transfusion reactions with a particular component / whole blood : ntrc / nc 100 where ntrc is the total number of reactions encountered with a particular component / whole blood , and nc is the total units of component / whole blood transfused [ table 1 ; column 5]risk of a particular reaction with a particular component / whole blood : nptr / nc 100 where nc is the total number of components / whole blood units transfused [ table 2 ; column 2 , 3 , and 4]frequency of a particular reaction in relation to total number of transfusions : nptr / nt 100 where nptr is the total number of the particular transfusion reaction and nt is the total number of transfusion units [ table 2 ; column 5]frequency of particular reaction in relation to total number of transfusion reactions : nptr / ntr 100 where nptr is the total number of the particular transfusion reaction and ntr is the total number of transfusion reactions [ figure 1 ] . nc is the number of component / whole blood units transfused and nt is the total number of transfusion units [ table 1 ; column 3 ] risk of transfusion reactions with a particular component / whole blood : ntrc / nc 100 where ntrc is the total number of reactions encountered with a particular component / whole blood , and nc is the total units of component / whole blood transfused [ table 1 ; column 5 ] risk of a particular reaction with a particular component / whole blood : nptr / nc 100 where nptr is the total number of a particular transfusion reaction , and nc is the total number of components / whole blood units transfused [ table 2 ; column 2 , 3 , and 4 ] frequency of a particular reaction in relation to total number of transfusions : nptr / nt 100 where nptr is the total number of the particular transfusion reaction and nt is the total number of transfusion units [ table 2 ; column 5 ] frequency of particular reaction in relation to total number of transfusion reactions : nptr / ntr 100 where nptr is the total number of the particular transfusion reaction and ntr is the total number of transfusion reactions [ figure 1 ] . frequency of wb / component transfusions and associated atrs type of transfusion reaction with respect to associated wb / component transfusions relative frequency of acute transfusion reactions the study was conducted in the department of transfusion medicine , ( hvpi centre ) , and the department of pharmacology , ( pvpi centre ) of this north east indian medical institute and attached teaching hospital . approval for conducting the study was obtained from the research protocol evaluation committee and the institutional ethical committee . this was a retrospective observational study in which all atrs reported to the blood bank over a period of 20 months ( may 2013 to january 2015 ) were reviewed and analyzed . the hospital is following the standard operating procedures adapted from the cdsco technical manual for transfusion , starting with donor selection , phlebotomy , component processing , testing for infectious diseases , storage , cross matching , issue of component , and follow - up of the recipient . the paramedical staff involved in blood transfusion has been trained in transfusion practices , enabling them to identify and manage any encountered transfusion reaction at the earliest . prior to the transfusion , they are required to cross - check for any clerical errors , abo - rh group of the patient and the blood bag , types of blood products and blood unit number , inspect the blood bag for hemolysis , clot and leakage , and expiry date . in the event of a transfusion reaction , the transfusion reaction form is being filled with information pertaining to : the date and time of initiation and cessation of the transfusion , time of the reaction , patients pre- and post - transfusion vital signs , approximate volume of blood transfused , as well as clinical signs and symptoms . the reaction form along with the posttransfusion blood sample ( 2 ml in citrate vial and 2 ml in plain vial ) , urine sample , and the leftover blood product bag , and transfusion set the blood bank performs a thorough evaluation of the suspected transfusion reaction and rechecks the blood requisition form , returned blood / component unit number , abo - rh grouping and screening for irregular antibodies . blood grouping of the patient is repeated and compared with the pretransfusion sample . the blood bag and posttransfusion blood sample is checked for hemolysis and compared with the pretransfusion sample . in case of a suspected hemolytic reaction , further investigations done include : qualitative and quantitative estimation of plasma hemoglobin % direct antiglobulin testhemoglobinuria : gross visual examination and urine hemoglobin by dipstickshematuria : microscopic examinationserum total and unconjugated bilirubinperipheral blood smears examination for the presence of schistocytes and spherocytes . qualitative and quantitative estimation of plasma hemoglobin % direct antiglobulin test hemoglobinuria : gross visual examination and urine hemoglobin by dipsticks hematuria : microscopic examination serum total and unconjugated bilirubin peripheral blood smears examination for the presence of schistocytes and spherocytes . compatibility testing is repeated on pre- and post - transfusion sample : diamed - id microtyping system . in case of a reaction such as transfusion - related acute lung injury ( trali ) and pulmonary embolism , chest x - ray report is cross checked . based on the clinical features mentioned in the transfusion reaction form , and the laboratory reports , the reactions are classified according to the standard criteria defined by the american association of blood banks . after completion of transfusion reaction workup and categorization of the transfusion reaction , data are submitted to the hvpi . the data collected for this study were analyzed for frequency , percentage , mean , and standard deviation . statistical software used was microsoft office excel 2007 and ibm spss statistics version 20 ( statistical package for the social sciences ibm corporation ) . the chi - square test was used where applicable , and p < 0.05 was considered significant . nc is the number of component / whole blood units transfused and nt is the total number of transfusion units [ table 1 ; column 3]risk of transfusion reactions with a particular component / whole blood : ntrc / nc 100 where ntrc is the total number of reactions encountered with a particular component / whole blood , and nc is the total units of component / whole blood transfused [ table 1 ; column 5]risk of a particular reaction with a particular component / whole blood : nptr / nc 100 where nptr is the total number of a particular transfusion reaction , and nc is the total number of components / whole blood units transfused [ table 2 ; column 2 , 3 , and 4]frequency of a particular reaction in relation to total number of transfusions : nptr / nt 100 where nptr is the total number of the particular transfusion reaction and nt is the total number of transfusion units [ table 2 ; column 5]frequency of particular reaction in relation to total number of transfusion reactions : nptr / ntr 100 where nptr is the total number of the particular transfusion reaction and ntr is the total number of transfusion reactions [ figure 1 ] . nc is the number of component / whole blood units transfused and nt is the total number of transfusion units [ table 1 ; column 3 ] risk of transfusion reactions with a particular component / whole blood : ntrc / nc 100 where ntrc is the total number of reactions encountered with a particular component / whole blood , and nc is the total units of component / whole blood transfused [ table 1 ; column 5 ] risk of a particular reaction with a particular component / whole blood : nptr / nc 100 where nc is the total number of components / whole blood units transfused [ table 2 ; column 2 , 3 , and 4 ] frequency of a particular reaction in relation to total number of transfusions : nptr / nt 100 where nptr is the total number of the particular transfusion reaction and nt is the total number of transfusion units [ table 2 ; column 5 ] frequency of particular reaction in relation to total number of transfusion reactions : nptr / ntr 100 where nptr is the total number of the particular transfusion reaction and ntr is the total number of transfusion reactions [ figure 1 ] . frequency of wb / component transfusions and associated atrs type of transfusion reaction with respect to associated wb / component transfusions relative frequency of acute transfusion reactions a total of 3455 units of whole blood and component transfusions were carried out in the study duration , out of which a total of 32 ( 0.92% ) atrs were encountered . the age of the recipients ranged from 14 to 88 years , with the mean age of females ( 43.7 years ) slightly lower than that of males ( 44.3 years ) . there was a female preponderance ( 59.4% ) in the frequency of transfusion reactions , over males ( 40.6% ) . however , this difference was not significant ( p = 0.13 , = 2.25 ) . transfusion with packed red blood cell ( prbc ) was most commonly associated with adverse reactions ( 15 reactions out of a total of 1042 transfusions ; p = 0.06 , = 3.52 ) , followed by whole blood ( wb ) transfusions ( 13 reactions out of a total of 1467 transfusions ; p = 0.83 , = 0.045 ) [ table 1 ] . a total of 650 units of fresh frozen plasma ( ffp ) transfusions were carried out that finally resulted in four reactions ( p = 0.36 , = 0.84 ) . allergic reaction was the most frequently encountered transfusion reaction ( 65.6% ) [ figure 1 ] , which was most commonly seen with prbcs ( risk of 0.76% ; p = 0.42 , = 0.63 ) and wb ( risk of 0.68% ; p = 0.63 , = 0.23 ) [ table 2 ] . this was followed by febrile nonhemolytic transfusion reactions ( fnhtrs ) ( 28.1% ) , which was seen more commonly with prbcs ( risk of 0.57% ; p = 0.016 , = 5.7 ) . there was one reaction each of anaphylaxis and pulmonary embolism , following wb and prbc transfusions , respectively . no hemolytic reactions were encountered in the study period . the mean volume of blood at which transfusion reaction had occurred was 192.5 95.7 ml [ table 4 ] . figure 2 shows the relative frequency of transfusion reactions according to the department in which the recipients were being treated , and the adverse event reported . most common manifestations included urticaria and itching ( n = 14 each ) , followed by fever ( n = 9 ) , dyspnea ( n = 7 ) , and increased pulse rate ( n = 6 ) [ figure 3 ] . demographic characteristics of the transfusion recipients reporting with atrs ( n=32 ) volume of blood at which atr initiated relative frequency of departments reporting acute transfusion reactions frequency of clinical manifestations related to the acute transfusion reactions the french blood agency had initially developed it as a national system of surveillance and alert , from blood collection to the follow - up of recipients . hemovigilance is currently recognized as an indispensable component of quality management in blood programs globally . an ideal hemovigilance system is designed to detect , gather , and analyze unexpected or undesirable effects associated with transfusion . the information obtained is intended to bring about required changes in transfusion policies , improve transfusion standards , assist in the formulation of transfusion guidelines , and thus improve safety and quality of the transfusion process . it is thus not only an avenue to analyze blood transfusion incidents , but also a tool to measure the effects of new processes or corrective actions implemented to remedy their causes and prevent their recurrence . noninfectious adverse transfusion reaction ( niatrs ) such as acute hemolytic transfusion reactions , fnhtrs , anaphylactic reactions , trali , and allergic reactions are recognized as predominant contributing factors of transfusion - related morbidity and mortality . atrs are those immune or nonimmune adverse reactions that occur within 24 h of the transfusion . the estimated frequency of atrs varies from 0.2% to 10% , and mortality is approximately , 1 in 250,000 . in this study , the frequency of atrs was observed to be 0.92% , which was comparable to that of a study carried out in punjab , where the incidence of atrs was 1.09% . two larger studies done in new delhi and chandigarh , however , showed a relatively lower frequency of transfusion reactions ( 0.05% and 0.18% , respectively ) . a study in switzerland and the incidence of transfusion reactions at our center was thus higher than that observed at some national and international centers . the frequency of atrs was more in females ( 59.4% ) than in males ( 40.6% ) . a similarly skewed incidence of transfusion reactions toward females was seen in studies done in saudi arabia ( 54.3% ) and zimbabwe ( 61.6% ) . however , studies were done in india by kumar et al . and bhattacharya et al . show a lower incidence of transfusion reactions in females ( 45.7% and 34.2% , respectively ) . the most common atr observed was an allergic reaction ( 65.6% of all atrs ) , which presented most commonly with urticaria and/or itching . there was no significant difference observed between the number of allergic reactions and type of transfusion ( p > 0.05 for wb , prbc , and ffp transfusions ) . allergic reactions can occur in up to 2% of transfusions as a result of recipient ige and donor antigen interactions triggering the release of histamine and de novo synthesis of leukotrienes and platelet activating factor . clinically , allergic reactions have been discerned from the more severe anaphylactoid reactions by the absence of clinical manifestations such as bronchospasm and/or hypotension . similar to the findings of this study , allergic reactions were also found to be the most common transfusion reaction in studies done in delhi ( 55.1% ) and iran ( 49.2% ) . however , the overall incidence of allergic reactions in the study done in delhi was only 0.02% , which was lower than the incidence of allergic reactions in our study ( 0.6% ) . fnhtr , which is defined as an otherwise unexplained rise in temperature of at least 1c during or shortly after transfusion , comprised of 28% of the total atrs , in this study ( 0.26% of total transfusions ) . fnhtrs are reported to be more common with platelet transfusions than prbcs because platelets require storage at 2024c , which results in donor leukocyte activation and pro - inflammatory cytokine accumulation . however , fnhtrs were found to be more with prbcs ( 0.57% of all prbc transfusions ) and wb ( 0.13% of all wb transfusions ) than platelets , with which no reactions were seen . the association of fnhtr 's with prbc transfusions was found to be significant ( p < 0.05 ) . a greater incidence of fnhtrs with prbcs was also observed in a study done by kumar et al . the relatively fewer number of platelet transfusions ( n = 246 ) as compared to whole blood ( n = 1467 ) and prbcs ( n = 1042 ) could be a reason for nondetection of any reactions with platelets . in a study done at aiims , the frequency of fnhtrs with prbcs was found to be 0.04% , which was lower than that observed in this study . the overall incidence of fnhtrs in the same study was 0.01% , which again was much lower than seen in our hospital . the higher incidence of both allergic reactions , as well as fnhtrs as compared to the premier institute in delhi could be a consequence of the lack of leukoreduction facilities at our hospital . the risk of fnhtr with transfusion of nonleukoreduced whole blood and prbcs was estimated to be 0.114% in a study done in chandigarh , which is closer to the incidence of fnhtrs seen in this study . though universal leukoreduction has reduced the incidence of febrile reactions , it has not been found to have a similar impact on that of allergic reactions because acellular plasma components rather than leukocytes in blood components contribute to allergic atrs . the introduction of leukoreduction at our institution could possibly enable us to reduce the incidence of atrs in general and febrile reactions in particular . there was one fatal transfusion event , in which a recipient suffering from deep vein thrombosis , developed severe respiratory distress , frothing from the mouth , collapsed , and eventually died . postmortem findings ascertained pulmonary embolism , which was , however , confirmed as not being due to air emboli . the overall incidence of transfusion reactions in this hospital is slightly higher than those having more advanced transfusion facilities in india . there was no significant difference among the overall incidence of atrs based on the type of component transfused . however , a significant association was observed between the number of prbc transfusions and febrile reactions . the use of leukoreduced wb and prbcs could possibly reduce the incidence of atrs in general and fnhtrs in particular .

objective : blood transfusions are inherently associated with risks ranging in severity from minor to life - threatening . continuous monitoring of transfusion related complications can promote understanding of factors contributing to transfusion reactions and help to formulate necessary remedial measures . this study was designed to analyze the frequency and nature of transfusion reactions reported to the blood bank of a remote north east indian teaching hospital.materials and methods : all acute transfusion reactions ( atrs ) reported to the blood bank over a period of 20 months ( may 2013 to january 2015 ) were reviewed and analyzed . the risk of transfusion reactions associated with each individual component was assessed.results:a total of 3455 units of whole blood and component transfusions were carried out of which a total of 32 ( 0.92% ) atrs were encountered . packed red blood cells ( prbcs ) ( n = 15 , p = 0.06 ) and whole blood ( wb ) ( n = 13 , p = 0.83 ) were most commonly implicated . allergic reaction was the most frequent transfusion reaction encountered ( 65.6% ) , seen most commonly with prbc ( risk of 0.76% , p = 0.42 ) , and wb ( risk of 0.68% , p = 0.63 ) transfusions . this was followed by febrile reactions ( 28.1% ) , which were seen more commonly with prbcs ( risk of 0.57% , p = 0.016 ) . no reactions were observed with platelet transfusions.conclusion:the overall incidence of transfusion reactions in this hospital is slightly higher than those having more advanced transfusion facilities in india . the lack of leukoreduction facilities in our hospital could be a likely cause for the same . the use of leukoreduced wb and prbcs could possibly reduce the overall incidence of atrs in general and febrile nonhemolytic transfusion reactions in particular .

five provinces in afghanistan were selected as a convenience sample ( accessibility ) that included both affected and unaffected areas . two accessible districts in each province were randomly selected by using a random number generator . random transects were used to select 10 households per village . to give an approximately equal male : female ratio , either the head of household , spouse ( woman ) , or the oldest person available at the time was selected . ethical approval was provided by the institutional review board , ministry of public health , afghanistan . a standardized , structured questionnaire collected information on demographic and socioeconomic measures , avian influenza information sources and knowledge of appropriate preventive measures , poultry and animal handling , food and generic hygiene , and human influenza knowledge and treatment seeking . the questions were ranked for importance in preventing avian influenza transmission in poultry or reducing human exposure and awarded 5 points , 3 points , or 1 point for correct answers . for each respondent , the sum of scores for correct answers divided by the sum of available points generated a percentage score . data were double - entered by using microsoft access ( microsoft , redmond , wa , usa ) and analyzed by using stata 8 software ( stata corporation , college station , tx , usa ) . percentage scores for each respondent were ranked and classified as above or below the median . the primary analysis was conducted to compare factors ( age , sex , socioeconomic status , provincial exposure to avian influenza iec campaigns , and poultry ownership ) associated with knowledge above the median . socioeconomic quintiles ( seqs ) were defined by principle components analysis using employment , education , and household assets as indicators ( 3 ) . factors independently associated by univariate regression at the 95% confidence level were included in a stepwise multivariate logistic regression model . to numerically evaluate kap levels , a secondary analysis assessed differences between mean percentage scores , stratified by factors identified by logistic regression analysis . data for 304 respondents were included in the analysis . of the 5 provinces , kabul and nangahar had had influenza outbreaks in poultry in 2007 . median age of respondents ( 38 years ) was high , but it reflected the age of heads of households and spouses . poultry ownership was reported by 65.2% of households ( > 95% backyard ownership ) and differed significantly between seqs ( poorest 53/62 [ 85.5% ] vs. least poor 20/55 [ 36.4% ] ; 30.0 , p<0.001 ) . provinces exposed to avian influenza and intensive information , education , and communication campaigns ( kabul , march 2007 , and nangahar , february 2007 ) . seq was positively associated with kap score above the median ( lowest vs. highest : adjusted odds ratio [ aor ] 14.3 , 95% confidence interval [ ci ] 5.239.9 ) , as was provincial exposure to avian influenza iec campaigns ( aor 9.5 , 95% ci 4.918.6 ) . backyard poultry ownership ( nonowners vs. owners : aor 0.3 , 95% ci 0.20.7 ) and older age group ( 1520 years vs. > 40 years : aor 0.3 , 95% ci 0.10.8 ) were both negatively associated . for secondary analysis , mean kap score differed between seq ( p<0.001 , by analysis of variance ) and was higher in provinces previously exposed to iec campaigns ( 50.2% vs. 40.1% ; p<0.001 , by t test ) . reporting of sick or dead poultry to authorities was less frequent among lowest seq ( 8/47 [ 13% ] ) than highest seq ( 20/49 [ 37% ] ; 6.6 , p = 0.02 ) where selling poultry in the event of a local outbreak was more commonly reported ( 21/66 [ 66% ] vs. 10/51 [ 18% ] ; 27.2 , p<0.001 ) . presence of coops was less frequent in lowest seq ( 9/49 [ 18.4% ] ) than in highest seq ( 21/46 [ 45.6% ] ; 8.2 , p = 0.004 ) . human cases of avian influenza ( h5n1 ) have resulted from contact between humans and infected backyard poultry ( 4 ) . risk to humans is also related to frequency of disease occurrence in the avian population ( 5 ) . recently , human - to - human transmission has been reported in the neighboring northwest frontier province of pakistan ( 2 ) . overall knowledge was low , although in provinces exposed to intensive iec campaigns , kap scores of the population were higher . the level of concern generated by the campaign , government response , media reports , and proximity to the outbreak are all likely to contribute to this association . despite this encouraging evidence , level of knowledge was far higher among persons with higher socioeconomic status . our results can be broadly generalized to the population , although we did not have access to unsafe districts ( most of the districts in southern and eastern afghanistan ) . this limitation may introduce selection bias , which would underestimate the effect of socioeconomic status because those living in inaccessible areas likely have a lower status than persons in accessible areas . preintervention and postintervention surveys would provide a more robust measure of effectiveness . in the immediacy of an outbreak , although there are limitations to the study design in concluding intervention effectiveness , the results provide evidence to support further intensive campaigns as a response to influenza outbreaks in poultry . several reports have examined kaps and behavior related to avian influenza ( h5n1 ) ( 69 ) . similar to the finding in the lao people s democratic republic ( 6 ) , our study suggests that conventional education and behavior change messages have a limited effect in populations with highest exposure . efforts to ensure that iec messages are suitable for lower socioeconomic groups should be adopted , specifically by improving the knowledge of community leaders , designing messages in a suitable format for the poor and illiterate , and ensuring that the most accessible channels are used . messages should carefully balance the risk for human disease against potential nutritional and economic consequences of high population concern ( e.g. , food scares ) . successfully promoting behavior change is a lengthy process and requires frequent reinforcement . the acuteness of avian influenza ( h5n1 ) outbreaks requires a concerted effort to enhance knowledge and change behavior among those most at risk in low - income countries .

from february through april 2007 , avian influenza ( h5n1 ) was confirmed in poultry in 4 of 34 afghan provinces . a survey conducted in 2 affected and 3 unaffected provinces found that greater knowledge about reducing exposure was associated with higher socioeconomic status , residence in affected provinces , and not owning backyard poultry .

regenerative periodontal therapy comprises of procedures which are specially designed to restore those parts of the tooth - supporting apparatus which have been lost due to periodontitis . the regeneration of osseous defects caused by inflammatory periodontal disease continues to provide an ongoing challenge in periodontal therapy . current alternative lines of treatment include autogenous bone grafts and allografts , guided tissue regeneration , alloplastics , or a combination of techniques . alloplastic materials are synthetic , inorganic , biocompatible , and/or bioactive bone graft substitutes which are claimed to promote bone healing through osteoconduction . alloplastic materials such as hydroxyapatite and tricalcium phosphate have been used in the treatment of intrabony defects . however , it has been shown that healing often occurs with encapsulation of the graft material by connective tissue and minimal or no bone formation . these materials differ from other bioactive ceramics in that the rate of bonding to bone can be controlled by varying the chemical composition . their bioactivity is attributed to the formation of a hydroxyl carbonated apatite ( hca ) layer on their surface similar to the mineral part of bone . the rate of tissue bonding appears to depend on the rate of hca formation , which follows a sequence of reactions between the implanted material and the surrounding tissues and physiologic fluids . the greater surface of nanostructure ceramics present an incomparable and promising character for orthopedic and dental implant formulations with better osseointegrative properties . by controlling the structural and particle size in the range of nanoscale , some properties of bioactive bioceramic such as osseoconductivity , solubility , and mechanical reliability can be improved . the grain size , large surface area to volume ratio , and the ultra fine structure of nanoscale engineering bioceramics similar to the biological apatite provide surprising functional properties for these materials . recently , nanoceramics have attracted interest because surface nanostructuring allows for improved cellular adhesion , enhances osteoblast proliferation and differentiation , and increases biomineralization . recently , researchers in isfahan university of technology have been succeeded to produce bioactive glass in nanoscale size that can be a desired substance in dentistry . considering the fact that interaction between periodontal cells and bone graft materials are important for periodontal regeneration and according to the possibility of cellular and genetic damage of implanted material for patient and clinician , it should be concerned about the safety of mentioned biomaterials . specifications and standards have been developed to aid producers , users , and consumers in the evaluation of the safety and effectiveness of dental products . according to iso specifications , implant devices are required to evaluate several tests such as cytotoxicity , subchronic systemic toxicity , skin irritation , intracutaneous reactivity , sensitization systemic toxicity , genotoxicity , chronic toxicity , systemic toxicity , and local effects after implantation . wilson et al . studied the test of solid bioglass implant in the soft tissues of rats and rabbits for time periods up to eight weeks . the surface activity in contact with bioglass which is so critical in bone adhesion was not toxic in rat tissues . studied the cytotoxicity of new bioglass composite after 24 hours in cell culture , using one of the biocompatibility tests . in addition , a significant increase in cellular activity suggested that bioglass composite was able to stimulate cellular activity by creating a favorable micromovement for cell proliferation and growth . the single cell gel electrophoresis ( scge ) assay , better known as comet assay , is a sensitive technique for detecting single and double strand break at the single cell level in dna of any kind of cells . this test is based on the ability of dna fragments to migrate in a weak electric field in direction of anode , given the nucleolus the appearance of comet tail when visualized by fluorescence microscopy.[1416 ] pelaez et al . estimated the genotoxicity of coatings containing bioactive particles on stainless steel by scge assay and significantly lower dna migration in the cells of the cultures was observed . evaluated the genotoxicity of hydroxyapatite bioceramics using comet assay and showed that dna break increased with increasing of biomaterial concentrations . the aim of this study was to evaluate the genotoxicity of a novel nano bioactive glass and novabone bioglass with control group in gingival fibroblasts cells using scge assay . in this in vitro experimental study , periodontal fibroblasts cells were cultured in their logarithmic phase and the genotoxicity of nano bioactive glass and novabone bioglass was studied in selected cultured cells at biotechnology laboratory of pharmacology of isfahan university of medical sciences . novel nano bioactive glass was made by sol - gel method in biomaterials laboratory of biomaterials research group at isfahan university of technology . prepared nano bioactive glass was bioactive and less than 100 nanometer in size based on previous researches . novabone bioglass ( novabone products llc , alachua , fl , usa ) , a silica - based biocompatible material with particle size ranged between 90 to710 m , has been examined in many studies . comet test or scge assay was used in order to evaluate the genotoxicity of mentioned biomaterials on c165 fibroblasts cells which was prepared from iran pastor institute in their logarithmic phase . cells were cultured in rpmi 1640 ( paa , vienna , austria ) and 10% fetal bovine serum , fbs ( gibco , ny , usa ) , and antibiotic ( penicillin , streptomycin ) , then incubated in atmosphere containing 5% co2 at the temperature of 37 c. before forming a confluent mono layer , the cells were harvested from the culture surface by incubation with 2 cc solution of trypsin ( gibco , ny , usa ) . the cells were counted and they were about 10 under microscope to be added experimental materials in the plates . nano bioactive glass and novabone bioglass powders were used for preparation of 10 mg / ml concentration . culture medium supplemented with penicillin and streptomycin the suspension was shaken for 72 hours in shaker incubator , then concentrated samples were centrifuged ( 10 minutes , 1 800 rpm ) . the culture medium was aspirated with a syringe and filtered and sterilized for 30 minutes under ultra violet ( uv ) light . each test concentration of biomaterials ( 1 , 2 , 4 , and 5 mg / ml ) was placed in a culture plate with total 3 ml cell and culture medium of rpmi1640 . as negative control ( c- ) , the cells were cultured with medium without adding biomaterials . after storing the samples at 37c in a 5% co2 atmosphere during 24 hours , they were centrifuged at 1800 rpm ( 10 minutes ) and supernatant was removed . finally , the cells were resuspended in culture medium and were used to measure dna damage in an individual cell using the alkaline protocol . detection of dna damage by alkaline comet assay was used for measurement of the genotoxic activity of the biomaterials using protocol of singh . cell suspension was mixed with low melting point agarose , lmp ( sigma , ny , usa ) in equal volume ( 250 l ) , and extended it on a slide previously coated with 100 l of 1% normal melting point agarose , nmp ( sigma , ny , usa ) . after gelling at 4 c , the cells were lysed for 60 minutes in a solution of 5 m nacl , 20 cc edta , 1 m tris , 10 ml dimethyl sulfoxide , dmso and 1 cc triton x-100 , at ph 10 and 4 c. the slides were washed three times in distilled water for 5 minutes and placed them on a horizontal electrophoresis unit , containing fresh electrophoresis buffer ( 300 mm naoh , 1 mm ethylene diamine tetraacetic acid , edta with ph 13 ) . after 45 minutes of unwinding at 4 c , electrophoresis was performed for 45 minutes ( 20 v , 300 ma ) . the slides were washed 3 times in 400 mm tris buffer ( ph 7.5 ) and stained with ethidium bromide ( 2 g / ml , 10 minutes ) . one hundred cells from each sample were selected randomly and analyzed by free comet score software . observations were made at magnification of x400 using an epifluorescent microscope ( ceti ) coupled with ccd camera ( sony dsc h9 ) . cells with damaged dna displayed high migration of dna fragments from the nucleus , forming a tail in comet form [ figure 1 ] . by using autocomet software 3 parameters ( tail length , % dna in tail , tail moment ) , microscopic view of concentration of ( a ) control sample , ( b ) 2 mg / ml nano bioactive glass , ( c ) 5 mg / ml nano bioactive glass ( magnification of 400 ) data were analyzed with statistical software spss 11.5 and statistical test , kruskal - wallis h and a complimentary test , mann - whitney test ( p = 0.05 ) . cells were cultured in rpmi 1640 ( paa , vienna , austria ) and 10% fetal bovine serum , fbs ( gibco , ny , usa ) , and antibiotic ( penicillin , streptomycin ) , then incubated in atmosphere containing 5% co2 at the temperature of 37 c. before forming a confluent mono layer , the cells were harvested from the culture surface by incubation with 2 cc solution of trypsin ( gibco , ny , usa ) . the cells were counted and they were about 10 under microscope to be added experimental materials in the plates . nano bioactive glass and novabone bioglass powders were used for preparation of 10 mg / ml concentration . culture medium supplemented with penicillin and streptomycin the suspension was shaken for 72 hours in shaker incubator , then concentrated samples were centrifuged ( 10 minutes , 1 800 rpm ) . the culture medium was aspirated with a syringe and filtered and sterilized for 30 minutes under ultra violet ( uv ) light . each test concentration of biomaterials ( 1 , 2 , 4 , and 5 mg / ml ) was placed in a culture plate with total 3 ml cell and culture medium of rpmi1640 . as negative control ( c- ) , the cells were cultured with medium without adding biomaterials . after storing the samples at 37c in a 5% co2 atmosphere during 24 hours , they were centrifuged at 1800 rpm ( 10 minutes ) and supernatant was removed . finally , the cells were resuspended in culture medium and were used to measure dna damage in an individual cell using the alkaline protocol . detection of dna damage by alkaline comet assay was used for measurement of the genotoxic activity of the biomaterials using protocol of singh . cell suspension was mixed with low melting point agarose , lmp ( sigma , ny , usa ) in equal volume ( 250 l ) , and extended it on a slide previously coated with 100 l of 1% normal melting point agarose , nmp ( sigma , ny , usa ) . after gelling at 4 c , the cells were lysed for 60 minutes in a solution of 5 m nacl , 20 cc edta , 1 m tris , 10 ml dimethyl sulfoxide , dmso and 1 cc triton x-100 , at ph 10 and 4 c. the slides were washed three times in distilled water for 5 minutes and placed them on a horizontal electrophoresis unit , containing fresh electrophoresis buffer ( 300 mm naoh , 1 mm ethylene diamine tetraacetic acid , edta with ph 13 ) . after 45 minutes of unwinding at 4 c , electrophoresis was performed for 45 minutes ( 20 v , 300 ma ) . the slides were washed 3 times in 400 mm tris buffer ( ph 7.5 ) and stained with ethidium bromide ( 2 g / ml , 10 minutes ) . one hundred cells from each sample were selected randomly and analyzed by free comet score software . observations were made at magnification of x400 using an epifluorescent microscope ( ceti ) coupled with ccd camera ( sony dsc h9 ) . cells with damaged dna displayed high migration of dna fragments from the nucleus , forming a tail in comet form [ figure 1 ] . by using autocomet software 3 parameters ( tail length , % dna in tail , tail moment ) , microscopic view of concentration of ( a ) control sample , ( b ) 2 mg / ml nano bioactive glass , ( c ) 5 mg / ml nano bioactive glass ( magnification of 400 ) data were analyzed with statistical software spss 11.5 and statistical test , kruskal - wallis h and a complimentary test , mann - whitney test ( p = 0.05 ) . in this study , maximum damaged % dna and longer tail length were observed in highest concentration of both materials . both higher number of comet and higher length of migrated damaged dna was found with increasing of concentrations [ figure 2 ] . mean value of comet parameter in different concentrations of nano bioactive glass and novabone bioglass no statistically significant difference was observed between the concentrations of novabone bioglass ( p value = 0.085 ) with control group and novel nano bioactive glass ( p value = 0.437 ) with control group in the evaluation of % dna in tail parameter . the comparison between cells corresponding to negative control and the concentrations of nano bioglass revealed statistically significant difference ( p 0.05 ) in concentrations of 4 mg / ml and 5 mg / ml in tail length and tail moment parameters [ table 1 ] . comparison of comet parameters in different concentrations of nano bioactive glass and novabone bioglass with control sample ( p 0.05 ) nano bioactive glass generated statistically significant amount of dna damaged at concentration of 5 mg / ml in tail length and tail moment parameters compared with negative control ( p 0.05 ) . the concentration 4 mg / ml revealed statistically significant difference only in tail moment parameter compared with negative control ( p 0.05 ) [ table 1 ] . the comparison between cells corresponding to different concentrations of nano bioactive glass revealed higher mean compared to novabone bioglass in all concentrations by evaluation of tail length parameter and difference was statistically significant ( p 0.05 ) [ table 2 ] . comparison of comet parameters in different concentrations of nano bioactive glass and novabone bioglass ( p 0.05 ) in comparison , two other parameters ( % dna in tail , tail moment ) for nano bioactive glass revealed higher mean compared to novabone bioglass in all concentrations but difference showed statistical significance ( p 0.05 ) only in 5 mg / ml concentration [ table 2 ] . in present experiment research , the comet assay was used to detect induction of dna damage in fibroblast cells after 24 hours treatment with novel nano bioactive glass and novabone bioglass . results showed that dna damage was induced in all tested biomaterials concentrations and the genotoxic effect increased with increasing the concentration [ figure 2 ] . evaluated the genotoxicity of hydroxyapatite bioceramics using comet assay and showed that biomaterials induced dna break , which increased with increasing the bioceramic concentration . according to this fact , it could be expected that the genotoxicity of biomaterials depends on the amount of concentration and it is so critical to detect the threshold . the comparison between cells corresponding to negative control and the concentrations of nano bioactive glass revealed statistically significant difference ( p 0.05 ) in concentrations of 4 mg / ml and 5 mg / ml in tail length and tail moment parameters [ table 1 ] . also , no statistically significant difference was observed between the concentrations of novabone bioglass ( p value = 0.085 ) and nano bioactive glass ( p value = 0.437 ) in the evaluation of % dna in tail parameter . according to the moller et al . 's study , the end point measured by the traditional comet assay is a mixture of the length of the comet tail and total dna in tail or by the tail moment ( the length of the comet tail multiplied by the intensity of fluorescence in the tail ) ; therefore , this parameter was highlighted in present study . according to the results , concentrations less than 4 mg / ml of novel nano bioactive glass have no genotoxicity effect , whereas biomaterials may have low , medium , or high potential risk to human safety , depending on the type and extent of patient contact . thus , for investigating the systemic toxic effect of nano bioactive glass and using it in dentistry , higher concentrations of the material are needed to be tested . pelaez et al . , by scge assay , estimated the genotoxicity of coatings containing bioactive particles on stainless steel and significantly lower dna migration was observed in the cells of the cultures . the present study has an advantage over pelaez 's study in evaluation of genotoxicity , because of using several concentrations of biomaterials and determining safety genotoxic threshold for tested materials . in our study , pelaez use olive tail moment parameter for detecting genotoxicity of materials and showed that the coating containing hydroxyapatite particles has lower values of olive tail moment , which indicates less dna damage without statistical difference with other materials . the comparison between cells corresponding to different concentrations of nano bioactive glass showed a higher mean value compared to novabone bioglass in all concentrations by evaluation of tail length parameter . the difference was statistically significant ( p 0.05 ) [ table 2 ] . in comparison , two other parameters ( % dna in tail , tail moment ) , nano bioactive glass showed a higher mean value compared to novabone bioglass in all concentrations but difference showed statistical significance ( p 0.05 ) only in 5 mg / ml concentration [ table 2 ] . these results show pieces of damaged dna , with lower dna percent , have migrated higher distance , so there is no statistical difference between nano bioactive glass and novabone bioglass in concentration less than 5 mg / ml [ table 2 ] , which can indicate safety of novel nano bioactive glass in genotoxicity according to the results of this study . haung et al.s and arun et al.s studies proved the biocompatibility of novel composite bioglasses by other biocompatibility evaluation tests . arun et al . investigated the effect of ha - bg on chromosomal aberrations in human lymphocytes which are the other approved biocompatibility tests . however , we applied comet assay , a sensitive technique , to detect induction of dna damage in fibroblast cells . more precise results are gained by choosing fibroblast cells for detecting genotoxicity which are the most periodontal connective tissue cells , and have more contact with our materials , whereas comet assay is a technique for detecting single and double strand break at the single cell level in dna of any kind of cells.[1416 ] the important point that must be considered in comparison of nano bioactive glass and novabone bioglass is the difference in particle size . nanoparticles have a higher surface area in comparison to microparticles and thus the amount and rate of ion release for nanoparticles are extremely higher . in other words , the bioactivity of nanoparticles is higher in comparison with the same mass of microparticles . this difference is the main reason for the different genotoxicity of nano bioactive glass and micro bioglass ( novabone bioglass ) in the concentrations higher than 4 mg / ml . according to the present research , concentrations less than 4 mg / ml of nano bioactive glass produced in iran have no genotoxic effect and there is no statistical difference between nano bioactive glass and novabone bioglass in concentrations less than 5 mg/ ml . therefore , other biocompatibility tests are needed to approve the application of new nano bioactive glass for regenerative therapy in periodontics .

background : the greater surface of bioactive glass nanoparticles presents an incomparable and promising feature similar to the biological apatite . nanoparticles improve cellular adhesion , enhance osteoblast proliferation and differentiation , and increase biomineralization for periodontal regeneration and dental implants . considering the fact that interaction between periodontal cells and bone graft materials are important for periodontal lesion regeneration , the present study was undertaken to investigate the genotoxicity of a novel synthesized nanoscale bioactive glass and compared it with novabone bioglass in periodontal fibroblasts cells , in order to approve the biocompatibility of nano bioactive glass.materials and methods : in this in vitro experimental study , periodontal c165 fibroblasts cells were cultured in their logarithmic phase and the genotoxicity of novel synthesized bioactive glass nanoparticles and novabone bioglass was studied in different concentrations and a control group using comet assay test . by using autocomet software , three parameters ( tail length , % dna in tail , tail moment ) were analyzed ; the genotoxicity of mentioned biomaterials and control group . obtained data were analyzed by spss 11.5 software , kruskal wallis h and mann whitney tests ( p = 0.05).results : no statistically significant difference was observed between the concentrations of novabone bioglass ( p value = 0.085 ) with control group and novel nano bioactive glass ( p value = 0.437 ) with control group in the evaluation of % dna in tail parameter . there was significant difference between genotoxicity of novel nano bioactive glass and control , and between novabone bioglass and control group in concentrations of 4 and 5 mg / ml . according to significance of the mean difference , novel nano bioactive glass showed higher genotoxicity compared to novabone bioglass in the concentration of 5 mg / ml ( p 0.05).conclusion : the findings of this study have demonstrated that novel nano bioactive glass had no genotoxicity in concentrations lower than 4 mg / ml . nanoparticles have a higher surface area in comparison to microparticles and thus , the amount and rate of ion release for nanoparticles are extremely higher . this difference is the main reason for the different genotoxicity of nano bioactive glass and micro novabone bioglass in the concentrations higher than 4 mg / ml .

the purpose of this study was to explore nurses attitudes to the barriers of patient education as a right for getting information based on work situation of nurses , educational facilities in hospitals , and patients situation . twenty questions were about their working situation , 4 questions about hospital educating facilities , and 12 questions were about patients situation in hospital . the type and frequency of education barriers were evaluated , and variables associated with reporting an obstacle were analyzed . in our questionnaire , we used a likert scale for determining severity of three domains as the barriers of patient education that ranged from 0 to 4 . generally , it was obvious that educational condition in our hospitals was not good and most of the nurses believed that patient education is not their duties , facilities in hospitals are not sufficient and shortness of time is the most important cause of insufficiency of patient education the interactions of patient , physician and systemic factors have implications for the implementation of patient education . the failure of adequate patient education may be attributed to the lack of patient adherence , the failure of nurses knowledge and skill level or the insufficient funding and organization of necessary programs in the current health care system . hospitalization , which is the major health care cost in community , consumes a considerable part of the health care budget in general . good education skills and strategies are particularly important in the diagnosis , treatment and management of diseases . few studies have explored the contextual dimensions and subsequent interactions that contribute to a lack of adherence in the application of guidelines for patient education that is the cornerstone of care for all patients with acute or chronic diseases . patients education is a fundamental aspect of patient care and yet poor education is the most common source of patient 's complaints in the health - care sector . other work indicates that miscommunication in education often occurs because of cultural differences between the communicator and recipient . problems of miscommunication and language may not only influence treatment but may also contribute to the reinforcement of stereotyped behavior . there are increasing pressures within primary care requiring a rethink of roles , responsibilities and skill mix . the use of suitably trained nurses to extend their sphere of responsibility may be an appropriate way to manage the demand without compromising quality or patient satisfaction . patient education is an essential nursing practice standard that meaningfully impacts a patient 's health and quality of life . education process is a systematic , sequential , logical , planned course of action consisting of two major interdependent operations , teaching and learning . the education process has been compared to the nursing process as the steps of each process run parallel to one another . to provide thorough and appropriate education education is used to empower the patient and is an important aspect of quality improvement given that it has been associated with improved health outcomes . the nurses role has undergone historical change , shifting from imparting disease - oriented health education toward empowering patients to use their own resources to attain health . essentials for effective patient education include use of an open communication style , written instructions and addressing barriers . demographic variables , such as ethnic background , formal education level , reading ability , and barriers to participation in education must be considered to maximize the effectiveness of self - management education outcomes . nurses attitude to patient education barriers can help to elimination of many problems for practitioners who are well suited to provide care that facilitates behavior change and health - oriented patient education . the process of patient education can be described in 5 steps : 1 ) assessment of the patient 's previous knowledge , misconceptions , learning abilities , learning styles , cognition , attitudes and motivation . 3 ) the planning of the education and goals are set and educational interventions are chosen . in the planning phase , the type of education , the frequency , who will deliver the education and when and how it should be given , should also be addressed . historically , the teaching role of nurses within medical education has been largely unrecognized , although in the clinical ward areas expert nurses frequently educate and induct newly qualified doctors into routines and procedures . barriers cited in the literature to adherence to guidelines for diseases management include : a need for education , lack of time and lack of confidence in clinical skills , complexity , and a need for effective charting systems . with patients requesting for information that is relevant to their own disease or recovery process , nurses must focus their attention on patient - tailored information resources , seeking information from a variety of resources including colleagues , the patient record , or other high quality sources . the purpose of our study was to assess nurses attitude to patient education barriers in educational hospitals . urmia city which is located in north - west of iran , has a population of 3200000 individuals . during 2009 - 2010 , this cross - sectional study was carried out . census method was used for sampling and all nurses who filled the questionnaire entered into the study . the data was gathered with a two part questionnaire : the first part included demographic variables such as age , marriage situation , ward , employment duration and kind of their shifts the second part assessed their attitudes to barriers of participation in education . three domains were studied ; 20 questions for their working situation , 4 questions for hospital educating facilities and 12 questions for patients situation in hospital . we used likert scale for determining severity of three domains as the barriers of patient education ranged from 0 to 4 . its reliability was assessed by internal consistency as cronbach 's alpha calculation was 0.89 and test - retest reliability was 0.91 . after explaining how to fill in the questionnaire , data were analyzed by spss version 16 and descriptive statistics used to show the barriers . in addition , we declare that have no conflict of interest in this study and subjects were surveyed in agreement with the research ethics . most of the participants ( 82.5% ) were female , and duration of working was 1 - 5years in the most subjects . the age average was 32.39 ( sd=6.2 ) years and most of them were in 30 - 35 years age group and 102 ( 72.8% ) subjects worked in rotating shifts . it was obvious that the education condition in our hospitals was not good and most of nurses believed that patient education was not their duty . participants believed hospital facilities were not sufficient and shortness of time was the most important cause of insufficiency of patient education . most of nurses ( 73.6% ) were unaware about patient education importance and patient education in their job promotion was not important . the most important barriers of patient education regarding nurses working situation were low knowledge of nurses about importance of education , feeling of ineffectiveness of it in quality of treatment , lack of interest of nurses to participation in patients education . the most important barriers of patient education in terms of hospital educating facilities were lack of educational resources and shift rotation . regarding patients situation in hospital , the most important barriers were lack of interest in patients for learning , insufficiency of hospitalization time , patient inconvenience and feeling of lack of importance of learning in hospital . table 1 shows the most common answers of nurses to patient educational barriers and scores . any combination of educational barriers might interfere with the plan being relevant and timely for the targeted learner . education , often delivered by nurses , is an important part of all management programs for patients , both in clinical practice and research . patient education includes all educational activities directed at patients , including aspects of therapeutic education , health education and clinical health promotion . it is obvious that interventions at multiple levels that address the demographic and socioeconomic obstacles to education are needed to ensure successful self - management training or self - efficacy . it is interesting to note that many study participants expressed concerns with aspects of the primary care process that are typical of more generally held concerns about health care changes resulting from the managed care revolution . perceptions that physicians and nurses are hurried and do not have the time to stop and talk with or listen to patients echo a common theme in discussions of contemporary health care quality . the interactions of patient , physician , nurse and systemic factors have implications for the implementation of patient education . the failure to education adequately for patients may be attributed to a lack of patient adherence , a failure of personnel knowledge and skill level , or insufficient funding and organization of necessary programs in the current health care system . however , our findings suggest that no single player is at fault and , with education ; the integration of the three factors relevant to patients care is achievable through implementation of a patient education model . interventions at multiple levels that address the demographic and socioeconomic obstacles to patient education are needed to ensure successful self - management training . in summary , essential principles for the education role include : acknowledge control as belonging to the patient , dialogue rather than monologue , use language the patient can understand , not overload with verbal instructions , use memory aids such as written instructions and mailed reminders , and suggest specific helps . patients in hospital need education in order to adapt to their condition and perform self - care behavior . despite the fact that many patients received education and perceived information about their treatment as important , they had low levels of knowledge and lacked a clear understanding of why they had developed diseases , how it was defined and what relevant self - care behavior should be performed . it is important to target barriers to learning such as functional and cognitive limitations , misconceptions , low motivation and self - esteem . health care professionals need to be skilled in assessing the requirements and the level of education given to the individual . education can be further improved by combining clinical experience with new technologies and nurse managers and must explicitly support the patient - teaching role of the inpatient nurses upon their employment , by providing the resources they need and rewarding their efforts . continuing education for nurses during new employee orientation programs and periodically thereafter can include information applicable to ambulatory care . this will allow hospital nurses to convey accurate information to patients regarding their care after discharge . continuing education offerings afford an opportunity to address the additional barriers for nurses who want to improve outcomes for their patients . it is important that nurses develop and master information seeking skills so that they can access and find information resources they can offer directly to patients and caregivers . some patients and caregivers , however , may doubt that their information needs are adequately addressed because the resources may not be available on the clinical unit . we believe our findings have exposed concerns that are likely to be held in similar general practice groups across the country and suggest that before nurses roles can become widespread , these concerns need to be addressed . the exploration of professional attitudes towards the employment of nurse practitioners is an essential precursor to a debate about how barriers may be overcome , and about the appropriate skill mix and employment arrangements required to manage primary health care services in the future .

background : the purpose of this study was to explore nurses attitudes to the barriers of patient education as a right for getting information based on work situation of nurses , educational facilities in hospitals , and patients situation.materials and methods : the study was conducted using a cross - sectional design . the populations consisted of 240 nurses affiliated in the educational hospitals . the data were gathered by a questionnaire . demographic variables and three domains were studied . twenty questions were about their working situation , 4 questions about hospital educating facilities , and 12 questions were about patients situation in hospital . the type and frequency of education barriers were evaluated , and variables associated with reporting an obstacle were analyzed.findings:in our questionnaire , we used a likert scale for determining severity of three domains as the barriers of patient education that ranged from 0 to 4 . generally , it was obvious that educational condition in our hospitals was not good and most of the nurses believed that patient education is not their duties , facilities in hospitals are not sufficient and shortness of time is the most important cause of insufficiency of patient educationconclusions : the interactions of patient , physician and systemic factors have implications for the implementation of patient education . the failure of adequate patient education may be attributed to the lack of patient adherence , the failure of nurses knowledge and skill level or the insufficient funding and organization of necessary programs in the current health care system .

a 2-year - old girl presented with partial seizures that manifested as twitching of the left angle of the mouth and jerking of the left upper limb but usually did not result in a loss of consciousness . the child s motor and cognitive development were within the normal ranges . at the age of 3 years , she was referred to our center with the aim of achieving control of her seizures . brain magnetic resonance imaging ( mri ) showed focal cortical dysplasia in the right centroparietal region ( figure 1a ) , while interictal tc - ethyl cysteinate dimer single - photon emission computed tomography ( spect ) did not detect any abnormalities ( data not shown ) . a , at the age of 3 years , a t2-weighted mri scan showed focal cortical dysplasia in the right centroparietal region ( ) . b , on day 2 after admission , a t2-weighted mri image showed a high signal intensity lesion ( ) adjacent to the focal cortical dysplasia lesion . c , apparent diffusion coefficient mapping also showed a high signal intensity lesion in the same region . d , the high signal intensity lesion ( ) was located close to the right insular cortex ( left ) . on day 69 , the high signal intensity lesion had diminished in size , and the patient s cluster seizures had improved ( right ) . dimer spect detected hyperperfusion in a region containing the high signal intensity lesion seen on mri , which was located close to the right insular cortex . interictal tc - ethyl cysteinate dimer spect performed on day 68 did not detect any abnormalities . mri indicates magnetic resonance imaging ; spect , single - photon emission computed tomography . at the age of 10 years and 5 months , the patient was admitted because of a sudden worsening of her seizures . on admission , she was taking phenytoin ( 250 mg / d ) in combination with valproate ( 400 mg / d ) . an ictal eeg showed rhythmic fast waves arising from the right central region , which sometimes evolved into diffuse slow waves corresponding to asymmetric bilateral tonic seizures ( left > right ) . the patient s seizures did not respond to the intravenous administration of midazolam , lidocaine , and phenobarbital . on the second day after admission , t2-weighted mri and apparent diffusion coefficient mapping revealed a high signal intensity lesion adjacent to the focal cortical dysplasia lesion ( figure 1b - d , left ) . diffusion - weighted imaging also revealed a high signal intensity lesion in the same region ( data not shown ) . an eeg performed at the onset of the seizure showed rhythmic fast waves arising from the right central region . ten seconds after the onset of the seizure , when the epileptic activity evolved into diffuse slow waves , the patient s baseline sinus heart rate of about 100 beats / min suddenly dropped to 40 to 50 beats / min , and she started to exhibit an idioventricular rhythm . immediately after the seizure activity disappeared , the patient s cardiac rhythm returned to a normal sinus rhythm ( figure 2 ) . oxygen desaturation ( an oxygen saturation value of < 92% ) due to apnea ( defined as breath arrest lasting > 10 seconds ) also occurred after the appearance of ictal bradycardia . cardiac assessments , including continuous 24-hour ambulatory electrocardiography ( ecg ) monitoring and echocardiography , did not detect any abnormalities between the seizures . a , an eeg performed at seizure onset showed rhythmic fast waves arising from the right central region . b , about 10 seconds after seizure onset , when the patient s epileptic activity evolved into diffuse slow waves , her baseline sinus heart rate of about 100 beats / min suddenly dropped to 40 to 50 beats / min and then exhibited an idioventricular rhythm . c , after the seizure activity disappeared ( 68 seconds after onset ) , the patient s cardiac rhythm returned to a normal sinus rhythm . between 3 and 5 days after the patient s admission , the frequency of her seizures increased to 5 to 10 an hour . the patient s ictal bradycardia and apnea were usually accompanied by seizures , despite the prompt discontinuation of phenytoin and lidocaine . the idioventricular rhythm was observed in about half of the seizures . on the sixth day after admission , when a brief period of ictal asystole occurred , barbiturate ( sodium thiopental ) therapy was started while the patient was under mechanical ventilation . the frequency of the patient s seizures decreased to once or twice an hour after the initiation of the barbiturate therapy ( 5 mg / kg / h ) , and her ictal bradycardia subsided . after the continuous intravenous infusion of sodium thiopental for 48 hours , the dose was gradually reduced . however , the patient s bradycardia was less severe than it had been previously , her heart rate only fell to around 60 to 70 beats / min , and she did not develop cardiac arrhythmia . on the 25th day after admission , ictal spect performed on day 27 showed hyperperfusion in the area containing both the focal cortical dysplasia lesion and the high signal intensity lesion seen on mri ( figure 1e ) . the frequency of the patient s seizures was reduced via the continuous infusion of midazolam and high - dose phenobarbital , and hence , her ictal bradycardia subsided again . interictal spect performed on day 68 did not reveal any abnormalities ( figure 1e ) . on day 69 , the high - intensity lesion adjacent to the focal cortical dysplasia lesion was found to have diminished in size on follow - up mri ( figure 1d , right ) . interictal f - fluorodeoxyglucose positron emission tomography / computed tomography was performed 2 months after discharge and detected hypometabolism in the right centroparietal region but not in the right insular cortex ( data not shown ) . she underwent surgery at the age of 13 years and 1 month , and the lesion was partially resected . after surgery , the patient had infrequent partial seizures that were similar to those observed before surgery but no longer developed cluster seizures . ictal bradycardia is considered to be rare in children because there have only been a few reported pediatric cases . however , a recent study found that ictal bradycardia occurred in 8.2% of the pediatric patients with epilepsy admitted to a pediatric epilepsy monitoring unit . another comparative study reported that ictal bradycardia occurred more often in children ( 23.8% of pediatric seizures ) than in adults ( 7.3% of adult seizures ) . in adults , ictal bradycardia was found to be more strongly associated with seizures originating from the temporal lobe , while in children it was more prevalent in patients having seizures of extratemporal origin . the risk factors for ictal bradycardia include seizure clustering , having suffered with epilepsy for a long period , taking multiple antiepileptic drugs , a younger age at seizure onset , and refractory epilepsy . the propagation of seizure activity into the autonomic centers that regulate the cardiovascular system is assumed to be the leading mechanism of ictal bradycardia . in 1990 , oppenheimer and cechetto demonstrated that stimulation of the left insular cortex evoked bradycardia in rats . the same authors later showed that perioperative stimulation of the insular cortex tended to result in heart rate changes ( left insular cortex : bradycardia and right insular cortex : tachycardia ) in patients with epilepsy . although conflicting results have been obtained regarding the left- or right - sided predominance of ictal bradycardia , several other studies have suggested that the insular cortex plays an important role in cardiac regulation in humans . however , to the best of our knowledge , there has only been 1 case report of ictal bradycardia due to an insular cortex lesion , which involved a 3-year - old girl with focal cortical dysplasia in her right insular cortex . some antiepileptic drugs , such as carbamazepine , lamotrigine , phenytoin , and lidocaine , are known to act as sodium channel blockers and can have side effects that affect the cardiac conduction system . our patient was receiving phenytoin and lidocaine for her cluster seizures when the first episode of ictal bradycardia occurred . however , phenytoin and lidocaine are unlikely to have caused the bradycardia experienced in the present case due to ( 1 ) the prolonged ( > 2 weeks ) persistence of the bradycardia , despite the prompt discontinuation of the above - mentioned drugs and ( 2 ) the absence of cardiac arrhythmia between seizures according to continuous 24-hour ambulatory ecg monitoring . focal cortical dysplasia constitutes the most important cause of intractable localization - related epilepsy in childhood . a recent study reported that ictal spect is useful for defining the epileptic zone in a high proportion of children with focal cortical dysplasia who undergo surgical evaluation . in our patient , the high signal intensity lesion seen on t2-weighted mri image appeared when the patient had cluster seizures and diminished in size as her seizures improved , suggesting that these mri changes were related to seizure clustering . ictal spect detected hyperperfusion in the area containing the high signal intensity lesion depicted on mri , which was located very close to the right insular cortex . the ictal bradycardia experienced by our patient might have been caused by the following mechanism : repetitive seizure activity might have expanded from the hyperperfusion zone into the right insular cortex , which controls cardiac rhythm , resulting in ictal bradycardia . the autonomic alterations and heart rate changes seen during epilepsy are complex and are not fully understood . although the presence of hyperperfusion on ictal spect might represent zones of seizure propagation , there have not been any reports about the ictal spect findings of patients with ictal bradycardia . in our patient , ictal spect demonstrated hyperperfusion in an area that was close to the insular cortex but did not involve the insular cortex itself . subtraction ictal spect coregistered to mri analysis also failed to detect ictal hyperperfusion in the right insular cortex ( data not shown ) . one possible reason for this is that the patient s bradycardia was less severe at the time of the ictal spect study than it had been previously . although the seizures experienced by the patient after the injection of the radiotracer used for the spect produced similar symptoms and paroxysmal eeg discharges to the previous seizures , the bradycardia induced was mild ( the patient s heart rate only fell to 60 - 70 beats / min ) and did not lead to cardiac arrhythmia . thus , seizures involving more severe ictal bradycardia might have been found to involve the insular cortex on spect . in this study , ictal and interictal spect were performed during the patient s cluster seizures ( day 27 ) , when ictal bradycardia was observed , and on day 68 , when the ictal bradycardia had gone into remission . however , no ictal spect scans were performed after the patient s ictal bradycardia went into remission because she did not experience seizures often during the follow - up period . comparing the 2 ictal spect scans might have helped us to clarify the origin of the patient s ictal bradycardia and why her ictal bradycardia was transient . further studies with ictal spect are necessary to elucidate the pathophysiology of ictal bradycardia in patients with epilepsy , providing that clinical conditions allow it .

ictal bradycardia , which is considered to be one of the causes of sudden unexplained death in epilepsy , is rare . a 10-year - old girl with focal cortical dysplasia in her right centroparietal region developed transient ictal bradycardia during cluster seizures . brain magnetic resonance imaging demonstrated a high signal intensity lesion adjacent to the focal cortical dysplasia lesion . ictal 99mtc - ethyl cysteinate dimer single - photon emission computed tomography ( spect ) detected hyperperfusion in an area containing the high signal intensity lesion , which was located close to the insular cortex . since the hyperperfusion zone observed on spect was considered to reflect seizure propagation , it is possible that the ictal bradycardia experienced in the present case was caused by the following mechanism : the repetitive seizure activity caused the high - intensity lesion seen on mri to expand into the right insular cortex , which controls cardiac rhythm , resulting in ictal bradycardia .

stroke is rare in children , however , a common cause of neurological disease , and it is a major cause of death ranks in the top ten in pediatric period11,15 ) . documented incidence has been reported as 2.5 - 8/100000 children / year12,13 ) . medical treatment includes maintaining cerebral perfusion pressure with hyperventilation and osmotherapy , barbiturate coma and in case of thrombotic ischemia , thrombolytic procedure , and anticoagulanttherapy . however , if all medical treatment is fail to drop the intracranial pressure ( icp ) and deterioration of patient progressed , alternative treatments , such as surgical decompression could be mandatory . the common rationale of decompressive craniectomy with or without duroplasty is to let the volume expansion of the swelling brain to extracranial space via removed skull flap and prevent cerebral herniation and secondary damage of brain parenchyme . in case of traumatic brain injury , however , there has not been a definite evidence or standard guideline for decompressive craniectomy for pediatric patient with non - traumatic acute stroke with uncontrolled refractory high intracranial pressure . we will introduce our series about the functional and clinical outcome after decompressive craniectomy to control refractory high icp due to non - traumatic acute stroke in pediatric patient . between march 2004 and december 2006 , decompressive - hemicraniectomy and duroplasty was performed in 5 toddlers and preschool children with non - traumatic , malignant refractory high icp . any patient have no trauma history , and refractory high icp was diagnosed as clinical evidence of acute stroke , massive infarction or hemorrhage with midline shift , compression of basal cisterns in conventional radiologic examination such as computed tomography ( ct ) and magnetic resonance image ( mri ) and , neurological deterioration consisting of decreased level of consciousness or increasing levels of sedation , or somnolence or stupor compared with baseline status at admission17 ) . and all operations were performed by one pediatric neurosurgeon and all patients received treatment in pediatric intensive care unit of one medical center . in all patients , ct or mri blood sugar , body temperature , ventilation / oxygenation , blood volume and trans - cutaneous oxygen saturation , arterial blood pressure was continuously monitored and controlled in pediatric intensive care unit . clinical and neurologic status was evaluated with the glasgow coma scale ( gcs ) . to reduce icp , hyperosmolar therapy craniectomy involved the removal of a largebone flap ipsilateral to the involved hemisphere includingfrontal , parietal , and temporal bone . a large skin flap was lifted from the skull with meticulous blood control , multipleburr holes were made , large bone flap removed . in the temporalregion , the craniectomies were extended toward the floor of the middlefossa to maximize decompression . multiple openings in the dura were made ; a dural patchwas placed and sutured . in every case , intra - cerebral pressure monitor probe was positioned epidural space of bone margin . the bone flaps were stored in a bone bank and then , following improvement of clinical and neurologic status , cranioplasty was performed 3 to 6 months later . glasgow outcome scale ( gos)5 ) , and pediatric cerebral performance category scale ( pcpcs)4 ) ( table 2 ) calculated every 6 months after discharge . in all patients , ct or mri was obtained immediately , after stabilization of ventilation and hemodynamicsat first . blood sugar , body temperature , ventilation / oxygenation , blood volume and trans - cutaneous oxygen saturation , arterial blood pressure was continuously monitored and controlled in pediatric intensive care unit . clinical and neurologic status was evaluated with the glasgow coma scale ( gcs ) . to reduce icp , hyperosmolar therapy craniectomy involved the removal of a largebone flap ipsilateral to the involved hemisphere includingfrontal , parietal , and temporal bone . a large skin flap was lifted from the skull with meticulous blood control , multipleburr holes were made , large bone flap removed . in the temporalregion , the craniectomies were extended toward the floor of the middlefossa to maximize decompression . multiple openings in the dura were made ; a dural patchwas placed and sutured . in every case , intra - cerebral pressure monitor probe was positioned epidural space of bone margin . the bone flaps were stored in a bone bank and then , following improvement of clinical and neurologic status , cranioplasty was performed 3 to 6 months later . glasgow outcome scale ( gos)5 ) , and pediatric cerebral performance category scale ( pcpcs)4 ) ( table 2 ) calculated every 6 months after discharge . five pediatric patients were treated with decompressive hemicraniectomy with duroplasty for non - traumatic and refractory intracranial hypertension after unilateral hemispheric stroke . four patients were toddler and one patient was pre - school girl . three were boy and two were girl . four cases were caused by ischemic stroke , and another one case was hemorrhagic stroke . one case of ischemic stroke , patient had atrial septal defect ( asd ) . and including this patient three cases of ischemic stroke patients were taken angiography ; however there was no abnormal findings in angiography . in all patients , conventional laboratory result and special serum anti - body test , including anti - phospholipid antibody were within normal rage . at the time of operation , all patients had a gcs score < 8 ( median 7 , range 6 - 8 ) and all patients hadunilateral mydriasis . surgical decompressive hemicraniectomies were performed at a mean of 12 hours ( range 4 - 19 hours ) . in all cases we had performed supratentorial hemicraniectomies . the mean peak icp was 28.6 mm hg ( range 25 - 30 mm hg ) . and the icp of all patients were dropped to normal range within one and half days after operation . there were no surgical complications , such as cerebrospinal fluid leak or wound infection , even after cranioplasty . one of ischemic stroke patient had got a ventriculoperitoneal shunt operation because of post - stroke hydrocephalus . the mean follow - up period was 47.6 months ( 36 - 68 months).gos and pcpcs for 5 patients had been measured to evaluate postoperative neurological outcome . based on the regular follow up gos scores , 5 patients had shown satisfied recoveries : 4 had good recoveries ( gos score of 5 ) , and 1 had moderate disabilities ( gos score of 4 ) . the pcpcs scores also had revealed contend results : 4 patients received scores of 2 , and only one patient scored of 3 . a 17-month - old girl was transferred to our emergency department with decreased mentality . hemiplegia , hyper - reflexia , and babinski 's sign were observed on the left side . in general physical examination , she had an ejection systolic murmur . by pediatriccardiologist consultation , patient was diagnosis with asd . initial brain ct scan was suggestive of acute infarction and hemorrhagic transformation atright temporal area . and emergency mri sturdy was performed with mr angiography and diffusion - weighted image . but there was appropriate for acute cerebral infarction with hemorrhagic transformation with significant mid - line shift . after surgery , she was treated in pediatric intensive care unit with be kept intubated for 7 days . a brain ct at the time of post - operative 7 days revealed sustained brain swelling , but midline shift did not noted anymore . the icp dropped to normal gradually within one day after surgery . at the time of postoperative 3-month , she returned for an elective cranioplasty . the bone flap was secured without any complications . at the end of follow - up , 41 months after surgery , this patient maintained a pcpcs of 2 , and gos of 5 , and she had showed mild left side hemiparesis , mild behavioral disorder and strabismus . a 17-month - old girl was transferred to our emergency department with decreased mentality . neurological examination revealed a stuporous child without occasional spontaneous eye opening . hemiplegia , hyper - reflexia , and babinski 's sign were observed on the left side . in general physical examination , she had an ejection systolic murmur . by pediatriccardiologist consultation , patient was diagnosis with asd . initial brain ct scan was suggestive of acute infarction and hemorrhagic transformation atright temporal area . and but there was appropriate for acute cerebral infarction with hemorrhagic transformation with significant mid - line shift . after surgery , she was treated in pediatric intensive care unit with be kept intubated for 7 days . a brain ct at the time of post - operative 7 days revealed sustained brain swelling , but midline shift did not noted anymore . the icp dropped to normal gradually within one day after surgery . at the time of postoperative 3-month , she returned for an elective cranioplasty . the bone flap was secured without any complications . at the end of follow - up , 41 months after surgery , this patient maintained a pcpcs of 2 , and gos of 5 , and she had showed mild left side hemiparesis , mild behavioral disorder and strabismus . in stroke patient , most common cause of death is due to uncontrolled icp associated with large hemispheric infarctions10,12 ) . when it is present , it is associated with a dramatic increase in mortality . in addition , early icp elevation in patients with large hemispheric infarctions is highly concluded with high mortality16 ) . it means that the one of most important goal of acute phase stroke treatment is control the icp and prevent a secondary damage due to brain swelling . in spite of many clinical and experimental trials on medical therapeutic method , there has been a widely accepted definite guideline for oxygen saturation , temperature , serum glucose level , blood pressure for adult stroke patient1 ) . however , for pediatric stroke patient , even in many of mostly basic and elementary medical guideline has not been established yet14 ) . to prevent and control icp , thrombolytic therapy such as clot lysis has an accepted role in acute stroke treatment . in adult stroke patient group , there are antithrombotic management of adult ischemic stroke protocol and guideline published by the american heart association1 ) , such as intravenous tissue plasminogen activator , intra - arterial thrombolysis and oral administration of aspirin . but , for pediatric stroke patient , there is no guideline of this useful treatment method either14 ) . to the best of our knowledge , the first report about the benefits of decompressive hemicraniectomy in pediatric patient was described by carter et al.3 ) at 1997 . however , from this time , only a few report deals with decompressive craniectomy for pediatric patient were published . in adult acute stroke patient group , the effect of decompressive craniectomy also has been established . vahedi et al.21 ) reported that in a meta - analysis combining the data of 93 subjects from three small , randomized , controlled trials , mortality for conservative management in adult ischemic stroke patients with early brain edema was 71% as compared with 22% for decompressive surgical intervention . nowadays , some case reports and review has been published favorable outcome after decompressive surgery in childhood acute stroke2,17 ) . it is not often however in some article , decompressive craniotomy have been regarded as emerging procedure can save life , for pediatric stroke patient6,11 ) , even guideline for craniectomy was introduced11 ) . in point of fact widely accepted proper operation timing is immediately after signs of herniation and a midline shift which was defined as a more than equals 5-mm contralateral shift of the midline structures on the preoperative cranial ct scan on septum pellucidum level , compressed basal cisterns , or uncal herniation -- had to be present on cranial ct had been noted . recent studies in adult stroke group have shown that this procedure not only reduces mortality but also improves neurological functional outcome . this procedure may be life - saving if done early in cases of impending herniation , is associated with good outcomes in pediatric stroke patients9,11 ) . maybe if operation was performed earlier , it may minimize the vicious circle of brain swelling , increased icp , ischemia , and infarction12 ) . in our series , four toddlers and one pre - school girl with refractory high icp due to non - traumatic , ischemic and hemorrhagic stroke four patient received pcpcs score of 2 ( mild disability ) and only one received that of 3 ( moderate disability ) after 3 years later post operation . these results showed that decompressive craniectomy can not only play a rescuer therapeutic role but also guaranty acceptable clinical outcome in the treatment regimen in non - traumatic , refractory high icp in pediatric field . in some articles , decompressive aghakhani et al.2 ) report that decompressive craniectomy was performed in pediatric patient with malignant intracranial hypertension due to infectious encephalitis . in adult patient group , there had been not infrequently reported that malignant intracranial hypertension due to encephalitis treated with decompressive craniectomy . now , even in case of pediatric patient with infectious disease in central nerve system , decompressive craniectomy could be considered as life - saving - treatment options . gordon et al.7 ) report that pediatric stroke can affect not only patient itself , but also his or her whole family . over half of stroke survivors have suffer from decrease in quality of life and affects the entire family , and relates to both neurological deficits and psychosocial factors11 ) . hence , if all medical treatment is fail to drop the icp in non - traumatic pediatric acute stroke patient , without hesitation , a decompressive craniectomy could be regarded as next step treatment option . in this study , we describes the young pediatric patients have undergone decompressivehemicraniectomy for a non - traumatic acute hemispheric stroke . it also shows that decompressive hemicraniectomy can be lifesaving and can besafely performed in toddler and pre - school children . and young children can get a survival and relatively independence and family satisfaction.decompressive hemicraniectomy should be considered as an alternative therapy for patients with life - threatening brains welling refractory to medical management .

objectivelife - threatening hemispheric stroke is associated with a high mortality and morbidity . decompressive hemicraniectomy has been regarded as an effective treatment option for refractory intracranial hypertension . here , we reported the clinical course of 5 children with decompressive craniectomy and duroplasty after non - traumatic refractory intracranial hypertension.methodsfour toddlers and one preschool - girl were included in this study ; there were 3 boys and 2 girls with a mean age of 34.6 months ( range 17 - 80 ) . decompressive craniectomy including duroplasty was performed in cases of dilatation of pupil size after intensified standard medical therapy had proven insufficient . all children had a pediatric glasgow coma scale score < 8 at pre - operation state . the mean time - point of craniectomy after stroke attack was 12 hours ( range 4 - 19).resultsduring the long - term follow - up period ( mean 47.6 months ) , no children died . one year later , when we checked their glasgow outcome scale scores , only one toddler received a score of 4 ( moderate disability ) . but the others had good recoveries although they had minor physical or mental deficits . according to the pediatric cerebral performance category scale , 4 children received a score of 2 ( mild disability).conclusiondespite our small cases , we suggest that decompressive hemicraniectomy and duroplasty is an acceptable and life - saving treatment for refractory intracranial hypertension after unilateral hemispheric stroke in toddlers and preschool children .

bourgelatia diducta ralliet , henry and bauche , 1919 is a small - sized , milky white - colored nematode parasite with a well - developed mouth and bursa . this nematode is a member of the subfamily oesophagostominae , family chabertiidae , and order strongylida . the genus bourgelatia comprises a single species , b. diducta , which was first found in the cecum and colon of pigs in 1919 . the species was formerly classified as phacochoerostrongylus schwartz , but later was reclassified into the genus bourgelatia by differences in the morphology of buccal capsule , ovejecter , vagina length , and dorsal ray . thus far , little is known about the pathogenesis and clinical symptoms of this parasite to its host , sus scrofa . previous studies indicated that the infection of this worm is common among domestic pigs in india ( 21.6% ) , papua new guinea and the solomon islands ( 13% ) . although the parasite was found in a relatively high prevalence among wild boars in japan ( 95% ) , the parasite so far has not been reported in the republic of korea ( south korea ) . it has been reported that korean domestic pigs are infected with oesophagostomum detatum only without b. diducta in some surveys performed by fecal examination [ 9 - 12 ] . recently , the population of wild boars in south korea has gradually increased due to the absence of predators such as panthera tigris . therefore , the growing number of wild boars has been creating serious problems for human residential life and local agriculture industry . however , there has been little systematic approach to investigate the disease status among wild boars in south korea . the purpose of this study , therefore , was to investigate parasitic diseases of wild boars in south korea . in this study , we examined the gastrointestinal tract of 87 wild boars over a 4-year period in the southwestern part of south korea and found that b. diducta was quite prevalent among wild boars . together with light and scanning electron microscopic evidences , we provided the identification key and measurements of various parts of the worm . gastrointestinal tracts of 87 korean wild boars ( sus scrofa coreanus ) hunted in mountains of suncheon - si , gwangyang - si , and boseong - gun between 2009 and 2012 by members of local hunting associations were collected and examined for visceral helminths . the gastrointestinal tract of each wild boar was removed from the body and brought to the laboratory to examine the presence of parasites . the cecum and colon were slit open lengthwise and the mucosa and contents were examined carefully in a separate container . worms were preserved in 70% ethanol , and were mounted on a slide glass using polyvinyl alcohol mounting medium . measurements were made under a light microscope ( axioscop , zeiss , city , country ) on body dimensions , lengths of corona radiata , eosophagus , spicules , and gubernaculum , width of pharynx and other body parts of adult male and female worms . species identification of the worm was based on the description by yamaguti and lichtenfels . scanning electron microscopy was used to identify b. diducta as previously described by yadav and tandon . briefly , worms were washed 3 times with 0.85% pbs for 20 min , fixed in 2.5% glutaraldehyde in 0.1 m phosphate buffer ( ph 7.2 ) for 24 hr in a refrigerator at 4 and were post - fixed in 1% oso4 for 2 hr in a refrigerator at 4. after serial dehydration steps in 30 , 50 , 70 , 80 , 90 , and 100% ethanol for 20 min each , critical - point drying in a hitachi hcp-2 ( hitachi ltd , tokyo , japan ) and sputter - coating with gold - palladium in an emitech k550 ( emitech ltd , ashford , kent , england ) , digital photography of worms was taken using a hitachi s-2400 scanning electron microscope ( hitachi ltd , tokyo , japan ) . gastrointestinal tracts of 87 korean wild boars ( sus scrofa coreanus ) hunted in mountains of suncheon - si , gwangyang - si , and boseong - gun between 2009 and 2012 by members of local hunting associations were collected and examined for visceral helminths . the gastrointestinal tract of each wild boar was removed from the body and brought to the laboratory to examine the presence of parasites . the cecum and colon were slit open lengthwise and the mucosa and contents were examined carefully in a separate container . worms were preserved in 70% ethanol , and were mounted on a slide glass using polyvinyl alcohol mounting medium . measurements were made under a light microscope ( axioscop , zeiss , city , country ) on body dimensions , lengths of corona radiata , eosophagus , spicules , and gubernaculum , width of pharynx and other body parts of adult male and female worms . species identification of the worm was based on the description by yamaguti and lichtenfels . scanning electron microscopy was used to identify b. diducta as previously described by yadav and tandon . briefly , worms were washed 3 times with 0.85% pbs for 20 min , fixed in 2.5% glutaraldehyde in 0.1 m phosphate buffer ( ph 7.2 ) for 24 hr in a refrigerator at 4 and were post - fixed in 1% oso4 for 2 hr in a refrigerator at 4. after serial dehydration steps in 30 , 50 , 70 , 80 , 90 , and 100% ethanol for 20 min each , critical - point drying in a hitachi hcp-2 ( hitachi ltd , tokyo , japan ) and sputter - coating with gold - palladium in an emitech k550 ( emitech ltd , ashford , kent , england ) , digital photography of worms was taken using a hitachi s-2400 scanning electron microscope ( hitachi ltd , tokyo , japan ) . the average length of adult females was 11.30.87 mm , and the thickest part of the body measured 0.540.04 mm in maximum width , while those of males were 9.80.72 and 0.450.03 mm , respectively . . 1 g , h ) , and males had the type ii dorsal ray with 2 rami ( fig . the buccal capsule was small , relatively thin - walled , cylindrical , very short , and ring - shaped ( fig . 2c ) , the cervical groove that can be recognized in the genus oesophagostomum was absent ( fig . the ovijector in the inside of the posterior extremity of females consisted of 3 parts as a thick - walled vestibule that connected to the vagina , a paired thick - walled sphincters and a thinner - walled infundibula which was connected to the uteri ( fig . the anterior extremity was equipped with 20 - 22 external corona radiata , a well - developed leaf - crown structure surrounding the mouth , 4 cephalic papillae and 2 lateral amphids around the mouth ( fig . there was an oral collar which was much deeper than transverse ridges of the body cuticle , and an excretory pore and a pair of cervical papillae were also present ( fig . the vulva was shown as a prominent circular protrusion with a semicircular opening while the anus was shown as a rather flat semicircular hole ( fig . the tapering tail bore a pair of minute caudal papillae and a spike ( fig . the posterior extremity of males had a well - developed bursa that consist of a long dorsal and 2 slightly shorter lateral lobes ( fig . there were 2 conspicuous protrusions at the end of externodorsal and anterolateral rays on its surface ( fig . the genital cone was observed at the point of the spicules flowing in and out ( fig . 3f ) . from 87 korean wild boars captured in the southwestern area of south korea , 47 ( 54.0% ) were found to harbour b. diducta in the large intestine , mainly in the cecum . the worm was found in 5 of 13 wild boars ( 38.5% ) from suncheon - si , 38 of 70 ( 54.3 ) from gwangyang - si , and all 4 ( 100% ) from boseong - gun . no differences between the gender were recognized on the rate of infection ( table 1 ) . a total of 938 worms ( 551 males and 387 females ) of b. diducta was collected from the cecum and colon with an average number of 20.0 per animal and the infection intensity being the highest in animals from boseong - gun . the number of female worms present was 1.3 times more prevalent than the number of male worms ( table 2 ) . from 87 korean wild boars captured from mountains of the suncheon - si , gwangyang - si and boseong - gun , south korea spanning 2009 to 2012 , the infection of b. diducta , with an average number of 20 worms per animal was confirmed in 47 ( 54% ) wild boars . although this is the first report in south korea , for instance , the prevalence of b. diducta in wild boars or domestic pigs reported from japan , india , and the solomon islands was 95% , 21.6% , and 13.0% , respectively . these reports suggest that the parasite may be distributed over a rather wide area ranging from asia and oceania . up to now , however , there have been few reports on the intensity and pathogenesis of the infection with the parasite among pigs in the rest of the world . although we carefully observed the large intestine , pathologic changes in the mucosa was hardly recognized . in the infection with oesophagostomum detatum which belongs to the same subfamily oesophagostominae as bourgelatia , infective larvae exsheath in the small intestine and enter the mucosa of the large intestine causing small nodules . although the parasite is normally regarded as being only mildly pathogenic to pigs , the intestinal walls become edematous and strongly hyperemic in heavy burdens to develop into necrotic enteritis . as observed during the autopsy in this study , the infection of b. diducta did not elicit pathologic lesions in pigs at the population density of 20 worms per animal . there is a possibility that pathologic changes in the mucosa were not recognized due to the relatively low worm burden . also , it is possible that the worm is relatively non - pathogenic in wild boars . the infection of domestic pigs with the genus oesophagostomum spp . has been frequently reported in south korea and the prevalences of o. dentatum among domestic pigs based on fecal examination of eggs were 10.5% , 14.8% , 6.4% , and 2.5% . however , eggs of the genus oesophagostomum and bourgelatia are indistinguishable by size and appearance , and therefore it is possible that previous surveys based on fecal examination might have made mistakes stemming from correct identification of the genus . similarly , the prevalence of b. diducta ( 17% ) previously reported in pigs from nigeria by fecal examination might not be based on correct identification . therefore , the infection status of o. dentatum among domestic pigs in korea requires identification of adult worms for a correct faunistic record in korea . previous studies on the morphological features of b. diducta are remarkably consistent with those of our present study . the measurements of the worm body in this study were practically identical with the report by yamaguti ( table 4 ) . studies on sem structures by yadav on external corona radiata , oral collar , cephalic papilla , lateral amphid , bursa , genital cone , externodorsal and anterolateral rays , vulva , anus , and caudal papillae match structurally up with our findings . in other members of the subfamily oesophagostominae in pigs , morphological structures are varied in shape and size . the cervical groove is present in daubneyia leroux , 1940 and oesophagostomum molin , 1861 while the buccal capsule of phacochoerostrongylus schwartz , 1928 is twice as wide as deep . the bourgelatioides traguli differs from b. diducta in lacking the anterior leaf - crown and in other details of the mouth capsule , in the presence of a groove and overlying flaps in the cervical region , and in the presence of terminal convoluted filaments on the spicules . we provided some identification key aspects regarding the morphological features of the superfamily strongyloidea and the genus bourgelatia ( fig . the identification keys in this article are adapted from lichtenfels . conclusively , in the present study , b. diducta ( nematoda : chabertiidae ) is recorded for the first time in south korea . in addition , morphological characteristics and differential keys provided in the present study will be helpful in the faunistic and taxonomic studies of strongylid nematodes related .

this study describes the first record of bourgelatia diducta ( nematoda : chabertiidae ) from wild boars in the republic of korea ( = south korea ) . gastrointestinal tracts of 87 korean wild boars ( sus scrofa coreanus ) hunted in mountains in the south - western part of south korea between 2009 and 2012 were examined for their visceral helminths . b. diducta , as identified by morphological characteristics of the head and tail , were recovered from the large intestine of 47 ( 54% ) wild boars . the average length of adult female worms was 11.30.87 mm and the thickest part of the body measured 0.540.04 mm in maximum width , while those of males were 9.80.72 and 0.450.03 mm , respectively . the characteristic j - shaped type ii ovejector was observed in females , and the type ii dorsal ray with 2 rami on each side of the median fissure was uniquely seen in males . the buccal capsule was small , relatively thin - walled , cylindrical , very short , and ring - shaped . the externodorsal ray arose from a common stem with the dorsal ray . the cervical groove was absent . the anterior extremity was equipped with 20 - 22 external corona radiata , 4 cephalic papillae and 2 lateral amphids around the mouth . the eggs were 66.038.9 m in average size . by the present study , b. diducta ( nematoda : chabertiidae ) is recorded for the first time in south korea . additionally , morphological characteristics and identification keys provided in the present study will be helpful in the faunistic or taxonomic studies for strongylid nematodes related .

ant was developed by bruno latour , michel callon and john law in the field of science and technology studies during the 1980s . although it carries theory in its name , ant is better understood as a range of methods for conducting research which aims to describe the connections that link humans and non - humans ( for example , objects , technologies , policies and ideas ) . in particular , ant seeks to describe how these connections come to be formed , what holds them together and what they produce . researchers using ant are interested in connections between humans and non - humans because they subscribe to the notion that everything that exists in the world is the outcome of an interaction between two or more human and/or non - human entities . using ant means ascribing equal agency to people and things . while it is true that people use data and do things with them , it is equally true that data make people do things , i.e. they influence their work , they structure organizational practices , make organizations take certain decisions rather than others and produce particular effects and affects . this system of mutual influence between people and objects is what ant calls an actor - network . in other words , ant states that actors ( for example , nurses in charge of collecting patient experience data ) act in the way they do and are able to produce effects only through their interactions with other human and non - human entities ( for example , the technical devices used to collect data , the protocols that regulate their work , the chains of authority they are accountable to , the targets they need to meet ) . from this perspective , data are what latour has called actants , entities that are endowed with the potential to produce change in , and in turn to be transformed by , the course of action of other actors . using ant to approach patient experience data has the potential to make two interrelated contributions to existing debates . firstly , it emphasizes the performative nature of quality improvement , bringing to the fore the ways in which quality improvement emerges or fails to emerge as a result of a contingent series of interactions between various human ( individual , institutional ) and non - human actors ( bureaucratic documents , policies , technologies , targets , etc . ) . in the case of patient experience , exploring such performativity of data would mean moving beyond dominant perspectives which see data as inert , open to infinite technical refinement in the service of quality improvement . rather , it would require recognizing that data collected by health care providers produce effects as a result of specific series of interactions with other actors , e.g. more or less competent hospital staff , sympathetic or indifferent policy makers , efficient or faulty technological devices used for their collection and analysis , etc . thus , it may be the case that the interaction of data with a particular presentational and analytical technology ( e.g. meridian or powerpoint ) , makes data more or less compelling to nurse managers , enabling or hampering its journeys to other hospital documents and meetings . secondly , tracing the movement of patient experience data through a health care organization by focusing on its performativity may also lead to important insights into the structure of an organization . as data travel , and translate into reports , narratives and interventions , they make and reveal alternative organizational relations to those which are officially recognized . for example , whereas hospitals are formally hierarchical institutions with a wide range of fixed roles and responsibilities , the contingent interactions in which data get embedded may reveal alternative decision - making processes , and may bring to the fore the role of certain actors ( such as health care assistants or receptionists ) who are conventionally marginal , but who nevertheless often come to play an unexpectedly central role in ensuring the quality of care . a flattened perspective such as this , which treats actors as equally important regardless of their assumed place in an institution , is key to more faithfully account for how quality improvement emerges in practice . by moving away from taken - for - granted organizational and institutional structures through which data are supposed to be gathered , analysed and deployed , such an approach requires that better attention is paid to alternative organizational arrangements as well as to forms of agency which would otherwise go undetected , including non - human agency . thus , in addition to allowing for the role unexpectedly played by certain people within hospitals , the flatness promoted by ant also requires us to pay attention to how quality improvement can be produced or hampered by the agency of specific non - human actors . this would , for instance , mean examining the unorthodox use of certain technologies , or of types of knowledge that are not usually labelled data. casting a panoramic view over the vast web of relations that practically shape quality improvement is essential to move forward the current debates on patient experience data and their role in health care . as mentioned at the beginning of the paper , it is widely recognized that a discrepancy exists between the proliferation of forms of data collection and the limited ways in which such data are used to inform quality improvement . with its flat approach to the mutually influencing relations between actors , ant holds out the promise of bringing to the surface the processes of interaction and negotiation between actors ; it keeps the messy , everyday mechanics of improvement centre stage . exploring these processes is key to helping organizations learn what data are and how they can be best put to use . if we understand data as not only the product of health care organizational structures but also as involved in creating and sustaining them , we may better show how such data shape quality improvement activities . recognizing this potentially creative character of data may offer alternative paths to improvements in patient care which would otherwise go undetected . the author(s ) declared no potential conflicts of interest with respect to the research , authorship , and/or publication of this article . the views and opinions expressed therein are those of the authors and do not necessarily reflect those of the health services and delivery research programme , nihr , nhs or the department of health . the author(s ) disclosed receipt of the following financial support for the research , authorship , and/or publication of this article : this work was funded by the national institute for health research health services and delivery research programme ( hs&dr 14/156/08 ) .

hospitals are awash with patient experience data , much of it collected with the ostensible purpose of improving the quality of patient care . however , there has been comparatively little consideration of the nature and capacities of data itself . using insights from actor - network theory , we propose that paying attention to patient experience data as having agency in particular hospital interactions allows us to better trace how and in what circumstances data lead ( or fail to lead ) to quality improvement .

the matricellular proteins are a heterogeneous group of extracellular matrix ( ecm ) proteins that interacts with either other ecm proteins and with cell - surface receptors , growth factors and cytokines . the periostin , also called osteoblast - specific factor 2 ( osf-2 ) , is a non - structural matricellular protein that directly interacts with type i collagen and fibronectin regulating the biomechanical properties of connective tissues . it is an 811 amino acid , 93 kda secreted ecm protein and four main isoforms have been identified which are not uniformly but differentially expressed in various cell lines ; a human periodontal ligament specific isoform has been recently characterized . periostin is present in collagen - rich connective tissues like bone , skeletal muscle , tendons , joint ligaments , periodontal ligament , heart valves , adipose tissue , or skin . at present the functions of periostin in several biomedical areas like osteology , oncology , cardiovascular and respiratory systems , inflammatory diseases or dentistry , in both normal and pathological conditions , are rather well known . as a whole its main functions seem to be during the development and tissue repair , because the predominant role in mesenchymal remode ling . a series of recent studies have detected expression of periostin in developing and mature dental tissues , alveolar bone or periodontal ligament . consistently , mice deficient in periostin display changes in ecm of dental tissues as well as defects in mineralized tissues . by contrast little information is available about the occurrence and distribution of periostin in the gingiva . it is known that gingival fibroblasts may be a source of periostin in response to some cytokines which are elevated in periodontitis , and decreased levels of periostin were found in gingival crevicular fluid proportionally with the progression and severity of periodontal disease . moreover , periostin is involved in signaling pathways of drug - induced gingival over - growth . since no data are available about the distribution of periostin in adult human gingival in normal conditions , and because its potential role in gingival pathologies , we used western blot and immunohistochemistry to analyze the occurrence and localization of periostin in these tissues . to establish whether periostin is cellular or extracellular we labeled in parallel keratinocytes and dermal fibroblasts samples of gingival tissues ( sized 2x2x2 mm , approximately ) were surgically excised from healthy patients undergoing orthodontic treatment from zones not exposed to orthodontic forces , and were obtained from the instituto asturiano de odontologia . the age range was 16 to 32 years , and were males ( n=8 ) and females ( n=4 ) . the tissue samples were washed with tap water followed with cold saline , then fixed in buffered 10% formalin for 24 h , and routinely processed for paraffin embedding . the pieces were cut 10 m thick and the sections mounted on gelatine - coated microscope slides . moreover , fresh samples ( n=6 from different subject ) were quickly frozen , stored at -80c and used for western blot . this study was approved by the ethics committee of instituto asturiano de odontologia ( oviedo , spain ) and informed consent was obtained from each subject . lysates prepared from gingival homogenates were processed as follows : representative samples of the free gingival , attached gingival and non - bone attached periodontal ligament were pooled and homogenized ( 1:2 , w / v ) in tris - hcl buffered saline ( tbs , 0.1 m , ph 7.5 ) containing 1 m leupeptin , 10 m pepstatin and 2 mm phenylmethylsulfonyl fluoride . the homogenates were centrifuged at 25,000 rpm for 15 min at 4c and the resulting pellet dissolved in tris hcl 10 mm ph 6.8 , 2% sds , 100 mm dithiothreitol , and 10% glycerol at 4c . after electrophoresis , proteins were transferred to a nitrocellulose membrane and antibody non - specific binding was blocked by immersion for 3 h in pbs containing 5% dry milk , and 0.1% tween-20 . the membranes were then incubated at 4c for 2h with a rabbit polyclonal antibody against a peptide from fasciclin domain 1 of mouse periostin ( ls - bl10443 , lifespan biosciences , inc . , after incubation , the membranes were washed with tbs ph 7.6 containing 20% tween-20 , and incubated again for 1 h with goat anti - rabbit igg ( diluted 1:100 ) at room temperature . membranes were washed again and incubated with the pap complex diluted 1:100 for 1 h at room temperature . finally , the reaction was developed using a chemiluminescent reagent ( ecl , amersham pharmacia biotech , buckinghamshire , uk ) and exposed to hyperfilm . relative values of periostin were estimated by imagej quantification in the three different segments of the gingiva . deparaffinized and rehydrated sections were processed for immunohistochemical detection of periostin using the envision antibody complex kit ( dako , copenhagen , denmark ) following the manufacturer s recommendations . moreover , anti - vimentin ( clone 334 , boehringer - mannheim , mannheim , germany ) , anti - pancytokeratin ( clone pck-26 , sigma - aldrich quimica , sl , madrid , spain ) mouse monoclonal antibodies , and anti - collagen type i rabbit monoclonal antibody ( erp778 , abcam , cambridge , uk ) were used to label dermal fibroblasts , epithelial cells and type i collagen fibrils , respectively . the sections were processed for simultaneous detection of periostin and vimentin , and periostin and pan - cytokeratin , as follows : the non - specific binding was reduced by incubation for 30 min with a solution of 1% bovine serum albumin in tbs . the sections were then incubated overnight , at 4c in a humid chamber with a 1:1 mixture of anti - periostin and anti - vimentin antibodies ( both diluted 1:100 in the blocking solution ) ; or anti - periostin and anti - pancytokeratin antibodies ( diluted 1:200 and 1:100 , respectively , in the blocking solution ) ; or anti - vimentin and anti - type i collagen ( both diluted 1:100 in the blocking solution ) . after rinsing with tbs , the sections were incubated for 1 hour with alexa fluor 488-conjugated goat anti - rabbit igg ( serotec , oxford , uk ) , diluted 1:1000 in tbs containing 5% mouse serum ( serotec ) , then rinsed again , and incubated for another hour with cy3-conjugated donkey anti - mouse antibody ( jackson - immunoresearch , baltimore , md , usa ) diluted 1:50 in tbs . both steps were performed at room temperature in a dark humid chamber . finally , to ascertain structural details sections were counterstained and mounted with dapi diluted in glycerol medium ( 10 ng / ml ) . triple fluorescence was detected using a leica dmr - xa automatic fluorescence microscope ( photonic microscopy service , university of oviedo ) coupled with a leica confocal software , ver . 2.5 ( leica microsystems gmbh , heidelberg , germany ) and the images captured were processed using the software image j version 1.43 g master biophotonics facility , mac master university ontario ( www.macbiophotonics.ca ) . for control purposes representative sections were processed in the same way as described above using non - immune rabbit or mouse sera instead of the primary antibodies , or omitting the primary antibodies in the incubation . under these conditions samples of gingival tissues ( sized 2x2x2 mm , approximately ) were surgically excised from healthy patients undergoing orthodontic treatment from zones not exposed to orthodontic forces , and were obtained from the instituto asturiano de odontologia . the age range was 16 to 32 years , and were males ( n=8 ) and females ( n=4 ) . the tissue samples were washed with tap water followed with cold saline , then fixed in buffered 10% formalin for 24 h , and routinely processed for paraffin embedding . the pieces were cut 10 m thick and the sections mounted on gelatine - coated microscope slides . moreover , fresh samples ( n=6 from different subject ) were quickly frozen , stored at -80c and used for western blot . this study was approved by the ethics committee of instituto asturiano de odontologia ( oviedo , spain ) and informed consent was obtained from each subject . lysates prepared from gingival homogenates were processed as follows : representative samples of the free gingival , attached gingival and non - bone attached periodontal ligament were pooled and homogenized ( 1:2 , w / v ) in tris - hcl buffered saline ( tbs , 0.1 m , ph 7.5 ) containing 1 m leupeptin , 10 m pepstatin and 2 mm phenylmethylsulfonyl fluoride . the homogenates were centrifuged at 25,000 rpm for 15 min at 4c and the resulting pellet dissolved in tris hcl 10 mm ph 6.8 , 2% sds , 100 mm dithiothreitol , and 10% glycerol at 4c . after electrophoresis , proteins were transferred to a nitrocellulose membrane and antibody non - specific binding was blocked by immersion for 3 h in pbs containing 5% dry milk , and 0.1% tween-20 . the membranes were then incubated at 4c for 2h with a rabbit polyclonal antibody against a peptide from fasciclin domain 1 of mouse periostin ( ls - bl10443 , lifespan biosciences , inc . , seattle , wa , usa ) , used diluted 1:200 . after incubation , the membranes were washed with tbs ph 7.6 containing 20% tween-20 , and incubated again for 1 h with goat anti - rabbit igg ( diluted 1:100 ) at room temperature . membranes were washed again and incubated with the pap complex diluted 1:100 for 1 h at room temperature . finally , the reaction was developed using a chemiluminescent reagent ( ecl , amersham pharmacia biotech , buckinghamshire , uk ) and exposed to hyperfilm . relative values of periostin were estimated by imagej quantification in the three different segments of the gingiva . deparaffinized and rehydrated sections were processed for immunohistochemical detection of periostin using the envision antibody complex kit ( dako , copenhagen , denmark ) following the manufacturer s recommendations . the anti - periostin antibody was the same described above . moreover , anti - vimentin ( clone 334 , boehringer - mannheim , mannheim , germany ) , anti - pancytokeratin ( clone pck-26 , sigma - aldrich quimica , sl , madrid , spain ) mouse monoclonal antibodies , and anti - collagen type i rabbit monoclonal antibody ( erp778 , abcam , cambridge , uk ) were used to label dermal fibroblasts , epithelial cells and type i collagen fibrils , respectively . the sections were processed for simultaneous detection of periostin and vimentin , and periostin and pan - cytokeratin , as follows : the non - specific binding was reduced by incubation for 30 min with a solution of 1% bovine serum albumin in tbs . the sections were then incubated overnight , at 4c in a humid chamber with a 1:1 mixture of anti - periostin and anti - vimentin antibodies ( both diluted 1:100 in the blocking solution ) ; or anti - periostin and anti - pancytokeratin antibodies ( diluted 1:200 and 1:100 , respectively , in the blocking solution ) ; or anti - vimentin and anti - type i collagen ( both diluted 1:100 in the blocking solution ) . after rinsing with tbs , the sections were incubated for 1 hour with alexa fluor 488-conjugated goat anti - rabbit igg ( serotec , oxford , uk ) , diluted 1:1000 in tbs containing 5% mouse serum ( serotec ) , then rinsed again , and incubated for another hour with cy3-conjugated donkey anti - mouse antibody ( jackson - immunoresearch , baltimore , md , usa ) diluted 1:50 in tbs . finally , to ascertain structural details sections were counterstained and mounted with dapi diluted in glycerol medium ( 10 ng / ml ) . triple fluorescence was detected using a leica dmr - xa automatic fluorescence microscope ( photonic microscopy service , university of oviedo ) coupled with a leica confocal software , ver . 2.5 ( leica microsystems gmbh , heidelberg , germany ) and the images captured were processed using the software image j version 1.43 g master biophotonics facility , mac master university ontario ( www.macbiophotonics.ca ) . for control purposes representative sections were processed in the same way as described above using non - immune rabbit or mouse sera instead of the primary antibodies , or omitting the primary antibodies in the incubation . under these conditions the expression of periostin at the protein level in the human gingiva was analyzed using western blot and immunohistochemistry . in gingival homogenates western blot associated to the anti - periostin antibody used throughout this study detected a single protein band with an estimated molecular weight of 94 kda ( figure 1a ) which is consistent with than expected for the human periostin . imagej densitometric analysis demonstrated that the higher relative levels of periostin with respect to -actin corresponded to the non - bone attached periodontal ligament segment , followed by free gingiva and attached gingiva ( figure 1b ) . to map the histological distribution of periostin in the human gingiva we used double immunofluorescence coupled with laser - confocal microscopy in three different sectors : the free gingiva , the attached gingiva , and the segment of connective tissue related to the junctional epithelium ( which is especially rich in fibers of the periodontal ligament ) . the results were compared with the pattern of localization of cytokeratin and vimentin . as a rule periostin was never localized within the cells but always in the extracellular space . in the sections processed for the simultaneous demonstration of periostin plus cytokeratin ( figure 1 c , d , g , h ) it was observed that periostin - immunoreactivity is concentrated at the epithelium - connective tissue junction , presumably associated to ecm proteins of the basal membrane and was never found within the cytoplasm of the basal epithelial cells . on the other hand , the distribution of the immunoreactivity for periostin and type i collagen were similar at the epithelium - connective tissue junction ( figure 1 e , f ) although a faint widespread immunostaining for type i collagen was also detected in the connective tissue . double immunofluorescence was also carried out for periostin and vimentin in order to determine the relation of periostin with the connective tissue fibroblasts . independently of the gingival segment analyzed , free ( figure 2 a - c ) or attached gingival ( figure 2 d - f ) , periostin immunoreactivity was dissociated from the vimentin positive fibroblast and was restricted to the epithelial - connective tissue junction . regarding to the gingival segment containing the non - bone attached periodontal ligament , disposed under the junctional epithelium , the distribution of the periostin immunoreactivity was irregular , and never co - localized with the vimentin - positive fibroblasts ( figure 2 g - i ) . although some images occasionally might suggest co - localization of periositin witn vimentin or cytokeratin 2d cytofluorograms from the two detection channels of periostin ( green ) and cytokeratin or vimentin ( red ) demonstrated absence of co - localization ( figure 2 j - l ) . as a summary , in human normal gingiva periostin is extracellular and is restricted to the epithelial - connective tissue junction , and among the fibroblasts forming the non - bone attached segments of the periodontal ligament . this study was designed to investigate the occurrence and distribution of the segregated matricellular protein periostin in the human gingiva . the localization of periostin in the dental tissues and the periodontal ligament in now rather well known , but no information is available about its distribution in the gingiva in spite of its potential role in the pathophysiology of some gingival disorders . the protein we have identified by western blot has a molecular weight of 94 kda . this is in good agreement with the expected molecular mass of this protein ( 93.3 kda ) , and was similar to that found in other human epithelial tissues like the cornea . the small differences between our results and others could be related to occurrence of tissue specific periostin isoforms , or post - translational processes of the protein . based on image densitometry the relative level of periostin varies among different segments of the gingiva . the reason for these differences might be related to the density of cells producing periostin , and the functional significance of these findings if any remain to be elucidated . we have regularly observed that periostin in normal gingiva is restricted to the epithelium - connective tissue junction , and is also present around the fibers of the non - bone attached portion of the periodontal ligament . as far as we know this is the first study reporting the distribution of periostin in the human gingiva . in agreement with the identification of periostin as a matricellular protein we observed that it is localized extracellularly nevertheless , other studies on epithelial tissues reported the presence of periostin immunoreactivity in the basal keratinocytes , basal lamina , and dermal fibroblasts in healthy human skin , or in the basal layer cells of the human corneal epithelium . according to those authors periostin only becomes extracellular during tissue remodeling of wound repair . since in normal gingiva periostin is exclusively extracellular , its function may be distinct from that of skin , and the presence of intracellular periostin probably reflects non - secreted protein . the role of periostin in the interactions of the connective tissue and the epithelial cells has been extensively studied , however it remains still unknown in the gingiva . in the skin periostin plays pivotal functions in collagen fibrillogenesis , collagen cross - linking , and the formation of ecm meshwork via interactions with other ecm components . whether or not it is the same for the gingiva remains to be investigated . in the skin periostin expression this is indicative of the physiologically protective functions of periostin , which promotes wound repair producing myofibroblast differentiation , keratinocytes proliferation and fibroblast proliferation and migration . the localization of periostin in the gingiva and the since it is an ecm secreted protein suggest it could signal in both epithelial cells . matricellular proteins appear to be of importance in collagen assembly , and the expression of periostin in the epithelial - connective tissue junction suggest it may influence the biology of the basal membrane , including collagen fibrillogenesis ( especially type i collagen which co - localizes ) but probably its presence may be also related to cell migration , proliferation and adhesion of fibroblasts . on the other hand , the free and sub - junctional epithelium segments of the gingival contain fibers of the periodontal ligament subject to mechanical stress which in turn may activate latent tgf- 1 and to increase periostin mrna in periodontal ligament fibroblasts . therefore , periostin may participate in the maintenance of the structure and reparative processes in adult human gingival as it occurs in the skin , although in these tissues have different localization , extracellular vs intracellular . as a summary , our study has demonstrated the localization of periostin in the ecm of the periodontal ligament and the cell - free zone of the dental pulp in adult human teeth .

the periostin is a matricellular protein expressed in collagen - rich tissues including some dental and periodontal tissues where it is regulated by mechanical forces , growth factors and cytokines . interestingly the expression of this protein has been found modified in different gingival pathologies although the expression of periostin in normal human gingiva was never investigated . here we used western blot and double immunofluorescence coupled to laser - confocal microscopy to investigated the occurrence and distribution of periostin in different segments of the human gingival in healthy subjects . by western blot a protein band with an estimated molecular mass of 94 kda was observed . periostin was localized at the epithelial - connective tissue junction , or among the fibers of the periodontal ligament , and never co - localized with cytokeratin or vimentin thus suggesting it is an extracellular protein . these results demonstrate the occurrence of periostin in adult human gingiva ; its localization suggests a role in the bidirectional interactions between the connective tissue and the epithelial cells , and therefore in the physiopathological conditions in which these interactions are altered .

we report a case of iga pemphigus with iga antibodies to desmoglein 1 ( dsg1 ) and desmoglein 3 ( dsg3 ) . we report the case of an 60-year - old man with intraepidermal neutrophilic iga pemphigus with iga antibodies to dsg1 and dsg3 . the disease was not effectively controlled by conventional therapeutic regimens ( colchicine , dapsone ) . systemic treatment with isotretinoin 25 mg / d and prednisone 20 mg / d achieved only a slight effect after six months . our case confirmed the recalcitrant nature of iga pemphigus in response to distinct therapies , indicating that further research focusing on therapeutic approaches for this type of pemphigus is needed . an otherwise healthy 60-year - old man was referred to our department with a one - year history of recurrent pruritic vesiculo - pustular lesions on both axillae and groin . the lesions improved after topical application of steroids but reappeared and gradually spread to trunk and extremities . examination revealed a symmetric eruption on the proximal extremities and trunk with prominent involvement of the axillae and groin . it was composed of grouped vesiculo - pustular lesions mostly on well - circumscribed erythematous patches ( figure 1 ) . direct immunofluorescence microscopy ( dif ) of perilesional skin detected intercellular deposits of iga throughout the epidermis ( figure 3 ) . igg - elisa for desmogleins ( dsg ) showed no igg antibodies to either dsg1 or dsg3 . iga - elisa for dsgs was then performed and the results indicated that the patient s serum was positive for iga anti - dsg1 and anti - dsg3 antibodies . colchicine , 0.5 mg 3 times daily was begun , but this had no effect after one month so it was substituted by dapsone 100 mg / d , which also failed to produce any improvement and his disease remained active . subsequently , isotretinoin 25 mg / d and prednisone 20 mg / d were initiated achieving only a slight effect after six months . based on pathology and dif findings , iga pemphigus can be further divided into two subtypes , namely , subcorneal pustular dermatosis ( spd ) and intraepidermal neutrophilic dermatosis ( ien ) types . clinically , as seen in our patient , both subtypes of iga pemphigus present with small blisters and pustules overlaying well - circumscribed erythemas . a herpetiform appearance has also been reported . the whole body can be involved , with a predilection for flexures , such as axilla , groin and submammary area . while spd - type iga pemphigus shows subcorneal pustules , the ien type is characterized by pustule formation throughout the entire epidermis . in dif , spd - type iga pemphigus involves cell surface iga binding only in the upper epidermis , where as ien - type iga pemphigus shows binding throughout the epidermis . although the histological features of our case are consistent with those of the spd type of iga pemphigus , ien - type iga pemphigus was diagnosed in the present patient because of iga deposits throughout the epidermis . in contrast , in the ien type no reactivity of auto antibodies with desmocollin 1 , 2 , and 3 has been found , whereas desmoglein 1 and 3 were suggested as putative target antigens of ien type in single case reports . the result of immunoelectron microscopic study revealed that the antigen of ien type may not be a desmosomal component . in our patient , iga - elisa for dsgs showed reactivity with dsg1 and dsg3 , suggesting that his pemphigus most likely belongs to the ien type . iep should be performed because iga pemphigus has been reported to be associated with monoclonal iga gammapathy . treatments of iga pemphigus are performed based on the disease pathomechanism and on anecdotal reports . dapsone is commonly the drug of choice due to its effect in suppressing neutrophilic infiltration . recently , adalimumab and mycophenolate mofetil , have also been reported to be useful in treating iga pemphigus . our case confirmed the recalcitrant nature of iga pemphigus in response to distinct therapies , indicating that further research focusing on therapeutic approaches for this type of pemphigus is needed .

backgroundiga pemphigus is a rare autoimmune vesiculo - pustular skin disease . only approximately 70 cases have been reported to date . we report a case of iga pemphigus with iga antibodies to desmoglein 1 ( dsg1 ) and desmoglein 3 ( dsg3).case reportwe report the case of an 60-year - old man with intraepidermal neutrophilic iga pemphigus with iga antibodies to dsg1 and dsg3 . histologic examination revealed subcorneal neutrophilic pustules with few acantholytic cells . the disease was not effectively controlled by conventional therapeutic regimens ( colchicine , dapsone ) . systemic treatment with isotretinoin 25 mg / d and prednisone 20 mg / d achieved only a slight effect after six months.conclusionsour case confirmed the recalcitrant nature of iga pemphigus in response to distinct therapies , indicating that further research focusing on therapeutic approaches for this type of pemphigus is needed . physicians should keep iga pemphigus in mind when approaching patients with bullous eruption .

segmental copy - number variations ( cnvs ) , involving the gain or loss of several hundreds of bases to several hundred kilobases ( kb ) of the genome , can be an important source of genetic variation among human populations of different ethnic groups as well as among individuals . molecular genetics analyses and cytogenetic analyses have provided significant information about these variations in the human genome , specifically as they relate to disease , such as cancer , and to congenital malformation ( see [ 1 , 2 ] , and review in ) . following the development of methodologies and the introduction of new research platforms [ 49 ] , information regarding the nature and pattern of cnvs from representative populations have accumulated . examinations of a relatively large number of individuals from various specific ethnic groups have recently been conducted using different array platforms , such as bac - arrays [ 1013 ] , oligo - arrays [ 1416 ] , and others . the results are not always consistent and it is likely that different human populations bear different cnvs . the numbers of japanese individuals examined to date are not so large compared to the studies for other ethnicities . polymorphic cnvs have received considerable attention since they might play an important role in the etiology of common diseases . therefore , more data regarding cnvs should be accumulated from japanese populations . in this report , we focus on cnvs which were observed at a high frequency ( 5.0% of the individuals ) in the population residing in hiroshima and nagasaki , japan by acgh with bac - clones as targets . in the study , the population studies were conducted at two stages : stage ( 1 ) : 80 unrelated japanese individuals were examined using bac - acgh with an array having 2,241 bac clones , and stage ( 2 ) : 133 unrelated japanese individuals were examined using bac - acgh that contained 2,622 bac - clones . the majority of the clones used in stage ( 1 ) of this study were selected from the set of cytogenetically mapped p1-artificial chromosome ( pac ) clones and bacterial artificial chromosome ( bac ) clones reported by the bac resource consortium and obtained from either the children 's hospital oakland research institute ( oakland , ca , usa ) or from invitrogen inc . , co. ( carlsbad , ca , usa ) . in stage ( 2 ) , in addition to bac clones used in stage ( 1 ) , an additional 381 bac clones were used , a majority of which were collaboratively obtained from dr . n. matsumoto of yokohama city university . the 2,241 clones of chromosomal fragments from chromosome 1 to chromosome 22 were used in stage ( 1 ) and 2,622 clones were used in stage ( 2 ) , respectively . that is , the additional 381 bac clones were examined for only 133 unrelated individuals in stage ( 2 ) . those clones are distributed every 1.2 mb across all of the human autosomes in stage ( 1 ) , and 1.1 mb in stage ( 2 ) , respectively . in addition to autosomal clones , four kinds of x - chromosomal clones were used as internal references . with respect to examination for stage ( 1 ) , three sets of arrays were constructed and imprinted : slide no.1 , consisted of 698 clones on chromosomes 1 to 4 ; slide no.2 consisted of 718 clones on chromosomes 5 to 10 , plus two bac clones on chromosome 3 , and six clones on chromosome 4 ; and slide no.3 consisted of 817 clones on chromosomes 11 to 22 . for stage ( 2 ) , all of the clones were printed onto one glass slid . the genomic dna samples used in this study were principally the same as those used in a previous study . the dna samples used for reference purposes were extracted from mononuclear cells of two physically and clinically normal volunteers ( a 57-year - old japanese male and a 54-year - old japanese female ) . the dna used for testing and analyses of this population was extracted from lymphoblastoid cell lines obtained from the offspring of atomic - bomb survivors . high molecular weight genomic dna was isolated using conventional methods as described in detail elsewhere . lymphoblastoid cell lines were derived from a cryopreserved archive of approximately 1000 families consisting of father , mother , and offspring from hiroshima and nagasaki for whom permanent cell lines have been established by epstein - barr ( eb ) virus transformation of peripheral b - lymphocytes . the composition of the families has been reported elsewhere . three hundred five offspring were initially screened . since the offspring include some siblings , we selected one representative offspring to construct unrelated individuals to avoid double counting of polymorphic cnvs from families containing two or more siblings . we selected the offspring who first visited our institution for donating blood rather than other siblings . the 213 offspring selected as unrelated individuals included 124 offspring from hiroshima and 89 from nagasaki . cloned dnas for microarray targets were isolated from bacterial cultures using nucleobond bac 100 ( nippon genetics , tokyo ) . with respect to stage ( 1 ) , dna was digested by noti followed by phenol - chloroform - isoamyl alcohol ( 25:24:1 ) extraction and ethanol - precipitation . on the other hand , in stage ( 2 ) , cloned dna was digested with msei followed by phenol - chloroform - isoamyl alcohol ( 25:24:1 ) extraction and ethanol precipitation . the fragmented dnas were amplified by ligation - mediated pcr carried out as described by snijders et al . . the target dnas ( 0.5 g/l ) were dissolved in 50%-dimethylsuloxide and printed in triplicate onto the glass slides ( matsunami glass co. ltd . ) using the affymetrix 417 arrayer ( affymetrix ) . the screenings of both stages were conducted following the procedures described previously . in brief , for labeling dna , test and reference genomic dna ( 1.25 g each ) was cut by bamhi , and labeled by a random priming method with cyanine-5- and cyanine-3-labeled dutp ( cy5- and cy3-dutp ; perkinelmer life sciences , wellesley , ma , usa ) . the labeled probes were mixed and centrifuged with microcon column ( millipore co. , bedford , ma , usa ) to purify the probes . subsequently , human coti dna ( 120 g ; roche diagnostic gmbh , mannheim , germany ) was added to the column , and recentrifuged . after the volume of the mixture became less than 20 l , it was transferred to microtubes with 100 l of hybridization solution ( 50% formamide , 10% dextran sulfate , 1% tween 20 , 2 ssc , 10 mm tris - hcl [ ph 7.4 ] and 800 g of yeast t - rna [ invitrogen , carlsbad , ca , usa ] ) . the hybridization mixture was then denatured at 70c for 10 minutes , and subsequently incubated at 37c for at least five hours to block repetitive sequences of the labeled probes . prehybridization was conducted in order to block repetitive sequence binding of target dna on the arrays , and to prevent nonspecific binding of probe dna to the targets . following the initial incubation ( overnight at 37c ) , the prehybridization solution was removed , and fresh hybridization solution with cy - labeled dna ( prepared as described above ) was added . again , hybridization processes were carried out . all of the procedures were conducted using the genetac hybridization station ( genomic solutions inc . , ann arbor , mi , usa ) . fluorescent images of the hybridized arrays were obtained using a scanarray 5000 confocal laser scanner ( perkinelmer life sciences ) . fairfax , va , usa ) in stage ( 1 ) and gene pix ( axon instruments , sunnyvale , ca ) in stage ( 2 ) , respectively , were used to quantify the fluorescence of each spot on the array images . fluorescent ratios of the total integrated cy3 and cy5 intensities for each target ( triplicate spots for each target ) were calculated , along with the mean ratios of the triplicate spots ( if the raw ratio of one of the triplicate spots differed by more than 10% from the other two , the value was automatically excluded from the mean ratio calculation ) . normalized ratios were computed by dividing each raw ratio by the mean raw ratios of every autosomally mapped target ( therefore , after normalization , the averaged ratio for all targets in one array was 1.0 ) . the spots whose ratios were 2.58 standard deviations ( sd ) below the mean ( 1.0 ) , or 2.58 sd above the mean , were marked cnv . to confirm the quality of each analysis , the ratio of x - linked clones was verified . dna in the plugs was cleaved by restriction enzymes ( paci , or sse8387i ) . the resulting fragments were then separated by pulse field gel electrophoreses ( pfge ) on 1% pulse field certified agarose ( biorad , hercules , ca , usa ) with 0.5 tbe ( tris - borate - ethylenediaminetetraacetic acid ) . using the chef - dr ii system ( biorad ) , electrophoresis was carried out using 6 v / cm at 14c for 22 hrs . the angle of pulse was 120. the switch time was used : ramped from 0.3 seconds to 15 seconds . southern blot analyses were carried out using conventional , well - described procedures . in brief , after completion of pfge , the dna in the gel was cleaved by uv irradiation and blotted onto nitrocellulose filters ( schleiche & schuell , dassel , germany ) . the filters were prehybridized with human coti dna ( 48 g / ml , roche diagnostic gmbh ) and salmon testis dna ( 14 g / ml , sigma - aldrich ) to decrease the background due to repetitive sequences . subsequently , the filters were hybridized with whole bac - dna as a probe . dna probes were labeled with [ -32p ] dctp ( amersham biosciences , piscataway , nj , usa ) and preannealed with human coti dna and salmon testis dna ( 10.5 g / ml ) . prehybridization and hybridization were performed overnight at 37c in a solution containing 50% formamide , 10% dextran sulfate , 1% tween 20 , 2 ssc , and 10 mm tris - hcl ( ph 7.4 ) . after hybridization , the filters were washed at 65c with 1.0 ssc containing 0.1% sds ( sodium dodecyl sulfate ) and 0.5 ssc containing 0.1% sds . banding patterns were obtained by either exposure to x - ray film ( fuji film , tokyo , japan ) or through use of the molecular imager fx ( biorad ) . the qpcr was performed using sybr premix ex taq ( takara - bio ) and the light cycler system ( roche diagnostics ) , according to the manufacturers ' protocols . primers were designed with primer3 software ( http://primer3.sourceforge.net ) , and the size of pcr products was confirmed by the pattern of restriction enzyme digested fragments using the labchip dna 500 kit on the 2100 bioanalyzer ( agilent technologies , waldbronn , germany ) . two , 0.5 , and 0.125 ng of genome dna from an individual with cnv were used and the quantification of each amplicon was carried out at 45 cycles of pcr . the results were analyzed with light cycler data analysis software using a second derivative maximum model . the main purpose of this paper is to report the accumulation of the data about highly polymorphic cnvs found in 5.0% of the individuals . ( the number of cnvs was 11 or more in each bac - spot . ) as shown in table 1 , 680 polymorphic cnvs were observed on 16 bac - regions . as described before , the results of two bac ( rp11 - 259n12 and rp11 - 121a8 ) were obtained from 133 unrelated individuals examined in stage ( 2 ) . southern blot analyses followed by pfge were carried out for the highly polymorphic cnvs . as shown by the typical cases in figures 1 and 2 , the patterns of these two bac clones ( rp11 - 79f15 and rp11 - 88l18 ) are shown in figures 1 ( rp11 - 79f15 ) and 2 ( rp11 - 88l18 ) as the typical examples . since each individual contained a different number of core segmental duplication units , each individual showed bands having different motilities . the results of qpcr conducted for two bac clone regions are described in figure 3 ( rp11 - 89b15 ) and figure 4 ( rp11 - 79o18 ) . for the former case , a part of a gene ( meox2 ) was deleted . on the contrary , for the latter case , the copy number of a part of the gene ( nsf ) increased , but the copy number of the gene ( wnt3 ) did not change . there are many segmental duplications which have already been summarized in a public data base , such as human genome segmental duplication database ( tcag database ; http://projects.tcag.ca/cgi-bin/variation/gbrowse ) , ucsc human genome browser ( ucsc database ; http:/genome.ucsc.edu / index.html ) , and ncbi map viewer ( ncbi database ; http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi ) . the cnv data obtained in our studies are summarized in table 1 in addition to the presence or absence of cnvs already reported in the databases . one bac clone , named rp11 - 115g22 , was mapped on two chromosomes , 6 and 15 in tcag database , so it is likely that this clone is present on two discrete chromosomes . on the contrary , however , we accept the reports from the latter two databases and described that this clone was mapped on only chromosome no.15 . with respect to segmental duplications , 10 out of 16 ( about 63% ) were present in the above databases ( ucsc database , and ncbi database ) . it was noteworthy that the majority of bac clones containing our cnvs were known to overlap to at least one cnv reported in the database . however , that does not mean that our highly polymorphic cnvs are exactly the same as those reported in previous reports , since precise comparisons between our study and the other studies were not carried out . as mentioned before , there was very little information about the cnvs of 45 japanese individuals for which relatively large sizes of population have been systematically screened . we compared our data with the data of japanese including the hapmap project as reported by redon et al . ten out of 16 cnvs identified in our study were reported in the data reported by redon et al . , although we should emphasize again that the cnvs identified in our bac region are not exactly the same as those reported by redon et al . . cnvs identified in our study with lower numbers tend to not be identified in redon 's report . on the other hand , when our data are compared with redon 's oligo - data from affymetrix 500ea array conducted for japanese , only two cnvs were overlapped to our cnvs . as redon et al . mentioned in their report , the reason appears to be that oligo arrays have some limitations for a complicated genome , such as segmental duplication areas . we summarized the genes and disease - related genes in omim which overlap to the bac - clone region with our cnvs ( table 2 ) . in addition to those two categories , mrnas have been also reported in the database , but they are too many to describe here . all bac - clone regions contained at least one mrna , although the functions of a majority of those mrnas are not known yet ( data not shown ) . we examined 213 unrelated japanese using bac - acgh and found a total of 680 cnvs on 16 bac clones . a large fraction of the regions involved in the cnvs observed in our study ( i.e. , 625 out of 680 ( 92% ) , table 1 ) have been reported previously in other studies listed in the database . a majority ( 63% ) of the cnvs had been found on the bac clones that overlapped with segmental duplication , suggesting the notion that segmental duplication might play a significant role in the creation of cnvs ( table 1 ) . in which they reported the sharing of cnvs among several populations , meaning those specific genomic imbalances either predated the dispersal of modern humans out of africa or arose independently in different populations . on the other hand , our cnvs , especially those showing high frequencies , were also identified by redon 's work . on the contrary , our cnvs showing low frequency , such as less than about 10% of individuals , were not observed in their work ( table 1 ) . that result suggests that those cnvs might be identified if the number of individuals examined by redon et al . were increased to the level of our study ( about 200 individuals ) . moreover , as described before , when our data compared with redon 's oligo - data conducted for japanese , only two of their cnvs were overlapped to our cnvs . although the oligo - based method tends to detect smaller cnvs ( about a few kilobases ) , this approach is less effective in tracking cnvs in genomic regions of complex structure , like segmental duplications , that are not sufficiently tagged by oligo targets . on the contrary , the bac platform can only identify larger cnvs ( > 40 kb ) , but this method has some advantage for detecting cnvs present in the regions of segmental duplications . the cnvs in the human genome are often associated with developmental disorders and susceptibility to diseases . large duplications and deletions have been known to be present within the human genome based initially on cytogenetic observations in the course of etiological studies of congenital malformations ( e.g. , [ 1 , 2 ] ) . the frequency of those duplications and deletions was presumed to be low and , for the most part , directly related to specific genetic disorders . a limited number of studies reported the presence of specific large duplications and deletions that were not apparently related to diseases ( e.g. , ) . reported that cgh on a bac - dna - based microarray could reliably detect single - copy gene decreases or increases from normal diploidy . following this , other array platforms , such as cdna , and oligo - nucleotide , have been developed and many data from them have been reported . as we mentioned before , many cnvs were reported to be closely related to disease phenotypes , and recent studies based on advanced molecular technologies , such as genomewide association studies [ 2729 ] and next generation sequencing [ 30 , 31 ] , reported that many genes appear to play important roles in the etiology of common diseases . we report our highly polymorphic cnvs in bac clones , which were reported to contain genes , expected to be related to phenotypic heterogeneity of each individual , based on the tcag database . we focused on genes reported in the above database , although many mrna were listed in the database in addition to genes ( table 2 ) . bac clone ( rp11 - 90a9 ) contains two genes : ankyrin repeat domain 34b ( ankrd34b ) and dihydrofolate reductase ( dhfr ) . a phosphoprotein encoded by ankrd34b is induced during bone marrow commitment to dendritic cells which play an important role in vertebrate immunity . dhfr genes were reported to be related to various malignancies including lymphoproliferative disorders such as systemic non - hodgkin 's lymphoma , primary central nervous system lymphoma ( pcnsl ) , and childhood acute lymphoblastic leukemia . those two genes are fully overlapped to the bac clone ( rp11 - 90a9 ) . it is likely that the cnvs might affect the copy number of those genes . one bac clone ( rp11 - 89b15 ) contains a gene mesenchyme homeobox 2 ( meox2 ) . the analyses by qpcr ( figure 3 ) demonstrated that the copy number of gene ( meox2 ) is deleted in the individual having cnvs detected by this bac clone . meox2 suppressed epithelial cell proliferation in cooperation with tgf - beta1 , and mediated induction of the cell - cycle inhibitor gene p21 . finally , the data from genome wide association study ( gwas ) reported that this is one of the candidate genes that might be associated with ischaemic stroke . another bac clone ( rp11 - 115g22 ) was mapped on the chromosomes 15 . ( chrna7 ) . that gene was used as a candidate target for examining interactions on the severity of adult attention deficit hyperactivity disorder ( adhd ) . moreover , it was reported that the gene is one of the candidates for alzheimer 's disease . the gene chrna7 is overlapped to the segmental duplication region of bac clone ( rp11 - 115g22 ) . many cnvs were listed in the databases mentioned as above , and those are overlapped to the gene . for those reasons , our cnv appears to be overlapped to the genes , and it might affect the copy number of gene chrna7 . a bac clone ( rp11 - 79o18 ) contains two genes which are n - ethylmaleimide - sensitive factor ( nsf ) and wingless - type mmtv integration site family , member 3 ( wnt3 ) . the qpcr results ( figure 4 ) demonstrated that the copy number of a gene ( nsf ) increased but no change was observed in the gene ( wnt3 ) . that result suggested that the cnv might affect the nsf gene , but not the wnt3 gene . however , there may be an opportunity for the cnvs to become a surrogate marker of wnt3 for the future association study between the cnv and some disease phenotype . the nsf gene is one of the essential components of membrane fusion machinery which is an important homeostatic process in eukaryotic cells . a recent study showed that the nsf gene is a good candidate marker for association studies for genetic risk underlying parkinson 's disease . the wnt3 gene 's single nucleotide polymorphisms ( snps ) were used as candidate markers for association studies of hemorrhagic stroke , hypertension , and chronic kidney disease . upregulation of the wnt gene family , including wnt3 , suggested involvement of the wnt 's canonical and/or noncanonical signaling pathway in chronic lymphocytic leukemia . a bac clone ( rp11 - 79f15 ) contains two genes : methyl - cpg binding domain protein 3-like 1 ( mbd3l1 ) and cell surface associated mucin 16 ( muc16 ) . the protein is localized to discrete areas in the nucleus , and expression appears to be restricted to round spermatids , suggesting that the protein plays a role in the postmeiotic stages of male germ cell development . on the other hand , muc16 is a member of the mucin gene family and encodes cancer antigen 125 ( ca125 ) which is a blood biomarker routinely used to monitor the progression of human epithelial ovarian cancer ( eoc ) , although its potential role in eoc is poorly understood . maintenance of an intact mucosal barrier , one of whose components is a gene product of muc16 , is critical to preventing damage and infection of wet - surfaced epithelia . as we demonstrated by southern blot analysis ( figure 1 ) , the polymorphism was caused by segmental duplications in the bac clone . the series of segmental duplications were on the 5-region of two genes ( mbd3l1 and muc16 ) . the cnvs do not affect the expression pattern of the genes , but these cnvs might be useful surrogate markers for future studies . glutamate receptor , ionotropic , ( ampa 2 ) . ampas are ligand - activated cation channels that mediate the fast component of excitatory postsynaptic currents in neurons of the central nervous system . since the size of the gene ( ampa 2 ) is larger than that of the bac clone ( rp11 - 231j7 ) , it is likely that the cnvs affect the copy number of the gene . a bac clone ( ctd-2100f13 ) contains two genes : rio kinase 3 ( yeast ) ( riok3 ) and niemann - pick disease , type c1 ( npc1 ) . npc1 can have a function in the egress of certain membrane - impermeable lysosomal cargo . the membrane - bound npc1 and soluble npc2 play an important role for the release of cholesterol from lysosomes . as a result of that mechanism , the gene is associated with obesity [ 5658 ] . the bac clone ( ctd-2100f13 ) is fully overlapped to both of the two genes ( riok3 and npc1 ) . the size of cnvs summarized in the databases shows that the reported cnvs were overlapped to both genes . we assumed that our cnvs may reflect the change of copy number of the genes themselves . since the genes mentioned are good candidate markers for enabling us to examine the etiology of common diseases and phenotypical heterogeneities among individuals , our highly polymorphic cnvs should be able to become good markers in future studies . we are currently planning to examine the same population using a high - density oligo - array platform to accumulate more cnv data for japanese . the reason , as mentioned before , is that the bac platform and oligo methods complement each other . the oligo platform is known to be more effective in detecting smaller cnvs ( around a few kilobases ) , even though this approach is less effective in tracing cnvs in genomic regions of complex structure that are covered by the bac approach conducted in this study . we expect to construct a more definite japanese cnv database by the combination of bac- and oligo - platform arrays . we conducted population screening for 213 unrelated japanese , and observed 680 highly polymorphic cnvs . the majority of the polymorphic cnvs presented on bac clones that overlapped with regions of segmental duplication , and had been previously reported in other publications . moreover , it is expected that the cnvs might be good surrogate markers for detecting etiological genes , even if cnvs did not directly affect the genes themselves .

segmental copy - number variations ( cnvs ) may contribute to genetic variation in humans . reports of the existence and characteristics of cnvs in a large japanese cohort are quite limited . we report the data from a large japanese population . we conducted population screening for 213 unrelated japanese individuals using comparative genomic hybridization based on a bacterial artificial chromosome microarray ( bac - acgh ) . we summarize the data by focusing on highly polymorphic cnvs in 5.0% of the individual , since they may be informative for demonstrating the relationships between genotypes and their phenotypes . we found a total of 680 cnvs at 16 different bac - regions in the genome . the majority of the polymorphic cnvs presented on bac - clones that overlapped with regions of segmental duplication , and the majority of the polymorphic cnvs observed in this population had been previously reported in other publications . some of the cnvs contained genes which might be related to phenotypic heterogeneity among individuals .

posterior reversible encephalopathy syndrome ( pres ) was first reported as reversible posterior leucoencephalopathy syndrome in 1996 . initially , it was described in acutely ill patients who had a reversible syndrome of headache , altered mental function , seizure , and loss of vision associated with findings indicating predominantly posterior leucoencephalopathy on imaging studies . literature consists of few case reports or few small series of patients in different clinical settings . the setting in which it was described included patients with hypertensive encephathalopathies , patients who were receiving immunosuppressive medicines after organ transplantation or other malignancies , patients with renal failure , and patients with eclampsia . other comorbid conditions later described included children with nephrotic syndrome , acute nephritis , hemolytic uremic syndrome , systemic lupus erythematosus , and patients with volume and electrolyte disturbances . pathogenetic mechanism is believed to be vasogenic edema and hyperperfusion of cerebral white matter affecting commonly occipito - parietal region due to autoregulatory failure or endothelial dysfunction . predilection for posterior brain regions could be due to pial and intracerebral vessels in anterior circulation which have higher concentration of adrenergic nerves when compared with posterior hemisphere . diagnosis was made in appropriate clinical setting with neurological symptoms supported by neurological imaging by computed tomography ( ct ) scan brain , magnetic resonance imaging ( mri ) , and diffusion weighted images . we describe a series of children with kidney disease who developed pres and describe their clinical and laboratory features . out of total 660 pediatric renal patients including renal transplant patients admitted at our institute during september 2010 to february 2012 ( 18 months ) , we identified 11 patients with characteristic neurological symptom complex in association with typical neurological images suggesting pres as described in introduction . all patients had undergone thorough evaluation clinically , neurologically , and biochemically to search for likely contributory factors with special attention to blood pressure , volume status , severity of anemia , presence or absence of hemolysis , and its severity by following peripheral smear , platelet count , ldh , level of renal function , electrolyte and acid base balance , severity of hypoproteinemia , character of underlying renal disease , and blood level of immunosuppressive medications . hypertension was staged according to fourth task force report on high blood pressure in children and adolescents by comparing patients bp reading to bp level for boys and girls by age and height percentile in bp table prepared by national heart lung and blood institute ( nhlbi ) . grade 1 hypertension is defined as systolic or diastolic bp from 95 percentile to 99 percentile , + 5 mm hg for age and height for individual gender . grade 2 hypertension is designated to bp levels that are more than 5 mmhg above 99 percentile for age and height for individual gender . possibility of other neurological disorders like cerebral venous thrombosis , meningeal infection , intracranial hemorrhage or space occupying lesion were ruled out in each case with help of appropriate investigations . initially , all patients were evaluated by in - house cranial ct scan without contrast . mri with or without contrast was obtained in patients where ct brain alone was inconclusive . all patients received appropriate treatment for renal disease , adequate dialysis for renal failure , timely correction of fluid electrolyte and acid base balance , nitroglycerine drip and other anti - hypertensives for hypertension , iv albumin for hypoalbuminemia , plasma exchanges in cases of hus , and dose adjustment of the medication was made . record of treatment received for renal disease and prevailing abnormal parameters and time frame of clinical and biochemical improvement was made and follow - up radio - images were compared ( usually taken at 4 week interval and more frequently in selected cases ) with original images . a total of 11 patients ( eight boys and three girls , ranging in age from 3 years to 15 years ) with pres , were evaluated and studied . presenting symptoms were seizures in ten cases ( 91% ) , headache in eight cases ( 72% ) , altered mental status in five cases ( 45% ) , and dimness of vision or cortical blindness in three cases ( 27% ) in different combinations . so far as underlying renal disease and setting of syndrome complex is considered among these patients , six patients ( 55% ) had acute renal failure ( four had hus , one had pauci - immune crescentic gn , one had solitary functioning kidney obstructed by a stone who developed pres in setting of post - obstructive diuresis and associated persistent hypocalcemia ) , four patients ( 36% ) had normal renal function [ one patient with diffuse proliferative lupus nephritis and uncontrolled hypertension , one patient of focal segmental glomerulosclerosis ( fsgs ) with high trough level of cyclosporine 300 ng / ml , one patient with mpgn who developed hypertensive encephalopathy due to non - compliance to medication , one with morbid nephrotic state with scrotal skin necrosis and severe hypoproteinemia ( serum albumin 1.1 gm / dl ) ] . one patient of chronic renal failure ( crf ) who was on continuous ambulatory peritoneal dialysis ( capd ) developed pres after receiving inadvertent blood transfusion due to misjudged anemia and his hb at the time of pres was 12.5 gm / dl . significant comorbid contributory factors found in these patients at the time of pres were uncontrolled hypertension in all 11 patients ( 100% ) , renal failure ( 55% ) , cni toxicity ( 9% ) , hypoproteinemia ( 9% ) , high hematocrit ( 9% ) , hypocalcemia ( 9% ) , steroid toxicity ( 9% ) , hus ( 36% ) , and lupus nephritis ( 9% ) [ figure 1 ] . 5 showing morbid nephrotic state with scrotal skin necrosis a five - year - old - male child weighing 14 kg was admitted with arf and serum creatinine of 6.4 mg / dl , and respiratory acidosis . he was prepared by acute peritoneal dialysis for pcn diversion of obstructed solitary functioning kidney . clinically , his pulse and hydration was normal and bp was 130/90 mmhg ( grade 2 ht ) which was controlled with oral nifedipine . biochemically , electrolytes were normal except for ionized serum calcium which was 2.26 mg / dl , phosphate 5.4 mg / dl , magnesium 1.77 mg / dl , ph of 7.32 and serum bicarbonate level was 17.5 patient was treated by sodium bicarbonate drip in one iv line and calcium gluconate drip in another iv line . in first 12 h the child received 15 ml of calcium gluconate and 10ml in next 12 h. he was given 0.5 ml of magnesium sulphate ( i.e. , 250 mg ; 25% w / v ) intramuscularly . the child 's venous blood gas and electrolytes were studied every 6 hours and child persisted to be severely hypocalcemic though he received 15 ml of calcium gluconate on second day and 10 ml for further two days . so in total 50 ml of calcium gluconate was given in three days of pcn diversion in a 14 kg child . childs hypocalcemia was within normal range on fourth day only when we reduced 24 h calcium dose in stepwise fashion . his bp was well controlled with oral nifedipine . throughout the course , his urine output , serum urea , and serum creatinine were improving day by day after diversion . we measured 24 h urinary calcium also on 2 day after diversion which was 30 mg/24 h which was high in face of very low ionized serum calcium level . the amount of calcium gluconate the child required parenterally to correct hypocalcemia was surprisingly high which we have observed first time in such a setting . his ipth , five days after pcn diversion was 408 pg / ml which was 540 pg / ml when he presented with arf . logically , we hypothesized that calcium gluconate given iv was increasingly being taken up by bone due to decreasing hyperparathyroidism in association with improving renal function and it was wasted to some extent in urine also because of defect in tubular reabsorption leading to persistent hypocalcemia . calcium has unique intracellular messanger role and it is complexed with numerous ligands in cytosol , including adenosine and vitamin d dependent calcium binding protein , calmodulin . persistent hypocalcemia may be expected to affect directly or indirectly mechanisms involved in autoregulation and endothelial function . an 8-year - old child , weighing 15 kg ( crf on capd ) was found to have pres with history of blood transfusion by private pediatrician . his hb was 12.5 g% with grade 2 ht ( bp 120/80 mm hg ) when he presented to us with pres . initially , we also intensified pd with 2.5% dextrose capd fluid , used diuretics , and controlled ht with oral medication for two days . child was on capd for 1.5 year without any neurological complaint . during his intensive pd with hypertonic fluid , child lost 300 g weight after 1 day and further 150 g on 2 day after admission . child was euvolemic with normal x - ray chest and was not improving neurologically for two days . so , observing the temporal relationship of blood transfusion and development of pres , we tapped 100 ml of blood on 3 day by femoral route and pd was switched to 1.5% dextrose solution again . temporal relationship of improvement with phlebotomy left no doubt about the fact that blood transfusion was causally related to pres in this child who was habituated to low hematocrit . sudden rise in hematocrit might have resulted in increased viscosity of blood and rise in vascular resistance , superimposed on possibly sensitive cerebral vasculature , may have resulted in hypertensive encephalopathy like picture . kdoqi clinical practice recommendation for anemia in ckd also have set target of hb in the range of 11 - 12 g / dl . initial cranial imaging revealed fairly symmetrical areas of edema not only involving occipito - parietal white matter regions in majority of cases but also extended to cortical gray matter in nine cases ( 81% ) and extended to fronto - temporal area in five cases ( 45% ) , involved basal ganglia also in two patients ( case 4 and 11 ) whereas cerebellum and medulla were also involved in one patient ( case 11 ) [ figure 2 ] . computed tomography / magnetic resonance imaging images of brain showing positive findings all patients had neurological recovery within 5 - 7 days except one patient of hus ( case four ) who persisted to have altered mentation even after a month of follow up , though his renal function and cranial imaging had recovered completely after a month . this was the patient who had neurological symptoms for six days before getting admitted in our hospital where specific management of peritoneal dialysis and plasma exchange was started . follow - up cranial images were normal after four to five weeks in all cases . all patients had normal renal function after four weeks except in two cases , one with hus who was put on capd and one patient with obstructed solitary functioning kidney who had serum creatinine of 1.9 mg / dl at the end of one month when he was stone free . a five - year - old - male child weighing 14 kg was admitted with arf and serum creatinine of 6.4 mg / dl , and respiratory acidosis . he was prepared by acute peritoneal dialysis for pcn diversion of obstructed solitary functioning kidney . clinically , his pulse and hydration was normal and bp was 130/90 mmhg ( grade 2 ht ) which was controlled with oral nifedipine . biochemically , electrolytes were normal except for ionized serum calcium which was 2.26 mg / dl , phosphate 5.4 mg / dl , magnesium 1.77 mg / dl , ph of 7.32 and serum bicarbonate level was 17.5 patient was treated by sodium bicarbonate drip in one iv line and calcium gluconate drip in another iv line . in first 12 h the child received 15 ml of calcium gluconate and 10ml in next 12 h. he was given 0.5 ml of magnesium sulphate ( i.e. , 250 mg ; 25% w / v ) intramuscularly . the child 's venous blood gas and electrolytes were studied every 6 hours and child persisted to be severely hypocalcemic though he received 15 ml of calcium gluconate on second day and 10 ml for further two days . so in total 50 ml of calcium gluconate was given in three days of pcn diversion in a 14 kg child . childs hypocalcemia was within normal range on fourth day only when we reduced 24 h calcium dose in stepwise fashion . his bp was well controlled with oral nifedipine . throughout the course , his urine output , serum urea , and serum creatinine were improving day by day after diversion . we measured 24 h urinary calcium also on 2 day after diversion which was 30 mg/24 h which was high in face of very low ionized serum calcium level . the amount of calcium gluconate the child required parenterally to correct hypocalcemia was surprisingly high which we have observed first time in such a setting . his ipth , five days after pcn diversion was 408 pg / ml which was 540 pg / ml when he presented with arf . logically , we hypothesized that calcium gluconate given iv was increasingly being taken up by bone due to decreasing hyperparathyroidism in association with improving renal function and it was wasted to some extent in urine also because of defect in tubular reabsorption leading to persistent hypocalcemia . calcium has unique intracellular messanger role and it is complexed with numerous ligands in cytosol , including adenosine and vitamin d dependent calcium binding protein , calmodulin . persistent hypocalcemia may be expected to affect directly or indirectly mechanisms involved in autoregulation and endothelial function . an 8-year - old child , weighing 15 kg ( crf on capd ) was found to have pres with history of blood transfusion by private pediatrician . his hb was 12.5 g% with grade 2 ht ( bp 120/80 mm hg ) when he presented to us with pres . initially , we also intensified pd with 2.5% dextrose capd fluid , used diuretics , and controlled ht with oral medication for two days . child was on capd for 1.5 year without any neurological complaint . during his intensive pd with hypertonic fluid , child lost 300 g weight after 1 day and further 150 g on 2 day after admission . child was euvolemic with normal x - ray chest and was not improving neurologically for two days . so , observing the temporal relationship of blood transfusion and development of pres , we tapped 100 ml of blood on 3 day by femoral route and pd was switched to 1.5% dextrose solution again . temporal relationship of improvement with phlebotomy left no doubt about the fact that blood transfusion was causally related to pres in this child who was habituated to low hematocrit . sudden rise in hematocrit might have resulted in increased viscosity of blood and rise in vascular resistance , superimposed on possibly sensitive cerebral vasculature , may have resulted in hypertensive encephalopathy like picture . kdoqi clinical practice recommendation for anemia in ckd also have set target of hb in the range of 11 - 12 g / dl . initial cranial imaging revealed fairly symmetrical areas of edema not only involving occipito - parietal white matter regions in majority of cases but also extended to cortical gray matter in nine cases ( 81% ) and extended to fronto - temporal area in five cases ( 45% ) , involved basal ganglia also in two patients ( case 4 and 11 ) whereas cerebellum and medulla were also involved in one patient ( case 11 ) [ figure 2 ] . computed tomography / magnetic resonance imaging images of brain showing positive findings all patients had neurological recovery within 5 - 7 days except one patient of hus ( case four ) who persisted to have altered mentation even after a month of follow up , though his renal function and cranial imaging had recovered completely after a month . this was the patient who had neurological symptoms for six days before getting admitted in our hospital where specific management of peritoneal dialysis and plasma exchange was started . follow - up cranial images were normal after four to five weeks in all cases . all patients had normal renal function after four weeks except in two cases , one with hus who was put on capd and one patient with obstructed solitary functioning kidney who had serum creatinine of 1.9 mg / dl at the end of one month when he was stone free . with easy availability of radio - imaging technique , mri in particular , there is increasing number of reports of pres in all age groups including pediatric patients over last ten years . with an objective of studying predisposing factors , clinical spectrum , cranial imaging , and course of pres in pediatric renal patients , renal patients are at particular risk of pres because renal disease and hypertension go hand in hand . higher chances of volume perturbation , electrolyte disturbances and mechanism of renal disease itself , its treatment - associated drug toxicities , and other complications of management put these patients at special risk for pres . table 2 shows comparison of our results with two other pediatric studies of pres in literature . it could be seen that in all three pediatric series there were more boys than girls with pres . other two pediatric series , recently published , also showed more boys than girls . in adult series comparative data of pres in pediatric patients seizure as a presenting symptom was most common in ishikura 's study ( 85% ) as well as in our study ( 91% ) . all the series had different combination of four clinical characteristic symptoms as a presenting picture . so far as level of renal function was considered among these patients , we found six patients ( 55% ) with acute renal failure . other two studies have not specified about level of renal function . but more and more patients with hypertension and renal failure of different etiologies are reported with pres . hypertension was almost always observed in pres but its level was not correlated to the severity of pres and cause effect relationship is yet to be established , as in many cases other comorbid conditions like drug toxicity , vacuities , auto - immune disease , or endothelial dysfunction from any cause may be involved . may have multiple factors involved because they are often hypertensive , receive steroid and cni , may have severe hypoproteinemia , and may have massive oedema with fluid retention and associated increased vascular permeability . ishikura 's series had seven cases ( 35% ) , kwon series had two cases ( 16% ) whereas our study had two cases ( 18% ) of nephrotic syndrome . like our series ( 36% hus ) , series of kwon et al . also had two cases of hus ( 16.5% ) who developed pres . here , pathological process itself in addition to renal failure and hypertension could be involved in pathogenesis like that of pre - eclampsia . our patient with pauci - immune crescentic gn ( case 1 ) had developed pres following three pulses of methylprednisolone . , in two cases ( 16% ) of nephrotic syndrome , whereas no specific allegation to steroid high dose was made in ishikura series . ishikura 's series had ten patients ( 50% ) with kidney transplantation on cni and kwon et al . 33% of cases on cni and our study had 9% ( 1 case ) of pres which was associated with cni overdose . immunosuppresive medicines and cni in particular are more frequently reported to be associated with pres in post - transplant scenario . after renal toxicity , vascular toxicity , and neurotoxicity are the most serious side effect of cni , affecting 25 - 59% of transplant patients . reduction in drug dosage or prompt withdrawal of cytotoxic drugs is usually recommended in cases of pres . when same agent is re - introduced , patient must be followed closely as recurrence has been reported in this setting . recently , de laat from netherlands described seven childhood cancer patients with clinical radiological findings of pres . he also reviewed 49 other documented cases of pres from literature during childhood cancer and concluded that hypertension and renal failure related to immunosuppressive treatment of leukemia and other solid tumor seems to be the most important triggers for occurrence of pres . similarly , faruk inceik from turkey in 2009 described nine cases of pediatric pres of whom seven were receiving immunosuppressive treatment and two were hypertensive crisis patients . involvement of occipital and parietal region was almost universal in all three studies and lesions were not restricted to white matter alone but involved gray matter also in majority of cases in all three studies . other areas like frontal lobe , temporal lobe , basal ganglia , cerebellum , and medulla were involved in lesser frequency in all series with individual incidence . follow - up cranial images were normal after four to five weeks in all cases in present study . one case of hus ( 9% ) persisted to have altered mentation even after a month of follow - up , though his renal function and cranial imaging had recovered completely . in a study by ishikura , hypertension , renal disease , immunosuppression , and chemotherapy of malignancies are triggers for pres . it is important to consider this diagnosis in children presenting with seizure , visual disturbances , headache , and altered mentation in appropriate clinical setting .

posterior reversible encephalopathy syndrome ( pres ) is a clinic - radiographic entity of heterogeneous etiologies that are grouped together because of similar findings on neuro - imaging and associated symptom complex of headache , vision loss , altered mentation , and seizures . although usually considered benign and reversible , characteristics of this syndrome in pediatric patients remain obscure . this case series included 11 patients ( 8 males , 3 females , age 3 - 15 years ) of pres during september 2010 to february 2012 out of a total 660 renal pediatric patients ( 1.66% ) . we studied their clinical profile , contributory factors , and outcome . presenting symptoms were headache in 73% , dimness of vision or cortical blindness in 36% , seizures in 91% , and altered mentation in 55% . the associated renal diseases were acute renal failure ( 55% ) , chronic renal failure ( 9% ) , and 36% had normal renal function . the contributory factors were uncontrolled hypertension ( 100% ) , severe hypoproteinemia ( 9% ) , persistent hypocalcemia ( 9% ) , hemolytic uremic syndrome ( 36% ) , cyclosporine toxicity ( 9% ) , lupus nephritis ( 9% ) , high hematocrit ( 9% ) , and pulse methylprednisolone ( 9% ) . brain imaging showed involvement of occipito - parietal area ( 100% ) and other brain areas ( 63% ) . all but one patient of hemolytic uremic syndrome had complete clinical neurological recovery in a week , and all had normal neurological imaging after 4 - 5 weeks . pres is an underdiagnosed entity in pediatric renal disease patients . associated hypertension , renal disease , and immunosuppressive treatment are important triggers . early diagnosis and treatment of comorbid conditions is of prime importance for early reversal of syndrome .

ocular parasitosis in human is more prevalent in geographical areas where environmental factors and poor sanitary conditions favor the parasitism between man and animals . in recent years , population shift and rapid transport have facilitated the spread of certain parasitic diseases from endemic to nonendemic areas . the routes of infection to man vary with species of the parasite and the animal hosts they infest . lesions in the eye can be due to damage directly caused by the infectious pathogen , indirect pathology caused by toxic products , immune response incited by infections , or ectopic parasitism of the preadult or adult stages . the epidemiology of parasitic ocular diseases reflects the habitat of the causative parasites as well as the habits and health status of the patient . additional consideration must include local sanitation and the presence of a vector for transmission as well as the more complicated life cycles of the parasites and definitive hosts . dietary history should be considered since most parasitic transmission is through food and water contamination . an awareness of these is therefore important to the clinician evaluating this group of patients . an ocular examination may provide clues to the underlying disease , and an awareness of the possibilities of travel - related pathology may shed light on an ocular presentation . the eye is involved both in a variety of systemic infections and may be the primary focus of other pathologies . the ophthalmic lesions occur as a result of antiparasitic treatment as it has been noticed in the prophylactic and therapeutic attempts to treat malaria [ 1 , 2 ] . drugs such as hydroxychloroquine and chloroquine can damage vision because of their toxic effects , which is due to slow accumulation of the drugs in the retinal epithelium that results in irreversible visual loss . much debate and confusion have taken place over the type and frequency of ocular examination in patients taking these drugs . despite improved understanding of the clinical features of inflammatory eye diseases and advances in diagnostic testing , clinicians should maintain a high index of suspicion for infective parasitic diseases in patients thought to have inflammatory eye involvement . because of this somehow more complex scenario , and the tendency for the parasites to cause a wider variety of pathologic lesions , the various parasitic etiologies of ocular diseases will be addressed individually , including epidemiology , pathology , diagnosis , and treatment . acanthamoeba spp . is ubiquitous free - living protozoa that have been isolated from several habitats , including soil , bottled water , eyewash stations , and air . there are two stages in the life cycle of this environmental ameba : the motile trophozoite ( 840 m ) and the dormant cyst ( 829 m ) . by encysting , acanthamoeba spp . can evade extreme environmental conditions such as hyperosmolarity , glucose starvation , desiccation , extreme temperatures , and extreme ph . the leading risk factors for acanthamoeba keratitis are contact - lens wear and corneal trauma [ 5 , 6 ] . although > 80% of the cases of acanthamoeba keratitis occur in contact lens , in other countries such as india , keratitis that is not related to contact lenses commonly occurs after corneal trauma or exposure to contaminated water . one clinical manifestation of acanthamoeba keratitis is radial neuritis and severe pain that is not commensurate with the extent of tissue damage . it typically presents as a unilateral central or paracentral corneal infiltrate , often with a ring - shaped peripheral infiltrate . other characteristic symptoms ( which appear in the early phases of infection ) include eyelid ptosis , conjunctival hyperemia , epithelial ulcers , and lack of discharge . these symptoms are often followed by the appearance of a ring - like stromal infiltrate in the later stages of disease . acanthamoeba keratitis can progress to scleritis , and , in severe cases , uncontrolled infections require the removal of the affected eye . interestingly , the pathogenic cascade of acanthamoeba keratitis has interesting parallels with the amebic colitis caused by e. histolytica . a provisional diagnosis of ak can be made using the clinical features and confocal microscopy although a definitive diagnosis requires culture , histology , or identification of acanthamoeba deoxyribonucleic acid by polymerase chain reaction . trophozoites and cysts can be identified in giemsa or periodic - acid - schiff - stained smears from scrapings or corneal biopsy specimens . long - term topical application of agents such as propamidine , miconazole , and neomycin has been successful in only few instances . once introduced , the trypomastigotes circulate throughout the body with a preference for invading muscle cells , neural tissue , and the reticuloendothelial system . if the initial bite is near the orbit , the patient may experience significant palpebral and periorbital oedema ( romana 's sign ) . the edema is usually painless and is frequently followed by constitutional symptoms of fever , malaise , and anorexia . the diagnosis of acute chagas ' disease is made by the detection of trypomastigotes in the bloodstream by direct examination of uncoagulated blood or buffy coat preparation . direct culturing of blood on novy , macneal , nicolle 's medium ( nnn ) or other suitable media may result in positive cultures in 7to 10days . serologic testing is of little value in the diagnosis of acute chagas ' disease as antibodies do not usually appear for 2to 40days following the onset of symptoms . additionally , serologic studies may falsely detect the cross - reactivity of antibodies to nonpathogenic trypanosoma rangeli . therapy of chagas ' disease with antitrypanosome therapy is most successful in the acute stage . therapy is usually extended for a period of months , and parasitologic cure rates are somewhat disappointing . the protozoan disease giardiasis can cause ocular complications , including salt and pepper retinal changes . one study showed that asymptomatic , nonprogressive retinal lesions are particularly common in younger children with giardiasis . this risk does not seem to be related to the severity of the infection , its duration , or the use of metronidazole but may reflect a genetic predisposition . there are numerous species within the genus , and disease manifestation is , in part , species specific . once injected into humans during the sandfly blood meal , the promastigote develops into an amastigote after being engulfed by tissue macrophages . within these cells , visceral leishmaniasis , or that which represents systemic disease , is known as kala - azar . the ocular manifestations of kala - azar are relatively uncommon and include chorioretinitis , central retinal vein thrombosis , iritis , papillitis , and keratitis . glaucoma has been reported to develop after the successful treatment of kala - azar . ocular findings in cutaneous leishmaniasis represent a local phenomenon resulting from the initial site of infection near the eye with occasional spread to the lacrimal duct . if the initial bite occurs on the conjunctival mucosa , the disease is termed mucocutaneous leishmaniasis . the diagnosis of leishmaniasis is made by direct demonstration of organisms on tissue smears or biopsy . amastigotes are usually demonstrated fairly easily in the case of cutaneous or mucocutaneous ocular disease . however , amastigotes have not been directly identified in cases of ocular disease associated with kala - azar . when present , leishmania spp . may be cultured on novy , macneal , nicolle 's medium ( n.n.n . ) as well as schneider 's drosophila medium supplemented with 30% fetal bovine serum . while available , serologic testing is not particularly useful for diagnosing cutaneous and mucocutaneous disease due to cross - reactivity with t.cruzi and mycobacterium leprosum . treatment of choice is pentavalent antimony , sodium stibogluconate 1520 mg per kg per day i m or iv for 1520 days . a second or even a third treatment course with pentavalent antimonal caused by the plasmodium species and transmitted via the bite of the female anopheles mosquito , this sometimes fatal infectious disease has characteristic findings in the eye . signs of falciparum malaria in the eye include retinal whitening , retinal haemorrhage , papilloedema , and cotton wool spots [ 15 , 16 ] . much research done in endemic areas has shown a correlation between papilloedema or extramacular retinal oedema ( retinopathy ) and poor outcome in children with cerebral malaria . studies show that retinopathy was associated with subsequent death , and the increasing severity of retinal signs was related to increasing risk of fatal outcome . other studies have shown that retinal changes related to microvascular obstruction were common in adults with severe falciparum malaria and correlated with disease , severity and comma [ 18 , 19 ] . it is important to emphasise that whilst these signs give a pointer to the severity of disease they do not alter the drug management of malaria . the outcome in terms of vision in patients with ophthalmological findings and severe malaria is usually good . insights from retinal investigations have furthered the understanding of cerebral malaria [ 21 , 22 ] . the photobiological effects of quinacrine and chloroquine are similar in model systems ; thus , development of a bull's - eye maculopathy with quinacrine ingestion is an unsurprising potential side effect . the definitive diagnosis of malaria is made by microscopic identification of the parasite in the blood smear . a thin blood film should be examined for at least 15 minutes , whereas a 5-minute search of a thick film should reveal parasites if present . the thick film is the most efficient method of detecting malarial parasites , but interpretation requires an experienced worker . antimalarial drugs may be classified as ( 1 ) suppressive , by acting upon asexual blood cell stages and preventing the development of clinical symptoms ; ( 2 ) therapeutic , by also acting on asexual forms to treat the acute attack ; ( 3 ) radical cure , for destruction of the ee forms ; ( 4 ) gametocytocidal , for destroying gametes ; ( 5 ) sporoniticidal , for drugs that render gametocytes noninfective in the mosquito . two genera appear to be important in the pathogenesis of ocular disease : encephalitozoon and nosema . it should be noted that knowledge of microsporidiosis seems to be rapidly expanding , given its important role as an opportunistic infection in patients with aids . the life cycle is somewhat complex , involving three general stages : infection , merogony , and sporogony . ocular infection is presumed to occur either by direct inoculation into eye structures or by dissemination systemically , with the latter proposed to be the pathogenesis in patients with aids . ocular findings are generally limited to the conjunctiva and cornea . with respect to diagnosis , spores have been demonstrated in most cases in which corneal scrapings or biopsy specimens are examined by light or electron microscopy . current recommendations for treatment include the use of albendazole , which has shown some promise in the treatment of corneal disease . rhinosporidiosis , caused by rhinosporidium seeberi , is a mucocutaneous disease that involves the palpebral conjunctiva in ~15% of all cases of rhinosporidiosis . reproduction of r.seeberi in tissue produces polypoid or papillary growths that arise from mucous epithelium . recent investigations of rna genes from this microorganism disclose that it may be more closely related to fish parasites than to fungi , and it is , therefore , included in protozoan diseases of the eye . the etiologic agent , rhinosporidium seeberi , has never been successfully propagated in vitro . at present , the treatment for rhinosporidiosis is the surgical excision . some authors proposed a medical therapy with dapsone , but the results are not convincing . antimicrobial therapy is ineffective , and the disease may recur after months or years . the majority of infections are asymptomatic and the prevalence of antitoxoplasma igg ( indicating past infection ) ranges from 15 and 20% in northern europe to 80% or more in parts of the developing world . some patients may present with a glandular fever - like systemic febrile illness with adenopathy . most cases of adult infection will not present with eye signs , those that do usually present with a focal necrotising retinitis occasionally associated with vascular occlusion . toxoplasmosis in immunosuppressed patients , for example , with aids can present with multiple , widespread lesions of differing chronicity and look different from classical toxoplasmosis . vitritis is a common feature of toxoplasmosis which often leads to symptomatic haze and floaters that lasts for months after the resolution of the acute attack . lesions are usually self - limiting , but where they threaten sight around the macula or optic nerve treatment with a combination of corticosteroids , pyrimethamine , and sulfadiazine is usually advocated . however , therapeutic trials suggest that there is little evidence that drug therapy alters the natural history of the disease . relapses are relatively common , occurring in around 80% of patients followed up for more than 5 years . the most serious consequences of toxoplasmosis are seen when acquisition occurs in pregnancy leading to congenital infection of the newborn . involvement of the macula is common in the developing foetus and has devastating effects on central vision . serologic tests are very important in the diagnosis of toxoplasmosis . because of the common occurrence of antibodies to the parasite in the general population , diagnosis by serologic means requires the demonstration of a significant increase in antibody titers . drug treatment for ocular and cerebral toxoplasmosis is the same and lesions will continue to grow without therapy . azithromycin has been shown to be effective in reducing the number of attacks in brazil ( see tables 1 and 2 ) . ocular angiostrongyliasis , caused by angiostrongylus cantonensis , is a nonfatal disease ; however , it can cause permanent damage to an affected eye . it was first reported in thailand in 1962 and since then has rarely presented in tropical countries . according to the a. cantonensis life cycle , the human is an accidental host . only a small number of worms remigrate to pulmonary arteries or move randomly to other tissues such as cranial nerves or orbits . although blood eosinophilia is demonstrated in most cases of eosinophilic meningitis , it has not been noticed in ocular angiostrongyliasis without eosinophilic meningitis . hence , it may indicate that ocular angiostrongyliasis occurs because a worm moves randomly from the bloodstream to an eye without invasion of the brain or meninges . in cases of ocular involvement ocular symptoms although a wide range of initial visual acuity was reported , from finger count to 6/6 , five cases had visual acuity less than 2/60 . the duration of visual impairment varied from 4 days to 8 weeks , mostly 2 - 3 weeks . additionally , indirect ophthalmoscopy should be recommended in any individual presenting with a history of eating raw pila spp . no dominant affected eye has been reported because of a nonspecific pattern of parasite movement . any types of laser , surgical removal , and corticosteroid treatment did not improve visual acuity . alteration of the retinal pigment epithelium or retinal inflammation caused directly by parasites was the main reasons for poor vision at presentation . furthermore , it produced permanent damage to an affected eye and gave a poor outcome . although corticosteroids and albendazole have been reported to be effective in ocular cysticercosis and neurocysticercosis , there are no specific anthelmintic therapies in ocular angiostrongyliasis even after a definite diagnosis has been made . human ocular infestation by live filarial worm is a rare occurrence and has been reported mostly from south - east asia . inflammation of the retinal pigment epithelium and retinal vasculitis decreased vision , and panuveitis with secondary glaucoma can occur . indirect ophthalmoscopy showed vitritis with plenty of vitreous membranes , and subretinal yellow lesions in the peripheral retina along with retinal pigment epithelial tracts . baylisascaris procyonis is the common intestinal raccoon roundworm in north america and is found in 82% of raccoons in illinois . it was identified in seven childhood cases manifesting as diffuse unilateral subacute neuroretinitis and choroidal infiltrates in association with neurologic disease . differences in inoculum level are likely responsible for isolated ocular larva migrans versus neural larva migrans in humans . indirect immunofluorescence assays on serum , and cerebrospinal fluid is usually positive or serially positive and increasing . dirofilarial zoonotic infections are caused by mosquito vectors that carry the parasites from their animal hosts to people . although these infections remain rare , they are increasing in incidence and human dirofilariasis may be considered an emergent zoonosis . as the worm matures , it elicits a host inflammatory response that ultimately produces the clinical presentation of a subcutaneous nodule . subcutaneous dirofilariasis appears as a small subcutaneous nodule that gradually grows over periods of weeks or months . when the location is ocular , the worms are situated in the conjunctiva and can be extracted by incision . the diagnosis of dirofilariasis is established histopathologically . both the gross and microscopic features of d. tenuis have been well described . once diagnosed , the recommended treatment is complete removal of the nematode . if the nematode is not removed , it eventually degenerates , and the mature granulomatous response results in either calcification or abscess formation with subsequent purulent expulsion of the parasite . infection is acquired by humans through the bite of the tabinid flies of the genus chrysops . when humans are bitten , larvae pass from the fly to the human , where they develop over 1year into mature adult worms . ocular disease may be due to both the presence of microfilaria and the presence of the adult worm . the diagnosis of loiasis is generally made by the detection of circulating microfilariae . in cases of conjunctival involvement , therapy of loiasis involves the manual removal of adult worms present in the conjunctiva in addition to the use of diethylcarbamazine ( dec ) . severe hypersensitivity responses may occur due to the killing of both microfilariae and adult worms . it appears that humans are the main reservoir of onchocerciasis , with infection occurring from the bite of an infected female blackfly , simulium spp . an estimated 37 million people in 34 countries in sub - sahara africa and south america are affected by it . after biting an infected person and ingesting microfilariae , the microfilariae mature to the larval stage as they migrate to the proboscis of the fly . there , the larvae may be injected into a human with the next bite , resulting in the formation of an adult worm capable of producing microfilariae . these microfilariae migrate throughout skin and connective tissue , where they die after several years . adult worms may live in the subcutaneous tissue for years , with a female producing one - half to one million microfilariae yearly . the site of the adult worm is usually found over a bony prominence and may develop into a firm , nontender nodule , or onchocercoma . it is the migration of microfilariae through skin and connective tissue which is responsible for the majority of clinical findings in onchocerciasis . ocular onchocerciasis is due to the presence and/or migration of microfilariae in and through ocular structures as well as the host 's response to the migration . there are five predominant ocular findings that correlate with the location of microfilariae : punctate keratitis , sclerosing keratitis , iridocyclitis , chorioretinitis , and optic atrophy . other findings may include distortion of the pupil , which may also be covered with exudate . wolbachia and wolbachia - derived molecules are bacterial symbionts of o. volvulus that is implicated in the pathogenesis . experiments using wolbachia - containing extracts of o. volvolus in a mouse model of onchocercal keratitis demonstrated that the presence of the bacteria was essential for neutrophil - mediated inflammation , opacity , and corneal haze . the diagnosis of onchocerciasis is accomplished by a combination of clinical symptoms and signs with histopathologic examination of specimens . pcr may aid in the diagnosis of disease associated with a low burden of microfilariae . xenodiagnosis , using laboratory - bred blackflies , may provide a clue as well . traditional therapy has centered on the use of dec , but this is active only against microfilariae , allowing adult worms to repopulate the microfilariae in several months . ivermectin is the treatment of choice and mass distributed by the who onchocerciasis control and the onchocerciasis elimination programme for the americas . this had led to dramatic improvements in disease control to the extent that elimination has become a realistic target . transmission of eyeworms occurs via nonbiting diptera that feed on the ocular secretions , tears , and conjunctiva of animals . the disease , thelaziasis , is characterized by a range of subclinical to clinical signs such as epiphora , conjunctivitis , keratitis , corneal opacity , and ulcers . asymptomatic , subclinical thelaziasis occurs mainly when only the male nematodes parasitize animals , whereas evident symptoms have been more frequently registered in the presence of gravid females . the lateral serration of the thelazia cuticle causes mechanical damage to the conjunctival and corneal epithelium . t. callipaeda nematodes have a serrated cuticle , buccal capsule , mouth opening with a hexagonal profile , and 6 festoons . fortreatment of human cases , the removal of the worm is suggested . topical treatment with thiabendazole hasalso larva migrans in man are a disease characterized by inflammatory reaction around or in the wake of migrating larvae , most commonly larvae of nematode parasites of other animals . for some of the larva migrans producing larvae , man is merely an accidental but more or less normal intermediate or paratenic host . toxocariasis is an important cause of unilateral visual loss and leukocoria in infants , and as a differential diagnosis of retinoblastoma . visceral larva migrans are best known in the form produced by the larvae of toxocara canis , these having been identified in autopsy specimens of lungs , liver , brain , and in several enucleated eyes . human infection by a spiruroid form of nematode gnathostoma spinigerum has been reported sporadically from thailand , the philippines , china , japan , and india . intraocular parasites occur so rarely that they are considered as ophthalmological curiosities , nevertheless , it can cause intraocular hemorrhage , uveitis , and loss of vision within 2 days . following surgical removal , treatment is with albendazole and topical corticosteroids . trichinosis is a parasitic disease which probably presents itself for diagnosis not infrequently . because of its varied symptomatology trichinosis is , unless by chance , almost as frequently undiagnosed ocular trichinosis can manifest itself as oedema of the face especially around the eyes , conjunctivitis , and exophthalmoses . diagnosis is only confirmed by finding the worm in a section of the excised muscle ( see tables 3 and 4 ) . this helminthic infection caused by the larval cysts of the pork tapeworm ( taenia solium ) . ocular involvement is well recognised and includes orbital , intraocular , subretinal , and optic nerve lesions [ 5457 ] . cysticercosis can be evident as a free - floating cyst with amoeboid movements within the vitreous or anterior chamber of the eye . gaze palsies may also occur secondary to intramuscular cysts or cranial nerve lesions from intracerebral cysts . diagnosis depends on imaging with ultrasound , mri , and ct scanning all being useful , depending on the location of the cysts [ 58 , 59 ] . serology can be useful but in cases of isolated cysts may be negative . treatment is largely with the antihelminthic albendazole . antihelminthic therapy may lead to an increased inflammatory reaction around the lesions , and for this reason corticosteroids are often used when treating neurological or ocular disease . spontaneous extrusion of cysts from the orbit may occur , and surgery may be required for isolated ocular lesions when they are growing and causing visual loss . fasciola hepatica is a zoonotic helminth that is prevalent in most sheep - raising countries . in 1989 , an outbreak of human infestation of more than 10 000 cases living in guilan province , iran was reported . the biliary duct of the liver is the main site of establishment of the parasite . however , immature flukes may deviate during migration , entering other organs , and causing an ectopic infestation . in humans , ectopic locations in the orbit [ 62 , 63 ] identification of the route of entry of the parasite larva into the anterior chamber of the eye is difficult . one possible route can be via the central retinal artery into the vitreous , causing vasculitis and endophthalmitis . severe intraocular reaction , haemorrhage , diffuse vasculitis , and retinal ischaemia of the patient may be caused as a result of the presence or irritation of the parasite . early vitrectomy and removal of the parasite resulted in a rapid response , with reasonable final visual acuity . hydatid cysts are most commonly seen in the liver ( 6070% ) and lungs ( 20% ) . hydatid disease involving the orbit represents < 1% of all cases of hydatid disease and requires surgical treatment . laboratory and immunologic tests are generally unhelpful . from the literature and our own observations , orbital hydatid cysts usually appear as a well - defined , thin - walled , oval shape lesions with fine peripheral rim enhancement of their fibrous capsule after contrast medium administration . various theories have been postulated as to the different routs by which the schistosoma ova or even the adult worms can reach the systemic circulation and then after lodged in ectopic sites such as the eyes . cercariae ( the infective stage ) develop to maturity and lay their eggs in the veins directly under the skin or the mucous membrane through which they have penetrated if the part is richly vascularised . the presence of schistosomal eggs in the eye can produce granuloma formation and inflammatory sequelae . considering how common the infection is in endemic areas , involvement of the eye is incredibly rare . effective treatment , using the drug praziquantel , has been available for 25 years , but the growth of human populations in high - risk areas , as well as the high probability of rapid re - infection after treatment , has thwarted efforts to control the number of human infections worldwide ( see tables 5 and 6 ) . genera important to human myiasis include dermatobia , gasterophilus , oestra , cordylobia , chrysomia , wohlfahrtia , cochliomyia , and hypoderma . ophthalmomyiasis may be categorized into three categories : ophthalmomyiasis externa , ophthalmomyiasis interna , and orbital myiasis . ophthalmomyiasis externa is usually seen in areas of shepherding and is typically due to larvae of the sheep nasal botfly , oestra ovis . a crawling or wriggling sensation accompanied by swelling and cellulitis orbital myiasis may be due to a number of fly species and is generally seen in patients who are unable to care for themselves . diagnosis of ophthalmomyiasis is made by demonstration of maggots , and histologic examination may show granuloma formation . capitis , the human head louse ; phthirus pubis , the crab louse . depending on the species , eggs , or nits , are laid and glued to body hairs or clothing fibers . following this , nymphs emerge to feed on the host , giving rise to symptoms of pruritis . of the species in addition to pruritis , small erythematous papules with evidence of excoriation may be present . involvement of the eyelash may cause crusting of the lid margins . in this case , diagnosis is relatively simple as nits are easily seen at the base of the eyelash . eyelid disease is treated with a thick layer of petrolatum twice a day for 8days , or the application of 1% yellow oxide of mercury four times a day for 2 weeks . there are a number of different species of ticks which may cause disease in humans and animals . ticks exist in three life stages larva , nymph , and adult all of which requires blood meals . most tick bites are uncomplicated , and prompt removal of the tick is all that is necessary . in one such case , the nymph was associated with a stinging sensation . following the removal of the tick , a firm nodule , representing a tick bite granuloma , may remain for several weeks . the majority of the clinically important species of parasites involved in eye infections are reviewed in this paper . emphases have been placed on literatures published within the past decade , but prior noteworthy reviews and case reports are included . we searched the medline database via pubmed and identified articles by cross - referencing the terms ocular , eye , ophthalmic , retinitis , endophthalmitis , conjunctivitis , and uveitis to specific infectious diseases in adults . we searched the cochrane database for systematic reviews on the treatment of specific parasitic ocular infections . additionally , we reviewed texts for completeness and to obtain other references of eye complications of systemic infections ( see tables 7 and 8) .

ocular parasitosis in human is more prevalent in geographical areas where environmental factors and poor sanitary conditions favor the parasitism between man and animals . lesions in the eye can be due to damage directly caused by the infectious pathogen , indirect pathology caused by toxic products , or the immune response incited by infections or ectopic parasitism . the epidemiology of parasitic ocular diseases reflects the habitat of the causative parasites as well as the habits and health status of the patient . an ocular examination may provide clues to the underlying disease / infection , and an awareness of the possibilities of travel - related pathology may shed light on an ocular presentation . this paper is a comprehensive review of the parasitic diseases of the eye . the majority of the clinically important species of parasites involved in eye infection are reviewed in this paper . parasites are discussed by the disease or infection they cause .

however , oxygen also poses a potential hazard via reactive oxygen species ( ros ) and reactive nitrogen species ( rns ) , with biological and functional alterations of lipids , proteins , and deoxyribonucleic acid ( dna ) [ 13 ] . therefore , ros / rns have been initially considered as harmful products of the normal aerobic metabolism . the control of ros / rns production plays physiological roles , especially , in regulating cell signaling to involve cell proliferation , differentiation , and apoptosis [ 13 ] . oxidative stress ( os ) mediated by free radicals is defined as an imbalance between the production of ros / rns and the antioxidant capacity of the cell [ 13 ] . the predominant inhibitory neurotransmitter in the brain is -aminobutyric acid ( gaba ) , and almost all researchers have focused on gaba or the regulation of gaba receptor ( gabar ) in the brain [ 48 ] . currently , gaba is considered to be a multifunctional molecule with various physiological effects throughout the body [ 9 , 10 ] . in the brain , many researchers have found that the regulation of gabar has preventive effects against os - induced damage [ 5 , 7 , 8 ] . these results in the brain were mainly explained via specific pathways against os ( i.e. , inhibition of the response to dna damage [ 5 , 11 , 12 ] and promotion of cell survival [ 13 , 14 ] or the free radical scavenging system [ 15 , 16 ] ) . however , in the liver , the effects of gabar regulation have not been reported . orthotopic liver transplantation ( olt ) is an accepted therapy for children and adults with end - stage liver disease , and it currently provides long - term survival and quality lifestyle . however , cold ischemia during organ storage and subsequent reperfusion severely damage the transplanted liver . during cold ischemic preservation , parenchymal cells swell and bleb , and then kupffer and endothelial cells trigger ros / rns production after warm reperfusion . this cold ischemia / warm reperfusion ( ciwr ) injury is still a major cause of morbidity and mortality after olt due to primary graft dysfunction or a nonfunctioning graft . reperfusion not only triggers the liver regeneration cascade but also causes fatal damage in the liver graft due to os [ 18 , 19 ] . proactive strategies through pharmacological pretreatment to limit graft damage from ciwr injury have the advantage of excellent graft function after olt . a small - for - size graft ( sfsg ) is also an issue in deceased - donor liver transplantation ( ddlt ) and living - donor liver transplantation ( ldlt ) . an sfsg is defined as a ratio of graft weight against standard liver volume < 40% [ 20 , 21 ] . an inevitable insufficiency of graft size can not be avoided in ldlt or split orthotopic liver transplantation ( solt ) for ddlt . shear stress not only triggers the liver regeneration cascade but also causes fatal damage in the sfsg by os [ 22 , 23 ] . an sfsg in ldlt or solt is accompanied by ciwr injury , as well as shear stress with portal hypertension . the choice of a left - side graft is preferred from the viewpoint of greater donor safety and expanded donor candidates in ldlt [ 20 , 24 ] . guaranteed solt with successful outcomes resolves a donor shortage in ddlt [ 24 , 25 ] . our laboratory has focused on the effect of gabar regulation on liver damage by using rodent models [ 2628 ] . we failed to show beneficial effects in gabar regulation ex vivo and in gabar regulation by a specific antagonist [ 27 , 28 ] . however , gabar regulation in vivo by a specific agonist showed a subtle reduction in liver damage in a murine hepatectomy model involving shear stress with portal hypertension and in a rat orthotopic liver transplantation model with a whole - liver graft involving ciwr injury . proactive strategies through pharmacological pretreatment to limit graft damage from ciwr injury and shear stress with portal hypertension have advantages for overcoming a current issue . as a final goal of gabar regulation in the liver , we investigated the strategic potential of graft pretreatment in vivo by a gabar agonist in the rat solt model with a 40%-sfsg , and we examined the possible pathways involved . graft donors and recipients were 812-week - old rats ( approximately 250 g ) . the experimental protocols were approved by the ethical committee of our institution ( mayo clinic , institutional animal care and use committee , no . rats were cared for in accordance with the institutional guidelines for animal welfare based on the national institutes of health guide for the care and use of laboratory animals . a dose of 43.56 nmol / g body weight of gabar agonist ( gabaa receptor agonist , muscimol , 114.10 g / mol ; 70015 , fluka , sigma - aldrich co. , st . four hours before graft harvest , the donor rat intravenously received 1.0 ml of gabar agonist into the penile vein . comprehensive details of the surgical procedures for rat solt and postoperative care in our institution have been previously described [ 29 , 30 ] . briefly , the syngeneic graft had a cold ischemic time of 2 h at 4c in normal ringer 's solution . the liver graft was washed twice by 10 ml of normal ringer 's solution , immediately after the graft harvest and before graft implantation . the 40%-sfsg was made by the left median and lateral segments at the back table [ 29 , 30 ] . to avoid any irrelevant signaling , the hepatic artery was reconstructed by ultramicrosurgery in this study [ 29 , 30 ] . each rat was housed separately after surgery , and body temperature was maintained by a heating pad . postoperative observation was performed every 30 min until 6 h after solt , and 1.0 ml of warm lactate ringer 's solution was routinely administered every 1 h until 6 h after solt . in this model , we previously demonstrated the importance of a shortened anhepatic phase and exclusion of unreliable samples based on autopsy findings [ 29 , 30 ] . in this study , the anhepatic phase was maintained within 20 min in each solt , and no surgical complications were observed in each case at autopsy . recipient rats were divided into three groups according to donor treatments and the recipient 's surgery as follows : ( i ) saline ( normal saline , 1.0 ml , i.v . ) and laparotomy , ( ii ) saline ( normal saline , 1.0 ml , i.v . ) and solt with 40%-sfsg , and ( iii ) gabar agonist ( muscimol , 43.56 nmol ( 4.98 g)/g body weight , 1.0 ml , i.v . ) and solt with 40%-sfsg . first , a survival study was performed ( n = 10 in each group ) . cell signalings involving cell proliferation , differentiation , and apoptosis were investigated from the early postoperative period [ 18 , 3133 ] , and subsequently , progressive necrosis was observed [ 18 , 3133 ] . serum , plasma , and liver samples for histopathological / immunohistological assessment and western blotting analyses were then collected 6 h after solt ( n = 5 in each group ) . aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , and total bilirubin ( t - bil ) levels , and the international normalized ratio of prothrombin time ( pt - inr ) were measured . serum ast , alt , and t - bil levels were assessed ( sgot , sgpt , and total bilirubin reagent , respectively , biotron , hemet , ca , usa ) . the pt - inr in plasma was measured by the i - stat system ( abbott , princeton , nj , usa ) . liver tissue was fixed in 10% neutral - buffered formalin , embedded in paraffin , and sliced into 4 m sections . morphological characteristics and graft injury scores were assessed after hematoxylin - eosin ( he ) staining . the graft damage score ( points ) has previously been described elsewhere [ 30 , 34 , 35 ] . scores were counted in 10 fields ( 100 ) in each slide , and then these scores were averaged . induction of apoptosis was assessed by immunostaining of terminal deoxynucleotidyl transferase - mediated deoxyuridine triphosphate nick - end labeling ( tunel ) ( apoptag peroxidase in situ apoptosis detection kit , s7100 , chemicon international , inc . , billerica , ma , usa ) and cysteine aspartic acid protease ( caspase ) 3 ( cleaved caspase-3 ( asp175 ) antibody , 9661s , cell signaling technology , inc . , danvers , ma , usa ) . tunel - positive nuclei were stained brown , and negative nuclei were counterstained light blue . slides were scanned with an automated high - throughput scanning system ( scanscope xt , aperio technologies , inc . , vista , ca , usa ) . to quantify the immunohistological findings , positively stained nuclei were counted by aperio imagescope software ( aperio technologies , inc . ) . all nuclei were classified into four color intensity levels , and the higher two levels were considered as positive . the ratio of positively stained nuclei to all nuclei was calculated , and the mean ratio per mm was determined . the primary antibodies for 4-hydroxynonenal ( 4-hne ) ( 4 hydroxynonenal antibody , ab46545 , abcam , cambridge , ma , usa ) , ataxia - telangiectasia mutated kinase ( atm ) ( phospho - atm / atr substrate rabbit mab , cell signaling technology ) , phosphorylated histone h2ax ( phospho - histone h2ax antibody , 2577 , cell signaling technology ) , phosphatidylinositol-3 kinase ( pi3k ) ( phospho - pi3k p85/p55 antibody , 4228 , cell signaling technology ) , akt ( phospho - akt rabbit mab , 4058 , cell signaling technology ) , superoxide dismutase ( sod ) 1 ( cu / zn superoxide dismutase , ls - b2907 , lifespan biosciences , seattle , wa , usa ) , sod 2 ( mn superoxide dismutase , ls - c62194 , lifespan biosciences ) , and catalase ( catalase , ls - b2554 , lifespan biosciences ) were used . liver samples were collected , homogenized , and centrifuged at high speed for 10 min at 4c . the supernatant was then collected and used for bicinchoninic acid protein determination ( bca protein assay reagent , thermo fisher scientific , rockford , il , usa ) and western blot analysis . forty micrograms of protein were run on 420% tris - glycine gels and transferred onto 0.45 m nitrocellulose membranes . the membranes were then blocked with 5% nonfat milk made up in a tris - buffered saline solution . after blocking , the next day , the membranes were washed three times for 10 min with tris - buffered saline solution and then incubated with the peroxidase - conjugated secondary antibody for 1 h , with shaking at room temperature . after incubation , the membranes were once again washed three times for 10 min with tris - buffered saline solution and then developed using chemiluminescence . signals were quantified by using imagequant 5.0 software ( molecular dynamics , sunnyvale , ca , usa ) . the student 's t - test was used for the comparison of unpaired continuous variables between groups . survival curves were constructed by the kaplan - meier method ( log - rank test ) . solt with a 40%-sfsg clearly showed poorer survival than laparotomy ( p < 0.0001 ) , and graft pretreatment by gabar agonist prolonged survival after solt ( p = 0.0369 ) . inflammatory cell infiltration , vacuolization , hepatocyte ballooning , and necrosis were confirmed after solt with a 40%-sfsg . actual histopathological findings in h - e staining are shown in each group in figures 1(b)1(d ) . there were significant differences between laparotomy and solt with saline ( 0.0 0.0 versus 5.8 1.1 points , p < 0.0001 ) and between solt with saline and solt with gabar agonist ( 5.8 1.1 versus 4.1 1.0 points ; p = 0.0280 ) ( figure 1(e ) ) . there were significant differences in serum ast levels between laparotomy and solt with saline ( 45.4 10.3 versus 387.4 36.8 u / l ; p < 0.0001 ) and between solt with saline and solt with gabar agonist ( 387.4 36.8 versus 296.0 32.3 there were significant differences in serum alt levels between laparotomy and solt with saline ( 54.2 9.2 versus 354.2 32.1 u / l ; p < 0.0001 ) and between solt with saline and solt with gabar agonist ( 354.2 32.1 versus 272.4 31.3 there were significant differences in serum t - bil levels between laparotomy and solt with saline ( 0.28 0.04 versus 1.37 0.29 mg / dl ; p < 0.0001 ) and between solt with saline and solt with gabar agonist ( 1.37 0.29 versus 1.02 0.15 mg / dl ; p = 0.0453 ) ( figure 2(c ) ) . there were significant differences in pt - inr between laparotomy and solt with saline ( 0.99 0.04 versus 1.22 0.06 ; p = 0.0001 ) and between solt with saline and solt with gabar agonist ( 1.22 0.06 versus 1.13 0.06 ; p = 0.0456 ) ( figure 2(d ) ) . the ratio of tunel - positive nuclei was significantly different between laparotomy and solt with saline ( 0.001 0.002 versus 0.166 0.052 ; p < 0.0001 ) and between solt with saline and solt with gabar agonist ( 0.166 0.052 versus 0.092 0.038 ; p = 0.0324 ) ( figure 3(d ) ) . the ratio of caspase 3-positive nuclei was significantly different between laparotomy and solt with saline ( 0.0001 0.0001 versus 0.115 0.019 ; p < 0.0001 ) and between solt with saline and solt with gabar agonist ( 0.115 0.019 versus 0.080 0.024 ; p = 0.0347 ) ( figure 4(d ) ) . normalized 4-hne showed significant differences between laparotomy and solt with saline ( 1.00 0.06 versus 1.38 0.22 ; p = 0.0068 ) and between solt with saline and solt with gabar agonist ( 1.38 0.22 versus 1.05 0.15 ; p = 0.0276 ) ( figure 5(b ) ) . actual intensities of atm and h2ax in each group are shown in figure 6(a ) . normalized atm showed significant differences between laparotomy and solt with saline ( 1.00 0.11 versus 1.21 0.11 ; p = 0.0131 ) and between solt with saline and solt with gabar agonist ( 1.21 0.11 versus 0.90 0.28 ; p = 0.0477 ) ( figure 6(b ) ) . normalized h2ax showed significant differences between laparotomy and solt with saline ( 1.00 0.10 versus 2.59 0.66 ; p = 0.0007 ) and between solt with saline and solt with gabar agonist ( 2.59 0.66 versus 0.83 0.25 ; p = 0.0005 ) ( figure 6(c ) ) . actual intensities of pi3k and akt in each group are shown in figure 7(a ) . normalized pi3k showed significant differences between laparotomy and solt with saline ( 1.00 0.11 versus 0.59 0.27 ; p = 0.0139 ) and between solt with saline and solt with gabar agonist ( 0.59 0.27 versus 0.92 0.13 ; p = 0.0443 ) ( figure 7(b ) ) . normalized akt showed significant differences between laparotomy and solt with saline ( 1.00 0.12 versus 0.34 0.24 ; p = 0.0006 ) and between solt with saline and solt with gabar agonist ( 0.34 0.24 versus 1.11 0.22 ; p = 0.0007 ) ( figure 7(c ) ) . actual intensities of sod 1 , sod 2 , and catalase in each group are shown in figure 8(a ) . normalized sod 1 showed significant differences between laparotomy and solt with saline ( 1.00 0.10 versus 0.81 0.16 ; p = 0.0445 ) but not between solt with saline and solt with gabar agonist ( 0.81 0.16 versus 0.82 0.12 ; p = 0.8248 ) ( figure 8(b ) ) . normalized sod 2 showed significant differences between laparotomy and solt with saline ( 1.00 0.13 versus 0.79 0.14 ; p = 0.0361 ) but not between solt with saline and solt with gabar agonist ( 0.79 0.14 versus 0.84 0.15 ; p = 0.5765 ) ( figure 8(c ) ) . normalized catalase showed no significant differences between laparotomy and solt with saline ( 1.00 0.14 versus 0.95 0.14 ; p = 0.6904 ) and between solt with saline and solt with gabar agonist ( 0.95 0.14 versus 0.96 0.26 ; p = 0.9764 ) ( figure 8(d ) ) . based on the current situation in the clinical field , the 40%-sfsg needs to be investigated in detail because successful solt overcomes a donor shortage in ddlt , and the shift to a left - lobe graft provides donor safety in ldlt [ 20 , 24 , 30 ] . however , the 40%-sfsg is prone to ischemia / reperfusion injury and shear stress with portal hypertension , and therefore , the os - induced damage after solt is more fatal [ 18 , 3638 ] . in our study , a survival study , biochemical assays , and histopathological assessment showed that the 40%-sfsg received the liver injury enough . os causes dna damage and subsequent apoptosis [ 13 ] , and in our study , immunohistochemistry showed that solt induced apoptosis in the 40%-sfsg . ros / rns can attack and damage a variety of critical biological molecules [ 13 ] , and the products of lipid peroxidation reliably and rapidly reflect sensitive and specific signals due to os occurring in vivo [ 39 , 40 ] . the fatty aldehyde 4-hne is an end product of lipoperoxidation [ 39 , 40 ] . therefore , os after solt with a 40%-sfsg resulted in apoptotic induction and subsequent necrosis . os mediated by free radicals is defined as an imbalance between the production of ros / rns and antioxidant capacity [ 13 ] . ros / rns have been suggested as a major contributing factor for dna damage in the progression of os . as a sensor of dna damage responses , the protein kinase atm can be initiated through rapid intermolecular autophosphorylation induced by dna damage [ 12 , 41 ] ; it phosphorylates various proteins , and subsequently amplifies the responses to dna damage . h2ax is required for cell cycle arrest and dna repair following double - stranded dna breaks [ 5 , 42 ] . dna damage results in the rapid phosphorylation of h2ax by atm at sites of dna damage [ 5 , 4345 ] . our study showed that this response to and repair of dna damage via atm / h2ax was clearly triggered after solt with a 40%-sfsg and that this cascade is a possible pathway in the process of os - induced injury after solt with sfsg . our preliminary data in the rat olt model with whole - liver grafts ( i.e. , a model for only ciwr injury ) suggested that gabar regulation by a specific agonist showed differences in atm / h2ax . we consider that gabar regulation may have a beneficial effect against ciwr injury via the atm / h2ax pathway in the liver . from the viewpoint of the production of ros / rns in the process of os , akt also plays a critical role in controlling apoptosis [ 41 , 46 , 47 ] and promotes cell survival [ 4750 ] . apoptotic machinery is inhibited by the activation of akt [ 46 , 51 , 52 ] . akt is a component of the antiapoptotic process related to the activation of pi3k , and pi3k is upstream from akt [ 47 , 53 ] . the cell survival pathway via pi3k / akt is also considered as an important signaling pathway to control apoptotic induction in the liver [ 54 , 55 ] . our study showed that this promotion of cell survival via pi3k / akt was disturbed after solt with a 40%-sfsg and that this cascade could be one of the possible pathways in the process of os - induced injury after solt with sfsg . our preliminary data in the murine hepatectomy model ( i.e. , a model for only shear stress with portal hypertension ) suggested that gabar regulation by a specific agonist showed differences in pi3k / akt . therefore , we consider that gabar regulation may have a beneficial effect against shear stress with portal hypertension via the pi3k / akt pathway in the liver . from the viewpoint of antioxidant defenses , free radical scavenging enzymes , such as sod and catalase , also play an important role in reducing dna damage and subsequent apoptosis [ 2 , 3 , 56 ] . normal cells are able to defend themselves against os through this scavenging system [ 3 , 56 ] . our study showed a decrease in sod 1 and sod 2 levels after solt with a 40%-sfsg , although we initially expected that antioxidant enzymes would increase . our results appear to be consistent with a previous opinion that os impairs mitochondrial importing and processing of sod . however , another possible explanation for our results may be that this scavenging system failed , and some reactive molecules evaded the detoxification process and damaged potential targets because of drastic damage after solt with a 40%-sfsg , even though these scavenging enzymes can handle large amounts of ros / rns . our results of the survival study , histopathological assessment , and biochemical assays showed that pretreatment for sfsg by gabar regulation in vivo affected graft damage after solt . moreover , immunohistochemistry showed that this pretreatment reduced apoptotic induction after solt . in the field of brain research , the effect of gabar regulation on the prevention of os has been reported [ 57 ] . although gaba was initially thought to be confined to the central nervous system , gaba is currently considered to be a multifunctional molecule with various physiological effects throughout the body [ 9 , 10 ] . although the liver contains gaba and hepatic gabar [ 10 , 17 ] , the effects of gabar regulation in the liver are unknown . our study suggests that gabar regulation may have a strategic potential for 40%-sfsgs as a pharmacological pretreatment for reducing os - induced damage after solt , although solt with a 40%-sfsg involves fatal os due to dual damage ( i.e. , ciwr injury and shear stress with portal hypertension ) . whether gabar regulation ex vivo ( i.e. , a procedure during organ storage ) is more suitable for ldlt is unknown . although our results showed the strategic potential of gabar regulation in vivo as a pretreatment for liver grafts , we failed to confirm a positive effect of gabar regulation ex vivo . therefore , some innovations are still required for clinical application . in previous reports on the brain , many investigators have suggested that gabar regulation by a specific agonist or antagonist affects the response to reduce os - induced injury [ 5 , 7 , 8 ] . their preventive effects in the brain have been mainly explained via specific pathways against os ( i.e. , inhibition of the response to dna damage [ 5 , 11 , 12 ] and promotion of cell survival [ 13 , 14 ] or the free radical scavenging system [ 15 , 16 ] ) . many previous investigators have suggested that gabar regulation in the brain has certain effects on the response to and repair of dna damage via the atm / h2ax pathway in vivo and in vitro in the process of os [ 5 , 11 , 12 ] . our study showed that the regulation of hepatic gabar also appeared to reduce os - induced dna damage via the atm / h2ax pathway as well as to have effects in the brain . with regard to the effects of gabar regulation on os in the brain , the pi3k / akt pathway promotes cell survival against dna damage [ 5 , 13 , 14 , 46 , 59 ] . our study showed that regulation of hepatic gabar appeared to promote cell survival via the pi3k / akt pathway against os - induced dna damage as well as to have effects in the brain . however , antioxidant enzymes reduce os - induced damage . from the viewpoint of this scavenging system , some researchers have shown that gabar regulation in the brain has preventive effects against os - induced damage via antioxidant enzymes [ 15 , 16 ] . although sod 2 plays an important role in preventing dna damage in the sfsg , our results suggested that the effects of the regulation of hepatic gabar against os did not depend on this scavenging system . overall , we speculate that the regulation of hepatic gabar has a preventive effect against os , by reducing dna damage via the atm / h2ax pathway and by promoting cell survival via the pi3k / akt pathway . however , antioxidant enzymes might be important for gabar regulation in the brain [ 15 , 16 ] . in conclusion , regulation of gabar by a specific agonist in vivo works well in the liver , as well as the brain . even though ciwr injury and shear stress with portal hypertension affect 40%-sfsgs after solt and results in fatal os , graft pretreatment in vivo by gabar regulation clearly improves graft damage after solt . this strategy may be advantageous for overcoming current issues in the ddlt and ldlt fields . the effects of gabar regulation on graft damage after solt with a 40%-sfsg appear to prevent os by reducing dna damage via the atm / h2ax pathway and by promoting cell survival via the pi3k / akt pathway .

background . graft pretreatment to limit postoperative damage has the advantage of overcoming a current issue in liver transplantation ( lt ) . the strategic potential of graft pretreatment in vivo by a specific agonist for -aminobutyric acid receptor ( gabar ) was investigated in the rat lt model with a small - for - size graft ( sfsg ) . methods . recipient rats were divided into three groups according to donor treatments and recipient surgeries : ( i ) saline and laparotomy , ( ii ) saline and split orthotopic liver transplantation ( solt ) with 40%-sfsg , and ( iii ) gabar agonist and solt with 40%-sfsg . survival was evaluated . blood and liver samples were collected 6 h after surgery . immunohistological assessment for apoptotic induction and western blotting for 4-hydroxynonenal , ataxia - telangiectasia mutated kinase ( atm ) , histone h2ax , phosphatidylinositol-3 kinase ( pi3k ) , akt , and free radical scavenging enzymes were performed . results . pretreatment by gabar showed improvement in survival , histopathological assessment , and biochemical tests . apoptotic induction and oxidative stress were observed after solt with an sfsg , and this damage was limited by gabar regulation . gabar regulation appeared to reduce dna damage via the atm / h2ax pathway and to promote cell survival via the pi3k / akt pathway . conclusions . pretreatment in vivo by gabar regulation improves graft damage after solt with an sfsg . this strategy may be advantageous in lt .

heart failure ( hf ) can result from cardiac overload or injury as well as from a complex interplay of genetic , neurohormonal , and inflammatory factors . hf is classified as being associated with a reduced ejection fraction ( hfref ) or with a normal ejection fraction ( hfnef ) . the percentages of patients and the mortality rates are similar in patients with hfref and hfnef . the likelihood of survival is higher in patients with hfref than in those with hfnef , and the survival rate in hfnef has not significantly improved in recent years . various biomarkers , including those for inflammation and neurohormones , provide important information about the pathogenesis , risk stratification , and diagnosis of hf and are used to monitor response to therapy . inflammatory markers are closely associated with pathogenesis , poor functional state , and adverse prognosis in patients with hf . pentraxin 3 ( ptx3 ) is a newly identified member of the pentraxin superfamily , which includes creactive protein ( crp ) and serum amyloid p. in contrast to crp , which is produced by the liver , ptx3 is produced by various cell types in various tissues , especially in the vasculature , in response to inflammatory stimuli . ptx3 may reflect local inflammatory status in tissues and thus may be a new biomarker of inflammation . we recently demonstrated that plasma levels of ptx3 , but not highsensitivity crp ( hscrp ) , were significantly higher in patients with hfnef than in nonhf patients . in addition , these elevated levels of ptx3 were significantly and independently correlated with the presence of hfnef in patients with normal left ventricular ejection fraction ( lvef ) . other studies have also shown that ptx3 is a predictor of adverse clinical outcome in patients with hfref . although much attention has been focused on hfref , less is known regarding factors that correlate with clinical outcome in patients with hfnef . the irbesartan in heart failure with preserved ejection fraction ( ipreserve ) study demonstrated that age , presence of diabetes mellitus , history of hospitalization for hf , new york heart association classification , nterminal probtype natriuretic peptide levels , and lvef were associated with adverse outcomes in patients with hfnef . this study was designed to determine the ability of plasma ptx3 levels to predict future cardiovascular events in patients with hfnef . we initially screened 747 chronic hf patients under a clinically stable condition with new york heart association functional class ii to iv who were referred to kumamoto university hospital , kumamoto , japan . none of the patients had any noncardiac causes of hflike symptoms , especially lung disease ( eg , chronic obstructive lung disease ) . the diagnosis of hfnef was based on the criteria formulated by the european working group on hfnef . hfref was defined as reduced systolic left ventricular ( lv ) function ( lvef50% ) . hfnef was defined as normal or mildly abnormal systolic lv function ( lvef>50% ) with lv diastolic dysfunction . lv diastolic dysfunction for hfnef was defined as ( 1 ) a ratio of mitral early diastolic peak flow velocity to tissue doppler early mitral annular diastolic velocity ( e / e ) 15 ; ( 2 ) e / e 8 and < 15 and btype natriuretic peptide ( bnp ) levels > 200 pg / ml ; or ( 3 ) e / e 8 and < 15 and lv mass index ( lvmi ) 122 g / m ( females ) or 149 g / m ( males ) . patients were excluded if they had active systemic inflammatory disease , chronic renal failure requiring hemodialysis , collagen disease , presence of malignancy , or acute coronary syndrome within 3 months preceding enrollment . the study complied with the declaration of helsinki regarding investigation in humans and was approved by the institutional review committee of each institution . the study was also conducted in accordance with the guidelines of the ethics committee of our institution . this study was registered at the university hospital medical information network clinical trials registry with the identification number umin000002170 . flow chart showing the protocol that was used for enrollment of hfnef patients in the present study . bnp indicates btype natriuretic peptide ; e / e , ratio of mitral early diastolic peak flow velocity to tissue doppler early mitral annular diastolic velocity ; hf , heart failure ; hfnef , hf with normal ejection fraction ; lvef , left ventricular ejection fraction ; lvmi , left ventricular mass index ; nyha , new york heart association . ptx3 levels were measured using a highsensitivity enzymelinked immunosorbent assay system ( values ranged from 0.1 to 20 ng / ml ; perseus proteomics ) . other factors that were measured included serum hscrp levels and plasma tumor necrosis factor , interleukin6 , and bnp levels . echocardiography was performed in standard parasternal and apical views by a specialized echocardiologist using commercially available ultrasound systems ( vivid 7 , gevingmed ultrasound ; aplio xg , toshiba ) . lvef was measured in biplane apical ( 2 and 4chamber ) views using a modification of simpson 's method . the e / e ratio is used as an index of lv filling pressure and abnormal lv relaxation . to assess lv diastolic function , e / e was measured , as described previously , as was the medial annulus of e / e. lv mass was calculated , as described previously , and the lvmi was expressed relative to body surface area . patients with hfnef were followed prospectively at the outpatient clinic until december 2012 or until an endpoint occurred . the endpoint was a composite of cardiovascular death , nonfatal mi , unstable angina pectoris , nonfatal ischemic stroke , hospitalization for hf decompensation , or coronary revascularization . cardiovascular events were ascertained from a review of medical records and confirmed by direct contact with the patients , their families , and physicians . patients were divided into low and highptx3 groups based on the median concentration of ptx3 ( 3.0 ng / ml ) . cardiovascular death was defined as death because of mi ( within 28 days ) , congestive heart failure , or documented sudden death without apparent noncardiovascular causes . mi was diagnosed as a rise or fall in cardiac biomarkers ( plasma creatine kinasemb or cardiac troponint ) greater than the 99th percentile of the upper limit of normal , together with evidence of myocardial ischemia with at least one of the following : ecg changes ( recent stt changes , left bundle branch block , or a pathological q wave ) or imaging evidence of new loss of viable myocardium or new regional wall motion abnormality . unstable angina pectoris was diagnosed from new or accelerating symptoms of myocardial ischemia accompanied by recent ischemic sttwave changes . ischemic stroke was diagnosed by both neurological symptoms and radiological evidence , excluding intracranial hemorrhage . hospitalization for hf decompensation was defined as admission for symptoms typical of hf with objective signs of worsening hf requiring intravenous drug administration . coronary revascularization was defined as coronary artery bypass grafting or percutaneous coronary intervention in patients with stable angina pectoris . for subjects experiencing 2 or more cardiovascular events , parameters with a normal distribution , as assessed by the shapirowilk test , are expressed as meansd . parameters with a skewed distribution , such as ptx3 , hscrp , tumor necrosis factor , interleukin6 , and bnp levels , as well as e / e and lvmi , are shown as median values ( interquartile range ) and were logarithmically transformed before linear regression analysis . differences between continuous variables were analyzed by the unpaired t test and the mannwhitney u test , as appropriate . linear regression analysis was used to determine the associations between ptx3 and bnp levels , lvmi , inflammatory makers , and cardiovascular events . survival data were analyzed by the kaplanmeier method and assessed by the logrank test . age and the ability of any marker to predict cardiovascular events was assessed by cox proportional hazards regression analysis . in cox proportional hazards regression analyses , associations between groups and all other parameters were first analyzed by univariate analysis followed by multivariable analysis after adjustment for factors that were significant with univariate analysis . model 1 incorporated the inflammatory makers ptx3 , hscrp , tumor necrosis factor , interleukin6 , and bnp . model 2 incorporated the 5 prognostic factors ( pf5 ) that were identified during the ipreserve study in patients with hfnef age , presence of diabetes mellitus , previous hospitalization for hf , new york heart association classification , and lvef as well as bnp and ptx3 . model 4 incorporated pf5 , bnp , hscrp , and ptx3 . estimates of the cstatistic for cox proportional hazards regression models were calculated . calibration of cox regression models was also performed by the grnnesby and borgan calibration test . the incremental effects of addition of ptx3 to pf5 and bnp levels to predict future cardiovascular events were evaluated using the net classification index ( nri ) , as previously described . patients were stratified into 1 of 3 risk categories based on pf5 and bnp levels measured during the mean 30month followup period : low risk ( 0% to < 10% ) , intermediate risk ( 10% to 20% ) , or high risk ( > 20% ) . ptx3 was subsequently used to reclassify the risk category for ascertaining whether there would be improvement in the nri . the nri was calculated using the following equation : nri=([number of events reclassified as highernumber of events reclassified as lower]/number of events)([number of nonevents reclassified as lowernumber of nonevents reclassified as higher]/number of nonevents ) . all analyses were performed using spss version 19.0j for windows ( ibm corporation ) , stata version 11 ( stata corporation ) , and sas version 9.1.3 ( sas institute inc ) . we initially screened 747 chronic hf patients under a clinically stable condition with new york heart association functional class ii to iv who were referred to kumamoto university hospital , kumamoto , japan . none of the patients had any noncardiac causes of hflike symptoms , especially lung disease ( eg , chronic obstructive lung disease ) . the diagnosis of hfnef was based on the criteria formulated by the european working group on hfnef . hfref was defined as reduced systolic left ventricular ( lv ) function ( lvef50% ) . hfnef was defined as normal or mildly abnormal systolic lv function ( lvef>50% ) with lv diastolic dysfunction . lv diastolic dysfunction for hfnef was defined as ( 1 ) a ratio of mitral early diastolic peak flow velocity to tissue doppler early mitral annular diastolic velocity ( e / e ) 15 ; ( 2 ) e / e 8 and < 15 and btype natriuretic peptide ( bnp ) levels > 200 pg / ml ; or ( 3 ) e / e 8 and < 15 and lv mass index ( lvmi ) 122 g / m ( females ) or 149 g / m ( males ) . patients were excluded if they had active systemic inflammatory disease , chronic renal failure requiring hemodialysis , collagen disease , presence of malignancy , or acute coronary syndrome within 3 months preceding enrollment . the study complied with the declaration of helsinki regarding investigation in humans and was approved by the institutional review committee of each institution . the study was also conducted in accordance with the guidelines of the ethics committee of our institution . this study was registered at the university hospital medical information network clinical trials registry with the identification number umin000002170 . flow chart showing the protocol that was used for enrollment of hfnef patients in the present study . bnp indicates btype natriuretic peptide ; e / e , ratio of mitral early diastolic peak flow velocity to tissue doppler early mitral annular diastolic velocity ; hf , heart failure ; hfnef , hf with normal ejection fraction ; lvef , left ventricular ejection fraction ; lvmi , left ventricular mass index ; nyha , new york heart association . ptx3 levels were measured using a highsensitivity enzymelinked immunosorbent assay system ( values ranged from 0.1 to 20 ng / ml ; perseus proteomics ) . other factors that were measured included serum hscrp levels and plasma tumor necrosis factor , interleukin6 , and bnp levels . echocardiography was performed in standard parasternal and apical views by a specialized echocardiologist using commercially available ultrasound systems ( vivid 7 , gevingmed ultrasound ; aplio xg , toshiba ) . lvef was measured in biplane apical ( 2 and 4chamber ) views using a modification of simpson 's method . the e / e ratio is used as an index of lv filling pressure and abnormal lv relaxation . to assess lv diastolic function , e / e was measured , as described previously , as was the medial annulus of e / e. lv mass was calculated , as described previously , and the lvmi was expressed relative to body surface area . patients with hfnef were followed prospectively at the outpatient clinic until december 2012 or until an endpoint occurred . the endpoint was a composite of cardiovascular death , nonfatal mi , unstable angina pectoris , nonfatal ischemic stroke , hospitalization for hf decompensation , or coronary revascularization . cardiovascular events were ascertained from a review of medical records and confirmed by direct contact with the patients , their families , and physicians . patients were divided into low and highptx3 groups based on the median concentration of ptx3 ( 3.0 ng / ml ) . cardiovascular death was defined as death because of mi ( within 28 days ) , congestive heart failure , or documented sudden death without apparent noncardiovascular causes . mi was diagnosed as a rise or fall in cardiac biomarkers ( plasma creatine kinasemb or cardiac troponint ) greater than the 99th percentile of the upper limit of normal , together with evidence of myocardial ischemia with at least one of the following : ecg changes ( recent stt changes , left bundle branch block , or a pathological q wave ) or imaging evidence of new loss of viable myocardium or new regional wall motion abnormality . unstable angina pectoris was diagnosed from new or accelerating symptoms of myocardial ischemia accompanied by recent ischemic sttwave changes . ischemic stroke was diagnosed by both neurological symptoms and radiological evidence , excluding intracranial hemorrhage . hospitalization for hf decompensation was defined as admission for symptoms typical of hf with objective signs of worsening hf requiring intravenous drug administration . coronary revascularization was defined as coronary artery bypass grafting or percutaneous coronary intervention in patients with stable angina pectoris . for subjects experiencing 2 or more cardiovascular events , parameters with a normal distribution , as assessed by the shapirowilk test , are expressed as meansd . parameters with a skewed distribution , such as ptx3 , hscrp , tumor necrosis factor , interleukin6 , and bnp levels , as well as e / e and lvmi , are shown as median values ( interquartile range ) and were logarithmically transformed before linear regression analysis . differences between continuous variables were analyzed by the unpaired t test and the mannwhitney u test , as appropriate . linear regression analysis was used to determine the associations between ptx3 and bnp levels , lvmi , inflammatory makers , and cardiovascular events . the ability of any marker to predict cardiovascular events was assessed by cox proportional hazards regression analysis . in cox proportional hazards regression analyses , associations between groups and all other parameters were first analyzed by univariate analysis followed by multivariable analysis after adjustment for factors that were significant with univariate analysis . model 1 incorporated the inflammatory makers ptx3 , hscrp , tumor necrosis factor , interleukin6 , and bnp . model 2 incorporated the 5 prognostic factors ( pf5 ) that were identified during the ipreserve study in patients with hfnef age , presence of diabetes mellitus , previous hospitalization for hf , new york heart association classification , and lvef as well as bnp and ptx3 . calibration of cox regression models was also performed by the grnnesby and borgan calibration test . the incremental effects of addition of ptx3 to pf5 and bnp levels to predict future cardiovascular events were evaluated using the net classification index ( nri ) , as previously described . patients were stratified into 1 of 3 risk categories based on pf5 and bnp levels measured during the mean 30month followup period : low risk ( 0% to < 10% ) , intermediate risk ( 10% to 20% ) , or high risk ( > 20% ) . ptx3 was subsequently used to reclassify the risk category for ascertaining whether there would be improvement in the nri . the nri was calculated using the following equation : nri=([number of events reclassified as highernumber of events reclassified as lower]/number of events)([number of nonevents reclassified as lowernumber of nonevents reclassified as higher]/number of nonevents ) . all analyses were performed using spss version 19.0j for windows ( ibm corporation ) , stata version 11 ( stata corporation ) , and sas version 9.1.3 ( sas institute inc ) . table 1 shows the clinical characteristics of the participating patients . the median ptx3 level was 3.0 ng / ml . mean age , new york heart association class , and bnp levels were higher and mean body mass index , waist circumference and estimated glomerular filtration rate were lower in the highptx3 group ( > 3.0 ng / ml ) than in the lowptx3 group ( 3.0 ng / ml ) . lvmi values were significantly higher in the highptx3 group than in the lowptx3 group , whereas lvef and e / e were similar in these 2 groups . simple linear regression analysis showed a positive and significant correlation between ln(ptx3 ) and ln(bnp ) ( r=0.295 , p<0.001 ) ( figure 2a ) and a significant but weak positive correlation between ln(ptx3 ) and ln(lvmi ) ( r=0.114 , p<0.05 ) ( figure 2b ) . furthermore , simple linear regression analysis showed a significant but weak positive correlation between ln(ptx3 ) and ln(tumor necrosis factor ) ( r=0.106 , p<0.05 ) but not ln(hscrp ) ( p=0.11 ) or ln(interleukin6 ) ( p=0.80 ) . demographic and clinical characteristics of patients with hfnef data are meansd , number of patients ( % ) , or median ( interquartile range ) . acei indicates angiotensinconverting enzyme inhibitors ; arb , angiotensin ii receptor blocker ; bnp , btype natriuretic peptide ; cad , coronary artery disease ; e / e , mitral early diastolic peak flow velocity to tissue doppler early mitral annular diastolic velocity ; gfr , glomerular filtration rate ; hdl , highdensity lipoprotein ; hf , heart failure ; hfnef , heart failure with normal ejection fraction ; hscrp , highsensitive creactive protein ; il6 , interleukin6 ; ldl , lowdensity lipoprotein ; lv , left ventricle ; lvef , left ventricular ejection fraction ; nyha , new york heart association ; ptx3 , pentraxin 3 ; tnf , tumor necrosis factor. relationships between ptx3 and bnp levels and ptx3 levels and lvmi . simple linear regression analysis shows a positive and significant correlation between ( a ) ln(ptx3 ) and ln(bnp ) ( r=0.295 , p<0.001 ) and ( b ) a significant but weak positive correlation between ln(ptx3 ) and ln(lvmi ) ( r=0.114 , p<0.05 ) . bnp indicates btype natriuretic peptide ; lvmi ; left ventricular mass index ; ptx3 , pentraxin 3 . the mean followup period was 30 months ( range : 1 to 59 months ) . during this time , 106 of these patients experienced cardiovascular events , including cardiovascular death ( n=9 ) , nonfatal mi ( n=4 ) , unstable angina pectoris ( n=13 ) , ischemic stroke ( n=9 ) , hospitalization for hf decompensation ( n=41 ) , and coronary revascularization ( n=30 ) . the frequency of cardiovascular events was significantly higher in the highptx3 group ( n=75 ) than in the lowptx3 group ( n=31 , p<0.001 ) ( table 2 ) . hfrelated cardiovascular events , including cardiovascular death and hospitalization for hf decompensation , were significantly higher in the highptx3 group ( n=41 ) than in the lowptx3 group ( n=9 ) ( p<0.001 ) ( table 2 ) . kaplanmeier analysis showed that the probability of cardiovascular events was also significantly higher in the highptx3 group than in the lowptx3 group ( p<0.001 , logrank test ) ( figure 3a ) . cardiovascular events in hfnef patients with low and high plasma ptx3 levels differences between the groups were assessed by the logrank test . hf indicates heart failure ; hfrelated cardiovascular events , cardiovascular death and hospitalization for hf decompensation ; hfnef , heart failure with normal left ventricular ejection fraction ; ptx3 , pentraxin 3 . kaplanmeier analysis for the probability of cardiovascular events . patients with ( a ) high ptx3 levels and low ptx3 levels , ( b ) subgroups of patients with high and low ptx3 levels plus bnp , and ( c ) subgroups of new york heart association class ii patients with high and low ptx3 levels are shown . bnp indicates btype natriuretic peptide ; ptx3 , pentraxin 3 . the combination of ptx3 and bnp levels led to identification of subgroups ( n=116 , highptx3 and bnp group ; n=64 , highptx3 and lowbnp group ; n=64 , lowptx3 and highbnp group ; and n=116 , lowptx3 and bnp group ) with significantly different probabilities of cardiovascular events ( p<0.001 , logrank test ) ( figure 3b ) . in the lowbnp ( p=0.02 ) and highbnp ( p<0.01 ) groups , the probability of cardiovascular events was significantly higher in those with high ptx3 levels than in those with low ptx3 levels . even among patients with mild hf symptoms ( new york heart association class ii ) , the outcomes were poorer in patients with high ptx3 levels than in those with low ptx3 levels ( p<0.001 , logrank test ) ( figure 3c ) . the results of univariate and multivariable cox proportional hazards analyses for cardiovascular events are shown in tables 3 and 4 . multivariable cox proportional hazards analysis identified ptx3 ( hazard ratio [ hr ] : 1.16 ; 95% ci : 1.05 to 1.27 ; p<0.01 ) , highdensity lipoprotein cholesterol ( hr : 0.99 ; 95% ci : 0.97 to 0.99 ; p<0.05 ) , lvmi ( hr : 1.03 ; 95% ci : 1.05 to 1.27 ) , and bnp ( hr : 1.08 ; 95% ci : 1.03 to 1.14 ; p<0.001 ) as predictors of future cardiovascular events after adjustment for significant factors that were identified by univariate analysis ( table 3 ) . using the forced inclusion model , which evaluated levels of inflammatory markers and bnp ( model 1 ) in multivariable cox hazards analysis , ptx3 , but not hscrp , significantly predicted cardiovascular events ( hr : 1.17 ; 95% ci : 1.07 to 1.28 ; p<0.001 ) . in the forced inclusion model , which included pf5 and bnp , ptx3 significantly predicted cardiovascular events ( model 2 : hr : 1.16 ; 95% ci : 1.05 to 1.27 ; p<0.01 ; model 4 : hr : 1.16 ; 95% ci : 1.06 to 1.27 ; p<0.01 ) ( table 4 ) . furthermore , multivariable cox proportional hazards analysis identified ptx3 ( hr : 1.16 ; 95% ci : 1.01 to 1.35 ; p<0.05 ) and bnp ( hr : 1.21 ; 95% ci : 1.11 to 1.31 ; p<0.001 ) as predictors of future hfrelated cardiovascular events , including cardiovascular death and hospitalization for hf decompensation , after adjustment for significant factors identified by univariate analysis . we classified patients with hfnef into 4 groups according to the levels of ln(ptx3 ) : first quartile ( < 0.59 , ptx3 : < 1.80 ng / ml ) , second quartile ( 0.59 to 1.10 , ptx3 : 1.80 to 3.00 ng / ml ) , third quartile ( 1.10 to 1.35 , ptx3 : 3.00 to 3.85 ng / ml ) , and fourth quartile ( > 1.35 , ptx3 : > 3.85 ng / ml ) ; the relative risk of total cardiovascular events was 1 ( reference ) , 1.36 , 2.71 ( p<0.001 versus the reference ) , and 3.17 ( p<0.001 versus the reference ) , respectively . the logtransformation values of ptx3 demonstrated a linear association with the occurrence of cardiovascular events in patients with hfnef ( r=0.965 , p=0.03 ) . cox proportional hazards analysis of factors that were predictive of future cardiovascular events in patients with hfnef after adjustment for significant factors identified by univariate analysis bnp indicates btype natriuretic peptide ; cad , coronary artery disease ; e / e , mitral early diastolic peak flow velocity to tissue doppler early mitral annular diastolic velocity ; gfr , glomerular filtration rate ; hdl , highdensity lipoprotein ; hf , heart failure ; hfnef , heart failure with normal ejection fraction ; hr , hazard ratio ; hscrp , highsensitivity creactive protein ; il6 , interleukin6 ; ldl , lowdensity lipoprotein ; lvef , left ventricular ejection fraction ; nyha , new york heart association ; ptx3 , pentraxin 3 ; tnf , tumor necrosis factor. cox proportional hazards analysis of factors that were associated with future cardiovascular events in hfnef patients using forced inclusion models bnp indicates btype natriuretic peptide ; ci , confidence interval ; hf , heart failure ; hfnef , heart failure with normal ejection fraction ; hr , hazard ratio ; hscrp , highsensitive creactive protein ; il6 , interleukin6 ; lvef , left ventricular ejection fraction ; nyha , new york heart association ; ptx3 , pentraxin 3 ; tnf , tumor necrosis factor. we also estimated the cstatistics of pf5 alone . separate incorporation of ptx3 and bnp into pf5 increased the cstatistics for the prediction of future cardiovascular events from 0.617 for pf5 alone to 0.656 for pf5 plus ptx3 and pf5 plus bnp ( table 5 ) . moreover , incorporation of ptx3 and bnp into pf5 increased the cstatistics to 0.683 ( p<0.05 ) . in addition , the pf5 plus bnp plus ptx3 model performed better than the pf5 plus bnp model ( cstatistics : 0.683 versus 0.656 ) . we also confirmed good calibration for the analysis by grnnesby and borgan statistics ( p=0.19 ) . cstatistics for cox proportional hazards analysis predicting future cardiovascular events in patients with heart failure with normal ejection fraction bnp indicates btype natriuretic peptide ; pf5 , the 5 factors identified in the ipreserve study as prognostic in patients with heart failure with normal ejection fraction ( age , presence of diabetes mellitus , previous hospitalization for heart failure , new york heart association classification , and left ventricular ejection fraction ) ; ptx3 , pentraxin 3 . the nri was significant after ptx3 was included : 12.2% for patients without cardiovascular events , 0% for those with cardiovascular events , and 12.2% overall ( p<0.01 ) ( table 6 ) . reclassification of the risk of pf5 and bnp for cardiovascular events after the addition of ptx3 bnp indicates btype natriuretic peptide ; pf5 , the 5 factors identified in the ipreserve study as prognostic in patients with heart failure with normal ejection fraction ( age , presence of diabetes mellitus , previous hospitalization for heart failure , new york heart association classification , and left ventricular ejection fraction ) ; ptx3 , pentraxin 3 . table 1 shows the clinical characteristics of the participating patients . the median ptx3 level was 3.0 ng / ml . mean age , new york heart association class , and bnp levels were higher and mean body mass index , waist circumference and estimated glomerular filtration rate were lower in the highptx3 group ( > 3.0 ng / ml ) than in the lowptx3 group ( 3.0 ng / ml ) . lvmi values were significantly higher in the highptx3 group than in the lowptx3 group , whereas lvef and e / e were similar in these 2 groups . simple linear regression analysis showed a positive and significant correlation between ln(ptx3 ) and ln(bnp ) ( r=0.295 , p<0.001 ) ( figure 2a ) and a significant but weak positive correlation between ln(ptx3 ) and ln(lvmi ) ( r=0.114 , p<0.05 ) ( figure 2b ) . furthermore , simple linear regression analysis showed a significant but weak positive correlation between ln(ptx3 ) and ln(tumor necrosis factor ) ( r=0.106 , p<0.05 ) but not ln(hscrp ) ( p=0.11 ) or ln(interleukin6 ) ( p=0.80 ) . demographic and clinical characteristics of patients with hfnef data are meansd , number of patients ( % ) , or median ( interquartile range ) . acei indicates angiotensinconverting enzyme inhibitors ; arb , angiotensin ii receptor blocker ; bnp , btype natriuretic peptide ; cad , coronary artery disease ; e / e , mitral early diastolic peak flow velocity to tissue doppler early mitral annular diastolic velocity ; gfr , glomerular filtration rate ; hdl , highdensity lipoprotein ; hf , heart failure ; hfnef , heart failure with normal ejection fraction ; hscrp , highsensitive creactive protein ; il6 , interleukin6 ; ldl , lowdensity lipoprotein ; lv , left ventricle ; lvef , left ventricular ejection fraction ; nyha , new york heart association ; ptx3 , pentraxin 3 ; tnf , tumor necrosis factor. relationships between ptx3 and bnp levels and ptx3 levels and lvmi . simple linear regression analysis shows a positive and significant correlation between ( a ) ln(ptx3 ) and ln(bnp ) ( r=0.295 , p<0.001 ) and ( b ) a significant but weak positive correlation between ln(ptx3 ) and ln(lvmi ) ( r=0.114 , p<0.05 ) . bnp indicates btype natriuretic peptide ; lvmi ; left ventricular mass index ; ptx3 , pentraxin 3 . the mean followup period was 30 months ( range : 1 to 59 months ) . during this time , 106 of these patients experienced cardiovascular events , including cardiovascular death ( n=9 ) , nonfatal mi ( n=4 ) , unstable angina pectoris ( n=13 ) , ischemic stroke ( n=9 ) , hospitalization for hf decompensation ( n=41 ) , and coronary revascularization ( n=30 ) . the frequency of cardiovascular events was significantly higher in the highptx3 group ( n=75 ) than in the lowptx3 group ( n=31 , p<0.001 ) ( table 2 ) . hfrelated cardiovascular events , including cardiovascular death and hospitalization for hf decompensation , were significantly higher in the highptx3 group ( n=41 ) than in the lowptx3 group ( n=9 ) ( p<0.001 ) ( table 2 ) . kaplanmeier analysis showed that the probability of cardiovascular events was also significantly higher in the highptx3 group than in the lowptx3 group ( p<0.001 , logrank test ) ( figure 3a ) . cardiovascular events in hfnef patients with low and high plasma ptx3 levels differences between the groups were assessed by the logrank test . hf indicates heart failure ; hfrelated cardiovascular events , cardiovascular death and hospitalization for hf decompensation ; hfnef , heart failure with normal left ventricular ejection fraction ; ptx3 , pentraxin 3 . patients with ( a ) high ptx3 levels and low ptx3 levels , ( b ) subgroups of patients with high and low ptx3 levels plus bnp , and ( c ) subgroups of new york heart association class ii patients with high and low ptx3 levels are shown . the combination of ptx3 and bnp levels led to identification of subgroups ( n=116 , highptx3 and bnp group ; n=64 , highptx3 and lowbnp group ; n=64 , lowptx3 and highbnp group ; and n=116 , lowptx3 and bnp group ) with significantly different probabilities of cardiovascular events ( p<0.001 , logrank test ) ( figure 3b ) . in the lowbnp ( p=0.02 ) and highbnp ( p<0.01 ) groups , the probability of cardiovascular events was significantly higher in those with high ptx3 levels than in those with low ptx3 levels . even among patients with mild hf symptoms ( new york heart association class ii ) , the outcomes were poorer in patients with high ptx3 levels than in those with low ptx3 levels ( p<0.001 , logrank test ) ( figure 3c ) . the results of univariate and multivariable cox proportional hazards analyses for cardiovascular events are shown in tables 3 and 4 . multivariable cox proportional hazards analysis identified ptx3 ( hazard ratio [ hr ] : 1.16 ; 95% ci : 1.05 to 1.27 ; p<0.01 ) , highdensity lipoprotein cholesterol ( hr : 0.99 ; 95% ci : 0.97 to 0.99 ; p<0.05 ) , lvmi ( hr : 1.03 ; 95% ci : 1.05 to 1.27 ) , and bnp ( hr : 1.08 ; 95% ci : 1.03 to 1.14 ; p<0.001 ) as predictors of future cardiovascular events after adjustment for significant factors that were identified by univariate analysis ( table 3 ) . using the forced inclusion model , which evaluated levels of inflammatory markers and bnp ( model 1 ) in multivariable cox hazards analysis , ptx3 , but not hscrp , significantly predicted cardiovascular events ( hr : 1.17 ; 95% ci : 1.07 to 1.28 ; p<0.001 ) . in the forced inclusion model , which included pf5 and bnp , ptx3 significantly predicted cardiovascular events ( model 2 : hr : 1.16 ; 95% ci : 1.05 to 1.27 ; p<0.01 ; model 4 : hr : 1.16 ; 95% ci : 1.06 to 1.27 ; p<0.01 ) ( table 4 ) . furthermore , multivariable cox proportional hazards analysis identified ptx3 ( hr : 1.16 ; 95% ci : 1.01 to 1.35 ; p<0.05 ) and bnp ( hr : 1.21 ; 95% ci : 1.11 to 1.31 ; p<0.001 ) as predictors of future hfrelated cardiovascular events , including cardiovascular death and hospitalization for hf decompensation , after adjustment for significant factors identified by univariate analysis . we classified patients with hfnef into 4 groups according to the levels of ln(ptx3 ) : first quartile ( < 0.59 , ptx3 : < 1.80 ng / ml ) , second quartile ( 0.59 to 1.10 , ptx3 : 1.80 to 3.00 ng / ml ) , third quartile ( 1.10 to 1.35 , ptx3 : 3.00 to 3.85 ng / ml ) , and fourth quartile ( > 1.35 , ptx3 : > 3.85 ng / ml ) ; the relative risk of total cardiovascular events was 1 ( reference ) , 1.36 , 2.71 ( p<0.001 versus the reference ) , and 3.17 ( p<0.001 versus the reference ) , respectively . the logtransformation values of ptx3 demonstrated a linear association with the occurrence of cardiovascular events in patients with hfnef ( r=0.965 , p=0.03 ) . cox proportional hazards analysis of factors that were predictive of future cardiovascular events in patients with hfnef after adjustment for significant factors identified by univariate analysis bnp indicates btype natriuretic peptide ; cad , coronary artery disease ; e / e , mitral early diastolic peak flow velocity to tissue doppler early mitral annular diastolic velocity ; gfr , glomerular filtration rate ; hdl , highdensity lipoprotein ; hf , heart failure ; hfnef , heart failure with normal ejection fraction ; hr , hazard ratio ; hscrp , highsensitivity creactive protein ; il6 , interleukin6 ; ldl , lowdensity lipoprotein ; lvef , left ventricular ejection fraction ; nyha , new york heart association ; ptx3 , pentraxin 3 ; tnf , tumor necrosis factor. cox proportional hazards analysis of factors that were associated with future cardiovascular events in hfnef patients using forced inclusion models bnp indicates btype natriuretic peptide ; ci , confidence interval ; hf , heart failure ; hfnef , heart failure with normal ejection fraction ; hr , hazard ratio ; hscrp , highsensitive creactive protein ; il6 , interleukin6 ; lvef , left ventricular ejection fraction ; nyha , new york heart association ; ptx3 , pentraxin 3 ; tnf , tumor necrosis factor. we also estimated the cstatistics of pf5 alone . separate incorporation of ptx3 and bnp into pf5 increased the cstatistics for the prediction of future cardiovascular events from 0.617 for pf5 alone to 0.656 for pf5 plus ptx3 and pf5 plus bnp ( table 5 ) . moreover , incorporation of ptx3 and bnp into pf5 increased the cstatistics to 0.683 ( p<0.05 ) . in addition , the pf5 plus bnp plus ptx3 model performed better than the pf5 plus bnp model ( cstatistics : 0.683 versus 0.656 ) . we also confirmed good calibration for the analysis by grnnesby and borgan statistics ( p=0.19 ) . cstatistics for cox proportional hazards analysis predicting future cardiovascular events in patients with heart failure with normal ejection fraction bnp indicates btype natriuretic peptide ; pf5 , the 5 factors identified in the ipreserve study as prognostic in patients with heart failure with normal ejection fraction ( age , presence of diabetes mellitus , previous hospitalization for heart failure , new york heart association classification , and left ventricular ejection fraction ) ; ptx3 , pentraxin 3 . the nri was significant after ptx3 was included : 12.2% for patients without cardiovascular events , 0% for those with cardiovascular events , and 12.2% overall ( p<0.01 ) ( table 6 ) . reclassification of the risk of pf5 and bnp for cardiovascular events after the addition of ptx3 bnp indicates btype natriuretic peptide ; pf5 , the 5 factors identified in the ipreserve study as prognostic in patients with heart failure with normal ejection fraction ( age , presence of diabetes mellitus , previous hospitalization for heart failure , new york heart association classification , and left ventricular ejection fraction ) ; ptx3 , pentraxin 3 . to the best of our knowledge , this report is the first to show a significant association between plasma levels of ptx3 , a marker of inflammation , and adverse cardiovascular outcomes in patients with hfnef . the addition of high ptx3 levels to the pf5 and bnp levels , which were previously found to be prognostic in patients with hfnef , improved their risk stratification , as indicated by a substantial increase in the cstatistics and significance of nri . activation of the inflammatory process is important in the pathogenesis of hf and in adverse prognosis of patients with this condition . several studies have investigated the role of inflammation as a therapeutic target , although initial trials had limited success . consequently , specific antiinflammatory approaches for the different types and stages of hf ( ie , hfnef and hfref ) remain a priority , as does a better understanding of the mechanisms underlying hfrelated inflammation . the established inflammatory marker hscrp is an independent predictor of morbidity and mortality in patients with hf . elevated crp levels predict hospitalization of hf patients , although the association between crp and hf events is no longer significant after adjustment for the presence of lv diastolic dysfunction . consequently , the relationships between inflammatory markers and the prognosis of patients with hfnef remain unclear . , remodeling is driven by the loss of cardiomyocytes , whereas in hfnef , a systemic inflammatory state induces oxidative stress in the coronary microvascular endothelium , which drives myocardial dysfunction and remodeling . other studies have shown that serum levels of inflammatory cytokines are high in patients with hfnef and that interleukin16 , a cytokine considered an important mediator in inflammation , promotes cardiac fibrosis and myocardial stiffening in hfnef . consequently , investigating the relationships between inflammatory markers and prognosis in hfnef is important . we observed a significant correlation between plasma levels of ptx3 , a novel inflammatory maker , and future cardiovascular events in patients with hfnef . furthermore , considerable evidence suggests that ptx3 may better reflect local inflammatory status in tissues than does liverderived crp . we previously reported that ptx3 was partly produced in the coronary circulation in patients with hfnef . these results suggest that ptx3 , rather than crp , reflects vascular and cardiac inflammation in patients with hfnef and might be a cardiovascular biomarker for the assessment and management of hf . in the present study , we showed that increased inflammation as assessed by ptx3 measurement was significantly associated with poor prognosis for hfnef patients by multivariable cox hazards analysis . ptx3 as an inflammation maker and bnp as a myocardial stress marker to the previously established prognostic factors in the ipreserve study of hfnef patients improved their risk stratification performance , as indicated by an increase in the cstatistics . these results suggest the possible relative merits of these 2 biomarkers for future risk management in hfnef . in nri analyses , the addition of ptx3 was able to provide a more appropriate risk assessment in patients who were evaluated as having a high risk state by previously described hfnef prognostic factors ( pf5 plus bnp ) in patients without cardiovascular events . furthermore , the addition of ptx3 never resulted in a worse risk classification in patients with cardiovascular events . consequently , the significance of nri was shown by incorporating ptx3 into the new riskassessment model . the site of ptx3 production in patients with hfnef remains unclear . under normal physiological conditions , ptx3 is expressed in atherosclerotic lesions , adipose tissue , and the heart with acute and subacutephase hf ; however , the types of cells that express ptx3 in failing hearts of patients with hfnef and chronic hf have not been determined . accumulation of the extracellular matrix ( ie , fibrosis ) may also be pathophysiologically important in progression of the hfnef disease process and be a contributor to subsequent events . hfnef may also be caused by structural and molecular abnormalities of the cardiovascular system , including cardiac and noncardiac factors , such as those associated with vascular functions . we recently demonstrated significant involvement of endothelial dysfunction in the prognosis of patients with hfnef , suggesting that improvement in endothelial function could be a potential therapeutic target in patients with hfnef . ptx3 is produced by fibrous tissues and endothelial cells in response to inflammatory stimuli and is considered an important marker of vascular pathology . in patients with acute mi and infectious myocarditis , ptx3 is produced by macrophages , endothelial cells , and , to a lesser extent , myocardiocytes and is localized in the interstitium . the interstitial localization of ptx3 in human failing hearts suggests that ptx3 is produced locally by fibroblasts in the cardiac interstitium . consequently , in patients with hfnef , ptx3 might be produced by myocardial fibroblasts and/or endothelial cells rather than in subclinical atherosclerotic lesions in patients with hfnef . we found that the frequency of hfrelated cardiovascular events , including cardiovascular death and hospitalization for hf decompensation , was significantly higher in patients with high ptx3 levels than in those with low ptx3 levels . this finding suggests that ptx3 is more predictive of future hfrelated cardiovascular events than of vascular events in patients with hfnef . we used the median value of ptx3 ( 3.0 ng / ml ) as the cutoff point in the present study . suzuki et al demonstrated that the median value of ptx3 was 3.7 ng / ml in patients with hfref and split the patients into 2 groups : those above and those below 4.0 ng / ml . in this study , patients with hfref were targeted , and it has already been demonstrated that patients with hfref have higher inflammatory activity than those with hfnef . we previously demonstrated that the 75% range value of ptx3 was 2.90 ng / ml in nonhf patients with risk factors ( hypertension , 67.8% ; diabetes mellitus , 37.4% ; dyslipidemia , 57.1% ; and coronary artery disease , 55.0% ) . furthermore , inoue et al showed that ptx3 levels were < 2.28 ng / ml in healthy volunteers . in previous studies of cardiovascular events in stable patients with coronary risk factors , thus , we consider that the median value of 3.0 ng / ml for ptx3 was a clinically meaningful cutoff point in our study . first , our study included only a relatively small number of patients in a single center . second , the population of this study was relatively young and predominantly male ; had a low prevalence of atrial fibrillation , a higher frequency of mild hf , relatively lower bnp levels ; and had less frequent use of diuretics . because our institution is an educational hospital and the design was a singlecenter study in japan , the recruited patients with hfnef might have had differences in characteristics compared with those in western studies . consequently , a large , multiracial , multicenter study is required to confirm our results . recent studies in mouse models have shown that ptx3 has a cardioprotective effect , suggesting that ptx3 might protect the cardiovascular system by modulating the immune inflammatory balance . further in vivo and in vitro experiments are required to determine the exact role of high ptx3 levels in hfnef . despite these limitations , a largescale multicenter trial is warranted to further examine the pathological role and clinical significance of ptx3 in hfnef . high plasma ptx3 levels , but not other inflammatory markers including hscrp , are significantly correlated with future cardiovascular events in patients with hfnef . we are grateful to megumi nagahiro and saeko tokunaga of the department of cardiovascular medicine , faculty of life sciences , kumamoto university , kumamoto , japan , for their skillful technical assistance .

backgroundpentraxin 3 ( ptx3 ) is a novel inflammatory marker produced by various cell types including those of the vasculature and the heart . the relationship between inflammatory markers and prognosis of patients with heart failure with normal ejection fraction ( hfnef ) remains unknown . we investigated whether plasma ptx3 levels can predict future cardiovascular events in patients with hfnef.methods and resultsplasma ptx3 , highsensitivity creactive protein , and btype natriuretic peptide levels were measured prospectively in 360 stable patients with hfnef . the subsequent incidence of cardiovascular events , including cardiovascular death , nonfatal myocardial infarction ( mi ) , unstable angina pectoris , nonfatal ischemic stroke , hospitalization for heart failure decompensation , and coronary revascularization , was determined . during a mean 30month followup , 106 patients experienced cardiovascular events . these events were more frequent in patients with high plasma ptx3 levels ( > 3.0 ng / ml ) than low levels ( 3.0 ng / ml ) . multivariable cox hazard analysis showed that ptx3 ( hazard ratio : 1.16 ; 95% ci : 1.05 to 1.27 ; p<0.01 ) and btype natriuretic peptide ( hazard ratio : 1.08 ; 95% ci : 1.03 to 1.14 ; p<0.001 ) , but not highsensitivity creactive protein levels , were significant predictors of future cardiovascular events . multivariable cox analysis with the forced inclusion model , including 5 previously identified prognostic factors , found that ptx3 was a significant predictor of cardiovascular events ( hazard ratio : 1.16 ; 95% ci : 1.06 to 1.27 ; p<0.01 ) . the cstatistics for cardiovascular events substantially increased from 0.617 to 0.683 when ptx3 was added to the 5 previously identified prognostic factors.conclusionshigh plasma ptx3 levels , but not other inflammatory markers , are correlated with future cardiovascular events in patients with hfnef . ptx3 may be a useful biomarker for assessment of risk stratification in hfnef.clinical trial registrationurl : http://www.umin.ac.jp ; unique identifier : umin000002170 .

achieving emmetropia in eyes with full thickness corneal grafts is a target that has long been pursued . for decades , a successful keratoplasty was judged in terms of preservation of a clear surviving graft as a final outcome . however , the ultimate goal of vision restoration was often hampered by the frequent association with postoperative ametropia and/or anisometropia [ 1 , 2 ] . astigmatism , the main refractive error following penetrating keratoplasty ( pkp ) , has been related to a variety of pre- , intra- , and postoperative factors , while the less commonly associated spherical errors as myopia and , rarely , hyperopia were attributed mainly to axial length abnormalities and postoperative suture manipulations [ 14 ] . a wide scope of therapeutic modalities has been proposed and employed for the correction of such refractive errors . these ranged from the very conservative spectacle prescription to the final possibility of repeating the whole grafting procedure . however , none of these techniques has proven itself as a sole ideal solution for the management of post - pkp ametropia and many are associated with graft survival and , even , vision threatening complications [ 1 , 2 , 5 ] . visual rehabilitation using spectacles represents a good choice but their use is limited by significant anisometropia especially with astigmatism more than 3 d or the presence of irregular astigmatism . contact lenses ( especially rigid gas permeable ) provide another conservative management option but dry - eye syndrome and fitting - related inconvenience as well as patient 's age , dexterity , and lifestyle are major concerns that may affect contact lens tolerance . they may also induce peripheral corneal neovascularization which can result in graft rejection [ 1 , 2 , 69 ] . initial surgical management options range from simple procedures like selective suture removal or suture adjustment [ 4 , 10 , 11 ] to the more sophisticated techniques of astigmatic keratotomy , relaxing incisions with or without compression sutures [ 1315 ] , and wedge resection . the latter two have low predictability and are associated with a high incidence of recurrence of astigmatism . they also carry the risk of inducing overcorrection , perforation , wound dehiscence , and graft rejection . moreover , they mainly aim for rectifying the astigmatic component of the refractive error but they do little , if any , when it comes to the spherical part of refraction [ 1 , 2 ] . intracorneal ring segment ( icr ) implantation has recently been reported in post - pkp eyes but with significant undercorrection and low predictability compared to their results in keratoconic eyes . immune reaction induction leading to graft rejection and ring migration and perforation into the anterior chamber are possible complications that can violate the future integrity of the grafted cornea [ 1 , 17 , 18 ] . excimer laser photorefractive keratectomy ( prk ) has also been proposed but was proven less predictable than when performed in previously nonoperated eyes . limited astigmatic correction , irregular astigmatism induction , significant regression , corneal haze , and photoablation induced graft rejection are other important disadvantages of such a technique [ 19 , 20 ] . the use of laser in situ keratomileusis ( lasik ) reduced haze and allowed for more refractive correction but showed an increased risk of flap complications compared to normal eyes . moreover , the lamellar keratectomy step causes thinning of the graft - host interface as the flap usually has a larger diameter than the corneal button . this disruption of the healing scar can add to the risk of having wound dehiscence . toric intraocular lens implantation ( phakic or pseudophakic ) provides a wider range of correction but lens rotation , increased endothelial cell loss , corneal decompensation , graft rejection , endophthalmitis , and secondary glaucoma are important drawbacks of such procedures [ 2732 ] . small incision lenticule extraction ( smile ) is a novel , all - in - one , corneal laser refractive surgical procedure in which a lenticule of stromal tissue of planned thickness and diameter is isolated between two intracorneal planes created using a femtosecond laser platform . the lenticule is , then , manually removed from the cornea through a small incision to change the corneal curvature and exert its refractive effect . it was reported to be safe , predictable , and effective for treating myopia and myopic astigmatism in previously nonoperated eyes . it has the advantages of being flapless and less invasive than other intraocular procedures together with having the ability to tailor and center the whole procedure as required within a specific area of the cornea [ 3335 ] . this study was performed with the aim of evaluation of visual and refractive outcomes after smile for treating post - pkp myopia and myopic astigmatism . this interventional case - series included ten eyes of 10 patients with previous pkp and residual compound myopic astigmatism . inclusion criteria consisted of patients who had had an 8.25 mm donor button transplanted to 8.00 mm trephination of the recipient cornea with a duration of at least 18 months since the time of keratoplasty and a residual myopic refractive error of up to 10.0 diopters ( d ) of spherical equivalent with astigmatism up to 6.0 d at the spectacle plane . a smooth postkeratoplasty course with no attacks of graft rejection or suture complications was mandatory . sutures were completely removed prior to performing the preoperative examination by at least three months during which patients were followed up monthly to ensure a stable refraction . only patients whose topography and anterior and posterior elevation maps ' data within the acceptable range for laser vision correction and a thinnest graft location of 500 m or greater were enrolled in the study . patients with graft apposition abnormalities ( override or underride ) , severe dry eye , ocular surface disease , abnormal topographies , thin grafts , elevated intraocular pressure ( iop ) , peripheral corneal neovascularization , nonsuture track related peripheral opacities , or central or paracentral opacities were excluded . patients experiencing post - smile interface inflammation , cellular infiltration , or any other reported smile complication were planned to be excluded , as well . also patients with other ocular or recorded eye - related systemic illnesses ( e.g. , diabetes mellitus ) were not included in the study . the study protocol was based on the tenets of the declaration of helsinki and was approved by the ethics committee of the faculty of medicine of alexandria university . the risks and advantages of the procedure were explained to all patients and an informed consent was obtained from each of them . full ophthalmic examination was performed including measurements of manifest refraction ( mr ) , uncorrected distance visual acuity ( udva ) , corrected distance visual acuity ( cdva ) ( using snellen decimal notation ) , and intraocular pressure as well as biomicroscopic fundus examination . keratometric data , corneal topography , thickness data , and height maps of the anterior and posterior corneal surfaces were obtained from the allegro oculyzer pentacam ( wavelight , gmbh , germany ) . following smile , postoperative follow - up visits were scheduled at 1 day , 1 week , and 1 , 3 , and 6 months postoperatively . during these visits , biomicroscopic examination of the anterior and posterior segments and udva , mr , and cdva testing and recording were performed . efficacy was expressed in terms of the cumulative udva at 6 months postoperatively as well as the efficacy index calculated as the ratio of the postoperative udva to the preoperative cdva . safety was judged by the change in the corrected distance visual acuity at 6 months postoperatively and also by the safety index calculated as the ratio of the postoperative cdva to the preoperative cdva . cases were operated by two surgeons ( osama ibrahim and tamer h. massoud ) in roayah vision correction centre , alexandria , egypt . preoperatively , refractive data was fed and revised on the computer system linked to the visumax femtosecond laser system ( carl zeiss meditec ag , germany ) with a 500 khz repetition rate . data entered included the mr to be corrected ( measured at 12 mm vertex distance ) , the mean corneal radius ( mm ) , or mean k - reading ( d ) in addition to pachymetry of the thinnest corneal ( graft ) location ( obtained from pentacam ) . for all cases , small suction cups were chosen as the patient - laser interface . the cap and the lenticule diameters were calculated to be smaller than those of the graft ( 8.0 mm ) so that they are centered within its margins . their diameters ranged from 6.9 to 7.5 mm depending on the clear area of the graft available for refractive correction . the width of the incision ranged from 3.4 to 3.6 mm and the side cut angle of the incision was set to 70 for all cases . the carved lenticules had optical zones ranging from 5.5 to 6.0 mm based on the residual stromal depth which was always kept above 300 m . standardized lenticule data for all cases included a transition zone of 0.1 mm , a minimum lenticule edge thickness of 10 m , and a circumferential side cut angle of 130. during surgery , the laser suction cup was centered relative to the pupil and the graft . the patient was asked to keep looking at the flickering green fixation light during laser application to the cornea . after creation of the cuts , the lenticule and the cap were manipulated using the usual techniques described for smile [ 34 , 36 ] . after extraction , the lenticule was spread on the corneal surface and stained with prednisolone acetate 1% eye drops to ensure its intactness as a complete disc and to detect the presence of any residual tissue remnants within the intrastromal pocket that can result in irregular astigmatism . the same postoperative treatment regimen consisting of topical prednisolone acetate 1% , gatifloxacin 0.3% , and nonpreserved artificial tears data analysis was performed using the software spss for windows version 20.0 ( spss inc . , chicago , usa ) and microsoft excel 2010 ( microsoft corp . , redmond , wa , usa ) . nonparametric tests were used as the sample size was less than that optimum for parametric analysis . the wilcoxon signed ranks test was used for comparison between the preoperative and postoperative data , and kruskal - wallis test was used for comparison between the postoperative data obtained from consecutive visits . differences were considered to be statistically significant when the associated p value was < 0.05 . bivariate regression analysis was carried out to predict achieved sphere , cylinder , and seq accuracy using the preoperative attempted data . standard graphs for reporting the outcomes in refractive surgery according to the waring protocol and its modifications [ 3840 ] were used for displaying and summarizing the outcomes of this study . for simplicity , only the preoperative and the 6-month follow - up data are demonstrated in the results . demographic and pre- and postoperative clinical ( refractive and visual ) data of the ten patients are shown in table 1 . the cap and the lenticule were centered in relation to the pupil within the corneal graft except in two cases ( n = 2 , 5 ) in whom the graft itself was slightly decentered . here the best centration in relation to the pupil was performed taking into consideration not to bisect the graft - host interface with the laser incisions . it is , however , worth mentioning that those two cases were the ones in whom astigmatic correction was unlikely , with almost the same amount of astigmatism remaining postoperatively . intraoperatively , some resistance was met during dissection of both the cap and the lenticule at the sites of the suture related fibrous tracks . however , this has not led to any complication and cases were completed as planned . none of the cases enrolled in the study suffered from any of the reported post - smile complications . ( ranging from 7.0 to 4.0 d ) and the preoperative mean refractive spherical equivalent ( mrse ) was 6.84 1.38 d ( ranging from 9.0 to 5.0 ) . on the other hand , the mean postoperative spherical refraction was 0.8 0.97 d ( ranging from 2.0 to + 1.0 d ) while the mean postoperative mrse was 1.39 0.9 d ( ranging from 2.63 to 0.0 d ) . the means of achieved correction values ( calculated by subtracting the 6-month postoperative refraction from the preoperative target refraction ) for sphere and mrse were 4.5 1.45 d and 5.45 1.59 d , respectively . on comparing these values to the values of the preoperative target refractive correction ( preoperative manifest refraction ) , statistically significant differences existed for both sphere ( p = 0.042 ) and mrse ( p = 0.008 ) . vector analysis of the results of astigmatism correction revealed a mean target induced astigmatism ( tia ) magnitude of 2.61 1.06 d at axis 66 49.1 degrees and a mean surgically induced astigmatism ( sia ) magnitude of 2.06 0.76 d at axis 91.6 59.3 degrees , while the mean of the magnitude of the difference vector was 1.14 0.75 at axis 72.5 53.8 degrees . on comparing the magnitudes of tia versus sia , a statistically significant difference was found ( p = 0.028 ) . the mean correction ratio ( induced / intended correction ) of sphere was 0.84 0.19 d and mrse was 0.79 0.13 d. this means that for each diopter of sphere about 84.3% correction was achieved and for each diopter in mrse 79% correction was achieved . on the other hand , vector analysis of astigmatic results showed a mean percentage of astigmatic correction of 80.66 20.9% ( correction index = 0.81 0.21 ) and a mean percentage of astigmatism reduction at the intended axis of 67 25.5% . the mean arithmetic angle of error was 10.4 15.4 degrees ( p = 0.05 ) and the mean absolute angle of error was 12.4 13.7 degrees ( p = 0.008 ) . assessment of the accuracy of the achieved correction values versus the attempted ones revealed a positive correlation for all three refraction elements with the sphere showing correlation values of p = 0.024 , r = 0.702 , and r = 0.4928 ( figure 1 ) , the cylinder ( tia versus sia ) of p = 0.002 , r = 0.855 , and r = 0.656 ( figure 2 ) , and the mrse of p = 0.004 , r = 0.815 , and r = 0.6642 ( figure 3 ) . as regards predictability , only one out of the ten eyes ( 10% ) had a mrse between zero and 0.5 d , 4 eyes ( 40% ) were between zero and 1.0 d , and 7 eyes ( 70% ) were between zero and 1.5 ( figure 4 ) . figure 5 displays the stability of the mrse values along the follow - up period with a fairly stable refraction . no statistically significant differences were found among the mrses measured at one week ( 1.26 0.9 ) , one month ( 1.33 0.86 ) , three months ( 1.35 0.85 ) , and six months ( 1.39 0.9 ) ( p = 0.937 ) . on assessing the predictability of astigmatism correction , five out of the ten eyes ( 50% ) had astigmatism above 3.0 d preoperatively , while six months after surgery 30% had astigmatism values within 0.5 d , 50% within 1.0 d , and 70% within 1.5 d ( figure 6 ) . preoperatively , the mean cdva was 0.73 0.15 while , 6 months postoperatively , the mean udva was 0.68 0.14 and the mean cdva was 0.82 0.1 . this resulted in an efficacy index ( e ) of 0.93 and a safety index ( s ) of 1.12 . a more detailed evaluation of efficacy showed a cumulative snellen preoperative cdva of 0.9 or better in 20% of eyes , 0.8 or better in 60% of eyes , and 0.7 or better in 70% of eyes . on the other hand , the cumulative snellen postoperative udva was 0.8 or better in 40% of eyes and 0.7 or better in 70% of eyes ( figure 7 ) . also , 90% of eyes had an udva within one line of the preoperative cdva . figure 8 shows the safety data of the procedure with 40% of eyes experiencing no change from the preoperative cdva , 50% gaining one line , and 10% ( one eye ) gaining more than 2 lines . the quest for the best unaided visual performance following pkp has entailed the exploration of a variety of conventional and novel refractive correction procedures . yet , none has proven enough refractive accuracy , predictability , efficacy , or safety to be adopted as the standard trustworthy technique . a wide variability as regards the obtained refractive and visual results in post - pkp eyes compared to results reported in healthy eyes with unoperated corneas has become a generally anticipated conclusion for all reports on such cases . in addition , the inherent complications of these refractive surgical correction techniques were found to have a higher incidence rate in grafted corneas adding an increased menace for the future viability of the graft and , rarely , the whole eye [ 132 ] . the introduction of the single step femtosecond laser small incision lenticule extraction ( smile ) for correction of myopia and astigmatism and its reported good results gave hope for a simple , fast , easily designed , readily centered , and theoretically safe refractive correction means that can be applied to corneal grafts while salvaging the circumferential graft - host interface scar as well as the endothelium from being violated [ 3335 , 4143 ] . this study was performed with the aim of evaluating visual and refractive outcomes after correcting postkeratoplasty myopia and myopic astigmatism using small incision lenticule extraction ( smile ) . to our knowledge , the only published data about smile after keratoplasty is a single case report in which the authors reported achieving the target refraction and an improved udva with a follow - up of 3 months . this study should , therefore , be one of the earliest clinical trials about the same subject . the timing of intervention has been a matter of debate among researchers ; however , it is generally agreed upon that the corneal graft - host junction heals completely about one year following transplantation and that further surgical interventions should not be done until three to four months has passed since all the sutures have been removed . our cases had a minimum of 18 months before complete suture removal and refractive stabilization were pursued for three months afterwards . as regards the refractive results , a statistically significant undercorrection was noted for sphere , cylinder , and mrse . smile , in otherwise healthy nonoperated eyes , was reported to result in a slight undercorrection by about 0.25 d of mrse as reported by hjortdal et al . . possible causes suggested to explain such an undercorrection after smile for treating myopia included a small difference in the achieved lenticule thickness of about 9 m ( due to a hypothesized elastic recoil of the lamella between the cap and the residual stromal bed ) and postoperative epithelial thickness changes . smile undercorrection of astigmatism was also reported by ivarsen and hjortdal in unoperated eyes especially for higher degrees of astigmatism ( up to 16% per diopter in highly astigmatic eyes ) . proposed mechanisms included lenticule decentration , inappropriate energy and spot spacing settings , and again postoperative epithelial hyperplasia . in addition to the previous causes , corneal button decentration as well as the release of the tension within the graft caused by dissecting the planar incisions and the sutures - related fibrous tracts can also have a role in undercorrection or induction of lower and/or higher order astigmatism and coma . therefore , centration of the treatment in relation to the graft and the pupil is of paramount importance for achieving the best possible astigmatic correction and reducing higher order coma induction . the statistically significant values of arithmetic and absolute angles of error of astigmatism correction denote the possibility cyclotorsion occurrence which can , also , aid the explanation of the relative imprecision of astigmatism correction at the intended angle as well as the induction of postoperative different axes cylindrical errors . means for prevention of , compensating for , or correction of intraoperative cyclotorsion should be adopted . compared to other techniques , undercorrection and , rarely , overcorrection have also been reported for almost all corneal refractive surgical methods of correcting post - pkp myopia and astigmatism including incisional surgeries , photoablation ( prk or lasik ) [ 1926 ] , and icr implantation [ 17 , 18 ] . despite this undercorrection reported for smile , stability of the achieved refraction was statistically proven on comparing the achieved mrses at 1 week , 1 month , 3 months , and six months . the same was reported by other studies which investigated smile for myopia and myopic astigmatism [ 3335 , 4143 ] . the early biomechanical stability status achieved after smile can aid the explanation of such a finding . our results also showed that smile for correcting post - pkp myopia and astigmatism is of high efficacy and safety . the value of the efficacy index ( e : 0.93 ) suggests that grafted patients undergoing smile can expect an udva of more than 90% of their preoperative cdva , while the value of the safety index ( s : 1.12 ) indicates a potential improvement of the cdva postoperatively for such patients . ( e : 0.9 , s : 1.07 ) who assessed smile for correcting myopia in nonoperated eyes . the fact that none of the eyes enrolled in the study had lost any of its preoperative cdva postoperatively adds more to the safety profile of the technique . the feasibility of centering the whole treatment within the graft had the advantage of avoiding the violation of the graft - host interface , thus , preserving the structural integrity of this potentially weak spot . on the contrary , in other nonfemtosecond laser dependent techniques , moreover , the keratoplasty scar is recognized as a new limbus [ 1 , 50 ] outside which any refractive correction should be almost worthless and unquestionably risky . any extension of the treatment procedure into the recipient 's possibly diseased corneal rim either through flap lifting or laser ablation is , therefore , considered undesirable and quite useless . also , the facts that the endothelium is spared in femtosecond refractive lenticule extraction procedures compared to other intraocular procedures with no extra - stress added on the endothelial cells to achieve proper flap adhesion as in lasik add another advantage to smile when compared to those refractive correction techniques [ 21 , 2732 ] . the drawbacks of this study , however , include the absence of controls , the lack of randomization , the few number of the enrolled eyes , and multiple surgeons . since higher order aberrations evaluation and visual quality assessment were beyond the scope of this study , we strongly advocate them to be done in future similar studies to ascertain the nature and the amount of induced higher order aberrations as well as the quality of vision provided following smile in grafted eyes . the need for more prolonged follow - up , evaluation of induced graft biomechanical changes , and comparison to other techniques used for the same indication can not be overlooked . to sum up , smile for correction of postkeratoplasty myopia and astigmatism can be considered a valuable addition to the armamentarium of procedures utilized to correct post - pkp myopia and astigmatism . the whole treatment can be centered within the graft preserving the viability of the healed graft - host interface . however , management of cyclotorsion as well as centration of both the graft initially and the lenticule afterwards is crucial for achieving the best refractive results .

purpose . to evaluate the visual and refractive outcomes after small incision lenticule extraction ( smile ) for treating myopia and myopic astigmatism after penetrating keratoplasty ( pkp ) . design . case - series . methods . ten eyes of 10 patients with previous pkp and residual myopic astigmatism for whom pentacam imaging and thickness measurements were acceptable for laser vision correction . manifest refraction ( mr ) , uncorrected distance visual acuity ( udva ) , and corrected distance visual acuity ( cdva ) were obtained preoperatively and one day , one week , and one , 3 , and 6 months postoperatively . cases were operated on the visumax femtosecond laser platform with 500 khz repetition rate . results . the mean correction ratio for spherical errors was 0.84 0.19 d and for the mean refractive spherical equivalent ( mrse ) was 0.79 0.13 d. vector analysis showed a mean astigmatism reduction at the intended axis of 67 25.25% , a correction index of 0.81 0.21 , and an overall mean percentage of success of astigmatism surgery of 53 37.9% . the postoperative mrse was stable throughout the 6-month follow - up period . the efficacy index was 0.93 and the safety index was 1.12 . conclusion . smile for correction of post - pkp myopia and astigmatism is effective , safe , and stable with moderate accuracy and predictability . centration of the treatment within the grafts was easily performed .

common sources include backyard burning , incineration of plastics , and chlorine bleaching of pulp in paper mills . documented health effects include acute and chronic effects , including chloracne , various types of cancers , reproductive diseases , circulatory and respiratory diseases , and diabetes . the traditional approach to environmental remediation includes a host of physical and chemical methods , depending on the characteristics of the polluted site and the extent of contamination present . bioremediation , which is the use of biogenic materials and organisms for environmental cleanup , has also been proposed , including phytoremediation using plants and microbial degradation using primarily bacteria and fungi . bioremediation is an attractive strategy , as it can destroy the pollutant rather than transferring it from one environmental compartment to another . common bioremediation strategies include the addition of nutrients , degradative microorganisms , or both . sphingomonas wittichii rw1 is a microorganism of great interest to the bioremediation community for its ability to biotransform a large number of toxic polychlorinated dioxins and to utilize both nonchlorinated dibenzo - p - dioxin and nonchlorinated dibenzofuran as a growth substrate and sole source of carbon and energy . one of the major challenges in bioaugmentation strategies relying on the addition of nonnative microbes is to ensure their viability and degradative activity toward the target compounds . monitoring bioremediation is critical to ensure the efficacy of the process and the reduction of contaminant mass to acceptable levels . traditionally , the most important characteristics investigated for microorganisms used in bioremediation were their ability to transform the substrate , the rate of substrate removal , and the resulting metabolites [ 69 ] . to optimize the bacterial degradation of pollutants , it is important to understand how these organisms function during growth on recalcitrant substrates and which factors influence their degradative abilities . this includes analyzing not only the degradative pathways [ 1012 ] , but also the peripheral processes and mechanisms that are involved in taxis ( i.e. , directed motion in a chemical gradient ) , uptake , and transport during exposures to specific substrates . analysis of dna and rna can shed light on an organism 's metabolic potential ; however , these measurements poorly correlate to actual protein expression profiles . therefore , global analyses of protein expression profiles may be a more informative tool for understanding the physiological mechanisms of biodegradation . in addition to identifying important degradative enzymes in a variety of important microbes [ 1517 ] , proteomic studies have opened the door to a better understanding of system - wide changes in response to differing substrates . the imperative to perform proteomic analyses is particularly true for s. wittichii rw1 because the enzymes in the dioxin degradation pathway are encoded on different loci throughout the genome , certain elements in the pathway are located on a plasmid , and there may be alternative pathways at work . the present study builds on previous work and utilized difference gel eletrophoresis ( dige ) coupled with mass spectrometry ( ms ) to exploit recently gathered rw1 genome data . when used together , these tools yield information on the response of cells of s. wittichii rw1 to dioxin exposure and the bacterium 's degradative activity toward this recalcitrant compound . the aim of this study was to investigate system - wide changes in protein expression during growth on dibenzofuran , a nontoxic surrogate for dibenzo - p - dioxin , as compared to nonselective growth media . acetate was selected as the nonselective alternate substrate , as growth on this compound was observed to influence expression of select proteins , including the dioxin dioxygenase . thus , any changes measured in response to cells grown on dibenzofuran should represent cell - wide effects related specifically to the growth substrate and not to unanticipated extraneous effects . cultures of s. wittichii strain rw1 ( 100 ml to 1.0 l ) were grown to mid log phase at 30c in m9 phosphate - buffered minimal medium ( ph 7.05 ) supplemented with either dibenzofuran crystals or 50 mm acetate as growth substrates . cells were grown overnight on dibenzofuran and acetate as sole carbon sources to an optical density of 0.40.6 absorbance units ( 560 nm ) . following biomass processing , protein levels in the samples were on the order of 75200 g / ml . protein concentrations were normalized prior to analysis by concentration and resuspension in dige sample preparation buffer . culture purity was confirmed by the streak plate method using luria bertani medium supplemented with 1.5% agar . harvested biomass was washed , spun again , and the resultant pellet suspended in a small volume of 100 mm ammonium bicarbonate ( ph ~7.0 ) . this microbial suspension was then sonicated under cooling with ice , using a microtip sonicator ( fisher scientific , pittsburgh , pa , usa ) in a sequence of three 10-second bursts delivered in thirty - second intervals . the sonicated cells were then immediately centrifuged at 10,000 xg for 10 minutes at 4c . briefly , 8 parts of 10% tca in acetone ( 20c ) were added per volume of supernatant and , following mixing on a vortex , the resultant dilution was incubated at 20c overnight . following centrifugation ( 10,000 xg , 10 minutes , 4c ) , harvested biomass was washed in cold acetone for 10 minutes at 20c . following a subsequent centrifugation , the pellet was resuspended in sample preparation buffer ( 7 m urea , 2 m thiourea , 2% chaps , 0.2% dtt , 0.02% bromophenol blue ) and stored at 20c until analyzed . protein concentrations were measured using the bicinchoninic acid assay ( pierce , rockford , il , usa ) following dilution to reduce the concentration of interfering agents . twenty - five g of crude cell lysates of rw1 biological replicates grown on dibenzofuran ( n = 3 ) and acetate ( n = 3 ) were labeled using cy dyes ( ge healthcare ) as described elsewhere . briefly , samples were adjusted to 1 g/l using sample preparation buffer and the ph checked . subsequently , 0.25 l of 1 pmol/l cy dyes were added to samples for 30 minutes in the dark on ice . to stop the labeling reaction , 0.5 l of 10 mm lysine was added to the samples , which were mixed and incubated on ice for 10 minutes prior to storage at 20c until analysis . unless stated otherwise , all procedures were carried out in the dark or minimal light to protect the integrity of the fluorescent dyes . samples were randomized to reduce the effect of dye bias and in - gel variations . a global pool consisting of fractions of each sample was labeled as outlined above using cy 2 and added to each sample as an internal standard . one cy-3- and one cy-5-labeled sample were added to each gel , as defined by the experimental randomizing procedures . to each sample , an additional 175 g of unlabeled sample were added ; the volume was increased to a total of 450 l using sample preparation buffer . the reducing agent dtt ( dithiothreitol ; 1.3 mg per tube ) and ipg ( immobilized ph gradient ) buffer ( 0.5% ) were added , and the samples incubated and mixed in the dark at room temperature for approximately 1 h. samples were then applied to 24 cm ph 47 ipg strips ( ge healthcare ) and focused for 60 kvh using the following protocol : 12 h rehydration at 30 v ; 1 h step and hold at 500 v ; 7 hour gradient to 1,000 v ; step and hold at 1,000 v for 1 hour ; gradient to 8,000 v for 3 h ; step and hold at 8,000 v until 60 kvh . strips were then reduced and equilibrated using 10 mg / ml dtt ( 15 min ) followed by 25 mg / ml iodoacetamide ( 15 min ) . the ipg strips were overlaid on 24 26 cm 816% gradient tris - hcl ph 8.8 precast gels ( nextgen sciences , ann arbor , approximately 1 ml of agarose was applied to fix the gels and a cy-2-labeled molecular weight marker was applied adjacent to the acidic side of the strip . the gels were then run 1 - 2 w per gel overnight ( ~2224 hours ) at 20c until the marker dye ran off the gel . gels were then imaged with a typhoon 9400 scanner and processed using decyder v6.5 ( ge healthcare ) bva batch processor tool . images were uploaded to decyder ( version 6.5 ) and spurious image objects ( water spots , streaks , and mismatches ) were identified and excluded from further analysis . following allocation of changed proteins , individual spots were manually inspected and excluded from analysis if they fell outside acceptable parameters for peak height , area , and slope . pick lists were generated by selecting proteins whose expression was statistically changed in the two growth conditions ( p < 0.05 ) following digital image analysis using decyder , and the corresponding spots were automatically picked using an ettan spot picker ( ge healthcare ) with ettan spot pick software v.1.1 . spots were delivered in 100 mm ammonium bicarbonate to a 96-well plate and digested using established protocols . briefly , gel pieces were sequentially dried using three exchanges of 100% acetonitrile followed by a 10-minute speedvac ( savant ) drying . gel pieces were rehydrated in 40 l of 10 ng/l trypsin in 100 mm nh4hco3 on ice for 45 minutes . the supernatant was removed and replaced with 100 mm nh4hco3 and digested at 37c overnight . peptides were then extracted using 50% acetonitrile/0.1% tfa ( trifluoroacetic acid ) for 30 minutes at 37c . the peptides were microextracted using omix c18 tips ( varian , palo alto , ca , usa ) following the manufacturer 's instructions and then deposited on a stainless - steel target plate in a matrix consisting of 10 mg / ml 2,5-dihydroxybenzoic acid . mass spectra were acquired using a voyager de - str matrix - assisted laser desorption / ionization time - of - flight ms ( applied biosystems , foster city , ca , usa ) in positive reflector mode with delayed extraction using the following parameters : laser energy , 1400 arbitrary units ; mass range , 5005,000 da ; 120 nsec delay , 100 laser shots per spectrum . external calibration was conducted using a standard peptide mixture ( bradykinin , insulin b chain , p14r , and acth ) , and internal calibration was carried out using trypsin autolysis peaks . data were processed in data explorer v1.1 ( applied biosystems , foster city , ca , usa ) using noise reduction ( 2 standard deviations ) and peak deisotoping . peak masses were searched using the mascot online search engine ( http://www.matrixscience.com/ ) with the following settings : database , ncbi entire database ( 5.6 million entries ) ; no missed cleavages ; monoisotopic peaks ; no fixed modifications ; variable modification of methionine oxidation ; error tolerance of 150 ppm . database and literature searches were used to further characterize and classify the proteins identified by maldi - tof ms . where ambiguous names were encountered , blastp searches were used to identify homologous proteins from orthologous species . image analysis of 24 cm 2d - dige gels loaded with protein of s. wittichii rw1 cells grown on either dibenzofuran or acetate revealed 937 unique spots . differential in - gel analysis of individual gels determined gel - specific parameters for selection criteria and allowed visual examination of changes between growth on the two substrates ( figure 1 ) . of the 937 identified spots , 595 were matched between all the gels used to statistically compare the quantitative abundance of proteins . statistical analysis compared triplicate biological observations for each condition , normalized to the internal pooled standard ( figure 2 ) . crude cell lysates from s. wittichii rw1 grown on dibenzofuran showed that , of all proteins observed , 22 proteins were modulated in response to changes in culture conditions . these candidate biomarkers of metabolic activity and phenotype were observed in at least 6 of 9 dige images and were modulated as follows : 16 showed an apparent increase and 6 an apparent decrease ( figure 2 ) . these proteins , along with 22 proteins selected due to their high abundance in both growth conditions , were further analyzed and identified using mass spectrometry ( figure 3 ) . a mascot search of the entire ncbi database using mass spectral data generated by peptide mass fingerprinting identified 23 of the 44 proteins ( 52% ) . all protein identifications corresponded to the genome of s. wittichii strain rw1 . among the 16 proteins upregulated during growth on dibenzofuran , 7 were successfully identified ( table 1 ) . among the 6 proteins downregulated during growth on dibenzofuran , an additional 13 proteins were identified whose expression level remained unchanged regardless of culture conditions ( table 3 ) . of the 7 identified proteins increased during growth on dibenzofuran , 2 were directly related to the dibenzofuran degradation pathway ( figure 4 ) ; the others were involved in downstream metabolic processes ( catechol 1,2-dioxygenase , adenosylhomocysteinase ) , cell growth ( elongation factor ts ) , and cell protection ( cold shock dna - binding domain protein , alkyl hydroperoxide reductase ) . the three identified proteins whose expression was decreased ( fumarylacetoacetate hydrolase , tonb - dependent receptor , and acyl - coa dehydrogenase ) are involved in biosynthesis , catabolism , and transport . the unchanged proteins represented basic cell functions , although biosynthesis , catabolism , and transport proteins dominated the identities . the alpha subunit of the dioxin dioxygenase , the first step in the dioxin degradation pathway , was also identified as unchanged . dige and 2d electrophoresis are an accepted strategy for mining microbial proteomes for biomarkers related to a number of processes [ 2830 ] . the complete protein content of s. wittichii rw1 consists of approximately 5,000 putative proteins from the bacterial chromosome and two megaplasmids . using simple extraction and purification techniques followed by dige , over 500 protein spots were resolved on a large ( 24 cm ) 2-dimensional gel and matched between the three biological replicates , representing approximately 10% of the entire protein content . of these 500 proteins , 22 were found to be regulated in response to growth condition changes . of the genes they identified as being changed in their experiments , only two overlapped with the proteins identified in our study : swit_3144 ( tonb - dependent receptor , downregulated ) and swit_3376 ( chaperonin groel , upregulated ) . both of these gene levels responded to short - term perturbation with peg8000 but not sodium chloride in the transcriptomic study and were unchanged in our study . in our study , we also identified a number of proteins that are related to dioxin / dibenzofuran degradation ( e.g. , dioxin dioxygenase , meta - cleavage product hydrolase , and 2,3-dihydroxybiphenyl 1,2-dioxygenase ) . other proteins were identified that showed increases in abundance but whose role was not directly related to the dibenzofuran degradation pathway . the increase in the presence of antioxidants such as alkyl hydroperoxide reductase suggests that there is an increasing stress upon the bacterial cell during growth on dibenzofuran , perhaps due to a change in catabolism resulting in an increase in endogenous peroxide generation . increases in a cold - shock dna - binding protein may be further evidence of an increased cellular stress . however , proteins of the cold shock family and related ones are also known to have transport and protein processing roles . among the proteins in the dioxin degradation pathway , the most prominent on the gel was the meta - cleavage product hydrolase . this identification was produced from two adjacent spots , likely representing an artifact due to the protein 's extremely high expression or a reflection of the presence of multiple isoforms or a modified enzyme . the one identified in the present study is the product of the swit_3055 locus , a gene also known as dxnb2 . the glyoxalase / bleomycin resistance protein / dioxygenase identified in this study is also annotated as a 2,3-dihydroxybiphenyl-1,2-dioxygenase located on the chromosome at the swit_3046 locus . again , there are multiple isoforms of this enzyme found both on the chromosome and the megaplasmids . the kegg dioxin degradation pathway identifies swit_4182 as the dihydroxybiphenyl dioxygenase involved in biphenyl metabolism and swit_4902 as the trihydroxybiphenyl dioxygenase in both dioxin and dibenzofuran metabolism . the increased expression in response to dibenzofuran suggests that the swit_3046 dioxygenase plays a more important role in dibenzofuran degradation in vivo . the high degree of redundancy in the dioxin and dibenzofuran degradation pathways , that is , the presence of multiple ring - hydroxylating alpha and beta subunits , glyoxalase / bleomycin resistance protein / dioxygenases , and meta - cleavage product hydrolases , remains to be explained . one possibility is that the various isoforms have different affinities for chlorinated metabolites that would result from chlorinated dioxins and furans . further experiments are needed to fully distinguish the roles of these enzymes in s. wittichii rw1 degradation pathways . although not directly implicated in dioxin degradation , the fumarylacetoacetate hydrolase is also of interest because the gene encoding this protein ( swit_5089 ) flanks the ferredoxin fdx1 ( swit_5088 ) that has been identified as part of the electron supply chain supporting dioxin dioxygenase activity . of the multiple isoforms of this enzyme , the electron supply chain also contains two isofunctional reductases . neither the ferredoxin itself nor the reductases could be identified . in previous studies , the reductase was present as a much smaller fraction of the soluble cell proteome than either the ferredoxin or the dioxin dioxygenase , so gel - based methods may not be sensitive enough to detect this protein . if transcription of the ferredoxin is linked to the other genes at that locus , as is predicted , the decreased expression in response to dibenzofuran suggests that another ferredoxin is more important in the dioxin degradation pathway in vivo . the detection of the dioxin dioxygenase alpha subunit and related enzymes in both acetate- and dibenzofuran - grown cells is potentially of importance for the field of bioremediation because it suggests an avenue of biostimulation . when utilizing s. wittichii rw1 as a bioremediation agent , it may be possible to induce the expression of the dioxin degradation pathway using acetate . induction of the dioxin degradation pathway has not been observed when s. wittichii rw1 is grown on glucose or rich medium , and growth in a complex environmental medium ( landfill leachate ) was correlated with a decrease in copy number of the gene encoding the dioxin dioxygenase alpha subunit . previous studies using s. wittichii rw1 to transform chlorinated dioxins in soil or fly ash have observed a progressive decrease in degradative activity or viable cells [ 38 , 39 ] , respectively . the addition of acetate may generate sufficient relevant protein biomass to catalyze the successful degradation of dioxin and dioxin - like compounds in environments bioaugmented with s. wittichii rw1 . proteomic technology has emerged in microbiology more rapidly than in other fields for several reasons . the relatively small genomes code for relatively limited proteomes featuring no or very limited posttranslational modifications compared to higher organisms . furthermore , microbes are easily controlled and manipulated in the laboratory , both during growth and gene expression . these factors will continue to drive biomarker discovery in microbial proteomes , including phenotypic biomarkers informing on the degradative activity of biomass produced for bioaugmentation of contaminated environments . furthermore , the field of bioremediation can benefit from methods suitable for monitoring microbial biomarkers in field samples to inform on progress in site bioremediation . this study highlights a number of proteins that were changed in response to dibenzofuran exposure , opens the door to a greater understanding of how s. wittichii rw1 performs and regulates the degradation of dioxins , and suggests ways to enhance the biodegradation of dioxins .

sphingomonas wittichii rw1 is a bacterium of interest due to its ability to degrade polychlorinated dioxins , which represent priority pollutants in the usa and worldwide . although its genome has been fully sequenced , many questions exist regarding changes in protein expression of s. wittichii rw1 in response to dioxin metabolism . we used difference gel electrophoresis ( dige ) and matrix - assisted laser desorption / ionization mass spectrometry ( maldi - ms ) to identify proteomic changes induced by growth on dibenzofuran , a surrogate for dioxin , as compared to acetate . approximately 10% of the entire putative proteome of rw1 could be observed . several components of the dioxin and dibenzofuran degradation pathway were shown to be upregulated , thereby highlighting the utility of using proteomic analyses for studying bioremediation agents . this is the first global protein analysis of a microorganism capable of utilizing the carbon backbone of both polychlorinated dioxins and dibenzofurans as the sole source for carbon and energy .

leptin plays an important role in the regulation of body weight due to reducing food intake and increasing energy expenditure ( 1).leptin can signal the body fat level to the brain to control energy homeostasis by regulating the activity of neurons in the hypothalamus . in a study , leptin in maternal plasma increased from 15.5 9.0g / l in the 18 week of pregnancy to 17.710.7 g / l in the 35 week ( 2 ) . also , leptin contributes greatly to the growth and development of the embryo , fetus and infant ( 3 ) , because it is synthesized by placental tissue and changes with adipocity and glucose metabolism in pregnancy ( 4 ) . neuropeptide y , a polypeptide containing 36 amino acids , is a potent orexigenic agent that regulates eating behaviour . neuropeptide y is synthesized in the hypothalamic arcuate nuclei and secreted by nerve terminals in the paraventricular nucleus . the major stimuli for neuropeptide y synthesis and secretion are food deprivation , reduced circulating insulin levels and elevated serum glucocorticoid levels . therefore , neuropeptide y initiates the outflow of efferent stimuli in the brain , resulting in increased appetite and food intake ( 5).previous studies have suggested that neuropeptide y is involved in hyperphagia during pregnancy and lactation ( 6).in a study , the mean plasma neuropeptide y concentration was higher in pregnant women during the first trimester of gestation compared to non - pregnant women . in another study , the mean plasma l in non - pregnant women ; this value was 144 13 and 156 24 pmol / l in the second and third trimesters of pregnancy , respectively ( 7 ) . it seems that the leptin - neuropeptide y mechanism counteracts the vasoconstriction attributable to the preeclamptic changes in the placenta and may cause trophic changes in placental tissue through its receptors . placental neuropeptide y production could not affect theneuropeptide y concentrations in the maternal or fetal circulation ( 8 , 9 ) . insulin is a hormone which plays a key role in the metabolism of blood glucose , causing the glucose level to decrease in the blood and increase in the liver . in addition , an increase in fat is usually accompanied with an increase in insulin ( 10 ) . insulin also inhibits food intake in animals through modulating neuropeptide y expression ( 11).on the other hand , the up - regulation of maternal islet function has a vital role in accommodating the heightened demand for insulin during pregnancy . in 0 , 45 , 90 , 135 and 180 minutes after glucose loading in pregnant women , the mean plasma insulin level was 25.2 , 77.2 , 57.0 , 57.8 and 44.7 u / ml , respectively . ramadan is the ninth month in themuslim lunar calendar.ramadan fasting is one of the five pillars of islam.the majority of muslims fast from dawn to dusk during the whole month of ramadan . the daily fast ( neither food nor drink ) in ramadan lasts nearly 12 - 19 hours every day depending on the geographic location and the season ( 12 ) . the fasting muslims usually have 2 meals per day , iftar at sunset and sahari before dawn ( 13 ) . many pregnant muslim women fast during ramadan every year worldwide . fasting during pregnancy in the form of skipping breakfast and other meals pregnancy is a state characterized by physiologicalhyperphagia ( 14 ) and modifications in maternal adiposity which causes an increase in adipose tissue mass during the early phase , followed by a decrease in the fat mass during the late phase . in a study conducted on animals , lipogenic pathway was predominant during the earlier phase , but it grew more active in the final phase ( 15).increased insulin secretion stimulates triglycerides synthesis ( 16 ) , and increased fat stores stimulate the release of leptin by the adipocytes ( 17 , 18 ) . consequently , leptin inhibits neuropeptide y secretion , while energy loss is accompanied by reduced leptin secretion and stimulated neuropeptide y synthesis ( 19 ) . very little research exists on those pregnant women who fast during ramadan , and no research has yet examined the effect of fasting on leptin , neuropeptide y and insulin level during pregnancy . because leptin , neuropeptide y and insulin are involved in the modulation of energy balance , we attempted to evaluate these parameters during ramadan and after ramadan in fasting pregnant women . thiscross sectionalstudy was conducted in the ramadan of 2012 ( from july 21 to august 18 ) in shahrekord university of medical sciences , iran . this study was conducted on 39 healthy volunteer fasting pregnant women aged 18 - 45 years , with a gestational age of 7 to 39 weeks and a mean ( sd ) of 22.4 ( 7.9 ) weeks . all the participants signed an informed written consentin which the experimentalprocedures was described in detail , and they were excluded from the study if they had diabetic mellitus , hypertension or if they were smokers . all the participants abstained from food and drink during the day from dawn to sunset and ate only during the night and completed fasting till the end of ramadan . this research was approved by the scientific advisory and ethics committee of shahrekord university of medical sciences . the participants were weighed barefoot with light clothes , standing on a standard pro - calibrated balance . to determine body mass index ( bmi ) , body mass was measured to the nearest one kg and height to the nearest one cm . at the end of ramadan , fasting venous samples were taken at 1:00 - 2:00 a.m. to measure the serum level of leptin , neuropeptide y and insulin . serum leptin levels were measured by radioimmunoassay according to the instructions of the manufacturer ( linco , st . neuropeptide y was assayed through competitive radioimmunoassay by direct assay without extraction using reagents ( euro - diagnostica ab , sweden ) . the human insulin specific radioimmunoassay detects insulin with a sensitivity of 2 m u / ml . blood glucose was measured by the hexokinase kit ( pars azmoon co. , iran ) . all measurements were done five times on 0 , 7th , 14th and 28th day of ramadan and on the14th day post - ramadan . the parametric repeated measures anovawas applied to determine whether any changes occurred among the variables of interest during the study . the multivariate f - tests of greenhouse - geisser were used for within the subject analysis due to the violation of sphericity assumptions . the tests of within - subject contrast were used to compare the mean of measurements . statistical significance was defined as p < 0.05 and analysis was performed by spss . thirty nine healthy fasting pregnant women voluntarily participated in this cross - sectional study with the following conditions : an age range of 17 to 42 years and a mean of 26.9 ( 6.4 ) , gestational age of 7 to 35 weeks with a mean of 21.9 ( 7.5 ) and a bmi of 18.3 to 35.9 kg / m2 with a mean of 25 ( 4.2 ) at the beginning of the study . the results relevant to the concerned variables including serum fasting blood sugar ( fbs ) , insulin , neuropeptide y and leptin are demonstrated in table 1 , and the trends are shown in figs . two weeks after compared with the fourth week ( p= 0.009 ) two weeks after compared with the second week ( p=0.018 ) two weeks after compared with the fourth week ( p=0.002 ) two week after compare to the fourth week ( p=0.001 ) during the study , the weight and bmi of women did not change significantly . fbs decreased significantly during ramadan and increased significantly after ramadan in such a way that the level of estrogen at the end of ramadan was the lowest of all . therefore , fbs was significantly higher two weeks after ramadan compared to the end of ramadan ( p= 0.009 ) ( fig . 1 ) . no significant change was found in the insulin level during the study . 3 ) : leptin level decreased until the second week , and then it increased at the end of ramadan and it decreased again significantly two weeks after ramadan compared to the fourth week of ramadan ( p= 0.02 ) . a monotonous increasing trend was found in neuropeptide y during ramadan and two weeks after ramadan on such a way that neuropeptide y was significantly higher two weeks after ramadan than its value at the end of ramadan ( p= 0.001 ) ( fig . serum leptin was correlated with bmi corresponding measurementat the second and fourth weeks of ramadan and at two weeks after ramadan . however the mean of leptin during the first , second and third trimesters and in the postnatal stage was higher in pregnant women compared to the non - pregnant because of the increase in food intake with the advancement of pregnancy ( 20 ) . leptin concentration gradually elevated during the first to third trimesters with a peak at 28th week , and its levels were significantly correlated with maternal weight and bmi ( 21).on the other hand , the increase of leptin levels during pregnancy may be due to a decrease in the rate of leptin clearance from maternal blood circulation ( 22 , 23 ) . the elevation of the serum leptin during pregnancy has also been reported by other investigators ( 24).also , previous studies indicated that short - term total fasting or a chronic reduction in caloric intake resulted in the reduction of leptin by 30% to 66% of basal level ( 25 , 26).leptin secretion decreases in response to fasting ( 27 ) and increases in response to positive energy balance induced by overfeeding ( 25 ) . because energy intake increases during ramadan , it is possible that the elevated levels of leptin in this study may have been due to the compensatory increase in food intake during the night ( 28 ) . our findings support those of previous studies.in our study , serum leptin level in the fourth week of ramadan significantly increased and was correlated with bmi in the second and fourth weeks of ramadan . a study showed that plasma leptin is secreted in a pulsatile fashion with peak levels at night and a nadir at noon ( 29).also , there is evidence that the diurnal rhythm of leptin secretion is related to the meal pattern and that shifting meal time causes a comparable shift in plasma leptin rhythm ( 30).because ramadan fasting was associated with a forward shifting of lunchtime by approximately 8 hours per day in our study , this shifting caused an increased level of leptin in the circadian pattern of leptin with progressive proximity toward the peak nocturnal level . therefore , the elevation of serum leptin level in our study may be due to the changes in shifting meal time because serum leptin is considered to reflect the state of nutrition and energy reserve ( 31).furthermore , serum leptin and its potential role in pregnancy bmi have been reported ( 32 ) . in the present study , during ramadan and two weeks after ramadan the serum level of neuropeptide this finding is in accordance with that of previous studies indicating that the level of neuropeptide y mrna content in the arcuate nucleus significantly increased during pregnancy ( 33).on the other hand , it has been demonstrated that diet restriction and starvation rapidly induce an accumulation of neuropeptide y in the paraventricular nucleus by increasing neuropeptide y mrna level in the arcuate nucleus ( 34).a study on rats also showed a very high concentration of prepro - neuropeptide y mrna following 96 hours of food deprivation ( 35).therefore , the elevation of neuropeptide y in our study may be due to the progression of pregnancy and fasting from dawn to dusk during the whole month of ramadan . nevertheless , in another study , the neuropeptide y decreased during ramadan in non - pregnant women ( 36 ) . in our study , insulin increases in pregnant and non - pregnant women in ramadan because fasting insulin and leptin are closely correlated with weight loss in obese women . in the present study , leptin , neuropeptide y and insulin were considerably higher in fasting pregnant women compared to the baseline levels obtained in the previous studies conducted on non - fasting pregnant women ; and this could be due to the need for more energy in fasting pregnant women who experience increased decomposition of lipid tissue . then , they consume more food and as a result their blood glucose and insulin increase , and the additional glucose causes the blood to turn into fat . having no control group was one of the limitations of this study . however , we measured the normal levels of serum leptin , neuropeptide y and insulin in non- fasting pregnant women as well . the results of this study revealed that circulating insulin and leptin relatively increased in pregnant women during ramadan fasting . in addition , it was found that serum neuropeptide y level progressively increased during ramadan and two weeks after ramadan . however , the increase in serum neuropeptide y was similar to that of leptin in fasting pregnant women . these data provide an insight into the important role of leptin and neuropeptide y in the long term regulation of energy balance in pregnant women , with chronic diurnal fasting . also , these results show that the level of factors considered in this study were higher in fasting pregnant women compared to non - fasting pregnant women . leptin levels were correlated significantly with bmi during and after fasting , but neuropeptide y had no relationship with bmi . the most important finding which was derived from this study was the lack of negative feedback between leptin and serum neuropeptide y concentrations in fasting pregnant women during ramadan . this study was supported by research and technology department of shahrekord university of medical sciences ( grant no . 963 ) .

background : many pregnant muslim women choose to fast during ramadan every year worldwide . this study aimed to examine the effect of ramadan fasting on serum leptin , neuropeptide y and insulin in pregnant women and find whether fasting during pregnancy could have a negative effect on the health of mothers and fetuses . methods : this cross - sectional study was conducted on 39 healthy volunteer fasting pregnant women . serum leptin , neuropeptide y , insulin levels , body mass index and weight were measured five times on 0 , 7th , 14th and 28th days of ramadan and on the 14th day post - ramadan . the data were analyzed by spss software ( version 11.5 ) using repeated measures anova to find whether any changes occurred in the variables of interest during the study , and pearson correlation coefficient was used to examine the relations among the variables . results : a significant change in fasting blood sugar , neuropeptide y and leptin was observed during the study ( p < 0.05 ) . fasting blood sugar decreased significantly during ramadan and increased after ramadan , with the lowest value at the end of ramadan . neuropeptide y increased both during ramadan and two weeks after ramadan . also , leptin decreased significantly two weeks after ramadan compared to the end of ramadan . no significant change was observed in insulin level during the study ( p>0.05 ) . conclusion : the result of this study revealed the important role of leptin and neuropeptide y in the long term regulation of energy balance in pregnant women with chronic diurnal fasting , and it further revealed that ramadan fasting did not significantly change the serum insulin level .

alopecias can be broadly classified into non - scarring or non - cicatricial and scarring or cicatricial forms . pathologically , a scar constitutes the end point of reparative fibrosis with permanent destruction of the preexisting tissue.1 scarring alopecias are further subdivided into primary and secondary types . in primary scarring alopecias , the hair follicle is the primary target of destruction , which is microscopically evident as preferential destruction of follicular epithelium and/or its associated advential dermis with relative sparing of the reticular dermis.2 this group includes the following clinical entities : chronic cutaneous lupus erythematosus ( ccle ) , lichen planopilaris ( lpp ) , pseudopelade of brocq ( pb ) , folliculitis decalvans ( fd ) , dissecting cellulitis / folliculitis ( df ) . if the follicular destruction is secondary to the scarring and occurs outside the follicular unit , e.g. , the reticular dermis , epidermis , or sub cutis , it can eventually impinge upon and even eradicate the whole follicle . the term secondary scarring alopecia implies that follicular destruction is not the primary pathologic event . exogenous factors , such as burns , and endogenous infiltrative and inflammatory diseases , such as sarcoidosis , pemphigus vulgaris and reticular dermal sclerosis , can result in secondary alopecias . in 2001 , a group of hair clinicians , pathologists and researchers , under the rubric of the north american hair research society ( nahrs),3 issued a consensus opinion on the classification of primary cicatricial alopecias . the proposed classification subdivides primary scarring alopecias on the basis of the predominant type of inflammatory cell component , an approach that had already published1,4,57 and was further refined by the workshop . in addition to the lymphocyte- and neutrophil - associated subgroups , a mixed and nonspecific group was differentiated and newly defined ( table 1 ) . in the late phase of cicatricial alopecias , a histopathologic diagnosis is more difficult because the main criteria of classification , i.e. , the type of inflammatory infiltrate , can not always be evaluated . in these cases , additional criteria , such as the evaluation of the perifollicular elastic sheet and the fibrosis , the goal of this study was to review clinical and histopathologic findings of 38 patients diagnosed with late , pauci or non - inflammatory phase of cicatricial alopecias at the dermatologic department of the hospital das clinicas , so paulo university medical school , over a six - year period in order to classify them by sub - types according to nahrs and evaluate the dermal elastic system and thickness of the epidermal basement membrane , using weigert and pas stains , respectively . biopsy specimens obtained using 5-mm punches from patients seen between 2000 and 2005 at the dermatologic department of hospital das clinicas , so paulo university medical school with cicatricial alopecias in the late , pauci or non - inflammatory phase were reevaluted . essential criteria included histopathologic diagnosis of cicatricial alopecia and accessibility of the patient s clinical records . slides stained with hematoxylin and eosin , pas and stain for elastic tissues ( weigert with previous oxidation by peracetic acid ) were reviewed , evaluating the presence or absence of criteria listed in the table 2 . a total of 38 cases with a histopathologic diagnosis of cicatricial alopecia were included in the study . these cases had been previously diagnosed as follows : eight cases of ccle , two cases of lpp , 13 cases of pb , two cases of fd , one case of scleroderma and 13 cases of non - specific cicatricial alopecia . twelve of 13 cases ( 92.3% ) that were initially classified as non - specific cicatricial alopecias could be specifically reclassified . chronic cutaneous lupus erythematosus was diagnosed in 17 cases , representing 43.6% of the total number of scarring alopecias . the female to male ratio was 4.7 : 1 ( 14 females and three males ) . the age at onset ranged from 29 to 75 years , with a mean age of 46.5 years . characteristic histopathologic findings of the late phase included hyperkeratosis , horn plugs , atrophy of the malpighian layer , slight vacuolar degeneration of the basal layer , and fibrous tract replacing the follicles . a thickened basal membrane could be seen in 58.8% of the cases on pas stained sections , and an incomplete elastic sheet occurred around the fibrous tracts . in all cases on weigert- stained sections . elastolytic foci were also found in areas of fibrosis outside the perifollicular zone ( figure 1d f ) . this category of cicatricial alopecia represented 10.2% of the total number of biopsies , totaling four cases . in one case , there was a lichenoid infiltrate around the infundibuloisthmic segment of one follicle in addition to fibrous tracts replacing follicles . in all the others , absence of inflammation , absence of sebaceous epithelium , and atrophy of the bulge area occurred , resulting in hourglass figures . fibrous tracts replacing the follicle , with or without the presence of colloid bodies , were also observed . the basal membrane was not thickened on pas - stained sections , and the perifollicular elastic sheet was partially destroyed , as observed with the weigert stain ( figure 1g pseudopelade of brocq was diagnosed in 12 cases , representing 30.8% of the total number of biopsies . age of onset ranged from 23 to 69 years , with a mean age of 54.4 years . characteristic histopathologic findings were the absence of criteria seen in ccle and lpp and preservation of the elastic sheet around the follicles ( figure 1a c ) . folliculitis decalvans was diagnosed in three cases , representing 7.7% of the total number of biopsies . the histopathologic hallmark was the presence of superficial suppurative folliculitis and fibrosis replacing the follicle and the perifollicular area , with elastolysis visible with the weigert stain ( figure 2a c ) . this category of cicatricial alopecia represented 2.6% of the total number of cicatrizing biopsies , totaling one case in a female 19 year - old patient . in this case , the inflammation was slight , and there was an extensive and deeper dermal fibrosis extending to the hypodermis , with elastolysis ( figure 2d f ) . one case of non - specific alopecia was diagnosed due to the destruction of the follicles being the only histopathologic finding . this category of cicatricial alopecia represented 10.2% of the total number of biopsies , totaling four cases . in one case , there was a lichenoid infiltrate around the infundibuloisthmic segment of one follicle in addition to fibrous tracts replacing follicles . in all the others , absence of inflammation , absence of sebaceous epithelium , and atrophy of the bulge area occurred , resulting in hourglass figures . fibrous tracts replacing the follicle , with or without the presence of colloid bodies , were also observed . the basal membrane was not thickened on pas - stained sections , and the perifollicular elastic sheet was partially destroyed , as observed with the weigert stain ( figure 1g pseudopelade of brocq was diagnosed in 12 cases , representing 30.8% of the total number of biopsies . age of onset ranged from 23 to 69 years , with a mean age of 54.4 years . characteristic histopathologic findings were the absence of criteria seen in ccle and lpp and preservation of the elastic sheet around the follicles ( figure 1a c ) . folliculitis decalvans was diagnosed in three cases , representing 7.7% of the total number of biopsies . the histopathologic hallmark was the presence of superficial suppurative folliculitis and fibrosis replacing the follicle and the perifollicular area , with elastolysis visible with the weigert stain ( figure 2a c ) . this category of cicatricial alopecia represented 2.6% of the total number of cicatrizing biopsies , totaling one case in a female 19 year - old patient . in this case , the inflammation was slight , and there was an extensive and deeper dermal fibrosis extending to the hypodermis , with elastolysis ( figure 2d f ) . one case of non - specific alopecia was diagnosed due to the destruction of the follicles being the only histopathologic finding . considering the group of primary lymphocytic cicatricial alopecias in the inflammatory phase , a differential diagnostic consideration for ccle is lpp . the superficial and deep perivascular and periecrine patterns of inflammation further aid in differentiating ccle from lpp . in the late pauci or non - inflammatory phase , as observed in our cases of ccle , interfollicular epidermis alterations were evident ( hyperkeratosis and atrophy ) , in an area usually spared in lpp cases . commonly , in the dermis in lpp , the only disturbance was the fibrous tract replacing the follicle , while in ccle , there was frequent extra follicular fibrosis and elastolysis ; even in this late phase , the basement membrane was thickened in 58.8% of cases . ccle is differentiated from pb by the presence of predominately vacuolar interface changes at the level of follicular infundibulum in the former and lack of interface alterations in the latter.1 in our cases , the perifollicular elastic sheet was preserved in pb cases , while it was partially destroyed in lpp and ccle , confirming the previous findings of pinkus et al.7 some authors believe that pb is not a distinct clinical - pathologic entity but a variant of certain primary cicatricial alopecias or , alternatively , a form of end - stage alopecia caused by other scarring alopecias , such as ccle , lpp and fd.79 according to stephen ( 1993)1 and other authors , with whom we agree , pb does indeed have sufficient distinct pathologic features to merit a separate classification.5,1012 klaus and wilma ( 2006)1 believe that the close clinicopathlogic correlation in the histologic absence of significant follicular plugging , as well as the use of elastic fiber stain in addition to evaluation of conventional he - stained sections , should enable the differentiation of classic pb from late stage lesions of lpp and ccle in most cases and further justify the classification of the condition as an entity sui generis . in relation to the neutrophilic cicatricial alopecias , it is remarkable that even in the late phases of this disorder , it seems that the inflammatory process persists in our cases , , and a neutrophilic folliculitis can frequently be seen , which helps make the correct histopathologic diagnosis . we could also see that the fibrotic process that is secondary to this neutrophilic folliculitis was more intense and proceeded by a granulation tissue , as is expected after a suppurative process . sinus tracts are the histopathologic hallmark of dissecting cellulites and are not seen in either folliculitis decalvans or acne keloidalis and were not observed in our late phase cases . bacterial or fungal folliculitis ( kerion and favus ) may have to be excluded in the group of neutrophilic cicatricial alopecias , with the use of special stains . acne keloidalis cases were not seen in our series because they are histopathologically reported in our laboratory as a superficial and deep suppurative and giant cellular granulomatous folliculitis with extensive fibrosis compatible with acne keloidalis , not as a cicatricial alopecia . besides , the clinical aspect of this disorder is so typical that histopathologic exam is seldom required . a scalp biopsy is mandatory in all cases of cicatricial alopecias , representing the clinically inflammatory area . if there is no evidence of inflammation , a biopsy at the border of the cicatricial zone should be performed . a precise diagnosis is possible , even in the late , pauci or non - inflammatory phase of cicatricial alopecias if a systematic evaluation of a constellation of criteria is employed , using routine he , pas and a stain for elastic tissue . in our cases , this method allowed a precise diagnosis in 97.4% of cases , even in those initially considered non - specific cicatricial alopecia .

backgroundscarring alopecias are classified into primary and secondary types according to the initial site of inflammation . in primary scarring alopecias , the hair follicle is the main target of destruction ; the term secondary cicatricial alopecia implies that follicular destruction is not the primary pathologic event.aimsto review the histopathologic diagnoses of cases of cicatricial alopecia in order to classify them according to the north american hair research society.patients and methodspatients with biopsy specimens diagnosed as cicatricial alopecia seen from 2000 to 2005 at the dermatologic department of hospital das clinicas , so paulo university medical school had hematoxylin and eosin , periodic acid - schiff and weigert stained slides reevaluated and sub - typed into different primary cicatricial alopecias.resultsthirty-eight cases of primary cicatricial alopecias were reclassified as : chronic cutaneous lupus ( 17 ) , lichen planus pilaris ( 4 ) , pseudopelade of brocq ( 12 ) , folliculitis decalvans ( 3 ) , dissecting folliculitis ( 1 ) , and non - specific scarring alopecia ( 1 ) . in our cases , the methods employed allowed an accurate diagnosis in 12 of 13 cases ( 92.3% ) previously classified as non - specific cicatricial alopecias.conclusionseven in the late , pauci or non - inflammatory phases , an approach with systematic evaluation of a constellation of criteria in routine hematoxylin and eosin stain , periodic acid - schiff and weigert stain allowed for a more accurate diagnosis of cicatricial alopecias .

tetralogy of fallot ( tof ) is a cyanotic congenital cardiac defect characterized by pulmonary outflow tract stenosis or obstruction , ventricular septal defect ( vsd ) , overriding aortic root , and right ventricular hypertrophy.1 left without surgical correction , the overall survival was poor and over 90% of the patients die by 40 years of age.2,3 there are some subsets of tof patients with advanced chronic kidney disease ( ckd ) ; however , the association between tof and renal failure severe enough to require dialysis treatment has been rarely reported in the literature.4,5 in this report , we describe the case of an end - stage kidney disease patient with tof . we also discuss the complex processes of how and why peritoneal dialysis ( pd ) was selected as a mode of chronic renal replacement therapy in this case . a 33-year - old female with tof managed by palliative surgery was admitted to our hospital with complaints of general fatigue and appetite loss at the end of december 2013 . taussig shunt at 2 years of age and palliative right ventricular outflow tract reconstruction , which is a procedure used to enlarge the ventricular outflow tract and main pulmonary artery , without inducing the closure of the concurrent vsd,6 at 4 years of age . there was no known family history of congenital cardiac disease . at 24 years of age , the patient s serum creatinine ( scr ) levels were increased at approximately 1.3 mg / dl , and her scr levels thereafter continued to increase gradually , but slowly . in 2012 , at 32 years of age , she became hypertensive , and her scr levels were approximately 3.1 to 3.4 mg / dl with 3 + for urine protein when the additional surgical correction for closure of the vsd was planned . she was then referred to our hospital . according to the clinical pictures and renal sonographic findings , which showed decreases in the renal long axis dimensions ( right : 80 mm , left : 85 mm ) with grade ii renal cortex echogenicity , the patient was diagnosed with ckd7 and subjected to contemporary and comprehensive renal care . despite the successful control of her blood pressure to the ranges of 130140/7080 mmhg with losartan potassium and azelnidipine , her renal function steadily worsened during the last year , and she finally became aware of the symptoms at the end of december 2013 when her scr and blood urea nitrogen ( bun ) levels elevated up to 8.25 and 99 mg / dl , respectively . as a result , she was admitted for further workup . a physical examination completed on the patient s blood pressure was 134/59 mmhg , her pulse was 78 beats / minute , and her temperature was 37.0 c . although the patient s oxygen saturation was 85% while she breathed ambient air , a chest x - ray film demonstrated neither an accumulation of fluid nor a sign of pulmonary infiltrates . a laboratory evaluation revealed the following results : white blood cell count , 9,000/l ; red blood cell count , 407 10/l ; hemoglobin ( hb ) , 12.1 g / dl ; hematocrit , 36.4% ; platelet count , 19.5 10/l ; bun , 111 mg / dl ; scr , 8.03 mg / dl ; total protein , 7.7 g / dl ; albumin , 4.7 g / dl ; sodium , 139 mmol / l ; potassium ( k ) , 5.3 mmol / l ; chloride , 108 mmol / l ; calcium ( ca ) , 10.5 mg / dl ; phosphorus ( pi ) , 7.2 mg / dl ; aspartate aminotransferase , 13 u / l ; alanine aminotransferase , 12 u / l ; c - reactive protein , 0.02 mg / dl ; and brain natriuretic peptide , 55.4 pg / ml . her urine was 3 + for protein and + for occult blood and contained 1.3 g of protein in a 24-hour specimen , while the patient s creatinine clearance was 5.02 ml / minute . transthoracic echocardiography disclosed a perimembranous vsd ( maximum diameter of 21 mm ) , a bidirectional shunt with left - to - right shunting ( 2.55 m / second ) predominance , and well contracting left ventricle ( lv ) with an lv ejection fraction of 63.8% ( fig . 1a ) , while chest computed tomography ( ct ) scans demonstrated right pulmonary artery stenosis ( fig . the patient was subjected to a transient session of hemodialysis ( hd ) treatment with repeated femoral vein puncture8 between hospital days 2 and 14 , during which time she began to feel well along with the decline in her scr level . finally , a tenckhoff pd catheter was placed on hospital day 17 through a classic transverse surgical incision with a favorable postoperative course . pd using two daily exchanges ( 1.5 l of midperiq135 2 , terumo co .. ) with a total dwell of 12 hours was then initiated ( fig . , the patient is still doing well with a daily urine output of approximately 1,100 to 1,300 ml . considering the current therapeutic guidance for pd adequacy,9 her solute clearance status expressed in terms of kt / vurea , where k is the clearance of urea , t is the treatment duration , and v is the urea distribution volume,10 also appears to be favorable ( a total kt / vurea : 1.9 [ peritoneal kt / vurea : 0.8 , residual renal kt / vurea : 1.1 ] ) . along with the decline in the mortality of patients with congenital heart diseases , it has become clear that various types of disturbed physiologies occur beyond the cardiovascular system.5,11 chronic renal insufficiency is one such late complication , and it may also be common even in adult subjects after obtaining the palliation of previous tof.5,11 several morphological alterations , including glomerular enlargement , mesangial hypercellularity , glomerular capillary congestion , and segmental glomerular sclerosis , have been focused on as pathologic bases of the disease.5,12,13 in the current patient , the absence of any pathological information precluded us from precisely evaluating the cause of end - stage kidney disease . however , we believe that numerous conditions , including previous surgical palliation and long - standing cyanotic conditions , could play a role in the establishment of her renal manifestations.5,11 alternatively , or in addition , the long - term use of various analgesics for relieving menstrual pain and a tension headache , which was revealed by our thorough clinical interview , might have played a role in our patient through their nephrotoxic nature.14 thus , the combination of tof and ckd may not be surprising ; however , the clinical significance of the current patient should be evaluated carefully in terms of the impact of the circulation disturbance due to cardiac anomaly on the therapeutic managements for end - stage kidney disease , including the choice of dialysis modality . for many patients with end - stage kidney disease , a renal transplant is the treatment of choice as it replicates the standard renal physiology much more closely than dialysis treatments and offers an improved quality of life as well as survival benefits.15,16 despite the high risk of cardiac death before and after renal transplantation,17 patients with cardiac disease or who are at high risk for cardiac disorders are still eligible for this procedure.18 moreover , it has been stated that patients with severe irreversible heart dysfunction should not be listed for kidney transplantation alone , while there may be select patients who are candidates for combined heart kidney transplants.18 the scarce information regarding renal transplantation among patients with unrepaired congenital heart disease precludes us from precisely evaluating the validity of such a strategy in the current case ; however , the lack of readily available cadaveric or living renal transplantation in a timely manner , which leaves many subjects requiring dialysis,15,16 obliged us to start dialysis treatment . considering the pathophysiologic characteristics of tof,1,19,20 we felt that avoiding the use of dialysis catheters as a means of either temporary or permanent vascular access for hd and the execution of periodic hd with peripheral vascular access should be mandatory in the current patient . indeed , these devices may predispose patients with tof to the detrimental pathologies such as pulmonary embolism , septicemia , infectious endocarditis , and paradoxical embolism,2127 although the available literature has seldom discussed pulmonary embolism in patients with tof.28 we must also recognize that in patients with an intrinsic cardiac anomaly , the creation of peripheral vascular access may result in severe cardiac failure.29 otherwise , it should be noted that bacteremia is less frequent in pd patients than in chronic hd patients with endovascular catheter , and no reports on infectious endocarditis in pd patients were available , despite the fact that septicemia is not exceptional in pd subjects30,31 ; therefore , pd does not appear to be an additional risk of infectious endocarditis among patients with end - stage kidney disease.31 alternatively , or in addition , the therapeutic nature of pd , including minimal variation in the intravascular volume status , reduction in cardiovascular stress , avoidance of peaks and troughs in uremic toxins , arrhythmia prevention , and the better preservation of residual renal function,10,32,33 also encouraged us to promote the procedure as a good modality of renal replacement therapy for the current patient . one may argue against our concerns about the potential risks associated with a dialysis catheter and hd . moreover , no one knows the impact of starting hd with repeated femoral vein puncturing for a transient vascular access on the risks of the concurrent development of thromboembolic and/or infectious events.8 nevertheless , it is necessary to take a proactive approach and not let lethal events become apparent . we believe that our policy regarding the application of tailored renal replacement therapy should be an appropriate therapeutic choice for the current patient . apparently , we are always facing , as do most physicians at various times , different types of clinical challenges , as described herein . there may be substantial variation in the type and intensity of managements provided to end - stage kidney disease patients with congenital heart disease , stemming from the uncertainty of the advantages and disadvantages of renal care in this population . a lack of prospective data suggests that numerous therapeutic decisions among such subjects are potentially empirical . indeed , only a few anecdotal reports describing patients with tof who began dialysis treatments during the observation periods for the disease are available.4,34,35 pd with reduced dialysate exchanges may be less advantageous than full - dose pd in terms of ultrafiltration and dialytic clearance , while such a pd protocol may permit early incremental dialysis and provide favorable clinical significance.33,36,37 at present , our patient appears to tolerate the pd program well with two daily dialysate exchanges ; however , the validity of our strategy should be determined only when more experience with additional cases similar to ours has been accumulated . thus , the establishment of an optimal management program for end - stage kidney disease patients with congenital heart disease should be a matter requiring continuous and careful attention .

in this report , we describe the case of an end - stage kidney disease patient with tetralogy of fallot ( tof ) . a 33-year - old female with tof was admitted to our hospital with complaints of general fatigue and appetite loss probably due to uremic milieu . she was ultimately treated with peritoneal dialysis ( pd ) with a favorable clinical course . tof patients with chronic kidney disease are not exceptional , although the currently available information regarding the association between tof and renal failure severe enough to require dialysis treatment is limited . we also discuss the complex processes of how and why pd was selected as a mode of chronic renal replacement therapy in this case .

chronic suppurative otitis media ( csom ) is an inflammatory disease of the middle ear and mastoid air cell system , which can result in irreversible changes not only in the middle ear structures but also in the inner ear.1 ) dysfunction of the eustachian tube and the bacterial infection are important factors in the multifactorial pathogenesis of csom . in order to eradicate inflammation , prevent recurrence and restore middle ear structures and hearing , both medical and surgical treatment modalities are important in csom . it has been emphasized that selection of appropriate antibiotics should be based on the bacteriologic studies to identify the pathogens and determine sensitivities.2,3 ) recent bacteriologic studies of the middle ear discharge in csom in korea have reported increasing prevalence of methicillin - resistant staphylococcus aureus ( mrsa).2,4,5 ) in addition , staphylococcus aureus ( s. aureus ) and pseudomonas species were most predominant from the postoperative otorrhea cultures.3 ) higher rates of postoperative otorrhea and reperforation after tympanomastoid surgeries were reported in csom patients with mrsa cultured from preoperative ear discharge.6 ) in addition to intrinsic mrsa infection of the mastoid air cell system , nasal colonization of mrsa may pose increased risk of postoperative infection . nasal colonization rate of mrsa and it is a known predictor of postoperative surgical site infection.7,8 ) previous studies have identified bacterial pathogens from the middle ear and the mastoid air cells , or nasopharynx as a bacterial reservoir in csom.4,5,9 - 12 ) however , few studies have focused on the effect of nasal mrsa colonization in the otologic surgery . the aims of this study are to examine the prevalence of nasal mrsa colonization in patients undergoing otologic surgeries and to determine the association between mrsa colonization and the pathogens identified from perioperative culture studies . the study included 66 consecutive patients ( 25 males and 41 females ) undergoing tympanomastoid surgeries from may 2010 to december 2011 at the yonsei university college of medicine gangnam severance hospital . patients with intracranial or extratemporal complications of csom , known history of external radiation , known history of corticosteroid therapy preoperatively , or those undergoing emergency operations were excluded . the mean age at operation was 51.715.7 years ( range 34 - 74 years ) , and the right ear was operated in 34 ears , the left in 32 ears . upon the initial visit in the outpatient department , the external auditory canal was cleaned , and the middle ear discharge was collected with collected with cotton swabs through sterile otoscopes , taking care not to touch the external auditory canal skin . to assess nasal colonization of s. aureus , the nasal samples were obtained by cotton swab from the anterior vestibular of the nares by the physician . during the operation , intraoperative culture specimens were taken in cases with overt discharge in the middle ear cavity . during postoperative period of 1 month , postoperative culture specimens were again taken in cases with otorrhea through the external auditory canal . all culture samples were in stuart transport medium and transported to the laboratory , where the samples were inoculated onto blood agar , macconkey 's agar , and chocolate agar plates . all plates were incubated at 37-c aerobically under 5% ( v / v ) co2 and examined 24 and 48 hours later . susceptibility testing was performed using disk diffusion . identified pathogen species and antimicrobial susceptibility were compared between nasal swab and perioperative ( preoperative , intraoperative and postoperative otorrhea ) cultures . statistical analysis was performed with fisher 's exact testusing spss statistical software ( version 15.0 ; spss inc . , among the 67 patients undergoing operation for csom , nasal swab confirmed bacterial colonization in 37 ( 55.2% ) patients ( table 1 ) . clinically importantly , nasal cavity colonization of s. aureus was noted in 10/67 ( 14.9% ) of patients , and 4/67 ( 6.0% ) patients were positive for mrsa . also , methicillin - sensitive staphylococcus aureus ( mssa ) was identified in 9/37 ( 23.4% ) and methicillin - resistant coagulase - negative staphylococcus ( mrcns ) in 11/67 ( 13.4% ) of patients . preoperative ear culture studies identified pathogens in 39/67 ( 58.2% ) of the patients : the most commonly identified species was staphylococcus [ mrcns 26.9% , methicillin - sensitive coagulase - negative staphylococcus ( mscns ) 1.5% , mrsa 13.4% and mssa 11.9% ] . mrsa was found in 3/40 ( 4.5% ) ears , mssa in 2 ears ( 3.0% ) , mrcns in 2 ears ( 3.0% ) and mscns in 1 ear ( 1.5% ) . after the surgery , 11 of the 67 patients developed otorrhea within 1 month postoperatively . the postoperative culture studies identified mrsa in 3/11 ( 27.3% ) and acinetobacter species in 1/11 ( 9.1% ) and fungus in 2/11 ( 18.1% ) patients ( table 2 ) . preoperative ear culture results and nasal swab culture results showed significant difference ( p=0.001 ) , as well as intraoperative middle ear cultures and postoperative otorrhea culture results showed significant difference ( p=0.023 ) . however , no significant difference was noted between preoperative ear and postoperative otorrhea culture results ( p=0.493 ) . then we compared the culture results according to the presence of bacterial growth and reports of no growth ( fig . no significant difference was noted between preoperative ear culture , and nasal swab culture results ( p=0.055 ) , between preoperative ear and postoperative otorrhea culture results ( p=0.068 ) , or nasal swab and postoperative otorrhea culture results ( p=0.094 ) . lastly , the culture results were compared according to the presence of antibiotic resistance ( fig . no significant difference was noted between the antibiotic - susceptible and resistant strains identified between preoperative ear culture and nasal swab cultures ( p=0.098 ) , or between preoperative ear and postoperative otorrhea cultures ( p=0.053 ) . then we analyzed the correlation of isolated organisms from the nasal swab and other cultures in patients with nasal colonization of antibiotic - resistant strains ( fig . 4 ) . among 4 patients with identified with nasal mrsa colonization , preoperative ear cultures were also positive for mrsa in 3 patients ( 75.0% ) . however , intraoperative culture identified mrsa in only one patient , and none of the patients developed mrsa infection postoperatively . preoperative ear cultures reported mrsa in 1 , mrcns in 1 , and mscns in 7 , ciprobay - resistant pseudomonas in 1 , and no growth in 2 patients . increasing incidence of antibiotic - resistant bacterial strains in csom patients poses a challenge to not only the selection of appropriate antibiotics therapy , but also to prevent the emergence of resistance to glycopeptides , the main drugs chosen for mrsa . it has been reported that patients who are colonized with s. aureus are the main source of s. aureus in hospitals , and that nasal carriage of mrsa increases the risk of postoperative infection . since the rate of methicillin resistance is higher in korean csom patients compared to other countries , we examined the rate of nasal mrsa colonization . the rate of nasal carriage of mrsa in csom patients was higher than among healthy individuals reported as 0.2 - 2.8% of the united states population.7 ) although this study was limited to small number of patients , the finding may suggest that in addition to nosocomial infection , community - acquired mrsa infection may contribute partially to the relatively higher rate of mrsa in csom patients in korea . on the other hand , the rates of bacterial isolation from preoperative ear discharge , intraoperative middle ear discharge , or postoperative otorrhea cultures in our patients were lower . also , the perioperative culture studies frequently reported no growth of pathogens . given that the patients were prescribed with antibiotics prior to surgery , it may signify that current antibiotics are effective in infection control during the tympanomastoid surgery . furthermore , the interpretation of the culture results from postoperative otorrhea is limited since antibiotics were routinely used postoperatively in all patients and culture samples were taken from the external auditory canal in cases with overt otorrhea or from wet surface of the grafted tympanic membrane immediately after removal of the packing material at 2 weeks postoperatively . the culture results can only reflect that mrsa infection that could not be controlled by routine antibiotics , which may be clinically significant . mrsa has been identified not only in csom patients,2,3,5,13 ) but also in patients with external otitis.14 ) s. aureus has been implicated in biofilm formation complicating cochlear implants.15 - 17 ) nasal mrsa colonization deserves attention since transmission from nasal carriage to surgical site infections or bacteremia is possible , and it is associated with increased morbidity and mortality . previous studies have reported that elimination of nasal mrsa colonization reduces the risk of postoperative infections.8,18,19 ) application of 2% mupirocin calcium ointment to the nares has been proven to be effective , and chlorhexidine - based solutions is used to reduce the density of skin colonization of mrsa preoperatively.18 - 20 ) a recent study compared the rates of mrsa infection in otolaryngology surgeries , before and after implementation of preoperative screening and treatment of nasal mrsa carriage.21 ) further studies are warranted to investigate the possible benefit of preoperative treatment of mrsa colonized patients undergoing middle ear surgeries . the rate of nasal mrsa colonization among patients with csom was higher than among the general community . further studies are warranted to investigate the possible benefit of preoperative treatment of mrsa colonized patients undergoing middle ear surgeries .

background and objectivesmethicillin - resistant staphylococcus aureus ( mrsa ) is one of major pathogens in patients with chronic suppurative otitis media ( csom ) . in addition to intrinsic mrsa infection of the mastoid air cell system , nasal colonization of mrsa , a known predictor of postoperative surgical site infection , may pose increased risk of postoperative complications . the purpose of this study is to describe microbiology of preoperative nasal swab screening and localized middle ear specimens in patients undergoing otologic surgeries.subjects and methodsforty - nine consecutive patients with csom who underwent middle ear surgery were included . preoperative nasal swabs for mrsa , and preoperative and intraoperative middle ear swabs were collected and compared for pathogens.resultspreoperative nasal swab screening confirmed mrsa colonization in 3/49 patients ( 6.1% ) and methicillin - resistant coagulase - negative staphylococcus ( mrcns ) in 9/49 patients ( 18.4% ) . correlation with preoperative culture results and nasal swab screening results were compatible in 2/4 patients with positive nasal swab for mrsa and 1/9 patients with positive nasal swab for mrcns . postoperative conversion to mrsa was observed in 3 patients.conclusionsthe rate of nasal mrsa colonization among patients with csom was higher than among the general community . preoperative mrsa colonization was associated with mrsa from middle ear specimens . further studies are warranted to investigate the possible benefit of preoperative treatment of mrsa colonized patients undergoing middle ear surgeries .

epidermolysis bullosa ( eb ) is a rare genetic disorder characterized by abnormal fragility of skin and mucosal surface . the separation of skin layers occurs after application of friction or shearing forces and results in intradermal fluid accumulation and bullae formation . in addition to considerations associated with positioning , monitoring , infection , and prevention of skin and mucosal trauma , anesthetic management of eb is uniquely challenging because of the effects on the airway . this case report describes the successful anesthetic management of a patient with eb presenting for syndactyly release . a 6-year - old male child , weighing 14 kg ; a known case of eb since birth presented with congenital left hand syndactyly [ figure 1 ] . significant preoperative findings were generalized scars , bed sores , pustules , and joint contractures . airway assessment revealed mallampatti class iii , microstomia , loose teeth , and poor oral hygiene with thyromental distance 3 cm . left hand shows syndactyly and right shows iv canula secured with nonadhesive technique airway assessment shows anticipated difficult airway oral antibiotics were administered preoperatively . intravenous ( iv ) line 22-g secured with use of emla and fixed with vaseline gauze . premedication with iv fentanyl 20 g , midazolam 0.4 mg , and dexamethasone 2 mg was given . were given with iv ranitidine 1 mg / kg and cefotaxime 50 mg / kg , respectively . intraoperative heart rate , rhythm , and oxygen saturation were monitored with lubricated clip on pulse oximeter . noninvasive blood pressure ( nibp ) and electrocardiogram monitoring were avoided [ figure 3 ] . patient was positioned supine and pressure points were padded with cotton . due to anticipated difficult airway , a difficult airway cart was prepared with various size masks , laryngoscope , endotracheal tubes ( ets ) , ventilating bougie , and stylet . though supraglotic airway devices were inappropriate in this patient they were kept ready for an emergency airway situation . minimal monitoring was used intraoperatively preoxygenation was followed by induction with iv propofol 20 mg along with oxygen and sevoflurane under gentle mask holding with vaseline gauze . after confirming ventilation , with use of lubricated macintosh laryngoscope trachea was intubated with uncuffed et no . 5 and tube secured with nonadhesive lubricated bandage [ figure 4 ] . et secure with nonadhesive technique anesthesia was maintained with oxygen , nitrous oxide , and sevoflurane with manual ventilation using jackson rees circuit . as rectal suppositories , subcutaneous and intradermal routes are generally not recommended ; postoperative analgesia was provided with iv diclofenac sodium 25 mg 8 hourly . regional block was avoided due to joint contracture and scars , which made location of anatomical landmarks difficult . neuromuscular blockade was antagonized at the end of the procedure , and trachea extubated when adequate signs of spontaneous recovery were evident . postoperatively , oral examination revealed small intra - oral blister formation not requiring active management . eb encompasses an array of autosomal dominant and recessive conditions that may have either localized or generalized dermatological manifestations . the loss or absence of normal intracellular bridges is due to a collagen abnormality , which makes patient susceptible for blister formation by friction / shearing forces and subsequent scarring . equipment and techniques routinely used in the induction and maintenance of general anesthesia can be the source of serious postoperative complications . anesthesia is frequently required for multiple surgeries like daily dressings , dental procedure , esophageal dilatation , gastrostomy , contracture release , and syndactyly release . malnutrition , anemia , and decreased immunity are present in patients due to decrease oral intake secondary to oropharyngeal and esophageal lesions . malnutrition can leads to hypoprotienemia , anemia and electrolyte imbalance which may affect pharmacokinetic effects of anesthetic agents . infection is common as patients often have both poor immunity and long - term corticosteroid treatment . antacid prophylaxis is required due to history of reflux , regurgitation , or esophageal stricture . patients with eb are prone for ophthalmic complications like corneal erosion , conjunctival symblepharon , and ectropion . dental problems like cleaning difficulty , poor eating patterns , enamel dysplasia , carious , and loose teeth are frequently associated . iv or intra - arterial access should be secured with bandage , cotton wrap , or suture . all persons involved in handling these children must be aware of the extreme vulnerability of skin . during transport or mobilization of the patient , the most important task is to maintain the integrity of the skin and avoid friction and trauma . direct pressure to the skin is not as damaging as frictional or shearing forces , so nibp and tourniquet can be used with pressure and duration limitations . as our case was of a short duration with no expected major blood loss and hemodynamic shifts , use of nibp and tourniquet therefore , clinical use of nibp and tourniquet in such patients depends on its merits and demerits . microstomia , neck contracture , oropharyngeal lesion , ankyloglossia ( decrease mobility of the tongue ) , thickened epiglottis , and possible tracheal stenosis make maintenance of a patent airway challenging . fiberoptic intubation is less traumatic to the mucosa than direct laryngoscopy and should be the first choice in eb patients with a difficult airway . laryngeal , tracheal involvement in eb is rare because that tissue is pseudostratified , columnar , ciliated epithelium and whereas oropharyngeal , esophageal mucosa is stratified squamous . supraglottic airway increases bullae formation but can be used in difficult airway scenario with appropriate care . after application of moisturizing ophthalmic gel , eyes should be covered with moistened gauze to protect from mechanical trauma . regional anesthesia should be considered whenever possible but contractures , scars , and infections are the issues associated with regional anesthesia . perioperative blister should be treated with liquid paraffin , silver sulphadiazine , or steroids . in ambulatory careful monitoring , transport and positioning was instituted in the present case to avoid undue skin trauma . atraumatic ventilation and intubation was possible with use of vaseline gauge and gentle mask holding . minimal mandatory monitoring and no touch principle remains the key to successful anesthesia management of eb cases . patients with eb present a unique challenge for all anesthesia - care providers . with maximal skin and mucous membrane protection , anesthesia in children with eb can be conducted with few sequelae . hence , meticulous execution of preoperative planning is indispensable for ensuring a favorable intraoperative and postoperative course .

epidermolysis bullosa ( eb ) is a rare genetic mechanobullous disorder , with excessive fragility of the skin and mucous membranes . avoiding mechanical injury to the skin and mucous membranes is essential in the anesthetic management . shearing forces applied to the skin result in bullae formation , while compressive forces to the skin are tolerated . the challenge is to use monitoring technology without damaging the epithelial surface . difficult airway , positioning issues , nutritional deficiencies , poor immunity , and carcinogenic potential add to the comorbidities . we managed a child with eb undergoing syndactyly release . ensuring maximal skin and mucous membrane protection , anesthesia in children with eb can be conducted with few sequelae .

stressful events activate the sympathetic nervous system and hypothalamic - pituitary - adrenal axis , which lead to the release of biochemical mediators of stress , such as cortisol , catecholamines , and neuropeptides [ 1 , 2 ] . these stress mediators trigger a variety of physiological changes meant to improve the performance of the organism , such as increasing blood pressure and heart rate and enhancing the immune response . thus , a short , acute stress has been shown to exert various beneficial effects . however , when stress becomes chronic , the prolonged exposure to the same stress mediators , which were beneficial in acute stress , often triggers pathological processes and contributes to the development or exacerbation of various diseases , including cancer . chronic stress has been implicated in the stimulation of tumor development and progression by both clinical and animal studies [ 46 ] . initially , stress - induced suppression of the immune response was suggested as the major mechanism of this phenomenon . as opposed to acute stress , which enhances immunity and has been shown to increase resistance to cancer , chronic stress impairs immune responses and in this way facilitates tumor growth [ 8 , 9 ] . however , there is also growing evidence indicating that stress mediators , such as glucocorticoids and sympathetic neurotransmitters , can directly affect tumor cell proliferation and survival as well as tumor angiogenesis . the direct effects on tumor cells vary significantly between different stress mediators and types of tumors [ 1013 ] . in contrast , their actions on tumor vascularization involve interactions with common angiogenic factors , such as vascular endothelial growth factor ( vegf ) , and seem to be universal between different tumor types [ 5 , 1416 ] . thus , stress mediators and their receptors can become novel targets in antiangiogenic tumor therapy . norepinephrine ( ne ) and epinephrine ( e ) belong to a family of catecholamines and are one of the best characterized stress neurohormones . ne is released primarily from the sympathetic nerves , while e is mainly secreted from the adrenal medulla . as the sympatho - adrenomedullary system is responsible for the body 's fight - or - flight stress response , circulating levels of both catecholamines ne and e activate the same and adrenoreceptors ( ar ) , which are widely distributed in all tissues . recently , ne and e have been implicated in stress - induced augmentation of tumor growth and progression . in an orthotopic model of ovarian carcinoma , the growth - promoting effect of stress was mimicked by a -ar agonist , isoproternol , and blocked by its antagonist , propranolol [ 5 , 6 ] . similarly , activation of -ar resulted in an increase in metastases in animal models of lung and breast cancer [ 18 , 19 ] . in all of the above models , the growth - promoting effects of stress , as well as direct activation of -ars , was associated with a significant increase in tumor vascularization , while -ar blockers reduced vessel density [ 5 , 6 ] . moreover , tumors derived from stressed animals had elevated levels of vegf and other angiogenic factors , and the growth promoting actions of -ar activation was reduced by blocking the vegf pathway . thus , an increase in angiogenesis appears to be the main mechanism of growth - promoting effects of ne and e. indeed , in various cancer cell types , such as ovarian cancer , colon cancer , melanoma , pharyngeal carcinoma , and multiple myeloma , activation of -ars present on tumor cells led to a dramatic increase in synthesis and release of angiogenic factors vegf , il-8 , and il-6 [ 5 , 16 , 2023 ] . these effects were mediated primarily via a -ar - dependent increase in camp levels , which resulted in the activation of protein kinase a ( pka ) and src [ 5 , 22 ] . adrenergic stimulation has also been shown to increase the secretion of metalloproteases , mmp-2 and mmp-9 , which further augment angiogenic and metastatic processes . interestingly , catecholamine - induced release of angiogenic factors from tumor cells can be further enhanced by its secretion from stromal cells , such as -ar - positive tumor - associated macrophages [ 24 , 25 ] . although the stimulatory effects of ne and e on the release of angiogenic factors seem to be the major mechanism of their tumor - promoting actions , these neurotransmitters can also exert direct trophic effects on endothelial cells ( ecs ) through -ars . phenylepinephrine , a non - vasoconstrictive -ar agonist , has been shown to induce ec proliferation and migration as well as promote capillary formation . since tissue ischemia is known to stimulate ne release from the sympathetic nerves , the direct angiogenic effect of ne can be significantly enhanced in hypoxic areas of tumors . thus far , the results of experimental studies have confirmed that ar agonists exert strong stimulatory effects on tumor growth and agree that the release of angiogenic factors is the main mechanism of these actions . these discoveries open new possibilities of treatment with well - known drugs , such as antagonists of ars . some clinical data indicating decreased incidence of prostate cancer among cardiovascular patients treated with -blockers corroborated the above findings [ 28 , 29 ] . however , it is important to remember that the indirect , pro - angiogenic effect of ar agonists mediated by other angiogenic factors depends on the presence of these receptors on tumor cells , thus it can be tumor - specific . moreover , the angiogenic actions of ne and e can be further modified by their direct effect on tumor cell proliferation and invasiveness , which in turn may differ among various tumors . in many cancer cell types , such as colon , ovarian , and prostate , . however , adrenergic stimulation can also inhibit proliferation of some tumor cells , as shown in melanoma and neuroblastoma [ 30 , 31 ] . in breast cancer , the adrenergic agonists seem to increase motility of cancer cells but at the same time inhibit their proliferation [ 13 , 32 ] . in agreement with these data , another clinical study indicated no effect of treatment with -blockers on the risk of breast cancer among cardiovascular patients [ 33 , 34 ] . thus , the success of potential cancer therapy targeting ars will depend on the type of tumor , its receptor expression pattern , and environmental factors , such as stress , which augment ne and e effects . dopamine ( da ) is not only a precursor of ne and e but is also an important neurotransmitter in the brain acting via two types of receptors d1 and d2 . in the periphery , da is synthesized in mesenteric organs as well as released from sympathetic neurons and adrenal medulla . levels of da are elevated during stress , but rather than mediating the fight - or - flight response , as ne and e do , its role involves coping with stress . it has been shown that administration of da inhibits the growth of various tumors , such as stomach , breast , and colon cancers [ 14 , 36 ] . consistently , in mice lacking the da transporter , which is normally responsible for uptake of this neurotransmitter , the elevated da levels were associated with reduced growth of lewis lung carcinoma . in gastric cancer , the endogenous levels of da were significantly lower than those in surrounding healthy tissue , indicating that the neurotransmitter acts as an endogenous tumor suppressant that needs to be inactivated to allow tumor progression . the main mechanism of these growth - inhibitory actions of da involves its direct antiangiogenic effect on ecs . in all animal models , treatment with da led to a significant reduction in tumor vascularization [ 14 , 36 , 37 ] . da has also been shown to block vegf - induced ec proliferation , migration , and vascular permeability . further studies revealed that da , acting through its d2 receptors , enhances endocytosis of vegf - r2 and decreases its membrane expression . this activity of da interferes with vegf signaling by reducing vegf - induced phosphorylation of its vegf - r2 and preventing the activation of downstream kinases fak and p42/44 mapk [ 38 , 39 ] . in addition to its effect on mature ecs , da has also been shown to block vegf signaling in endothelial progenitor cells ( epcs ) . as a consequence , da not only inhibits trophic functions of vegf in these cells but also blocks their recruitment from bone marrow . it has been shown that da levels are decreased in the bone marrow of tumor - bearing mice , which facilitates epc mobilization . since recent data strongly support a role for epcs in the tumor vascularization , da effect on epc function may significantly contribute to its growth - inhibitory effect . the role of da in stress - induced changes in tumor growth and progression has not been characterized . it seems that da is an endogenous inhibitory factor which requires inactivation for tumor growth , rather than sympathetic activation . however , in contrast to ne and e acting on specific tumors , da effects appear to be more universal , influencing various tumor types , via its direct actions on ecs and epcs . neuropeptide y ( npy ) is a 36-amino - acid peptide coreleased with ne from sympathetic nerves . npy is mainly known due to its anxiolytic effect in the brain and central regulation of food intake . in the periphery , npy inhibits the release of ne after sympathetic stimulation and acts as a vasoconstrictor . there is also a growing number of evidences that npy is a growth factor for variety of cells . the peptide has been shown to stimulate proliferation of vascular smooth muscle cells and neuronal precursors , while the trophic effect of npy on ecs revealed its angiogenic properties [ 4247 ] . the peptide stimulates proliferation and migration of ecs and promotes capillary tube formation , while in vivo , endogenous npy facilitates vascularization of ischemic tissues [ 43 , 46 , 47 ] . these actions are dependent on endothelial nitric oxide synthase ( enos ) activation and , partially , on the vegf pathway . the angiogenic activities of npy are mediated mainly by its y2rs , since npy - induced angiogenesis is severely impaired in y2r / mice [ 48 , 49 ] . due to its angiogenic properties , npy has been implicated in various pathological conditions associated with a deregulation of tissue vascularization , such as retinopathy , wound healing , atherosclerosis , and obesity [ 48 , 5052 ] . recently , its role in tumor angiogenesis has also been shown . in malignancies originating from neuroendocrine tissues , such as neuroblastoma and ewing 's sarcoma , npy released from tumor cells seems to be an essential factor involved in their vascularization . antagonists to npy receptors blocked the effect of both neuroblastoma and ewing 's sarcoma - conditioned media on ec proliferation . consequently , treatment with exogenous npy significantly increased vascularization of subcutaneous xenografts derived from both tumor cell types . as in the case of ne and e , the angiogenesis - related growth - stimulatory actions of npy are further modified by its direct effect on tumor cell growth and survival . for example , in neuroblastoma , the peptide stimulates proliferation of tumor cells via the same angiogenic y2rs , thereby further augmenting the growth of neuroblastoma xenografts . in contrast , in ewing 's sarcoma , npy induces tumor cell apoptosis via y1 and y5rs . as a result , exogenous npy inhibits growth of ewing 's sarcoma xenografts in vivo , despite increase in their vascularization . although neuroendocrine tumors , which synthesize and release endogenous npy , seem the most susceptible to tumor growth regulation by this peptide , npy and its receptors have also been implicated in nonneuronal types of tumors . for example , peptide yy ( pyy ) , which belongs to the same family of peptides and acts through the same receptors as npy , has been shown to inhibit proliferation of breast and prostate cancer cells via y4rs and pancreatic cancer cells via y2rs [ 5356 ] . thus , these direct effects on tumor cell proliferation and survival are an important aspect of npy actions in tumors and are often potent enough to overcome its angiogenesis - mediated growth - promoting effect . thus far , most of the studies addressing the role of stress in promoting cancer growth focus on the best known stress mediators catecholamines and glucocorticoids . there are no studies directly linking npy with stress - induced tumor growth and progression . however , systemic npy levels are also upregulated during stress , particularly those intensive and prolonged in nature . hence , once stimulated , the elevated levels of npy persist for a longer period of time . the physiological role of npy is to help cope with stress due to its central , anxiolytic effects [ 58 , 59 ] . however , it has been shown that elevated peripheral circulating levels of npy induced by intensive chronic stress can result in significant deleterious effects , such as enhanced atherosclerosis and diet - induced obesity , both of which are diseases associated with intensive tissue growth and upregulated angiogenesis [ 52 , 60 ] . thus , while high levels of npy in the brain improve stress coping , chronically elevated levels of the peptide in the circulation can result in a variety of side effects . as summarized above , the discoveries of recent years provided a significant body of evidence confirming an important role of sympathetic neurotransmitters and , consequently , chronic stress in regulating of tumor vascularization ( figure 1 ) . this research opens new avenues for developing novel therapeutics , as well as using already existing and well - characterized drugs , such as -blockers and da receptor agonists , in new clinical settings . this seems to be particularly important , since cancer diagnosis per se is usually a stressful event for the patient . however , careful consideration needs to be given to other actions of stress mediators , such as cancer - specific effects on tumor cells themselves , as well as changes in immune system , which can indirectly affect tumor development and progression . finally , since patterns of neuro - hormonal activation vary with different types of stress , tumor exposure to particular stress mediators would vary , too . thus , potential therapeutic value of modifying particular stress pathways may be dependent on a variety of factors .

recent evidence supports a longstanding hypothesis that chronic stress can influence tumor growth and progression . it has been shown that sympathetic neurotransmitters , such as catecholamines and neuropeptides , can affect both cancer cell growth and tumor vascularization . depending on neurotransmitter and type of tumor , these effects can be both stimulatory and inhibitory . norepinephrine ( ne ) and epinephrine ( e ) are potent stimulators of vascularization , acting both by inducing the release of angiogenic factors from tumor cells and directly on endothelial cell ( ec ) functions . as a result , activation of the adrenergic system increases growth of various types of tumors and has been shown to mediate stress - induced augmentation of tumor progression . dopamine ( da ) , on the other hand , interferes with vegf signaling in endothelial cells , blocks its angiogenic functions and inhibits tumor growth . another sympathetic neurotransmitter coreleased with ne , neuropeptide y ( npy ) , directly stimulates angiogenesis . however , proangiogenic actions of npy can be altered by its direct effect on tumor cell proliferation and survival . in consequence , npy can either stimulate or inhibit tumor growth , depending on tumor type . hence , sympathetic neurotransmitters are powerful modulators of tumor growth and can become new targets in cancer therapy .

the treatment of lung cancer with radiation has undergone significant improvements over the past decade . first , it is possible to better delineate the target volume through the advancement of imaging such as positron emission tomography ( pet ) fusion [ 1 , 2 ] . second , there has been an increase in the utilization of radiation planning and delivery systems such as intensity - modulated radiation therapy ( imrt ) , stereotactic body radiation therapy ( sbrt ) , and proton therapy that allow more conformal delivery of radiation to achieve dose escalation while minimizing toxicity to normal structures [ 6 , 7 ] . finally , the advancement of imaging modalities available during the planning and delivery process has made it feasible to adapt the target volume , both within a given fraction of treatment and between fractions , for such factors as internal motion , tumor response , anatomical changes , and weight loss . with the availability of 4d ct(four - dimensional computerized tomography)based radiotherapy planning and on - board imaging ( obi ) , accurate positioning of the target using daily image guidance may result in many advantages , such as a decreased probability of target miss , smaller setup margins , and less normal tissue exposed to high radiation doses . in addition , novel treatment adaptation algorithms can provide more effective modification of treatment plans to adapt to the changes in a patient 's anatomy and organ motion during and/or between ( intra- and/or inter- ) treatment fractions . adaptive radiotherapy , and will be the focus of this paper . in this examination of adaptive radiation in the setting of lung cancer , we will assess the impact of this development in sbrt , conventionally fractionated imrt , and proton therapy . we will first define key terms in adaptive radiation , and will then review prior studies assessing the magnitude of intrafractional changes , which we define as changes within a given fraction of radiation , from setup to delivery . this discussion will be followed by the effect of these changes on delivered dose and specific planning techniques that have been utilized to decrease this variation , specifically cone beam computed tomography ( cbct ) and respiratory gating . we will then focus on interfraction changes that occur during the length of treatment ( over a period of several weeks ) , such as changes in tumor mobility , tumor volume , anatomy , and body weight , and examine specific studies that assess the impact of adaptive planning on improving dose distributions in these setting . finally , we will appraise future directions of adaptive radiation , including selective dose escalation through the visualization of nonresponsive regions and internal tracking to monitor intrafraction motion in real - time , both of which may be able to allow for further sparing of critical structures while maintaining accurate delivery for treatment of this aggressive disease . 50 : . tumor visible by any imaging modality , to include both the primary tumor and any involved lymph nodes . lymph nodes greater than 1 cm in size in the shortest axis are generally considered positive for disease , but functional imaging such as pet scanning is critical for target delineation [ 10 , 11 ] . this region can not be visualized as a discrete structure with radiographic imaging , and the extent has been defined by surgical series with pathologic correlates , as well as autopsy series . as defined by giraud et al . , based on nsclc surgical specimens , an appropriate gtv to ctv margin for adenocarcinoma is 8 mm , and for squamous cell carcinoma , 6 mm . another study found that in stage i adenocarcinoma , a margin of 9 mm is sufficient to cover 90% of disease . in terms of mediastinal disease , a gtv to ctv margin has not been rigorously analyzed , but based on an abstract presented at the 2006 meeting of the american society for therapeutic radiology and oncology that found a maximal microscopic extension of lymph nodes is between 0.5 and 8.9 mm , we routinely use margins of 8 mm for nodal disease . ctv with a margin to account for organ motion ; or , in other words , changes in the size , shape , and position of the target during treatment . delineating the itv from 4d ct images involves assessing the target volume ( ctv ) on expiratory - phase images and then registering the outline to the images from other phases to create a union of target contours enclosing all possible positions of the target . an alternative method is to create a maximum intensity projection ( mip ) image by combining data from the multiple ct data sets with data from the whole - breath cycle and modifying the itv by visually verifying the target volume throughout the breathing phases ( typically 10 ) . in this process , attention should be paid to irregular breathing and breathing pattern variations during each treatment session and over the entire treatment course , as well as to the effects of these irregularities on the itv margin . because it is often more straightforward to delineate the boundaries of the gross tumor volume with motion on 4d ct image data sets as opposed to the clinical target volume , we previously proposed the concept of the internal gross tumor volume ( igtv ) , which envelops the gtv motion throughout the whole - breath cycle . in this process , rather than delineating the gtv , expanding to the ctv , and then adding the itv , the gtv is contoured , motion is assessed as outlined above ( i.e. , through the mip images or through all breathing phases ) , and this target is expanded to the igtv . then , the igtv is expanded to the itv ( itv = igtv + ctv ) . this latter method is what is commonly utilized at our institution to account for organ motion in the treatment of nsclc . ctv plus a margin to account for both organ motion and daily setup . or , the itv with a margin to account for daily setup . the application and revision of margins for the ptv and itv will be discussed in detail below . bissonnette et al . examined cbct images during each fraction of 18 patients receiving sbrt for medically inoperable stage i nonsmall cell lung cancer ( nsclc ) . the cbct images were performed at the beginning , midpoint , and end of each fraction to determine differences in tumor motion amplitude . the authors found that at a mean time of 35 minutes ( from the beginning of each treatment to the end ) , the mean change in tumor amplitude was 0.4 , 1.0 , and 0.4 mm in the medial - lateral ( ml ) , superior - inferior ( si ) , and anterior - posterior ( ap ) directions , respectively . these values were not statistically significantly different when compared to the initial respiratory correlated ( 4d ) ct scan , other than in patients in which abdominal compression was used , in which longer times on the couch were hypothesized to increase these differences . in another study , michalski et al . compared the amount of motion between the three aforementioned directional axes ( ml , si , and ap ) in 23 patients undergoing 4d ct scans . the authors found that the largest intrafractional extent of motion was in the si direction , with the largest range being 3.59 cm . a similar study from our institution also assessed respiration - induced tumor motion during radiation for lung cancer . this study by liu et al . assessed 166 tumors in 152 lung cancer patients , 57% of whom had stage iii or iv disease . the authors also found that the largest axis of motion was in the si direction , and that 39% of tumors moved > 0.5 cm in this direction , compared to 1.8% and 5.4% in the ml and ap directions , respectively . tumor motion was also found to be correlated with the amount of diaphragm motion , the si location of the tumor , and the size of the gtv , with smaller tumors exhibiting a greater degree of intrafractional motion . a study by thomas et al . demonstrated that mediastinal nodal regions also move substantially with the respiratory cycle , generally posteriorly and superiorly with exhalation , and that inferior nodal stations exhibit a greater degree of motion , such that the same issues with respiratory variation in parenchymal tumors can also be applied to mediastinal disease . thus , it can be concluded that : ( 1 ) inferior tumors move more than superior tumors , ( 2 ) the largest axis of motion is in the si direction , and ( 3 ) mediastinal lymph nodes are also subject to a significant degree of tumor motion . there are several methods to detect setup error and intrafractional variation during radiation therapy for nsclc . in this technique , two - dimensional images are composed in the treatment position , and the patient is set up on patient anatomical landmarks such as bony anatomy . historically , megavoltage ( mv ) imaging had been used , but the images produced with this technique were generally of poor image quality and exposed the patient to high radiation doses . in the past decade , the utilization of kilovoltage ( kv ) imaging has increased , which has improved image quality and decreased radiation exposure . in this method , a ct image is reconstructed on the treatment table from a set of projection images acquired at multiple angles around the patient . image reconstruction with this modality differs from conventional ct scans in that rather than a linear array of detectors being back projected to construct a 2d slice , a detector array ( e.g. , a flat - panel portal imager ) is used to reconstruct a 3d data set . ( figure 1 ) it follows that the ct images that are a result of this process are theoretically superior in improving intrafractional error as compared to portal images , and multiple studies have exhibited this improvement , particularly in sbrt where longer treatment times can lead to greater instability and less reproducibility of the setup process . intrafraction treatment variation has two components in lung cancer : uncertainties in patient positioning and variations in internal motion . nelson et al . examined the effect of lung tumor motion and setup uncertainties using implanted fiducial markers . the authors found that systematic and random uncertainties ranged between 4 and 6 mm in all three directions . with daily portal imaging , the authors found in a subsequent study that alignment based on implanted fiducials reduced systematic errors in the left - right and superior - inferior direction each by 3 mm . as a result , daily portal imaging is often utilized to decrease setup error in conventionally fractionated regimens , with a daily setup error of up to 5 mm . borst et al . compared cbct setup with portal imaging in daily setup for 62 patients in 524 scans with nsclc , and found that cbct reduced the setup error to less than 5 mm , from 51% of patients with setup errors more than 5 mm to 2% with cbct . bissonnette et al . assessed the accuracy of cbct in rt for lung cancer in patients receiving both sbrt for early - stage malignancies and in those patients undergoing conventionally fractionated treatment for locally advanced disease . the couch position was adjusted for discrepancies greater than 3 mm between the initial setup and treatment images . the accuracy of this adjustment was then verified with a second cbct . without cbct adjustments , positioning errors were found to exceed 5 mm in approximately 55% of patients . however , cbct decreased this error , such that systematic and random setup margin was within 3 mm for 82% of fractions in the sbrt group , and between 76% and 84% of the patients in the conventionally fractionated group . similar findings were published by grills et al . , who found that cbct image guidance significantly decreased setup margins in sbrt , with calculated precorrection population margins of 913 mm and 1014 mm with a stereotactic body frame and alpha cradle , respectively , while these same margins were 1 - 2 mm and 2 - 3 mm postcorrection , and 24 mm and 25 mm postreatment , respectively . the conclusion of these studies is that consistent cbct imaging can decrease the amount of systematic and random error on a daily basis ( to within 3 mm ) , and thus decrease intrafraction variation . lung tumors differ from many other treatment sites in that internal motion can account for large variations in tumor position , and as a result , dosimetry . mechalakos et al . examined treatment plans for 12 patients receiving radiation therapy for nsclc . the authors found that the dose to 95% of the gross tumor volume ( gtv ) , also known as the d95 , changed on an average of only 1.4% when normal breathing effects were incorporated . therefore , the authors concluded that while the chance of a 10% or greater decrease in d95 was less than 4% , patients with a large degree of respiratory motion could have significant effects , and thus these patients should be identified . a study from the university hospital rotterdam in the netherlands found that with a gtv to planning target volume ( ptv ) margin of 1.5 cm , approximately 11% of the tumor was not covered in mobile tumors . engelsman et al . found that when combined with setup error , respiratory motion reduced the tumor control probability ( tcp ) by almost 9% ( from 50% to 42% ) in patients receiving conventionally fractionated radiation to 70 gy in lung tumors . there are several methods in which tumor motion can be taken into account with the delivery of radiation therapy and which are utilized at our institution . first , breathing can be monitored during simulation and the igtv / itv can then be added as a margin to ensure that the tumor is treated adequately throughout all phases of the respiratory cycles as outlined above ( e.g. , if the tumor is noted to move 1.5 cm during treatment , then a 1.5 cm margin , termed the itv / igtv , is added to account for this respiratory motion . ) . if 4d ct imaging is not available at the institution , then an alternative method to account for respiratory motion is through the use of breath - hold spiral ct simulation . in this method , the patient is instructed to hold his breath during simulation , both at end inspiration and expiration . images are then taken at end - inspiration and end - expiration , such that an itv can be generated by combining the two ctvs from the inspiratory and expiratory scans . a disadvantage to the free - breathing method is that in tumors that have a large magnitude of motion , the amount of normal tissue that is treated can be relatively large , since radiation is being delivered throughout the breathing cycle . therefore , an alternative method of radiation delivery is to instruct the patient to hold their breath during treatment and activate the radiation while the tumor is in this full inspiratory , fixed position . the disadvantage to this technique ( often termed deep inspiratory breath hold , or disb ) is that it requires full cooperation of the patient , in that the patient will need to hold their breath for 15 seconds or longer . as an alternative to this method , radiation could be delivered in either a relaxed inspiratory position or in expiration , which is often more reproducible than disb . in patients that are not able to comply with any of these instructions , a third method of delivering radiation in tumors that have a great deal of motion is through a ventilatory - gated approach , during which the radiation beam is coordinated with the respiratory cycle through the placement of externally placed fiducial markers . or , in other words , the radiation is only delivered at certain phases , most typically full expiration . this type of therapy is generally most useful for tumors less than 5 cm in diameter and for a tumor motion of greater than 1 cm . figure 2 demonstrates the motion of a tumor in the inferior portion of the lung . it is evident that with timed radiation delivery , a great deal of normal tissue could be spared by reducing the treatment margins . vlachaki et al . assessed 10 patients with lung tumors to determine the effect of respiratory gating on dosimetry . gated images were acquired at full inspiration , full expiration , and at each quartile of respiratory movement . the authors found that gating led to higher minimum target volume doses , and that the v20 was reduced from 35% to 26% for gated plans . the mean lung , heart , and esophageal doses were also lower with gated plans . the authors concluded that gating could improve the dose to normal structures while maintaining target tissue coverage . underberg et al . examined 31 patients simulated with 4d ct scans and compared the dose to normal tissue in three different techniques to account for breathing motion in target delineation : ( 1 ) standard , population - based margins to account for internal motion , ( 2 ) the generation of an itv based on tumor mobility in three consecutive phases , and ( 3 ) a ptv generated from respiratory gating . margins led to unnecessary normal tissue irradiation , and that the risk was best minimized if gating was utilized . a recent study from fox chase cancer center arrived at a similar conclusion , in that 4d ct - based treatment planning maintains target coverage while reducing normal tissue dose . it is important to note that the intrafractional adaptive radiotherapy techniques of cbct and breathing control are not mutually exclusive , but in contrast should be utilized together to optimize the therapeutic ratio , as demonstrated by several studies . koch et al . found that the relationship between internal lung motion and skin fiducial motion is complex and unpredictable , and that the ap motion of tumors correlated poorly with skin surface markers . they also found that there is significant intersubject variability . in a follow - up study , liu et al . found that this variability ( and in turn , the target volume margins ) could be substantially reduced with the use of respiratory gating techniques . and in a study by nelson et al . , the authors assessed the margins necessary to account for uncertainties in tumor position with respiratory gating , image - guided patient setup , neither , or both . the authors found that utilizing both methods simultaneously allowed for the greatest reduction in margins that completely encompassed the tumor , and therefore concluded that when respiratory motion management is used , it should be used in conjunction with image - guided patient setup in order to reduce the overall treatment margin effectively . another delivery system of increasingly widespread use is cyberknife therapy , which utilizes a stereotactic guidance system designed primarily for radiosurgery in multiple organ systems . the premise behind this system is that the linear accelerator is mounted on a robotic arm , which can then track the target during treatment . cyberknife has been reported in single institution studies in the setting of sbrt for lung cancers . for example , le et al . reported 32 patients with lung tumors treated in a dose escalation study using single fractions , and found a 1-year local control rate of 91% for doses greater than 20 gy and at doses less than 25 gy there was no significant toxicity . brown et al . assessed the efficacy of this technique in peripherally located stage i nonsmall cell lung cancers . in a cohort of 31 patients , the authors reported no grade 3 or higher toxicities and 1-year local control rates of 93.2% . further studies on this technique will continue to define its role in adaptive radiotherapy and the definitive treatment of nsclc . more recently , novalis tx provides stereo x - ray targeting and adaptive gating using exactrac x - ray 6d and snap verification system . reported 94.4% local control with minimal toxicities with 50 gy delivered in 5 fractions using the novalis / brain lab system . all of these novel systems may provide an optimal treatment for clinical challenging cases such as patients with poor lung function and/or lesions close to critical structures . several factors change during the course of radiation can affect target and normal tissue dose . these factors include but are not limited to changes in tumor size , alterations in tissue anatomy , variations in respiratory patterns , and reductions in patient weight . in this setting , adaptive radiotherapy refers to repeat assessment of the target volume , either through repeat cbct , 4d imaging , or both . multiple studies have been performed examining causative factors of interfractional changes and the effect of these variations on dosimetry throughout treatment . examined 10 patients to determine whether tumor excursion due to respiratory motion was stable when comparing images acquired at the time of simulation with those during treatment . the authors found that while there was interfraction consistency in tumor excursion during treatment , the relationship between the gtv and other anatomic structures between respiratory changes varied with rescanning . the authors therefore expressed caution in relying on surrogate anatomic markers to assess tumor motion throughout treatment . bosmans et al . assessed 23 patients with locally advanced nsclc who underwent ct - pet and respiration - correlated imaging prior to treatment , and repeated at the first and second weeks after the start of radiation . the authors observed that while changes in tumor motion were relatively small , there was a great deal of variation in tumor size during therapy . these changes ranged from an increase in greater than 30% to a decrease of the same magnitude . the authors concluded that the changes in tumor size warranted assessment for replanning during the course of therapy . van zwienen et al . found that clinically evident regression occurred in approximately 40% of patients undergoing definitive treatment for nsclc , with approximately 10% of patients undergoing > 25% reduction in size by week three and in 24 out of 114 patients by week four . a study from johns hopkins university supported this finding of large variations in the gtv , and thus also recommended an adaptive approach in conventionally fractionated patients . britton et al . analyzed the effect of gross tumor volume regression and motion changes during the course of radiation therapy in locally advanced nsclc at md anderson cancer center . the authors found that in 8 patients with weekly 4dct data sets , the tumor volume was reduced by a range of 1571% . there was also noted to be increased tumor mobility in the si and ap directions throughout treatment , without any clear trends in tumor motion . in a follow - up study at the same institution , the authors found that the dose to 95% of the ptv and itv with weekly ct scans changed by approximately 12% and 2.5% , respectively . while the lung v20 and mean lung dose only increased by a mean of 3.1% and 2.2% , respectively , the spinal cord dose changed by an average of 34.3% . these studies together imply that continued assessment of the target volume is recommended and is essential in tumors that lie near the spinal cord . figure 3 demonstrates the significant reduction in size of a tumor throughout treatment , and the large effect that this reduction has on the dose distribution . a study from denmark demonstrated that anatomical and motion changes persist even with respiratory gating . in a study by juher - nottrup et al . , ten patients receiving 60 gy in 2 gy fractions underwent serial 4d ct scans during treatment . the authors found that the interfractional overlap of lung tumors was only 80%87% when bony landmarks were used with a gating technique , and that this overlap decreased to 70%76% when skin tattoos were present . with mediastinal tumors , it can therefore be concluded that gating does not preclude interfractional adaptive planning in patients with lung or mediastinal tumors . weight loss can be a factor in changing anatomy and in turn the dose to target volume , such as in the case of a patient that loses a great deal of weight that causes the effective beam path to change . some investigators have questioned whether weight loss in itself can be a cause of setup error during the course of treatment due to factors such as changes in the position of skin marks . johansen et al . attempted to answer this question by evaluating the relationship of interfractional setup errors with body mass index and weight loss . the authors assessed 34 head and neck cancer patients and 20 lung cancer patients who received serial cbct images to evaluate whether there was a change in 3d position between the initial cbct scans and those from the 10th and 20th treatment session . the study did not find a statistically significant correlation between setup error and either patient body mass index or weight loss . therefore , while it is still recommended that replanning is performed on the basis of weight changes during treatment , interfractional changes as a result of weight loss may be more a function of subsequent anatomical variations rather than true setup error . similar studies as above quantitating interfractional dosimetric error have been performed in patients being treated with sbrt . matsugi et al . examined 4d ct scans for 8 patients being treated with sbrt , to measure interfraction variations in position and the size of target volumes with this technique . in contrast to a conventionally fractionated treatment which is several weeks in duration , the authors found that the size of the gtv did not change significantly during treatment and that variations in motion range and position were also small . . however , the target volume is usually small in sbrt and a large fraction size is delivered in less than 5 fractions ; therefore , missing a small volume of target even just in one fraction can cause significant underdosing of the tumor and/or overdosing surrounding normal tissues . we therefore reiterate that we recommend daily volumetric verification for target coverage in sbrt . with the advent of proton therapy , investigators have recently begun to examine the effect of interfractional motion and anatomic changes on dose distribution with this technique . this issue is particularly important with protons because the impact of motion and anatomy changes is more significant in proton compared with photon ( figure 3 ) . hui and chang et al . acquired weekly 4d - ct scans on 8 patients with locally advanced nsclc treated with imrt . a conformal passive scattering plan was generated for each patient and compared with imrt plan over 7 weeks of radiotherapy . the authors found that normal tissue doses were increased and ctv coverage was significantly compromised in one patient ( with a decrease in approximately 8% ) with proton therapy but much less significantly in the imrt plan . the authors concluded that proton therapy is more sensitive to motion and anatomy changes compared with photon and interfractional adaptive planning and is indicated in select patients . at our institution , all patients undergo a 4d ct simulation to assess for internal motion . patients are immobilized with an upper body cradleand t - bar , which has a daily setup uncertainty of approximately 7 mm . at the time of simulation , whose tumor motion is less than 1 cm , a free - breathing technique is typically used , with the creation of an itv or igtv and radiation treatment delivery in all phases of the breathing cycle . if the target volume moves more than 1 cm and the patient can breathe reproducibly , then radiation is either timed with certain phases of the breathing cycle while the patient breathes freely ( ventilatory gated technique ) , or the patient is instructed to hold their breath for at least 15 seconds while radiation is delivered at deep inspiration ( disb ) . we utilize visual and/or audio feedback guidance for patients who can comply with these devices . patients undergoing standard fractionated regimens are initially set up daily with kv imaging for verification , which reduces the setup margin to 5 mm . all patients are assessed for concordance between bony / skin landmarks and tumor setup . if it is found that these setup parameters are discordant , or that the tumor is changing rapidly , cbct is employed , which reduces the itv to ptv margin to 3 mm . all patients receiving sbrt undergo volumetric verification of set up and motion such as cbct prior to each treatment . repeat 4d simulations are performed selectively at the physician 's discretion in patients with nsclc undergoing conventional fractionation for 6 - 7 weeks and are routinely performed in patients with small cell lung carcinoma undergoing hyperfractionated ( twice a day ) or dose escalated / accelerated radiotherapy for 3 to 7 weeks . adaptive 4d replanning is performed if motion / anatomy changes may change the target coverage and/or increase dose to surrounding critical structures . interfractional adaptive 4d planning is not routinely utilized on patients undergoing sbrt ( 4 - 10 fractions ) , as these patients receive daily volumetric imaging . it is notable that it is still unclear if target volume reductions are warranted in the scenario of gtv shrinkage during the course of radiotherapy [ 5052 ] . some physicians advocate an approach in which the target volume remains constant due to concerns for residual microscopic disease . to address these concerns , one option is to deliver at least 50 gy , the standard dose for microscopic disease , to the original target volume , and then to boost the reduced volume to the full dose . new technologies are evolving to improve adaptive radiation therapy , such that the high dose region is focused on the target while sparing critical normal tissues . treatment planning based on 4d ct images and on - board image - guided adaptive treatment delivery assists the radiation oncologist in tracking tumor motion and targeting the tumor precisely . as advancements in technology have allowed for both intra- and interfraction replanning , investigators have begun to assess whether or not the response of tumor during treatment can lead to changes in total dose to portions of the target volume based on the magnitude of tumor response to the initial phase of treatment ( image - guided adaptive treatment ) . this strategy is particularly relevant because several studies have shown that dose escalation in lung cancer improves local control . furthermore , prior studies have demonstrated that regions with high suvs and hypoxic areas are more likely to exhibit local failure when treated with radiation [ 54 , 55 ] . the information thus suggests that the radioresistance and propensity for distant metastasis in gross disease varies based on factors other than histologic type , stage , and tumor grade . it is in this realm that adaptive dose escalation may have a role in addition to the utility of imrt dose painting . performed a pilot study in which 14 patients with nsclc underwent repeat pet - ct scans prior to the start of radiation therapy and mid - way through treatment . boost fields were then designed based on residual pet avidity . in this dosimetric study , the authors found that this method allowed for a mean dose escalation of 58 gy or a reduction in normal tissue complication probability ( ntcp ) of up to 3% in patients with a reduction in tumor size . in a similar study , gillham et al . performed an additional pet - ct scan in week 5 or 6 of radiotherapy for patients being treated with localized inoperable nsclc to 66 gy in 2 gy fractions . the authors found that there was a median ptv reduction of 20% , and that in this dosimetric study , dose escalation to 78 gy was feasible in four out of ten patients without exceeding normal tissue constraints . future studies will determine whether this technique of adaptive dose escalation is feasible from a clinical standpoint without increasing toxicity and while maintaining tumor control . an additional technologic advancement in the context of adaptive radiotherapy is the development of real - time tracking of tumor motion so that the treatment can be actively and variably adapted in concordance with intrafractional changes . in addition to cyberknife , which has been used clinically as discussed above , another commercially available system is the calypso medical three- or four - dimensional electromagnetic tracking system . the system has undergone initial assessment in a wide variety of tumor sites and several studies have been published describing the technique . for example , smith et al . compared dosimetric results with and without real - time tracking . the authors found that the dose profiles were comparable with an idealized gating algorithm while minimizing the uncertainties inherent in the use of anatomical surrogates for target location , with a high level of efficiency . in a follow - up study , the same authors proposed a method for linear accelerator gating with wireless internal fiducial markers without the requirement for ionizing radiation for imaging , leading to improvements in the dose distribution . adaptive radiotherapy may improve tumor control in lung cancer by reducing target misses , escalating target dose , and minimizing side effects by avoiding critical structures over the course of radiotherapy .

the challenges of lung cancer radiotherapy are intra / inter - fraction tumor / organ anatomy / motion changes and the need to spare surrounding critical structures . evolving radiotherapy technologies , such as four - dimensional ( 4d ) image - based motion management , daily on - board imaging and adaptive radiotherapy based on volumetric images over the course of radiotherapy , have enabled us to deliver higher dose to target while minimizing normal tissue toxicities . the image - guided radiotherapy adapted to changes of motion and anatomy has made the radiotherapy more precise and allowed ablative dose delivered to the target using novel treatment approaches such as intensity - modulated radiation therapy , stereotactic body radiation therapy , and proton therapy in lung cancer , techniques used to be considered very sensitive to motion change . future clinical trials using real time tracking and biological adaptive radiotherapy based on functional images are proposed .

drug - induced dermatological reactions are common with the antiepiletic drugs such as carbamazepine ( cbz ) , phenytoin , lamotrigine , ethosuximide , and phenobarbital . these reactions may occur in the mild form as benign rash or may be severe and life - threatening as erythema multiforme major or toxic epidermal necrolysis ( ten ) . erythema multiforme major , also known as stevens - johnson syndrome , is usually caused by reactions to medications , rather than infections . cbz was originally introduced in therapeutic armamentarium as an anticonvulsant , and is known to produce such adverse drug reaction ( adr ) , but the reports are rare . cbz is still used as a first - line agent along with lithium and valproic acid in the treatment of bipolar disorder . although there are case reports of stevens johnson syndrome ( sjs ) occurring in schizophrenia , bipolar affective disorder , and during a manic episode when treated with cbz , we were unable to find reports of sjs in patients having epilepsy with bipolar affective disorder . sjs is a blistering disorder , characterized by mucosal erosions at two or more sites with small blisters and purpuric macules . a 17-year - old woman was brought to psychiatry opd with complaints of episodes of decreased sleep , irritability , and occasional aggressive and violent behavior . she was already on sodium valproate 600 mg / day for generalized tonic - clonic seizures , since the last 2 years from a primary health centre of uttarakhand state . she had a history of 510 convulsions per day for 23 days and then seizure free period of 1015 days . she accepted noncompliance to the regular treatment , thrice for 1520 days duration each , during the last 2 years , and admitted that a further increase in seizure frequency during such breaks compelled her to resume the treatment subsequently . on examination , she had history of frequent mood swings of mania and depression associated with episodic behavioral changes which was undoubtedly suggestive of bipolar affective disorder . she was diagnosed as a case of bipolar affective disorder , currently in remission , with comorbid epilepsy . the patient was initiated on higher dose ( 800 mg ) of sodium valproate which was further increased gradually to 1600 mg / day . at this stage , a further increase in dose caused significant sedation and was intolerable . at this stage , she was referred to a neurologist which she refused to comply with . cbz 200 mg once daily was added to her regimen of valproate 1600 mg / day in consultation with the hospital physician , which was increased to twice daily after 5 days and then three times daily after 10 days of initiation . she tolerated the treatment well until 19 day when she returned with high - grade fever , redness of eyes , swelling all over body , eruptions on lips , face in butterfly pattern [ figure 1 , 2 ] and was admitted in the dermatology ward . on examination her axillary temperature was 39c , blood pressure was 90/60 mmhg , and the pulse rate was 70 per min and regular . she had multiple bullae formation all over the body in the symmetrical pattern which gradually increased to involve > 6065% of the body surface area . she had severe pain during deglutition , generalized body edema , and bleeding per vagina . examination by a gynecologist revealed that she was in the menstruating phase and vaginal mucosa was found ulcerated . lesions on patient 's face in the butterfly pattern lesions on patient 's trunk her lab investigations showed that heamogloblin was 13 g/ dl , total leukocyte count 6000/mm , platelets 1.5 lakhs / mm , creatinine 0.9 mg / dl , urea 49 mg / dl , sodium 139 meq / l , and potassium 4.8 meq / l . examination of urine revealed albumin ( + ) and full field rbcs , which correlated with her menstruation phase . biopsy was not done as her clinical presentation was compatible with diagnosis of erythema multiforme major which was confirmed with an opinion from the pharmacologist . she was treated with methylprednisolone , chlorpheniramine , and ceftriaxone parenterally , and clobetasol gentamicin combination topically , along with iv fluids and other supportive measures . after about 2 weeks of intensive indoor management , her conditions started improving and during third week she was again put on oral valproate 200 mg with an incremental increase of 200 mg per day till it reached 1600 mg / day on eighth day of initiation . the patient again started feeling drowsy , which may have had a psychological component as the patient was already anticipating the effect due to past experience . she refused any other addition to her treatment for fear of similar reaction and agreed to bear the possibility of few convulsions per month . two months after discharge , the patient returned for review and the dose of sodium valproate was increased since the sedation was tolerable . her dose was increased to 1800 mg and then to 2000 mg in the next month . with this dose she started having about 34 months of seizure free period and maintained on the same dose since the last 6 months . cutaneous lesions mostly occur on the palms , soles , dorsum of the hands , and extensor surfaces . mucosal involvement may include erythema , edema , sloughing , blistering , ulceration , and necrosis . this patient had been taking valproate for 2 years , and her investigations were not suggestive of any other etiology for causation of these adrs . cutaneous reactions started within 3 weeks of administration of cbz , which is the usual risk period for this adr . the causality assessment by naranjo 's algorithm was done which revealed a score 7 suggesting a probable adr to cbz . in one such a report , a 6-year - old boy developed sjs five weeks after cbz was added to valproic acid , which he had been taking as sole antiepileptic therapy for several weeks . another study showed the incidence of skin rashes with the same dose of cbz in psychiatric patients ( 1215% ) was nearly three times more than that in neurological patients ( 5% ) . however , no study showing incidence in comorbid neurological and psychiatric patients was found during our search , hence we lack any comparison with our case report . our patient also received sodium valproate which is also known to cause hypersensitivity reactions , but the patient had been taking valproate since the last 2 years without any adrs . it was only after the administration of cbz along with a higher dose of valproic acid that this patient developed severe adrs . immunological reactions due to hormonal changes during menstruation trigger various hypersensitivity reactions such as mucosal ulcerations , skin rashes , asthma , etc . , so it may be an important factor in drug reactions which is yet an unreported factor and should be considered in further studies . approximately three persons per million per week may experience life - threatening dermatological syndromes with the use of cbz . the incidence of adrs may increase when cbz is given along with higher doses of valproic acid because of increased plasma concentrations of cbz . this was a rare case of life - threatening erythema multiforme major / stevens johnson 's syndrome with the use of cbz when given along with higher doses of valproic acid . it was perceived that menstruation , and comorbidity of neurological and psychiatric illnesses might have had predisposing roles . there is a need of continued adr monitoring and reporting of adrs , more so with the use of cbz .

carbamazepine ( cbz ) is frequently used for epilepsy and various psychiatric illnesses . it is known for its dermatological side effects which may range from mild rash to life - threatening reactions as stevens johnson syndrome or toxic epidermal necrolysis . we hereby report a rare case of 17-year - old woman suffering from generalized tonic clonic epilepsy with comorbid bipolar affective disorder , who was initially treated with sodium valproate with partial improvement . after 19 days of addition of cbz to the therapy , the patient developed erythema multiforme major with > 60% skin involvement and oral , conjunctival , intestinal , and vaginal mucosal involvement .

dengue fever is the most significant arbovirus infection in malaysia and tropical countries , with an upward trend from 44.3 cases/100,000 population in 1999 reaching 181 cases/100,000 population in 2007 . the world health organization ( who ) ranks dengue as the most crucial mosquito - borne viral disease in the world . this infectious disease affects more than 100 endemic countries including regions of asia , americas , africa , and eastern mediterranean , many of which are rural and developing nations , causing about 50100 million new dengue infections occurring each year . the dengue virus is a global pandemic threat which has increased incidence of 30-fold in the past five decades , with multiple new outbreaks . although the reported number of patients having intracranial hemorrhage ( ich ) caused by dengue virus is rare and only 20 cases excluding those in our series have been reported so far , it is likely that its incidence is under - reported or under - diagnosed with the current global disease burden . there were 5866 reported dengue cases and 17 dengue - related deaths in penang , malaysia , from january 2014 till june 2015 . the dengue virus , a member of the flavivirus group in the family flaviviridae , is a single - stranded enveloped rna virus , of which there are four known serotypes , namely denv 14 . dengue infection understanding has improved tremendously , but still no cure has been devised and the best strategy so far to curb the disease is through prevention and control of its mosquito vectors . dengue vaccines have been created but most are still undergoing the clinical development stage , and only mexico and philippines have approved its use so far . with better understanding of the disease , who has proposed new classification schemes . the term expanded dengue syndrome was introduced to include unusual presentations of dengue fever with severe organ involvement including severe neurologic complications . the incidence of dengue encephalopathy ranges from 0.5% to 6.2% . out of the 5400 patients with dengue fever reported by cam et al . the mechanism of which patients with dengue infection may be more prone to ich is complex , but the main contributors are coagulopathy , platelet dysfunction , thrombocytopenia , and vasculopathy . unfortunately , dengue fever with ich has high morbidity and fatality , but little is known about it . we collected data of all patients with ich diagnosed via a plain computed tomography ( ct ) of the brain with positive dengue virus type 1 nonstructural protein ( ns1 ) antigen test or positive dengue serology igm with thrombocytopenia from january 2014 till june 2015 at our center . a total of nine patients ( patient a to patient i ) were analyzed at our center . the data included were basic demographic data , existing comorbid , presenting symptoms , other dengue - related complications , the day ich was diagnosed in relation to onset of fever , glasgow coma scale ( gcs ) , and pupils size and light response on presentation , platelet counts during diagnosis of ich , lowest platelet counts recorded , international normalized ratio ( inr ) , activated partial thromboplastin time ( aptt ) , dengue igm and igg seropositivity , type of ich , volume of ich , presence of midline shift and patency of basal cisterns , surgical intervention , and outcome . the volumes of intracerebral hematomas were calculated using the well - accepted abc/2 ellipsoid method . in cases where there is a subdural hemorrhage , the maximum thickness of the hemorrhage was measured . we defined an intracranial lesion needing surgery as : ( 1 ) any intracranial lesion causing a midline shift of 5 mm or more , and/or , ( 2 ) effacement of basal cisterns on ct brain . there are three methods of laboratory detection in our center , namely , panbio dengue igm capture elisa ( sensitivity of 94.7% for primary infections , 55.7% for secondary infections and specificity of 100% ) , diaxis dengue ns1 antigen test ( sensitivity of 97.9% and specificity of 99% ) , and acco dengue igg / igm - ns1 combo rapid detection kit ( igg / igm : sensitivity 90% and specificity 100% , ns1 : sensitivity 96% and specificity 100% ) . for our literature review , we performed a pubmed and google scholar search using the keywords dengue , intracranial hemorrhage , and intracranial bleed without specifying a time interval . reports where diagnosis of dengue infection is questionable or very limited information of the case is explained were excluded from the study . fever and vomiting were seen in all patients , and surprisingly , only 55% of them had a history of headache at presentation . this of course may be due to the low gcs of patients where history from family members may not be accurate . the mean age was 59.3 years ( range 4386 years old ) and seven of the patients were males . the day of detection of ich from fever onset ranged from 4 to 22 days . the ich of patient i was most likely detected late as the ct of the brain was done only after the patient had poor gcs recovery when sedation was stopped . four of the patients had other bleeding tendencies and three of the patients were in clinical shock at the time of diagnosis of ich . the mean platelet count , inr , and aptt were 31 10/l ( range 280 10/l ) , 1.2 ( range 0.92.3 ) , and 37.3 ( range 27.7 more than 180 ) , respectively . the lowest platelet count was the platelet level at diagnosis for three of the patients but was even lower for six of the patients . seven patients had raised alanine transaminase ( alt ) levels ( range 30784 u / l ) . out of the nine patients , eight had positive ns1 test , two had positive igm test , and three had positive igg test . it is interesting that three of the patients with positive igg had ich requiring surgery and all of them died whereas two patients with negative igg did not have ich requiring surgery and survived . four patients had cerebral convexity subdural hemorrhage , two patients had intracerebral hemorrhage , two patients had subarachnoid hemorrhage , and two had tentorium cerebelli subdural hemorrhage . presenting symptoms of the patients characteristics of patients from our center all patients with intracranial lesions requiring surgery had gcs of eight or less and died . the other 4 patients had gcs of 14 or 15 and was discharged from the hospital , albeit one patient with a glasgow outcome scale ( gos ) of 3 . the other three patients were poor surgical candidates in terms of hemodynamic instability or persistent low platelet counts below 100 10/l despite multiple units of platelet transfusions . patient b 's platelet levels remained low with the highest platelet level of 46 10/l during admission and the patient required 4 types of inotropic support for shock [ figure 1 ] . patient f underwent cardiopulmonary resuscitation on presentation for cardiac arrest and required 2 types of inotropes . patient g required 3 inotropes and the highest recorded platelet count was 68 10/l [ figure 2 ] . patient a received platelet and fresh frozen plasma ( ffp ) transfusion till platelet level reached 101 10/l , inr 12 , and aptt 33.8 . the patient then underwent a right decompressive craniectomy , evacuation of subdural hemorrhage , and external ventricular drainage catheter insertion for intracranial pressure monitoring 8 h after admission [ figure 3 ] . patient c also received multiple units of platelet transfusion till platelet levels reached 109 10/l . the patient then underwent a parietal craniectomy 8 h after admission , but excessive bleeding complicated the procedure and the attempt to evacuate the entire clot was abandoned [ figure 4 ] . both patients who underwent surgery were nursed in the intensive care unit with cerebral resuscitation care . computed tomography of the brain of patient b computed tomography of the brain of patient g computed tomography of the brain of patient a computed tomography of the brain of patient c overall , fever , reduced or altered consciousness , vomiting , and headache are the top complaints . platelet counts on diagnosis ranged from 15 to 100 10/l . as in our series , it is seen again that patients with lower platelet levels did not necessarily have worse outcomes and that higher platelet levels did not seem to protect patients from fatal ich . dengue igg was detected in two patients where one died and another had outcome of gos of 3 or 4 . intraparenchymal hemorrhage was seen in 13 patients , pontine hemorrhage in two patients , intraventricular hemorrhage in two patients , medullary hemorrhage in one patient , cerebellar hemorrhage in one patient , and subdural hemorrhage in four patients . all the case reports did not state the volume or thickness of the bleeds , or whether the basal cisterns were effaced or patent . midline shift was detected in six patients , but the extent of the shift was not specified to determine if surgery was required . it is highly possible these six patients required surgery by reviewing the pictures that were included in the reports . out of 20 patients , 11 died , six had gos of 24 , and three had gos of 5 [ table 3 ] . there are no specific symptoms that clearly differentiate dengue fever from other common febrile infectious diseases such as chikungunya , leptospirosis , malaria , and influenza . while chikungunya infection may be distinguished from dengue fever , as chikungunya may cause significant postillness arthralgia and potentially debilitating polyarthralgia , objective assessment lies with antibody detection . initial suspicion of dengue fever usually rests upon the finding of leukopenia and thrombocytopenia from a full blood count . the most common tests used routinely are dengue igm , igg , and ns1 , but only ns1 seems useful in making an early and accurate diagnosis . each of these tests has its own disadvantages and can not be used in isolation for every case . in primary infection , igm antibodies are detectable in 50% of patients by days 35 , increasing to 80% by day 5 , 99% by day 10 , and peak about 2 weeks of illness before declining to undetectable levels over 23 months . igg antibodies are generally detectable at low titers at the end of the 1 week of illness and increasing slowly thereafter . during a secondary dengue infection , iggs are the main antibodies and are detectable at high levels even in the acute phase . early convalescent stage , igm levels are significantly lower in secondary infections than in primary ones and may be undetectable . in secondary dengue infections , igm was only detected in 78% of patients after day 7 in one study . if dengue igm was the only test performed , it is reported that 28% of secondary dengue infections would be missed . this drawback limits the usefulness of igm detection for diagnosing dengue infection in the acute phase of the illness when most patients present with complications such as ich . unfortunately , it is found that its usefulness declines on day 45 of illness but there are studies which show that ns1 antigen may be still be detected until the 14 day of illness . a primary dengue infection invokes the immune system to protect the patient from a secondary infection from the same serotype . however , a phenomenon known as antibody - dependent enhancement of dengue virus infection may actually cause a more severe form of dengue infection . it is well accepted that patients with secondary dengue infections have a higher risk of developing more severe forms of dengue infection . multiple methods such as using the igm / igg antibody ratios have thus been created to distinguish primary from secondary infections more accurately during the acute phase . in our series , detection of igg early in the course of the disease with a positive ns1 antigen test and negative igm would most likely mean that the patient has a secondary infection . it is unfortunate that we do not have the titers of igm and igg to confirm this observation . in this series , all the patients had a positive ns1 test except for one , which could be due to sampling time after day 5 of illness . it is highly likely that the three patients with positive igg had secondary dengue infection and thus were predisposed to have a more severe ich . even though sound clinical judgment would still be the best guidance for now , patients with a suspected secondary dengue infection should be monitored more closely and thresholds for diagnostic ct of the brains lowered when suspicion of ich is present . whether surgery or optimal medical management should be the treatment of choice remains as the age - old question of all ich . dengue infections causing ich are inherently harder to manage in that the causative factors such as vasculopathy and platelet dysfunction are usually still present and irreversible while surgery is undertaken . some measures of correcting platelet deficits and coagulation derangements with massive transfusions may be carried out but risks delay of surgery . when the availability of neurosurgical centers is limited and transportation of a critically ill patient entails long - distance hazardous travel , the question of cost to benefit ratio becomes an issue . considering the fact that the number of reported patients suffering ich due to dengue infection is low , it is too early to advocate if these patients should be treated like any other spontaneous ich . the latest guidelines from the american stroke association recommends that supratentorial hematoma evacuation in deteriorating patients might be a life - saving surgery and that decompressive craniectomy may reduce mortality when there are large hematomas with significant midline shift or elevated intracranial pressure refractory to medical management . some authors have illustrated that surgical treatment may be of benefit compared to medical management , but it is too premature to conclude with these limited data . the debate remains on whether surgery should be done for these patients immediately with only platelet cover despite the low platelet levels or should adequate platelet levels as recommended by the guidelines be achieved first . the american stroke association advocates that early hematoma evacuation compared to hematoma evacuation when patient deteriorates does not show a clear benefit at the moment . there is conflicting evidence from the available studies such as the stich ii trial that showed no statistical significance in outcome between early surgery compared with initial medical management with delayed surgery if the patient deteriorates and the meta - analysis by another group which showed an improved outcome if surgery was performed within 8 h of hemorrhage . it would be difficult to determine if these recommendations could apply to ich in patients with dengue infection . since thrombocytopenia and coagulopathy are usually severe in patients with dengue - related ich , neurosurgeons and physicians alike would have difficulty in determining the target levels and amount of blood product transfusion before surgery is undertaken . when ich is present in patients with dengue infection , there is no study as to whether the large amounts of platelets and ffp given would change the outcome . as bleeding in dengue fever is multifactorial and still poorly understood , contributing factors such as vasculopathy and platelet dysfunction may be a cause of the hemorrhage , and the efforts for correcting the deficits may prove to be a waste of precious blood product resources and prolong the time from diagnosis to surgery . moreover , it is shown that duration of survival of transfused platelets ranges from only a few hours to a day in cases with shock and that would mean that great amounts of blood products would be needed to keep the levels up . a recent randomized control trial on platelet transfusion in dengue fever showed that 53.6% of their patients did not respond to platelet transfusion and these patients were those with a lower baseline platelet count . unfortunately , current guidelines for platelet transfusion thresholds in thrombocytopenic surgical patients are mainly based on expert opinion and clinical experience . the current recommendation for platelet levels for neurosurgical procedures is 100 10/l . in dengue patients , it is difficult to keep the perioperative platelet levels as recommended and it is shown that a perioperative platelet count below 100 10/l in patients who failed to respond to platelet transfusions had a higher risk of postoperative intracranial hematoma formation . one group suggests prophylactic transfusion of platelets when levels are under a certain threshold , of which the threshold values still vary at the moment . the other group prefers transfusion only when significant hemorrhage occurs . one study showed that a platelet count of < 50 10/l and hematocrit of more than 50 are associated with bleeding manifestations . one more study of 225 dengue patients suggested that bleeding occurred more often in patients with platelet counts below 20 10/l and recommended prophylactic transfusion of platelets . in contrast , there was no correlation between bleeding tendencies and platelet counts in dengue patients in other studies . for dengue fever without bleeding , one study showed that prophylactic transfusions of platelet are not recommended as it does not change or reduce the bleeding outcome in patients with dengue hemorrhagic fever and may increase risk of pulmonary edema . platelet transfusion in adult patients with dengue in the absence of bleeding when the platelet count was < 20 10/l did not reduce bleeding or hasten platelet recovery , but there was potential harm by hampering recovery of platelet count to > 50 10/l and prolonging of hospitalization . in a retrospective analysis of 256 dengue - infected patients without prior bleeding and platelet counts < 20 10/l , 188 were given platelet transfusion ; it was found that subsequent bleeding , platelet increment , and platelet recovery were similar between the two groups . a review article in 2011 strongly concluded that prophylactic platelet transfusion is associated with hazards and wastage , which is not justified by any potential hematological benefit and therefore , should not be adopted as a routine clinical practice . then again , a salient point that must be considered in patients with ich is the significant morbidity that could be worse than mortality itself at times . the question arises as to whether prophylactic transfusion should be given to patients with very low platelet levels just to avoid devastating consequences of ich even though it is a rare event . the range of lowest recorded platelet levels in our series was 276 10/l and five of the patients had coagulopathy . our series shows that severe ich may still occur even with a platelet count of 66 ( patient f ) and a platelet count of 3 does not cause a more severe ich ( patient d ) . it has long been established that there is abnormal hemostasis in patients with dengue fever with platelet dysfunction , coagulopathy , vasculopathy , and immune- or virus - related destruction of platelets . it may be possible that our approach of determining the absolute platelet counts and inr might not be entirely enough as we might be missing out on the actual ability of platelet function and coagulation in vivo . there are laboratory techniques to assess platelet function and blood coagulation , namely , thromboelastography , thromboelastometry , and platelet aggregometry . although not widely available yet in developing countries , these tests might prove useful in determining a subset of patients that may benefit from prophylactic transfusions to prevent life - threatening hemorrhages . to our knowledge , there has been no trial in the use of thromboelastography , thromboelastometry , or platelet aggregometry in predicting bleeding tendencies in patients with dengue fever . it is important to consider that these symptoms may also be the first symptom of an ongoing ich . unequivocal symptoms such as seizures , weakness , numbness , altered consciousness , and seizures would warrant ct of the brains , but these presentations may only be present when the ich has expanded beyond treatment . multiple methods of noninvasive monitoring and detection of raised intracranial pressure have been developed , but most are expensive and require much training . in view of this , a simple noninvasive method to detect increased intracranial pressure is needed . in the setting where dengue infections are highest in developing nations and ct of the brains are not widely available , we can only recommend that fundoscopy to detect papilledema should be a routine assessment for dengue - infected patients with suspected ich . it would seem from our series that prevention of ich in patients with dengue should be the main aim as all patients who had a severe bleed warranting surgery deteriorated very quickly and both surgical and medical managements failed to stop the inevitable . if specific risk factors could be identified , perhaps , prophylactic transfusion and closer monitoring could bring about earlier detection or prevent ich altogether . in terms of risk of bleeding , a study done in malaysia found that prolonged duration of shock and low - normal hematocrit at the time of shock suggesting blood loss in addition to capillary leakage were the strongest predictors of hemorrhage . only patient g in our series who had upper gastrointestinal bleeding had a low hematocrit and shock . it is possible that patients with only ich as the only bleeding tendency would not show a drop in hematocrit associated with shock as the volume of intracranial blood loss alone would not be sufficient enough to reduce intravascular volume . other studies found that there was significant association between bleeding tendencies and older patients , secondary dengue infection , high baseline hematocrit levels , low platelet levels , prolonged aptt , female gender , vomiting , high absolute lymphocyte count , and high aspartate aminotransferase level . even though these may be generalized to include ich although not specific to ich , sahu et al . outlined the possible predictors for the central nervous system involvement that includes higher mean body temperature , elevated hematocrit , low platelet count , and liver dysfunction . three of our patients with positive igg had ich requiring surgery and all of them died , whereas two patients with negative igg did not have ich requiring surgery and survived ; thus , it is possible that secondary dengue infection is also associated with a tendency to develop severe ich . the mechanisms of which patients with dengue infection may be more prone to ich is complex and are postulated to be caused by coagulopathy , platelet dysfunction , thrombocytopenia , and vasculopathy . to date , there is no proven explanation for ich occurring in only a rare minority of patients with severe dengue . the blood brain barrier and blood cerebrospinal fluid ( csf ) barrier have been shown to breakdown during dengue virus infection . dengue igm , igg , and ns1 ag were also detected in csf of patients with dengue . it is plausible that ich follows the same general mechanisms as bleeding elsewhere in the body and focal cerebral vasculopathy is worsened by the presence of igg and ns1 ag immunopathological - related mechanisms . in general , immunopathological events involving t cells , cytokines such as tumor necrosis factor - alpha ( tnf- ) , and cross - reactive antibody that enhances dengue virus replication leading to vasculopathy are still incompletely understood . thrombocytopenia may be caused by disseminated intravascular coagulation , bone marrow suppression , increased apoptosis , and destruction by the complement system . interestingly , ns1 antigens are found to induce production of plasminogen cross - reactive antibodies and thus enhance plasminogen activation leading to increased plasmin and promoting fibrinolysis . platelet dysfunction and destruction have been attributed to the dengue virus and early studies reveal that dengue virus viremia seems associated with the megakaryocytic lineage and replication in platelets . much have been learned and platelets have been increasingly considered to play a key role in inflammation through inflammasome synthesis of interleukin-1 beta ( il-1 ) which is a cytokine closely associated with endothelial dysfunction and coagulation disorders . platelets activation by dengue virus through lectin receptor dendritic cell - specific icam-3-grabbing nonintegrin resulting in production of il-1 has been shown using mouse models , chen et al . observed that high viral titer , macrophage infiltration , and tnf- are three important events that lead to hemorrhage . chuang et al . found that dengue virus infection induced macrophage migration inhibitory factor ( mif ) production may contribute to vascular hyperpermeability and hemostatic abnormality and thus blocking of mif may be an answer for prevention of dengue hemorrhage . there are no specific symptoms that clearly differentiate dengue fever from other common febrile infectious diseases such as chikungunya , leptospirosis , malaria , and influenza . while chikungunya infection may be distinguished from dengue fever , as chikungunya may cause significant postillness arthralgia and potentially debilitating polyarthralgia , objective assessment lies with antibody detection . initial suspicion of dengue fever usually rests upon the finding of leukopenia and thrombocytopenia from a full blood count . the most common tests used routinely are dengue igm , igg , and ns1 , but only ns1 seems useful in making an early and accurate diagnosis . each of these tests has its own disadvantages and can not be used in isolation for every case . in primary infection , igm antibodies are detectable in 50% of patients by days 35 , increasing to 80% by day 5 , 99% by day 10 , and peak about 2 weeks of illness before declining to undetectable levels over 23 months . igg antibodies are generally detectable at low titers at the end of the 1 week of illness and increasing slowly thereafter . during a secondary dengue infection , iggs are the main antibodies and are detectable at high levels even in the acute phase . early convalescent stage , igm levels are significantly lower in secondary infections than in primary ones and may be undetectable . in secondary dengue infections , igm was only detected in 78% of patients after day 7 in one study . if dengue igm was the only test performed , it is reported that 28% of secondary dengue infections would be missed . this drawback limits the usefulness of igm detection for diagnosing dengue infection in the acute phase of the illness when most patients present with complications such as ich . unfortunately , it is found that its usefulness declines on day 45 of illness but there are studies which show that ns1 antigen may be still be detected until the 14 day of illness . a primary dengue infection invokes the immune system to protect the patient from a secondary infection from the same serotype . however , a phenomenon known as antibody - dependent enhancement of dengue virus infection may actually cause a more severe form of dengue infection . it is well accepted that patients with secondary dengue infections have a higher risk of developing more severe forms of dengue infection . multiple methods such as using the igm / igg antibody ratios have thus been created to distinguish primary from secondary infections more accurately during the acute phase . in our series , detection of igg early in the course of the disease with a positive ns1 antigen test and negative igm would most likely mean that the patient has a secondary infection . it is unfortunate that we do not have the titers of igm and igg to confirm this observation . in this series , all the patients had a positive ns1 test except for one , which could be due to sampling time after day 5 of illness . it is highly likely that the three patients with positive igg had secondary dengue infection and thus were predisposed to have a more severe ich . even though sound clinical judgment would still be the best guidance for now , patients with a suspected secondary dengue infection should be monitored more closely and thresholds for diagnostic ct of the brains lowered when suspicion of ich is present . whether surgery or optimal medical management should be the treatment of choice remains as the age - old question of all ich . dengue infections causing ich are inherently harder to manage in that the causative factors such as vasculopathy and platelet dysfunction are usually still present and irreversible while surgery is undertaken . some measures of correcting platelet deficits and coagulation derangements with massive transfusions may be carried out but risks delay of surgery . when the availability of neurosurgical centers is limited and transportation of a critically ill patient entails long - distance hazardous travel , the question of cost to benefit ratio becomes an issue . considering the fact that the number of reported patients suffering ich due to dengue infection is low , it is too early to advocate if these patients should be treated like any other spontaneous ich . the latest guidelines from the american stroke association recommends that supratentorial hematoma evacuation in deteriorating patients might be a life - saving surgery and that decompressive craniectomy may reduce mortality when there are large hematomas with significant midline shift or elevated intracranial pressure refractory to medical management . some authors have illustrated that surgical treatment may be of benefit compared to medical management , but it is too premature to conclude with these limited data . the other question that needs to be answered is the timing of surgery . the debate remains on whether surgery should be done for these patients immediately with only platelet cover despite the low platelet levels or should adequate platelet levels as recommended by the guidelines be achieved first . the american stroke association advocates that early hematoma evacuation compared to hematoma evacuation when patient deteriorates does not show a clear benefit at the moment . there is conflicting evidence from the available studies such as the stich ii trial that showed no statistical significance in outcome between early surgery compared with initial medical management with delayed surgery if the patient deteriorates and the meta - analysis by another group which showed an improved outcome if surgery was performed within 8 h of hemorrhage . it would be difficult to determine if these recommendations could apply to ich in patients with dengue infection . since thrombocytopenia and coagulopathy are usually severe in patients with dengue - related ich , neurosurgeons and physicians alike would have difficulty in determining the target levels and amount of blood product transfusion before surgery is undertaken . when ich is present in patients with dengue infection , there is no study as to whether the large amounts of platelets and ffp given would change the outcome . as bleeding in dengue fever is multifactorial and still poorly understood , contributing factors such as vasculopathy and platelet dysfunction may be a cause of the hemorrhage , and the efforts for correcting the deficits may prove to be a waste of precious blood product resources and prolong the time from diagnosis to surgery . moreover , it is shown that duration of survival of transfused platelets ranges from only a few hours to a day in cases with shock and that would mean that great amounts of blood products would be needed to keep the levels up . a recent randomized control trial on platelet transfusion in dengue fever showed that 53.6% of their patients did not respond to platelet transfusion and these patients were those with a lower baseline platelet count . unfortunately , current guidelines for platelet transfusion thresholds in thrombocytopenic surgical patients are mainly based on expert opinion and clinical experience . the current recommendation for platelet levels for neurosurgical procedures is 100 10/l . in dengue patients , it is difficult to keep the perioperative platelet levels as recommended and it is shown that a perioperative platelet count below 100 10/l in patients who failed to respond to platelet transfusions had a higher risk of postoperative intracranial hematoma formation . one group suggests prophylactic transfusion of platelets when levels are under a certain threshold , of which the threshold values still vary at the moment . one study showed that a platelet count of < 50 10/l and hematocrit of more than 50 are associated with bleeding manifestations . one more study of 225 dengue patients suggested that bleeding occurred more often in patients with platelet counts below 20 10/l and recommended prophylactic transfusion of platelets . in contrast , there was no correlation between bleeding tendencies and platelet counts in dengue patients in other studies . for dengue fever without bleeding , one study showed that prophylactic transfusions of platelet are not recommended as it does not change or reduce the bleeding outcome in patients with dengue hemorrhagic fever and may increase risk of pulmonary edema . platelet transfusion in adult patients with dengue in the absence of bleeding when the platelet count was < 20 10/l did not reduce bleeding or hasten platelet recovery , but there was potential harm by hampering recovery of platelet count to > 50 10/l and prolonging of hospitalization . in a retrospective analysis of 256 dengue - infected patients without prior bleeding and platelet counts < 20 10/l , 188 were given platelet transfusion ; it was found that subsequent bleeding , platelet increment , and platelet recovery were similar between the two groups . a review article in 2011 strongly concluded that prophylactic platelet transfusion is associated with hazards and wastage , which is not justified by any potential hematological benefit and therefore , should not be adopted as a routine clinical practice . then again , a salient point that must be considered in patients with ich is the significant morbidity that could be worse than mortality itself at times . the question arises as to whether prophylactic transfusion should be given to patients with very low platelet levels just to avoid devastating consequences of ich even though it is a rare event . the range of lowest recorded platelet levels in our series was 276 10/l and five of the patients had coagulopathy . our series shows that severe ich may still occur even with a platelet count of 66 ( patient f ) and a platelet count of 3 does not cause a more severe ich ( patient d ) . it has long been established that there is abnormal hemostasis in patients with dengue fever with platelet dysfunction , coagulopathy , vasculopathy , and immune- or virus - related destruction of platelets . it may be possible that our approach of determining the absolute platelet counts and inr might not be entirely enough as we might be missing out on the actual ability of platelet function and coagulation in vivo . there are laboratory techniques to assess platelet function and blood coagulation , namely , thromboelastography , thromboelastometry , and platelet aggregometry . although not widely available yet in developing countries , these tests might prove useful in determining a subset of patients that may benefit from prophylactic transfusions to prevent life - threatening hemorrhages . to our knowledge , there has been no trial in the use of thromboelastography , thromboelastometry , or platelet aggregometry in predicting bleeding tendencies in patients with dengue fever . it is important to consider that these symptoms may also be the first symptom of an ongoing ich . unequivocal symptoms such as seizures , weakness , numbness , altered consciousness , and seizures would warrant ct of the brains , but these presentations may only be present when the ich has expanded beyond treatment . multiple methods of noninvasive monitoring and detection of raised intracranial pressure have been developed , but most are expensive and require much training . in view of this , a simple noninvasive method to detect increased intracranial pressure is needed . in the setting where dengue infections are highest in developing nations and ct of the brains are not widely available , we can only recommend that fundoscopy to detect papilledema should be a routine assessment for dengue - infected patients with suspected ich . it would seem from our series that prevention of ich in patients with dengue should be the main aim as all patients who had a severe bleed warranting surgery deteriorated very quickly and both surgical and medical managements failed to stop the inevitable . if specific risk factors could be identified , perhaps , prophylactic transfusion and closer monitoring could bring about earlier detection or prevent ich altogether . in terms of risk of bleeding , a study done in malaysia found that prolonged duration of shock and low - normal hematocrit at the time of shock suggesting blood loss in addition to capillary leakage were the strongest predictors of hemorrhage . only patient g in our series who had upper gastrointestinal bleeding had a low hematocrit and shock . it is possible that patients with only ich as the only bleeding tendency would not show a drop in hematocrit associated with shock as the volume of intracranial blood loss alone would not be sufficient enough to reduce intravascular volume . other studies found that there was significant association between bleeding tendencies and older patients , secondary dengue infection , high baseline hematocrit levels , low platelet levels , prolonged aptt , female gender , vomiting , high absolute lymphocyte count , and high aspartate aminotransferase level . even though these may be generalized to include ich , it would be ideal if independent risk factors for ich could be determined . although not specific to ich , sahu et al . outlined the possible predictors for the central nervous system involvement that includes higher mean body temperature , elevated hematocrit , low platelet count , and liver dysfunction . three of our patients with positive igg had ich requiring surgery and all of them died , whereas two patients with negative igg did not have ich requiring surgery and survived ; thus , it is possible that secondary dengue infection is also associated with a tendency to develop severe ich . the mechanisms of which patients with dengue infection may be more prone to ich is complex and are postulated to be caused by coagulopathy , platelet dysfunction , thrombocytopenia , and vasculopathy . to date , there is no proven explanation for ich occurring in only a rare minority of patients with severe dengue . the blood brain barrier and blood cerebrospinal fluid ( csf ) barrier have been shown to breakdown during dengue virus infection . dengue igm , igg , and ns1 ag were also detected in csf of patients with dengue . it is plausible that ich follows the same general mechanisms as bleeding elsewhere in the body and focal cerebral vasculopathy is worsened by the presence of igg and ns1 ag immunopathological - related mechanisms . in general , immunopathological events involving t cells , cytokines such as tumor necrosis factor - alpha ( tnf- ) , and cross - reactive antibody that enhances dengue virus replication leading to vasculopathy are still incompletely understood . thrombocytopenia may be caused by disseminated intravascular coagulation , bone marrow suppression , increased apoptosis , and destruction by the complement system . interestingly , ns1 antigens are found to induce production of plasminogen cross - reactive antibodies and thus enhance plasminogen activation leading to increased plasmin and promoting fibrinolysis . platelet dysfunction and destruction have been attributed to the dengue virus and early studies reveal that dengue virus viremia seems associated with the megakaryocytic lineage and replication in platelets . much have been learned and platelets have been increasingly considered to play a key role in inflammation through inflammasome synthesis of interleukin-1 beta ( il-1 ) which is a cytokine closely associated with endothelial dysfunction and coagulation disorders . platelets activation by dengue virus through lectin receptor dendritic cell - specific icam-3-grabbing nonintegrin resulting in production of il-1 has been shown using mouse models , chen et al . observed that high viral titer , macrophage infiltration , and tnf- are three important events that lead to hemorrhage . chuang et al . found that dengue virus infection induced macrophage migration inhibitory factor ( mif ) production may contribute to vascular hyperpermeability and hemostatic abnormality and thus blocking of mif may be an answer for prevention of dengue hemorrhage . this article highlights the deadly effect of dengue hemorrhagic encephalopathy and the limited weapons and knowledge we have against it . more research is needed in this field to determine possible predictors of ich such as dengue igg in dengue infection .

introduction : dengue fever is a global pandemic threat with increasing incidence . to date , there are no cures and the effectiveness of dengue vaccines is still uncertain . world heath organization introduced expanded dengue syndrome to include unusual presentations of dengue fever including severe neurologic complications . one of the deadly complications is intracranial hemorrhage ( ich).methodology : we collected data of patients with ich diagnosed via a plain computed tomography of the brain ( ct brain ) with thrombocytopenia and positive dengue virus type 1 nonstructural protein ( ns1 ) antigen test or positive dengue serology igm from january 2014 till june 2015 at our center . nine patients were included and all 20 other remaining patients reported in literature so far are discussed.discussion:we found that all patients in our center requiring neurosurgical intervention died . another interesting observation is that detection of dengue igg usually meant more severe ich and poorer outcomes . from our series , platelet levels did not seem to influence the outcome.conclusion:we recommend that for early detection of ich , dengue igg should be routinely screened and a high index of suspicion be maintained . future research should be focused on determining predictors of ich in patients with dengue fever so that preventive steps can be taken as mortality is high and no treatment seems beneficial at the moment once severe ich occurs .

pyrazolines are nitrogen - containing heterocyclic compounds , well known for their pronounced biological activity . it was demonstrated that the combination of pyrazole with azole ring , linked to each by one sigma bond , led to more biologically active targets ; for example , pyrazolylthiazoles showed excellent antimicrobial activities . continuing our work in this research field [ 69 ] and in an attempt to identify new and potent antimicrobial agents , we tried here to generate new benzofuryl 2-pyrazolin-1-ylthiazoles as antimicrobial agents using simple methods . the starting pyrazoline-1-carbothioamide 5 was prepared by treatment of 2-acetylbenzofuran 1 with equivalent of 4-flurobenzaldehyde 2 in the presence of 10% alcoholic naoh in 90% ethanol with stirring at room temperature to give chaconne 3 . reaction of chalcone 3 with equivalent amount of thiosemicarbazide was performed in ethanol in the presence of 2.5 equivalent of sodium hydroxide to the target precursor 5 . the resulting pyrazoline-1-carbothioamide 5 was cyclized to the corresponding 2-(3-(benzofuran-2-yl)-5-(4-fluorophenyl)-4,5-dihydro-1h - pyrazol-1-yl)-4-methyl-5-(p - subs.phenyldiazenyl)thiazole derivatives 8a d by reaction with hydrazonoyl halides 6a d in anhydrous ethanol and in the presence of an equivalent of triethylamine ( scheme 1 ) . the reaction product structures were elucidated by means of nmr , ms spectroscopy , and elemental analyses ( table 1 ) . h nmr spectra of 8a d contained two doublet - doublet and one triplet signals due to the presence of ch2 adjacent asymmetric carbon . the mass spectra of 8a d showed the molecular ion peaks at m / z 481 , 499 , 515 and 561 , respectively in agreement with the calculated masses . the reaction between pyrazoline-1-carbothioamide 5 and the equivalent amount of -haloketones , for example , phenacyl bromides derivatives , 2-bromoacetylbenzofuran , and 3-bromoacetylcoumarin , was performed in refluxing ethanol to yield pyrazol-1-ylthiazoles 1315 in good yields via the intermediates a ( scheme 2 ) . the h nmr of compounds 1316 indicate the disappearance of nh2 signal due to blocking of nh2 with thiazole nucleus . the mass spectra of 13a , b16 showed the molecular ion peaks at m / z 473 , 518 , 479 , 507 , and 465 , respectively in agreement with the calculated masses . all the new synthesized compounds were screened for their antibacterial and antifungal activities at 100 g / ml concentration against four gram - positive bacteria ( staphylococcus aureus atcc 29213 ; b. subtilis atcc6633 ; b. megaterium atcc 9885 ; sarcina lutea ) , three gram - negative bacteria ( klebsiella pneumoniae atcc13883 ; pseudomonas aeruginosa atcc27953 ; e. coli atcc 25922 ) , and two yeast ( saccharomyces cerevisiae and candida albicans nrrl y-477 ) . most of the newly synthesized compounds showed good antimicrobial activities with respect to the control drugs . compound 8c exhibited the highest potency against all tested organisms with respect to reference drugs . compounds 8d and 13b inhibited the growth of staphylococcus aureus atcc 29213 with inhibition zones 23 , 22 mm , respectively , while compound 5 showed excellent activities against klebsiella pneumoniae atcc13883 ; pseudomonas aeruginosa atcc27953 ; and e. coli atcc 25922 with inhibition zone about 24 mm . also , compound 8c showed the highest activity against staphylococcus aureus atcc 29213 , saccharomyces cerevisiae , and candida albicans nrrl y-477 with inhibition zone about 23 mm . the minimum inhibitory concentration ( mic ) of the synthesized compounds against highly inhibited organisms is reported in table 3 . compounds 5 revealed low mic ( 200 g / ml ) against staphylococcus aureus atcc 29213 , b. megaterium atcc 9885 , and candida albicans nrrl , compound 8a exhibited high mic ( 16 g / ml ) against b. subtilis atcc6633 ( table 3 ) . elemental analytical data were carried from the microanalytical unit , cairo university , giza , egypt . the ir spectra were recorded in potassium bromide disks on a jasco ft / ir-6100 . h nmr spectra were run on joel - eca 500 mhz in deuterateddimethyl sulphoxide ( dmso - d6 ) . chemical shifts values ( ) are given in parts per million ( ppm ) . the mass spectra were performed using mass varian mat ch-5 spectrometer at 70 ev . ( e)-1-(benzofuran-2-yl)-3-(4-fluorophenyl)prop-2-en-1-one 3 ; hydrazonoyl halides ; 1-(benzofuran-2-yl)-2-bromoethanone 9 ; 3-(2-bromoacetyl)-2h - chromen-2-one 10 ; and 2,3-dichloroquinoxaline were prepared according to the literature . to a suspension of chalcone 3 ( 10 mmol , 2.66 g ) and sodium hydroxide ( 25 mmol , 1.0 g ) in ethanol ( 50 ml ) , thiosemicarbazide ( 12 mmol , 1.1 g ) was added . the mixture was refluxed for 12 h , then left to cool ; the solid product was filtered off , washed with ethanol , and dried . yield 52% ; m.p . ) max / cm 3460 , 3335 ( nh2 ) ; h nmr ( dmso - d6 ) 3.09 , 3.14 ( dd , 1h , ch , j = 3.05 hz , j = 3.05 hz ) , 3.94 , 4.06 ( dd , 1h , ch , j = 11.45 hz , j = 11.5 hz ) , 5.88 ( t , 1h , ch , j = 3.05 hz , j = 7.65 hz ) , 7.137.43 ( m , 9h , ar - h ) , 9.44 ( s , 2h , nh2 , d2o - exchangeable ) ; ms m / z ( % ) : 339 ( m , 75 ) , 60 ( 100 ) . to a suspension of compound 5 ( 1 mmol , 0.34 g ) in ethanol ( 20 ml ) , the 1 mmol of appropriate reagent { ( appropriate hydrozonoyl chlorides 6 + et3n ) ; ( appropriate phenacyl bromides 9 ) ; ( 2-bromoacetylbenzofuran 10 ) ; ( 3-bromoacetylcoumarin 11 ) ; or ( 3,4-dichloroquinoxaline 12 ) } was added and heated under reflux for 4 h. after cooling , the precipitate was collected by suction filtration . yield 58% ; m.p.180 - 2c ; h nmr ( dmso - d6 ) 2.54 ( s , 3h , ch3 ) , 4.09 , 4.11 ( 2dd , 2h , ch , j = 10.7 hz , j = 9.95 hz ) , 5.87 ( t , 1h , ch , j = 10.7 hz , j = 9.95 hz ) , 7.187.73 ( m , 14h , ar - h ) ; ms m / z ( % ) : 481 ( m , 75 ) , 95 ( 100 ) . yield 66% ; m.p . 201 - 3c ; h nmr ( dmso - d6 ) 2.54 ( s , 3h , ch3 ) , 4.07 , 4.12 ( 2dd , 2h , ch , j = 10.7 hz , j = 9.95 hz ) , 5.89 ( t , 1h , ch , j = 10.7 hz , j = 9.95 hz ) , 7.197.73 ( m , 13h , ar - h ) ; ms m / z ( % ) : 499 ( m , 80 ) , 95 ( 100 ) . yield 72% ; m.p . 206 - 8c ; h nmr ( dmso - d6 ) 2.53 ( s , 3h , ch3 ) , 4.10 , 4.34 ( 2dd , 2h , ch , j = 10.7 hz , j = 9.95 hz ) , 5.89 ( t , 1h , ch , j = 10.7 hz , j = 9.95 hz ) , 7.197.73 ( m , 13h , ar - h ) ; ms m / z ( % ) : 515 ( m , 70 ) , 95 ( 100 ) . 128 - 30c ; h nmr ( dmso - d6 ) 2.55 ( s , 3h , ch3 ) , 4.10 , 4.34 ( 2dd , 2h , ch , j = 10.7 hz , j = 9.95 hz ) , 5.87 ( t , 1h , ch , j = 10.7 hz , j = 9.95 hz ) , 7.197.72 ( m , 13h , ar - h ) ; ms m / z ( % ) : 561 ( m , 62 ) , 95 ( 100 ) . yield 49% ; m.p . 4.014.05 ( dd , 2h , ch2 , j = 11.45 hz , j = 10.33 hz ) , 5.73 ( t , 1h , ch , j = 5.35 hz , j = 6.1 hz ) , 7.177.92 ( m , 14h , ar - h ) ; ms m / z ( % ) : 473 ( m , 80 ) , 91 ( 100 ) . yield 58% ; m.p . 208 - 10c ; h nmr ( dmso - d6 ) 4.014.08 ( dd , 2h , ch2 , j = 11.45 hz , j = 10.33 hz ) , 5.71 ( t , 1h , ch , j = 5.35 hz , j = 6.1 hz ) , 7.177.92 ( m , 14h , ar - h ) ; ms m / z ( % ) : 518 ( m , 49 ) , 91 ( 100 ) . yield 63% ; m.p . 252 - 4c ; h nmr ( dmso - d6 ) 4.044.07 ( dd , 2h , ch2 , j = 11.45 hz , j = 10.33 hz ) , 5.73 ( t , 1h , ch , j = 5.35 hz , j = 6.1 hz ) , 6.88 ( s , 2h , benzofuryl - h ) , 7.197.70 ( m , 13h , ar - h ) ; ms m / z ( % ) : 479 ( m , 100 ) . 233 - 4c ; h nmr ( dmso - d6 ) 4.05 , 4.08 ( dd , 2h , ch2 , j = 11.45 hz , j = 10.33 hz ) , 5.72 ( t , 1h , ch , j = 5.35 hz , j = 6.1 hz ) , 7.227.72 ( m , 14h , ar - h ) , 8.23 ( s , 1h , coumarinyl - h ) ; ms m / z ( % ) : 507 ( m , 100 ) . ; h nmr ( dmso - d6 ) 4.02 , 4.06 ( dd , 2h , ch2 , j = 11.45 hz , j = 10.33 hz ) , 5.70 ( t , 1h , ch , j = 5.35 hz , j = 6.1 hz ) , 7.227.72 ( m , 13h , ar - h ) ; ms m / z ( % ) : 465 ( m , 100 ) . chemical compounds were individually tested against a panel of gram - positive and gram - negative bacterial pathogens and yeast . antimicrobial tests were carried out by the agar well diffusion method using 100 l of suspension containing 1 10 cfu / ml of pathological tested bacteria and 1 10 cfu / ml of yeast spread on nutrient agar ( na ) and sabourund dextrose agar ( sda ) , respectively . after the media had cooled and solidified , wells ( 10 mm in diameter ) were made in the solidified agar and loaded with 100 l of tested compound solution prepared by dissolving 100 mg of the chemical compound in one ml of dimethyl sulfoxide ( dmso ) . the inculcated plates were then incubated for 24 h at 37c for bacteria and at 28c for yeast . ciprofloxacin ( 50 g / ml ) and ketoconazole ( 50 g / ml ) were used as standard for antibacterial and antifungal activities respectively . after incubation time , antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms and compared with that of the standard . the experiment was carried out in triplicate , and the average zone of inhibition was calculated . the bacteriostatic activity of the active compounds ( having inhibition zones ( iz ) 18 mm ) was then evaluated using the twofold serial dilution technique . twofold serial dilutions of the tested compounds solutions were prepared using the proper nutrient broth . the final concentration of the solutions was 200 , 100 , 50 , and 25 g / ml . the tubes were then inoculated with the test organisms , grown in their suitable broth at 37c for 24 hours for bacteria ( about 1 10 cfu / ml and 1 10 cfu / ml of yeast ) , and each 5 ml received 0.1 ml of the above inoculum and incubated at 37c for 24 hours . the lowest concentration showing no growth was taken as the minimum inhibitory concentration ( mic ) . this precursor reacted with thiosemicarbazide in strong basic medium to afford the n - pyrazoline thioamide 5 . the new compounds were tested for their antimicrobial activities and significant activities due to presence of three nucleuses : benzofuran , pyrazole , and thiazole . also , some substituent increases the antimicrobial activities such as chloro substituent in compounds 8c and 13a .

a new series of 2-pyrazolin-1-ylthiazoles 8a d and 1316 was synthesized by cyclization of n - thiocarboxamide-2-pyrazoline with different haloketones and 2,3-dichloroquinoxaline . the structures of the new compounds were confirmed by elemental analyses as well as nmr , ir , and mass spectral data . the newly synthesized compounds were evaluated for their antimicrobial activities , and also their minimum inhibitory concentration ( mic ) against most of test organisms was performed . amongst the tested ones , compound 8c displayed excellent antimicrobial activity .

several diseases are associated with the extracellular deposition of protein aggregates , including the alzheimer 's disease , the transthyretin , and light chain amyloidoses as well as type ii diabetes . accumulation of insoluble protein in the extracellular space results from the aberrant assembly of proteins into aggregates , usually with a quaternary structure rich in cross--sheets , known as amyloid . the causative link between the observed pathophysiology and amyloid formation is now supported by numerous genetic , biochemical , and pharmacological studies [ 1 , 35 ] . more than 30 human endogenous proteins have been identified as precursors of amyloid fibrils whose deposition is associated with tissue degeneration . although these amyloidogenic precursors share no sequence and native state structure homologies , amyloids extracted from patients share several structural , chemical , and biological features , including an extensive cross -sheet structure and the capacity to bind specific dyes , such as congo red and thioflavin t ( tht ) . particularly , fibrils are virtually always associated with nonfibrillar biomolecules , including the serum amyloid p component , apolipoprotein e , collagen , metals , glycosaminoglycans ( gags ) , and various lipids . however , over the last two decades , several studies have instead highlighted that these amyloid cofactors can promote and/or modulate the amyloidogenic process . in this view , amyloid formation might not be simply a consequence of a protein misfolding event but may be more a consequence of the interaction of the amyloidogenic protein precursor with extrinsic factors and/or its ( bio)chemical microenvironment . the deposition of amyloid fibrils in the islets of langerhans of patients afflicted by type ii diabetes was originally described at the beginning of last century . over the 20th century , it was confirmed that islet hyalinization , that is , tissue degeneration into a classy translucent material , was closely associated with diabetes mellitus , particularly in elderly individuals [ 13 , 14 ] . it is only in 1987 that the major component of islet amyloids was identified as a 37-residue peptide , the islet amyloid polypeptide ( iapp ) or amylin . as iapp is coexpressed , copackaged , and cosecreted with insulin by the pancreatic -cells , the overproduction of insulin often associated with type ii diabetes will lead to an increased release of iapp . this elevated local concentration of iapp in the islets of langerhans should , in theory , promote the formation of amyloid . nonetheless , although iapp is expressed in nondiabetic subjects at levels higher than those required to form amyloids in vitro , iapp rarely deposits in the pancreas of normal individuals . this suggests that iapp concentration is not the critical factor contributing to its aggregation and proposes that other elements could play a determinant role in the amyloidogenic process and , accordingly , in the etiology of type ii diabetes . in this review , we will initially describe iapp structure and normal physiological functions and briefly present its proposed mechanisms of aggregation . we will mainly focus on the roles of amyloid cofactors and/or the biological environment in amyloid formation . as the role of model membranes in iapp fibrillogenesis has been previously discussed in outstanding reviews [ 2023 ] , the present paper will mainly put an emphasis on other factors , such as gags and metals . finally , we will discuss the potential roles of amyloid cofactors in -cells degeneration associated with iapp aggregation and amyloid deposition . characterization of the peptide isolated from human islet amyloids led to the identification of a c--amidated 37-residue peptide . iapp is expressed as an 89-residue polypeptide , called preproiapp , containing a 22-residue signal peptide that is cleaved off in the reticulum endoplasmic to form proiapp . subsequent posttranslational modifications of proiapp involving the action of prohormone convertase ( pc ) enzymes and carboxypeptidase e ( cpe ) lead to the formation of a c--amidated , cyclized , and biologically active peptide . the primary structure of iapp has been determined in several mammalian species , including monkey , dog , mouse , and rat ( figure 1(a ) ) . the n- and c - terminal regions of iapp have been well conserved in all mammalian species , whereas the central 2129 domain is more variable and shows important interspecies variations . particularly , iapp sequences found in mice and rat contain pro residues at positions 25 , 28 , and 29 whereas the human sequence encompasses ala , ser , and ser , respectively . this variation is significant for the amyloidogenesis process , as rat ( riapp ) and mice ( miapp ) peptide are less prone to aggregation and these two species do not form islet amyloids . in solution , human iapp ( hiapp ) exhibits a conformational ensemble mainly populated by disordered conformations , although it diverges from an absolute random coil by the presence of local and transient ordered structures . for instance , the segment 519 of riapp , which exhibits a high homology with hiapp , appears to transiently populate -helix in its monomeric form [ 28 , 29 ] . besides , from molecular dynamics simulations , it was reported that hiapp monomers could form ordered and extended -hairpins . in presence of lipid membrane models or organic solvent , such as hexafluoroisopropanol ( hfip ) , hiapp readily adopts an -helical conformation ( figure 1(b ) ) . for example , in dodecylphosphocholine ( dpc ) micelles , riapp exhibits a structure characterized by a single helical region spanning from residues ala-5 to ser-23 followed by a disordered c - terminal domain . when incubated with sodium dodecyl sulfate ( sds ) micelles , hiapp forms , instead , two -helical segments spanning from residues ala-7 to val-17 and asn-21 to ser-28 and a short 310 helix from gly-33 to asn-35 . both rat and human 119 iapp fragments show a helical conformation in dpc micelles , although they adopt different orientation on the micelle surface . iapp is a member of the calcitonin peptide family , which includes calcitonin , calcitonin - gene - related peptides ( cgrps ) , and adrenomedullin . these peptide hormones mediate their biological activities by binding and activating class b g protein - coupled receptors ( gpcrs ) . interestingly , no specific gpcr per se for iapp has been identified so far . instead , iapp shares the calcitonin receptor ( ct ) with calcitonin , although it binds to ct with a relatively low affinity . the function , pharmacology , and selectivity of the ct receptor are altered by its association with receptor activity - modifying proteins ( ramps ) . ramps constitute a family of single transmembrane proteins with 3 members : ramp1 , ramp2 , and ramp3 . association of the ct receptor with ramp1 or ramp3 changes the selectivity of the receptor and increases significantly the affinity for iapp . iapp specific binding sites were initially identified in the brain and the renal cortex and have now been identified in several peripheral tissues . under normal physiological conditions , iapp is cosecreted with insulin from -pancreatic cells in response to an elevated blood glucose concentration . in skeletal muscles , iapp inhibits basal and insulin - stimulated glycogen synthesis , resulting in an increase of glucose-6-phosphate level . studies have also shown that iapp suppresses glucagon secretion , decreases gastric emptying , and induces satiety [ 25 , 39 , 40 ] . iapp may also be involved in the process of tissues calcification and could play a critical role in the inhibition of bone resorption . like other members of the calcitonin family , iapp is a potent vasodilator and causes systemic hypotension and tachycardia [ 25 , 42 ] . however , these effects were observed at much higher concentrations than the circulating physiological concentration of iapp , normally ranging in the low picomolar ( 320 pm ) [ 23 , 43 ] . thus , these effects should be interpreted with precaution since they could result from the activation of the ct receptor not associated with a ramp and/or of the ct - receptor - like receptor . taken together , the biological functions of iapp are still far from being clearly understood . amyloid fibrils , including iapp amyloids , are highly ordered assemblies that predominantly adopt a characteristic cross--sheet quaternary structure . this structural motif provides the most favorable organization for these supramolecular assemblies and can accommodate a high diversity of polypeptide sequences . amyloids are characterized by an x - ray diffraction pattern with two characteristic signals , a clear reflection at 4.7 along the direction of the fibril , and a diffuse reflection around 10 perpendicular to the fibril axis ( figure 2 ) . by atomic force microscopy ( afm ) and electron microscopy ( em ) , amyloids extracted from patients or prepared in vitro appear as long ( 0.5 to 10 m ) and unbranched filaments having 4 to 15 nm of diameter ( figure 2 ) . until recently , the structure of amyloids at the atomic level was unclear , since amyloids do not form crystals and are insoluble , precluding their characterization by x - ray crystallography and solution nuclear magnetic resonance ( nmr ) . thanks to recent advances in techniques such as solid state nmr and the ability of growing nanocrystals of peptide fragments , it has been possible to elucidate the structure of several amyloids . these approaches , along with cryoelectron microscopy , have suggested that amyloid fibrils present a core sharing several characteristics . these differences can be seen in ( i ) the length of the -strands , ( ii ) the arrangement ( parallel versus antiparallel ) of the constituting sheets of the strand , ( iii ) the length and arrangement of structures which are not inside the fibril core , and ( iv ) the number of -sheets per each protofilament . thus , although amyloid fibrils display similar characteristics , a marked polymorphism exists not only between fibrils from different precursors , but also between amyloids assembled from the same polypeptide but in different conditions . the atomic structure of iapp in its fibrillar form has been studied by a variety of approaches , including solid state nmr , x - ray crystallography , and electron paramagnetic resonance ( epr ) spectroscopy . according to the technique used and/or the conditions in which iapp amyloids were assembled , three main atomic models have been proposed . firstly , in the model derived from solid state nmr study , iapp protofibrils consist of two columns of symmetry related monomers packed against each other . each polypeptide monomer adopts a u - shaped structure and contains two -strands connected by a bend - loop . these -strands comprise , respectively , residues 817 and 2837 whereas the loop involves residues 1827 . residues 1 to 7 do not participate in the -structure , most likely because of the conformational constraints imposed by the disulfide bridge . secondly , the eisenberg group has proposed a model based on the crystallographic studies of iapp fragments that shares many features with the solid state nmr model described above but differs in how the two columns of iapp monomers pack against each other and in the length of the c - terminal -strand . thirdly , epr studies of iapp variants lacking the cys cys disulfide bond have led to a slight variation of these two models . the protofibrils are still built up of u - shaped stacks of monomers , but the planes of the two -strands within one iapp molecule are staggered by around 15 . interestingly , in these three models , the 2029 amyloidogenic segment is not part of a -sheet . instead , it forms a partially ordered bend that connects the two -strands , questioning the sensitivity of hiapp amyloid formation to substitutions and/or modifications within this amyloidogenic prone region . structural analysis of iapp fibrils was so far exclusively performed using homogenous peptide assemblies , although amyloid deposits in islets of langerhans of diabetic patients contain a variety of biomolecules , including gags , lipids , and other proteins . thus , it will be interesting to study the molecular architecture of iapp amyloids assembled in a biologically relevant heterogeneous environment . while the mechanism by which proteins self - assemble into amyloids has been intensively studied over the last two decades , mechanistic details remain partially elusive and still the matter of controversy . amyloidogenic polypeptides can be divided into two different structural classes : those that are intrinsically ( or partially ) disordered in their native state and those that show a well - defined tertiary structure in their monomeric soluble state . generally , natively folded amyloidogenic proteins , such as transthyretin and 2-microglobulin , have to unfold ( or misfold ) , at least partially , to form amyloids . in contrast , intrinsically disordered polypeptides , such as iapp and a peptide , need to undergo conformational rearrangements allowing the formation of locally ordered structure(s ) to initiate the amyloidogenic process . the formation of amyloid fibrils is often described as a nucleation - dependent polymerization , although other models have been suggested , including the nucleated conformational conversion and the monomer - directed conversion . the nucleated polymerization model is characterized by the rate - limiting formation of the nucleus , which results from the equilibrium between monomers that are and are not assembly competent . as soon as the nucleus is formed , assembly rapidly occurs by the addition of competent monomers to the growing end of the protofibrils . firstly , a low amount of dynamic and transient oligomeric species is produced in the lag phase . this phase takes place slowly because of the unfavorable interactions between monomers to form oligomers . secondly , once the nucleus ( competent oligomer ) is formed , the elongation phase begins , leading to the rapid growth of the ( bio)polymers . amyloid formation kinetics , seeding experiments as well as the difficulty of detecting low ordered oligomers , suggest that iapp amyloidogenesis could be ascribed to a nucleated polymerization . recent studies performed with different amyloidogenic proteins have suggested that oligomers could be the most proteotoxic species of the aggregation cascade [ 5658 ] . for iapp , two major models have been proposed for its oligomerization in homogenous solution : the helical intermediates model and the -hairpins model . as inferred from nmr analysis and in silico prediction , monomeric soluble iapp transiently adopts an -helix between residues 519 and it has been suggested that this helical intermediate could be on - pathway to amyloid formation . for instance , by analyzing the kinetics of -sheet formation using two - dimensional infrared ( 2d ir ) spectroscopy , it was observed that the disappearance of the random coil conformation was associated with the emergence of a -helix . besides , the presence of a low percentage of hfip , a solvent known to promote helical formation , in the aggregation solution of iapp accelerates the rate of amyloid formation [ 62 , 63 ] . similarly , whereas the binding of iapp to model membranes favors its initial conformational conversion from a random coil to a -helix , it is well known that lipid vesicles significantly accelerate iapp amyloid formation . according to the helical intermediates hypothesis , self - association would be thermodynamically associated with helix formation within the 520 segment , in a similar way of the driven forces of coiled - coil motif formation . in turn , this transient helical oligomer would generate a high local concentration of the c - terminal amyloidogenic segment of iapp , favoring the intermolecular -sheets formation . taking into account this model , several molecules have been recently designed to target and stabilize helical intermediates with the ultimate goal of inhibiting iapp amyloid formation [ 6568 ] . by combining ion mobility mass spectrometry and molecular dynamics simulations , it was instead proposed that iapp early oligomerization steps include the formation of -strand rich dimers [ 29 , 60 ] . bowers and colleagues have suggested that iapp -sheet - rich assemblies are formed from ordered beta - hairpins rather than from coiled structures . the discrepancy between these two models indicates that the initial events of iapp amyloidogenesis still remain unclear . it is worth mentioning that in contrast to in vitro homogenous aqueous solution , the mechanisms of amyloid formation in vivo are most likely to be different and could involve alternative pathways . iapp amyloidogenesis takes place in a heterogeneous and crowded environment with the potential interactions with several components of the extracellular matrix and the plasma membrane . thus , mechanistic examinations of amyloid formation in heterogeneous environments constitute an important issue and relevant studies will now be discussed . amyloid formation has been originally perceived as a self - assembly homogeneous process in which the inherent physicochemical and structural properties of the amyloidogenic proteic precursor as well as its concentration constitute the major driving forces to fibrillation . accordingly , the presence of biomolecules tightly associated with the amyloids in vivo , including gags , metals , glycoproteins , and lipids , was seen as a contamination of the fibrils occurring after aggregation and/or deposition . however , numerous biophysical investigations as well as in vivo biochemical studies have shown a prominent role of these extrinsic factors in amyloid deposition associated with the etiology of various diseases , including type ii diabetes [ 1 , 8 , 9 ] . it is now evident that the biochemical microenvironment in which amyloid formation occurs and the interactions of the polypeptide precursor with various biomolecules not only modulate the rate and extent of aggregation , but also remodel the mechanisms as well as the structure , toxicity and stability of the resulting fibrils . immunohistochemical analysis revealed that the basement membrane heparan sulfate proteoglycan ( hspg ) , perlecan , was present within islet amyloid deposits , suggesting a causative role of sulfated gags in iapp fibrillogenesis . besides , incubation of hiapp transgenic mouse isolated islets with was-406 , an inhibitor of hspgs synthesis , resulted in a dose - dependent decrease in amyloid formation . similarly , the westermark group has established a mouse strain that overexpresses both hiapp and heparanase , an enzyme that catalyzes the cleavage of cell surface heparan sulfate . they reported that isolated islets from these mice showed a marked reduction in amyloid accumulation upon a 2-week high glucose treatment ; these conditions simulate the hyperglycemia observed in type ii diabetes and stimulate iapp expression and secretion . in addition , since the original work by castillo et al . , several reports have shown that sulfated gags , including heparin , heparan sulfate , and heparin derivatives , accelerate dramatically the rate of iapp and pro - iapp amyloid formation in vitro [ 7377 ] . overall , these studies constitute a clear testimony that sulfated gags could play an active role in islet amyloid deposition associated with type ii diabetes . gags are long and linear polysaccharides composed of repeating disaccharide units and some gags can contain up to 200 repeating disaccharide units . they are abundant on the outer leaflet of the plasma membrane of every cell type of metazoan organisms and in their basement membrane and extracellular matrix ( ecm ) . according to the structure of their carbohydrate backbone the most ubiquitous class of gag is heparan sulfate ( figure 3(a ) ) which is expressed at the cell surface of nearly every cells , constituting more than 50% of total proteoglycans [ 80 , 81 ] . other types of gags include heparin , chondroitin sulfate , dermatan sulfate , keratan sulfate , and hyaluronic acid . owing to their high density of carboxylate and sulfate groups , gags are highly negatively charged biopolymers that constitute a major reservoir of polyanions surrounding cells . with exception of hyaluronic acid and heparin , gags are usually covalently o - linked to a protein core , forming a structure known as proteoglycans . hspgs , which constitute approximately 95% of all proteoglycans , are present in all tissues and comprise five types of protein core , including the cell surface syndecan and the ecm perlecan , the latter being a major constituent of pancreatic islet amyloids . over the last 15 years , several studies have demonstrated that the addition of sulfated gags to amyloidogenic proteins accelerates their fibrillogenesis in vitro . these polypeptides include both intrinsically disordered polypeptides such as the a peptide , -synuclein , and iapp and natively folded proteins such as transthyretin , gelsolin , and 2-microglobulin . it has been proposed that gags hasten amyloidogenesis by a scaffold - based mechanism , in which the amyloidogenic protein , either in its monomeric or oligomeric form , interacts with the sulfated polysaccharides mainly through electrostatic interactions , increasing its local concentration and promoting aggregation [ 85 , 86 , 88 ] . it was also reported that the interaction with gags induces the conformational transition of the 3 kda fragment of gelsolin and a peptide from a random coil to -sheet . however , this structural modification is most likely related to the aggregation process rather than to a conformational conversion within the monomeric protein . the mechanisms by which sulfated gags accelerate iapp amyloid formation have been studied by a combination of biophysical approaches and are similar to the one described for other amyloidogenic polypeptides . owing to its net positive charge at physiological ph , iapp can bind by means of electrostatic interactions with polyanionic sulfated gags . as a matter of fact , it was observed by nmr spectroscopy that heparin binds to the n - terminal segment of iapp , which includes the only four potential positive charges in iapp sequence : the -amino group , lys-1 , arg-11 , and his-18 . besides , it was reported by isothermal titration calorimetry ( itc ) that the affinity of iapp to sulfated gags was dependent on the protonation state of his-18 and that the binding was predominately enthalpy - driven , most related to electrostatic interactions . a heparin binding site was characterized within the n - terminal domain of proiapp [ 77 , 90 ] and it was suggested that the interaction of unprocessed proiapp with sulfated gags could have strong implications for amyloid formation in pancreatic islets . fret analyses between tht and fluorescein - labelled heparin ( fh ) [ 73 , 74 ] showed that iapp binds to sulfated gags before amyloid formation , most likely in its monomeric form . as reported for other amyloidogenic polypeptides , heparin is incorporated into the fibrils and/or is tightly associated with the mature amyloids . by cd spectroscopy , iapp and proiapp association to sulfated gags induces a random coil to -helix conformational conversion [ 75 , 77 ] and this helical structure is rapidly converted into a -sheet structure . as the binding of iapp and proiapp accelerates the rate of amyloid formation , this secondary conformational conversion supports the helical intermediates hypothesis described above . by using heparin analogs of different length and/or degree of sulfation , it was reported that the effects of gags on iapp amyloidogenesis were dependent on the oligosaccharide length and sulfate content and not on the amount of charged monomers [ 72 , 73 ] . nonetheless , it was observed that the degree of sulfation of heparan sulfate isolated from pancreatic -tc3 cells does not determine all aspects of gag - mediated amyloid formation . besides , the nature of gag backbone also affects , to some extent , the enhancement of iapp fibril formation [ 72 , 92 ] . overall , these studies suggest a model for gags - accelerated iapp amyloidogenesis in which the positively charged n - terminal segment of the peptide binds to the sulfate moieties of gags , inducing the formation of a -helix . in turn , this generates a high local concentration of peptide on gag scaffold that drives the association of iapp amyloidogenic segment , accelerating drastically the formation of -sheet rich assemblies ( figure 3(b ) ) . several reports have suggested that the dysregulation of metal ion homeostasis could be implicated in the pathogenesis of amyloid diseases , comprising type ii diabetes . binding sites for transition metals , including zinc , copper , and iron , have been characterized in numerous amyloidogenic polypeptides , such as a peptide , -synuclein , and 2-microglobulin . most of mechanistic studies have been so far performed with the a peptides and have shown that physiological concentrations of metals , particularly zn , are sufficient to accelerate the rate of amyloid formation , although divergent results were reported . while it is known for more than 20 years that the secretory granules in pancreatic islets of langerhans , which store iapp and insulin , are characterized by a high concentration of zinc , the role of this metal in iapp amyloidogenesis has not been addressed until recently [ 98 , 99 ] . particularly , it has been reported that zinc transport into -cells secretory granules , involving the pancreas - restricted zinc transporter znt8 , could play a significant role in the etiology of type ii diabetes [ 100 , 101 ] . this observation suggests that zinc homeostasis could be associated with iapp misfolding / aggregation , although this avenue has not been explored in vivo so far . the modulation of iapp amyloidogenesis in vitro by zinc is complex and is dependent on zinc concentration as well as the ph and peptide concentration . at ph 7.5 , the presence of a low concentration of zn in the incubation solution decreases the rate of amyloid formation whereas at higher concentration the fibril elongation rate increases . it was also observed that while the total amount of fibrils is greatly reduced by zinc at all concentrations , the general morphology of the individual fibrils remained somewhat similar . notably , typical concentrations of zinc reported in the extracellular space where iapp deposition occurs , ranging from 10 to 25 m , decrease significantly amyloid formation at physiological ph . in sharp contrast , at ph 5.5 , at which the residue his-18 is protonated , zinc accelerated iapp fibrillogenesis . brender and colleagues have observed that iapp in an organic solvent undergoes a structural conversion upon zinc binding characterized by a local disruption of the helical structure around residue his-18 . thus , the inhibitory effect of zinc observed at low concentrations was initially ascribed to the unfavorable incorporation of a charge inside the loops , as the imidazole ring of his-18 is located in the hydrophobic core of the fiber . by combining itc , nmr , and esi mass spectrometry , it was observed that zinc favors the formation of off - pathway hexameric species while creating an energetic barrier for the formation of amyloids . thus , zinc binding to nonfibrillar iapp with an affinity in the micromolar range promotes the formation of prefibrillar aggregates , ultimately inhibiting the formation of amyloid fibrils . the inhibition of amyloid formation by metals appears to be restricted to metals that are known as good ligands for histidine , such as zn and cu , whereas mg and ca , which are poor imidazole ligands , have no significant effect on iapp amyloidogenesis [ 98 , 104 ] . the effect of the buffer ion composition on the kinetics of iapp amyloid formation was recently examined and it was reported that iapp fibrillogenesis was dependent on the anion identity , while the nature of the cationic species has little effect on the rate of fibrils formation . overall , whereas the modulation of -synuclein and a-peptide amyloidogenesis by metals is well - documented , the role of metal homeostasis in islet iapp deposition has been so far less studied and deserves further attention . particularly , it will be interesting to probe the effects of zinc and copper on the kinetics of iapp self - assembly in heterogeneous environment , that is , in presence of other biological factors such as gags and lipid membrane models . virtually all amyloid deposits , including islet amyloids [ 106 , 107 ] , are associated with apolipoprotein e ( apoe ) , a protein involved in lipid transport and metabolism . the importance of this protein in amyloid deposition has been highlighted in alzheimer 's disease as transgenic mice lacking the apoe gene form only diffuse plaques but not mature neuritic plaques . in sharp contrast , transgenic mice expressing hiapp crossbred with apoe deficient mice showed similar prevalence and severity of islet amyloids , indicating that apoe is not a critical factor for islet amyloid deposition . nonetheless , it was observed in vitro that apoe4 binds iapp and inhibits amyloid formation . insulin , which is stored with iapp in -cell secretory granules , is one of the most potent inhibitors of iapp aggregation . insulin binds to the putative helical domain of iapp , stabilizing the compact isoform of iapp and inhibiting the formation of -sheets [ 112 , 113 ] . the postulated mechanism of fibrillogenesis inhibition by insulin is consistent with the helical intermediates hypothesis . anionic model membranes are the most studied biological cofactors in the context of iapp amyloidogenesis , since they not only accelerate iapp amyloid formation but they also recapitulate the postulated initial site of iapp - induced cell death . the mechanisms of lipid - accelerated iapp amyloidogenesis have been previously addressed in several excellent reviews [ 2023 , 114 ] and readers are invited to consult them for additional information . the presence of insoluble protein deposition in the pancreatic islets of patients suffering from type ii diabetes has initially led to the postulate that amyloid fibrils cause -cell degeneration . this hypothesis was later reinforced by the work of lorenzo and colleagues demonstrating the potential toxicity of iapp fibrils on human pancreatic islet cells . this cell death event was associated with membrane blebbing , chromatin condensation , and dna fragmentation , indicating that iapp amyloids trigger -cell apoptosis . however , over the last fifteen years , several studies have instead suggested that nonfibrillar intermediates are the most toxic species of iapp amyloid cascade . for instance , it was observed that the inhibition of amyloid fibril formation with rifampicin did not reduce iapp - induced pancreatic cell death . furthermore , in a homozygous hiapp transgenic mouse model , selective -cell death and impaired insulin secretion were associated with the formation of early , small amorphous intra- and extracellular aggregates rather than with large amyloid deposits . bram and colleagues have recently reported the isolation of antibodies from diabetic patients that specifically recognized iapp oligomers . remarkably , these antibodies were shown to neutralize the apoptotic effect induced by iapp cytotoxic species on -cell . moreover , dynamic light scattering revealed that cytotoxicity corresponds to iapp aggregates containing between 25 and 6 000 iapp molecules . thus , as for other amyloid - related diseases , the scientific community generally agrees on the hypothesis that prefibrillar aggregates might be the toxic species causing -cell death . however , considering that pancreatic islets from patients afflicted with type ii diabetes are almost all converted into amyloids , this massive iapp deposition most likely interferes with normal -cell functions , such as insulin release . noteworthy , the search for the culprit species of the amyloidogenic cascade has been so far exclusively performed with aggregates prepared in iapp homogenous solution . however , as described above , amyloid cofactors such as metals , gags , and lipids can remodel the pathway of aggregation and can lead to the formation of oligomer species with unusual morphological , physicochemical , and/or biological properties . thus , it will be crucial in the nearest future to characterize the cytotoxicity of iapp oligomers prepared in heterogeneous environment that reconstitutes , as possible , the extracellular environment of pancreatic islets . although the mechanisms by which iapp induced -cell death have been intensively investigated since iapp discovery , the subject is very complex and is still the matter of debate . this topic has been recently discussed in excellent reviews [ 23 , 121 , 122 ] and , accordingly , we will briefly present the main postulated mechanisms . one of the most studied and accepted mechanisms is membrane disruption and transmembrane pore formation . iapp is a cationic peptide , favoring its electrostatic interaction with anionic lipids of the plasma membrane . experiments performed with planar phospholipid bilayer membranes showed the formation of nonselective ion - permeable channels , suggesting that channel - like formation could trigger iapp - induced cell death . similarly , the formation of abnormal vesicle - like membrane structures was observed when freshly dissolved iapp was added to mouse and human islet cells . apoptosis , or programmed cell death , is another mechanism by which iapp can cause -cell death and is closely associated with membrane disruption . actually , nonspecific channel - like formation by iapp causes a high influx of ca inside the cell that can engage apoptosis [ 125 , 126 ] . dna fragmentation , a key apoptosis characteristic , was observable for rinm5f cells exposed to iapp . moreover , iapp induces p53 activation , a well - known tumor suppressing gene that regulates the cycle and increases the transcription of proapoptotic factors . similarly , it was observed that the gene encoding the g1 inhibitor p21 is upregulated when cells are incubated in presence of iapp aggregates . besides , iapp expression in islets upregulates the expression of the fas receptor , a transmembrane protein able to engage programmed cell death , whereas the deletion of fas reduced iapp - induced toxicity , suggesting the involvement of specific apoptotic pathways . several studies have indicated that iapp can induce pancreatic cell death by inducing the generation of reactive oxygen species ( ros ) . for instance , an increased level of ros was observed when cells were exposed to iapp oligomers . interestingly , it was observed that phycocyanin , a natural compound known for its antioxidant properties , protects pancreatic -cells against iapp - induced apoptosis by attenuating oxidative stress and modulating apoptotic pathways . in contrast , treatment with the antioxidant n - acetyl - l - cysteine ( nac ) prevented the rise of ros induced by iapp but did not prevent -cell apoptosis . due to peripheral insulin resistance associated with type ii diabetes , insulin and iapp expression , maturation , and secretion by pancreatic -cells are significantly increased . this can overload the endoplasmic reticulum ( er ) , leading to er stress and the activation of the unfolded protein response ( upr ) . for instance , an elevated expression of iapp in hiapp transgenic rats induces er - stress , ultimately leading to -cells apoptosis . interestingly , by establishing a mouse model overexpressing riapp at a comparable rate as the transgenic hiapp mouse model , it was reported that the elevated er stress depends on the propensity of iapp to aggregate but is not the consequence of protein overexpression . it was recently showed that the expression of hiapp in mice with a cell - specific autophagy defect results in an increase of -cell dysfunction associated with iapp - toxicity , suggesting a protective role of autophagy in type ii diabetes . overall , these studies indicate that iapp - mediated cytotoxicity is multifaceted and is triggered by multiple mechanisms that are intrinsically related to each other . whereas biophysical studies have indicated that amyloid cofactors , including gags , metals , and lipids , can remodel iapp aggregation landscape and biochemical investigations have suggested that oligomeric species induce -cell death , it appears crucial to address the roles of these cofactors in iapp - induced toxicity . it was observed that the coinjection of sulfated gags with iapp in the culture media protects -pancreatic cells against iapp - mediated cytotoxicity [ 73 , 75 ] . this result suggests that , by hastening amyloid formation , sulfated gags stimulate the formation of nontoxic fibrillar species , in agreement with the toxic oligomeric species hypothesis . the role of cell surface proteoglycans in iapp - mediated cell death has been recently investigated . ins-1 cells treated with heparinase and chondroitinase in order to cleave polysaccharide chains of proteoglycans showed a similar vulnerability to iapp to their nontreated counterpart . this data indicates that the lack of gags on the outer leaflet of the plasma membrane does not prevent nor increases iapp toxicity . this result was confirmed by means of the mutant cho cell pgsa-745 , which lacks cell surface gags as a result of a deficiency in xylosyltransferase , a key enzyme in proteoglycans biosynthesis . these observations are not in line with previous studies performed with the a peptide showing that heparan sulfate deficient cells were essentially resistant to a cytotoxicity . similarly , a toxicity is attenuated in embryonic kidney cells overexpressing heparinase . nonetheless , as reported for iapp , the removal of cell surface gags did not prevent hypf - n aggregates toxicity , suggesting some heterogeneity among the mechanisms of cell death induced by amyloidogenic polypeptides . as described above , membrane disruption , including pore formation and membrane fragmentation , appears to play a key mechanistic role in the toxicity induced by iapp on -pancreatic cells . however , the contribution of the plasma membrane lipid composition and of its physicochemical properties on the cellular susceptibility towards iapp has not been directly addressed so far . in an elegant work , evangelisti and co - workers have recently shown that the extent of cytotoxicity of hypf - n oligomers is the result of a complex interplay between the physicochemical features of both the cell membrane and the oligomeric species . regarding iapp , it was reported that depletion of cholesterol from plasma membrane of rat insulinoma cells inhibits the internalization of oligomers , which in turn potentiates iapp cytotoxicity . by means of real - time single particle tracking , it was shown that iapp aggregates interact with gm1 gangliosides and decrease their lateral diffusion in neuroblastoma cell membrane . as gm1 is a major constituent of membrane lipid rafts , which are known for their contribution to cell signaling pathways , it will be interesting to probe the role of gm1 in iapp - induced toxicity . by combining biophysical approaches , it was shown that phosphatidylethanolamine ( pe ) phospholipids modulate the in vitro membrane disruption induced by iapp . this result suggests a possible role of pe in iapp plasma membrane disruption , although this possibility has not been addressed in vivo so far . it was recently observed that copper interacts with iapp to form metallopeptide complexes showing low toxicity towards pancreatic rat -cells , indicating that metal ions can also modulate iapp - induced cell death . as summarized in this review , the role of the so - called accessory amyloid biomolecules in iapp amyloidogenesis has been recently investigated by a combination of biophysical approaches . regardless of the complexity of the microenvironment in which iapp deposition occurs , the effects of several biological cofactors on amyloid formation are being increasingly recognized . nonetheless , several issues should be addressed in order to better appreciate the implication of these biomolecules in the development of amyloid deposition . in turn , this knowledge should lead to deeper understanding of the mechanisms by which iapp induced -cell degeneration . taking into account the prominent role of gags , metals , and lipids in iapp amyloidogenesis , it will be particularly important that the identification of amyloid inhibitors in vitro is performed in milieu that recapitulates , as much as possible , the complex biological environment in which iapp aggregation occurs . for instance , hebda and colleagues have recently performed the screening of small molecules in presence of lipid membrane model and identified 36 molecules that were not previously reported as active toward iapp fibril formation in homogenous solution . similarly , it was observed that the capacity of insulin to inhibit iapp amyloidogenesis is significantly reduced in presence of sulfated gags whereas the inhibition of iapp fibrillogenesis by is5 , a small molecule alpha helix mimetic , is increased in presence of heparin . considering that the simplistic model of iapp fibrillogenesis as a homogenous self - assembly process does not recapitulate amyloid deposition associated with the etiology of type ii diabetes , it will be important in the future to develop in vitro experimental conditions to study iapp aggregation that resemble the complexity of the pancreatic islet environment .

type ii diabetes mellitus is associated with the deposition of fibrillar aggregates in pancreatic islets . the major protein component of islet amyloids is the glucomodulatory hormone islet amyloid polypeptide ( iapp ) . islet amyloid fibrils are virtually always associated with several biomolecules , including apolipoprotein e , metals , glycosaminoglycans , and various lipids . iapp amyloidogenesis has been originally perceived as a self - assembly homogeneous process in which the inherent aggregation propensity of the peptide and its local concentration constitute the major driving forces to fibrillization . however , over the last two decades , numerous studies have shown a prominent role of amyloid cofactors in iapp fibrillogenesis associated with the etiology of type ii diabetes . it is increasingly evident that the biochemical microenvironment in which iapp amyloid formation occurs and the interactions of the polypeptide with various biomolecules not only modulate the rate and extent of aggregation , but could also remodel the amyloidogenesis process as well as the structure , toxicity , and stability of the resulting fibrils .

the large number of photoswitchable biomolecules discovered and developed in recent years covers a great variety of cellular functions , like catalysis of metabolic processes , cytoskeletal polymerization and motors , nucleic acids dynamics , intracellular signaling , and , perhaps most dazzling , membrane excitability , which has been at the focus of optogenetics and optopharmacology . the dream of precisely and remotely photocontrolling every aspect of the cell s inner workings in intact tissue appears within reach and offers the promise of interrogating complex cellular processes to discover their molecular mechanisms . in order to take full advantage of light - regulated proteins , multiphoton excitation with near - infrared ( nir ) light provides sub - micrometric resolution in three dimensions , deep penetration into tissue , and patterned illumination . however , to be adapted to two - photon stimulation technology , the light response of natural photoswitchable proteins like channelrhodopsin-2 ( chr2 ) must often be adjusted by mutating the tight binding pocket of the natural chromophore , which has fixed photochemical characteristics . in contrast , synthetic photoswitches developed by optochemical genetics and optopharmacology are based on chromophores that act on the protein surface and thus offer excellent opportunities for rationally tuning their photochemical behavior by chemical substitutions that do not affect the functional properties of the protein . remarkably , two - photon stimulation of synthetic photoswitchable proteins has not been investigated despite the advances of neurotransmitter uncaging and optogenetics using pulsed nir illumination . to demonstrate the multiphoton activation of synthetic photoswitches , we chose ion channels because they constitute highly sensitive transducers of chromophore isomerization ( potentially up to the single channel level ) . in particular , we focused on the well - characterized light - gated glutamate receptor ( liglur ) , a gluk2 kainate receptor - channel that is chemically conjugated to a maleimide azobenzene cis photoisomerization of this photoswitchable tethered ligand ( ptl ) allows the efficient activation of the receptor upon one - photon absorption of violet or blue radiation ( open liglur , figure 1b ) , a process that can be reverted back either by absorption of green light or thermal relaxation in the dark ( closed liglur , figure 1b ) . ( a ) structures of the photoswitchable tethered ligands applied to the two - photon control of liglur : mag ( 1 ) , mag2p ( 2 ) , and maga2p ( 3 ) . violet ( one - photon ) or nir ( two - photon ) light excitation induces glutamate recognition and channel opening via trans this process is reverted by illumination with visible light ( one - photon excitation ) for liglur - mag and by thermal back - isomerization for liglur - mag2p and liglur - maga2p . to control liglur using multiphoton excitation , here we have investigated the performance of mag and two new mag derivatives ( 2 and 3 , figure 1a ) upon pulsed nir illumination . compounds 2 and 3 were devised to enhance the two - photon excitation response of the symmetrically substituted azobenzene chromophore in mag , which is expected to be poor . we introduced an asymmetric aminoazobenzene with sufficiently strong push pull character as to enhance its two - photon absorption cross - section ( mag2p ) . in addition , the presence of the electron - donating tertiary amine in the 4-position should dramatically decrease the thermal stability of its cis state in physiological conditions , thus resulting in fast spontaneous cis trans back - isomerization and , as such , enabling single - wavelength operation of the switch . this behavior is also expected for 3 containing the same azobenzene core as mag2p . however , a novel scheme was exploited in this compound to enhance its nonlinear optical response , which consists in the introduction of a light - harvesting antenna to sensitize the trans cis isomerization of the system by absorption of nir radiation and subsequent resonant electronic energy transfer ( ret ) to the trans - azobenzene group . because of its maleimide azobenzene glutamate antenna structure , we named compound 3 as maga2p . a naphthalene derivative was selected as antenna because of ( i ) its high two - photon absorption cross - section , ( ii ) the large spectral overlap between its emission and the absorption of the trans isomer of the aminoazobenzene group in 3 , and ( iii ) its reduced size , to minimize potential steric hindrance effects on the glutamate - binding site of the receptor . the preparation of compounds mag2p and maga2p was achieved via a multistep modular synthetic sequence allowing structural diversity in the final compounds as well as the additional incorporation of a photo - harvesting antenna in 3 ( scheme 1 ) . in both cases , we took the n , n - orthogonally diprotected l - lysine 4 as scaffold , to which the different functional fragments of the target compounds were sequentially introduced : o - protected aminoazobenzene 5 , fully protected glutamate derivative 6 , naphthalene derivative 7 , and furan - protected maleimide 8 . these fragments were obtained from commercial products as described in the supporting information . with respect to the synthesis of mag , several changes first , a branching point was inserted between the glutamate and azobenzene moieties to facilitate the incorporation of additional functional units to the ptl structure . second , we introduce herein the use of 6 and 8 as more robust , versatile , and convenient precursors of glutamate and maleimide moieties during the multistep synthesis of novel mag derivatives . reagents and conditions : ( a ) 5 , hatu , dipea , thf ( 89% ) ; ( b ) 37% hcl , meoh ( 93% ) ; ( c ) 6 , edci , hobt , dipea , thf ( 88% ) ; ( d ) 20% piperidine / dmf ( 87% ) ; ( e ) 7 , edci , dipea , thf ( 81% ) ; ( f ) 20% piperidine / dmf ( 64% ) ; ( g ) clcoch3 , pyridine , thf ( 69% ) ; ( h ) 37% hcl , meoh ( 93% ) ; ( i ) 6 , edci , hobt , dipea , thf ( 71% ) ; ( j ) rhcl(pph3)3 , etoh / h2o , reflux ; ( k ) hgo , hgcl2 , acetone / h2o , reflux ; ( l ) 8 , ph3p , diad , thf ( 81% , over the three steps , for 13a , 27% for 13b ) ; ( m ) toluene , reflux ; ( n ) tfa , ch2cl2 ( 81% over the two steps for 2 , 86% for 3 ) . abbreviations : hatu , o-(7-azabenzotriazol-1-yl)-n , n , n,n-tetramethyluronium hexafluorophosphate ; dipea , diisopropylethylamine ; edci , n - ethyl - n-(3-dimethyldiaminopropyl)-carbodiimide hcl ; hobt , 1-hydroxybenzotriazole hydrate ; diad , diisopropyl azodicarboxylate . the synthesis of both mag2p and maga2p began by the coupling reaction of 4 with aminoazobenzene 5 to afford the common intermediate 9 , from which the synthetic pathways diverged . for the synthesis of mag2p , acid removal of the tert - butyl carbamate protection of 9 was followed by the coupling reaction of the resulting amine with glutamate derivative 6 , basic deprotection of the terminal amine , and its acetylation to furnish intermediate 12a . in the case of maga2p , the best results were obtained by deprotecting first the amino terminus and proceeding through its reaction with the antenna fragment 7 to deliver 11 . removal of the carbamate protection and coupling with 6 then furnished compound 12b . from intermediates 12a and 12b , the next synthetic steps were analogous for both ligands : removal of the allyl protecting group , introduction of the furan - protected maleimide 8 under mitsunobu conditions , release of the maleimide moiety via a retro - diels alder reaction , and cleavage of the tert - butyl carbamate and ester protections , thus finally affording the target compounds mag2p and maga2p . figure 2a plots the electronic absorption spectra of the initial trans state of compounds 13 and of the photo - harvesting antenna tethered to maga2p ( see also figures s1 and s2 in the supporting information ) . owing to the 4-amino substituent introduced in the azobenzene core of trans - mag2p and trans - maga2p , the absorption maximum of the azoaromatic * electronic transition of these compounds clearly bathochromically shifts with respect to trans - mag ( 50 nm in dmso ) . this allows the trans cis photoisomerization of mag2p and maga2p to occur upon illumination with blue light instead of violet radiation . as shown in figure 2b , excitation of both ligands at 473 nm led to a noticeable decrease of their * absorption band , a typical signature of photoinduced cis isomer formation . this was further confirmed by h nmr measurements in dmso - d6 , which revealed that the relative concentration of cis - mag2p and cis - maga2p in the resulting photostationary mixtures was 58% in both cases . such photoproducts can be transformed into their corresponding trans isomers by irradiation with green light , as previously reported for mag ( figure s3 in the supporting information ) . in the case of mag2p and maga2p , however , spontaneous thermal cis trans isomerization plays a significant role in the recovery of the initial state of the ligands , and it strongly competes with cis this effect is ascribed to the introduction of a 4-amino substituent in the azobenzene moiety of mag2p and maga2p , and it is expected to be dramatically enhanced in aqueous media . thus , while the lifetimes of cis - mag2p and cis - maga2p in the dark at room temperature are 75 min in dmso ( see figure s4 in the supporting information ) , they further drop off down to the millisecond time scale in aqueous buffer ( = 118 and 96 ms in 80% pbs : 20% dmso , respectively ; figure 2c ) . cis isomerization of mag2p and maga2p at high frequencies in aqueous media with a single irradiation source , which we have exploited to demonstrate the high photostability of these light - responsive ligands ( figure s5 in the supporting information ) . ( a ) absorption spectra in dmso of trans - mag , trans - mag2p , trans - maga2p , and the free naphthalene photo - harvesting antenna . ( b ) absorption spectra of trans - mag2p and trans - maga2p ( solid lines ) , and the photostationary states obtained upon photoexcitation of these compounds in dmso at exc = 473 nm ( dashed lines ) . ( c ) variation of the absorption at = 450 nm of trans cis mixtures of mag2p and maga2p in the dark at 25 c in 80% pbs:20% dmso . at these conditions , thermal cis trans back - isomerization takes place , thus restoring the initial concentration of the trans state of the ligands , which presents a larger extinction coefficient at abs = 450 nm . solid lines correspond to the experimental data , while dashed lines were obtained from monoexponential fits . although the incorporation of a photo - harvesting antenna negligibly affects the intrinsic photochemical behavior of the azobenzene group of maga2p with respect to mag2p , it provides ligand 3 with some additional optical properties . thus , trans - maga2p displays an extra band in the absorption spectrum ( max = 385 and 380 nm in dmso and 80% pbs:20% dmso , respectively ) , which arises from the naphthalene sensitizer ( figure 2a and figure s1 in the supporting information ) . the fluorescence emission of this group is however strongly quenched upon covalent attachment to the ligand , with a 20-fold decrease in fluorescence quantum yield measured in aqueous buffer ( antenna = 0.43 and trans - maga2p = 0.02 ; figure 3a ) . this indicates the occurrence of efficient ret processes from the photoexcited naphthalene antenna to the azo moiety of trans - maga2p , in agreement with the large frster radius calculated for this donor acceptor pair ( see figure s6 in the supporting information ) . cis isomerization should take place in this ligand , as demonstrated in figure 3b : 60% increase in trans cis photoconversion was determined for maga2p with respect to mag2p upon selective irradiation of the naphthalene antenna at exc= 355 nm . ( a ) fluorescence emission spectra in 80% pbs:20% dmso of trans - maga2p and the photo - harvesting antenna tethered to this ligand . the spectra are normalized relative to the excitation intensity and the absorption at the excitation wavelength ( exc = 355 nm ) . ( b ) trans cis photoconversion efficiency of trans - mag2p and trans - maga2p upon irradiation at = 355 nm in dmso , which allows nearly selective excitation of trans - maga2p sensitizer ( see figure 2a ) . we next tested mag , mag2p , and maga2p to photoswitch liglur in living cells using one- and two - photon stimulation . we expressed gluk2-l439c - egfp in hek293 cells and incubated them in mag , mag2p , or maga2p in order to allow the selective conjugation of the ptls to the cysteine introduced at position l439c of the receptor . for each compound , we recorded the corresponding photocurrents generated upon light - induced opening of liglur channels using whole - cell patch clamp ( see the supporting information ) . one - photon liglur currents were obtained when the receptor was conjugated with the new compounds ( figures 4 and 5a ) . the magnitude of the photocurrent response was not reduced after repeated stimulations , demonstrating the photostability of these compounds after protein conjugation ( figure 4b , c and figure s8 in the supporting information ) . figure 4 shows that for one - photon excitation , the wavelength dependence of current amplitude measured is different for each ptl . photocurrent amplitudes at different wavelengths were quantified from electrophysiological recordings obtained for the three compounds ( figure 4a c ) , and the corresponding one - photon action spectra were calculated ( figure 4d ) . introduction of the 4-amino substituent in the azo core allows the one - photon action spectra of mag2p and maga2p to red - shift 60 nm with respect to that of mag , as recently reported for a similar compound ( figure 4d and table s1 in the supporting information ) . an additional peak is observed for maga2p at = 360 nm , which lies very close to the absorbance band of the naphthalene moiety ( see figure 2a ) . thus , sensitization of the azobenzene photoisomerization by the antenna also occurs when the photoswitch is conjugated to liglur . in addition , the time course of the mag2p and maga2p one - photon currents ( blue and red traces in figure 5a ) confirms that fast spontaneous cis trans back - isomerization and channel closure takes place after the illumination is switched off , while it requires irradiation with green light for mag ( black trace in figure 5a ) . by fitting the one - photon current decays in the dark with monoexponential functions , the lifetimes of cis - mag2p and cis - maga2p tethered to liglur were determined to be 150 and 265 ms , respectively ( table s1 in the supporting information ) . these values are larger than those measured in solution ( see above ) , which suggests that the ligand - binding site interaction slows down the thermal cis this effect is enhanced for maga2p probably due to additional hydrophobic interactions and/or steric hindrance effects arising from the tethered naphthalene antenna . whole - cell voltage - clamp current recordings in hek293 cells expressing gluk2-l439c after conjugation to ( a ) mag , ( b ) mag2p , and ( c ) maga2p . current responses to one - photon light pulses of wavelengths ranging from 325 to 575 nm were quantified . ( note that in ( a ) resting = 500 nm induces liglur deactivation , and in ( b ) and ( c ) resting = 690 nm is not absorbed and allows thermal relaxation of these photoswitches . ) ( d ) normalized one - photon action spectra corresponding to mag ( black ) , mag2p ( blue ) , and maga2p ( red ) ( n = 2 cells , n = 38 cells , and n = 410 cells , respectively ) . before averaging over different cells , wavelength - dependent current amplitudes were normalized to the maximum photocurrent along the spectral range measured for each cell . errors are sem . ( a ) one- and ( b ) two - photon whole - cell voltage - clamp recordings on hek293 cells expressing liglur conjugated with mag ( black ) , mag2p ( blue ) , and maga2p ( red ) . bars indicate stimulation pulses applied to open ( one - photon pulses in violet and blue , two - photon pulses in gray ) and close liglur ( one - photon pulses in green ) . two - photon excitation conditions : mag ( = 820 nm , 10 scans of 0.4 s , 38 mw on sample ) , mag2p ( = 900 nm , 0.4 s scan , 30 mw on sample ) , and maga2p ( = 880 nm , 0.4 s scan , 42 mw on sample ) . using a custom - built multiphoton setup where a tightly focused fs laser is raster scanned over the cells of interest , all three ptls display robust and liglur - specific photocurrents in living cells that first demonstrate two - photon stimulation with nir light of a synthetic photoswitchable protein ( figure 5b and figures s9 and s10 in the supporting information ) . the amplitude of the responses follows the characteristic power dependence of two - photon absorption processes ( figure s11 in the supporting information ) and corresponds to 1020% of the photocurrent under one - photon excitation ( table s2 in the supporting information ) . in order to optimize the multiphoton stimulation conditions we characterized the two - photon action spectrum of each ptl ( figure 6a ) . the wavelength that yields maximal two - photon responses of mag is around 820 nm . repeated cell raster scans are required to get a saturating photocurrent from all available receptors ( black trace in figure 5b ) . then , the current remains stable without laser illumination until liglur is closed with 500 nm light via one - photon cis the mag2p two - photon action spectrum is red - shifted and yields maximum current amplitude around 900 nm . the reduced currents obtained from maga2p hindered the acquisition of a detailed action spectrum , but are sufficient to identify two spectral ranges allowing two - photon activation of liglur : the first can be found at 880 nm ( corresponding to the direct absorbance of azobenzene , as in mag2p ) , and the second is located around 740 nm and is consistent with the naphthalene - sensitized photoisomerization . ( a ) two - photon action spectra of liglur - mag ( black ) and liglur - mag2p ( blue ) and two - photon activation of liglur - maga2p ( red ) at selected wavelengths . photocurrent amplitudes were corrected for the different power densities used ( pd ) , averaged over all cells measured , and normalized to the spectral maximum . ( b ) absolute two - photon ( 2p ) responses at the optimal wavelength . for maga2p values are given for sensitized ( = 740 nm ) and direct ( = 880 nm ) azobenzene excitation . ( c ) ratio between the two- and one - photon responses ( 2p/1p ) . to compare between different liglur - tethers , two - photon responses were corrected for the distinct power densities and excitation times used and averaged over all cells measured . in all spectra here , remarkably , multiphoton currents mediated by mag2p and maga2p completely saturate after a few laser scans of the recorded cell ( blue and red traces in figure 5b ) . in addition , their rapid relaxation allows liglur to close immediately after the end of each stimulus , with time constants similar to those obtained with one - photon illumination ( table s2 in the supporting information ) , which enable fast , repeated activation of the receptor without requiring a second irradiation source for deactivation . thus , the novel compounds mag2p and maga2p enable single - wavelength , multiphoton gating of liglur . however , mag achieves higher two - photon current amplitudes than mag2p and maga2p in the long term ( figure 6b ) , because the thermal stability of its cis isomer allows building up a larger population of open - state channels upon repeated cell raster scans ( figure s12 in the supporting information ) . to compare the efficacy of liglur activation between ptls , we calculated the ratio between two - photon and one - photon maximal responses ( figure 6c ) . noticeably , mag2p and maga2p ( both via direct and sensitized azobenzene excitation ) display a higher ratio than mag , thereby demonstrating that the efficiency of multiphoton isomerization was enhanced by the design of the new photoswitches . after characterizing the two - photon stimulation of liglur , we pursued physiological applications that exploited the ability of this receptor to rapidly activate neurons and trigger calcium - regulated processes . the stimulation of individual neurons in micrometric volumes and millisecond time scales has been demonstrated using two - photon neurotransmitter uncaging and optogenetics . to complement this set of tools for investigating brain connectivity , we applied two - photon activation of liglur in neuronal and non - neuronal cells of the brain using the high photocurrents provided by mag and mag2p . we expressed gluk2-l439c - egfp in cultured hippocampal neurons , incubated them in mag2p and recorded neuronal activity using whole - cell patch clamp ( figures 7 ) . excitation of the soma with 900 nm light elicits inward currents in voltage - clamp experiments ( figure 7c ) . in current - clamp mode , these photocurrents triggered action potentials in two out of three tested neurons ( figure 7d ) . although several properties of liglur - mag2p must be improved in order to reliably photocontrol whole neurons and individual presynaptic terminals ( lifetime of the cis isomer , receptor expression level , and subcellular localization ) , these results indicate that it is possible to activate neurons using two - photon stimulation of synthetic photoswitchable proteins . ( a ) two - photon image ( = 1000 nm ) of a cultured liglur - mag2p hippocampal neuron filled with alexa fluor 594 . ( b ) voltage - clamp recording during one - photon stimulation ( blue bar ) . ( c , d ) two - photon raster scan ( gray bars ) of the same neuron during ( c ) voltage - clamp recording , which shows two - photon current with a transient current spike ( two - photon mean current amplitude : 21 3 pa , 19 2% of one - photon current , n = 3 ) , and ( d ) current - clamp recording ( resting potential = 45 mv ) . two - photon excitation conditions : = 900 nm , 0.25 s scan , and 24 mw on sample . in the same experimental conditions , no spikes were elicited by two - photon stimulation of liglur - mag , probably due to the slow photoresponses shown in figure 5b . however , the large , step - function photocurrents provided by mag and the calcium permeability of gluk2 make liglur - mag more attractive to trigger calcium - regulated processes including astrocyte activation ( see also figure s7 in the supporting information ) . in cultured astrocytes expressing liglur - mag ( figures 8) , two - photon excitation at 820 nm triggered bistable currents ( figure 8c ) . interestingly , whole - cell photocurrents can also be measured during the stimulation of a subcellular region ( figure 8d ) or a spot ( figures 8e g ) , and these responses are reversible by illuminating the cell at 500 nm ( figures 8c , d , h ) . in order to verify whether such stimuli were enough to activate an intracellular calcium response in the astrocyte , we performed two - photon calcium imaging together with two - photon stimulation of astrocytes expressing liglur - mag . when we stimulated an expressing astrocyte , liglur activation caused a calcium increase that propagated to neighboring cells , generating a calcium wave that expanded to astrocytes throughout the field of view ( figure 8i k and movie s1 in the supporting information ) . this effect , which is not observed when locally stimulating non - expressing astrocytes ( figure s13 and movie s2 in the supporting information ) , demonstrates that two - photon liglur activation can be used to manipulate cytosolic calcium levels in cultured astrocytes . ( a ) two - photon image ( = 1000 nm ) of a cultured liglur - mag astrocyte filled with alexa fluor 594 . red and blue squares define the whole cell and subcellular raster scan areas , respectively . ( b ) whole - cell voltage - clamp recording of the astrocyte during one - photon stimulation ( liglur opening , purple bar ; liglur closing , green bar ) . ( c h ) two - photon currents measured on the same astrocyte ( liglur opening , gray bar ; one - photon liglur closing , green bar ) : ( c ) cell scan stimulation at = 820 nm , 10 scans of 0.7 s and 37 mw on sample ( mean current amplitude : 60 20 pa , 30 10% of one - photon current , n = 2 ) ; ( d ) subcellular scan stimulation at = 820 nm , 10 scans of 0.25 s and of 68 mw on sample ( mean current amplitude : 17 9 pa , n = 2 ) ; ( e g ) single - point stimulation ( gray bar ) at = 820 nm , 50 ms and 68 mw on sample ( points 1 and 2 are on the cytoplasm , and point 3 is out of the cell as control ) ; ( h ) liglur closing at the end of the stimulation protocol ( green bar ) . ( i k ) two - photon calcium imaging of cultured astrocytes loaded with fura-2 ( in purple , = 800 nm ) overlapped with an image of gfp fluorescence ( in green , = 900 nm ) to identify astrocytes expressing liglur - mag . ( j ) images at 1.55 , 4.65 , and 7.75 s after targeting the astrocyte to which the arrow points in ( i ) with two - photon stimulation ( 20 targets , 16-pixel diameter , 10 ms per point at = 800 nm , 60 mw on sample ) . we have demonstrated the two - photon activation of azobenzene - based photoswitches in living cells expressing the light - gated receptor liglur . although a symmetrically substituted azobenzene was reported to photoisomerize under continuous - wave nir excitation , in general these chromophores present low two - photon absorption cross sections . however , synthetic ptls like mag offer great flexibility to adjust their photochemical properties without altering protein function . we have rationally designed mag derivatives with visible absorption , fast thermal relaxation , and high two - photon isomerization efficacy based on push pull substitutions and sensitization of the azobenzene photoisomerization . these modifications and the reported multiphoton excitation conditions should be directly applicable to all azobenzene - based bioactive ligands , including intracellular photoswitches known to penetrate into cells directly or through specific ion channels , and hyperpolarizing step - function photoswitchable channels like spark or ligaba . our findings thus enable the use of synthetic photoswitches to manipulate extra- and intracellular biochemical processes with the spatiotemporal precision provided by two - photon stimulation . a detailed description of the synthesis of target photoswitchable tethered ligands is given in the supporting information . cis isomerization of mag2p and maga2p in solution was investigated by ( i ) h nmr for the elucidation of the photostationary - state mixtures ; ( ii ) steady - state uv vis absorption spectroscopy for trans trans thermal back - isomerization processes ; and ( iii ) transient absorption spectroscopy for fast cis tsa201 cell line , cultured hippocampal neurons , and astrocytes plated on glass coverslips were transfected with gluk2-l439c - egfp . prior to each experiment , they were incubated with one of the ptls to allow the chemical conjugation with the receptor channel and light sensitization . a second incubation with concavalin a was done in order to inhibit desensitization of the glutamate receptor . for two - photon stimulation , voltage - clamp and current - clamp recordings under whole - cell configuration were done with an axon multiclamp 700b amplifier ( molecular devices ) , and data were acquired at 10 khz . borosilicate glass pipettes were pulled with a typical resistance of 46 m for hek293 tsa201 cells and neurons or 78 m for astrocytes . bath solution was composed of 140 mm nacl , 1 mm mgcl , 2.5 mm kcl , 10 mm hepes , 2.5 mm cacl2 , and 1020 mm glucose to fix osmolarity to 310 mosmkg , ph 7.42 adjusted with naoh . for hek293 tsa201 cell line , pipet solution contained 120 mm cesium methanosulfonate , 10 mm tea - cl , 5 mm mgcl2 , 3 mm na2atp , 1 mm na2gtp , 20 mm hepes , and 0.5 mm egta , 290 mosmkg , ph 7.2 adjusted with csoh . for neurons and astrocytes it consisted of 130 mm potassium gluconate , 5 mm nacl , 10 mm hepes , 0.1 mm egta , 2 mm mgso4 , 4 mm mg - atp , 0.4 mm naxgtp , 7 mm na2-phosphocreatine , 2 mm pyruvic acid , and 0.1 mm alexa fluor 594 ( molecular probes ) , ph 7.3 adjusted with koh . all two - photon experiments were performed in the yuste laboratory with a custom - made two - photon laser scanning microscope based on a modified olympus bx50wi microscope with a ti : sapphire laser as light source ( coherent chameleon ultra ii , 140 fs pulses , 80-mhz repetition rate ) . laser power was modulated by a pockels cell ( conoptics ) and adjusted for each wavelength to be close to 40 mw on sample for mag2p and maga2p and 50 mw on sample for mag , if not specified otherwise . in experiments with mag , we used a 20x/0.5-na objective ( olympus ) , and with the red - shifted compounds , we used a 20x/0.95-na objective ( olympus ) in figures 5 and 6 and a 40x/0.8-na objective ( olympus ) in figures 7 and 8 . for two - photon stimulation we defined a roi and applied a unidirectional raster scan using fluoview software , or we performed point stimulations with custom - written labview software . first , 50 l of dmso was added to a 50 g aliquot of fura-2-am ( life technologies ) . next , 0.2 l of this solution and 0.2 l of pluronic acid ( 20% in dmso ) in 2 ml supplemental media were added to the culture dish and incubated at 37 c for 30 min , before washing and liglur conjugation with mag and concanavalin a treatment . we raster - scanned fura-2 ( 100 frames , 1.55 s / frame ) at 800 nm and 40 mw on sample with a 20x/0.5-na objective for recording the activity of astrocytes and stimulated single nonexpressing or gfp - positive astrocytes using custom written software , with a protocol of 20 stimulation targets on the cell with a16-pixel diameter , corresponding to approximately 11 m diameter . amplitudes of liglur currents were analyzed using igorpro ( wavemetrics ) , and closing time constants of liglur were determined with a custom - made software using labview . in the two - photon action spectrum of each compound , every set of data from one cell was normalized to the action spectrum integral from a chosen representative before cell average . calcium imaging of astrocytes was analyzed using custom written software ( caltracer ) and imagej .

synthetic photochromic compounds can be designed to control a variety of proteins and their biochemical functions in living cells , but the high spatiotemporal precision and tissue penetration of two - photon stimulation have never been investigated in these molecules . here we demonstrate two - photon excitation of azobenzene - based protein switches and versatile strategies to enhance their photochemical responses . this enables new applications to control the activation of neurons and astrocytes with cellular and subcellular resolution .

for patients with non - small cell lung cancer ( nsclc ) , physicians have long been puzzled by the task of identifying candidates who may suffer major postoperative cardiopulmonary complications if they were to undergo lung resection surgery . among the few available tests , the cardiopulmonary exercise test ( cpet ) is a standard tool to assess the surgical risk of patients with nsclc [ 13 ] . specifically , maximal oxygen uptake ( vo2max ) during symptom - limited exercise was found to be a more reliable than other parameters of spirometry , such as forced expiratory volume in 1 second ( fev1 ) or the ratio of fev1 against forced vital capacity ( fvc ) , to predict postoperative morbidity and mortality among nsclc patients . as an alternative to cpet , excise - based diagnostic tests , such as stair - climbing test ( sct ) , have been reported to be a safe , economical way to assess risk - associated factors of patients [ 47 ] . interestingly , for patients with nsclc , since sct requires patients to ride cycles , it is more stressful than cardiopulmonary exercise and can be considered as valid tool to detect any abnormalities in the oxygen transport system . thus , it is plausible that sct may be helpful in predicting cardiopulmonary complications after lung resection . however , definitive evidence is lacking to determine whether sct is superior to cpet to predict postoperative cardiopulmonary complications for patients with nsclc . in this study , we used statistical methods to assess whether the key parameters of sct , including exercise oxygen desaturation ( eod ) , may be independent and proficient prognostic factors predicting postoperative cardiopulmonary complications among patients with nsclc who received various extents of lung resection . in this study , all protocols were reviewed and approved by the ethics committee at the cancer hospital ( institute ) , chinese academy of medical sciences in beijing , china . a total of 413 patients with non - small cell lung cancer ( nsclc ) performer the stair - climbing test between january 2010 and july 2015 . we excluded 74 patients due to lack of complete clinical data , 53 patients were excluded because they failed to correctly perform the pulmonary function test , 12 patients were excluded as they received lung resection and esophagectomy at the same time , and 103 patients were excluded because they did not receive surgery afterwards . thus , 171 eligible nsclc patients were enrolled in this study , including 41 females and 130 males . none of the patients had received any antineoplastic therapy such as radiotherapy and/or chemotherapy in the 6 months prior to surgery . all patients in the study participated in the symptom - limited stair - climbing test preoperatively by climbing 5 floors from the 1 floor to the 6 floor in our surgical building . there are 20 steps and 1 intermediate landing between each floor , and each step is 0.153 m in height . the patients were instructed before the stair - climbing test and encouraged to climb the stairs as fast as they could without stopping to rest until they reached the highest floor possible . the maximum height ( to the 6 floor ) was 18.4 m. the test was complete when patients reached either the 6 floor or the patients presented an apparent symptom such as severe dyspnea with oxygen desaturation , severe leg fatigue , or chest pain , which indicated exhaustion and they could not climb any more . all patients were accompanied by 2 physicians all the time during the test , who would monitor their symptoms and recorded test results simultaneously . heart rate and capillary oxygen saturation were recorded before the test , at each floor during the test , and after resting for 15 min after completing the test , using a portable pulse - oxy meter with finger probe , called the autobox ( newtech , shenzhen , china ) . the resting oxygen saturation ( osat ) and lowest osat during the test were recorded and used to calculate the desaturation level . for air leak management , single thoracic drainage in the chest was induced into the 2 intercostal space , midclavicular line ( pneumonectomy ) , and the 7 intercostal axillary line ( wedge resection and lobectomy ) . with the exception of pneumonectomy , all the drainage tubes were put into the chest with the front - most end reaching the top of the chest . at the end of the operation , residual lung tissues were carefully examined , and then air - leak tissue was stitched by prolene thread until there was no air leak from the residual lung tissue . spirometry , flow - volume curves , and absolute lung volumes were measured using a pulmonary function test apparatus . the diffusion capacity for carbon monoxide ( dlco ) was determined using a standard single - breath technique . best results were selected in 2 acceptable measuring results at intervals of at least 510 min between 2 measurements . all patients underwent surgical resection for lung cancer by either thoracotomy ( n=129 ) or video - assisted thoracoscopic surgery ( vats , n=42 ) . procedures included pneumonectomy in 22 cases , lobectomy in 122 cases , and pulmonary wedge resection in 27 cases . postoperative treatment was focused on early mobilization , chest physiotherapy and physical rehabilitation , pain control , and antibiotic and anti - thrombotic prophylaxis . postoperative complications and deaths were defined as those occurring 030 days postoperatively , according to the guideline reported by bolliger et al . , smith et al . , and brutsche et al . . statistical analyses were performed using sas software ( sas institute , cary , nc ) . categorical variables of clinical characteristics are shown as percentages and were compared using the chi - square test . continuous variables are presented as means standard deviations and compared by use of the t test . the utility of the complication as a predictive marker for drainage tube length of postoperative stay and hospital stay was investigated using kaplan - meier analysis . logistic regression analysis was used to determine the association of preoperative factors with postoperative complications . factors with a significant difference in univariate analysis ( p value < 0.05 ) were measured again by multivariate analysis to determine their independence . a p value in this study , all protocols were reviewed and approved by the ethics committee at the cancer hospital ( institute ) , chinese academy of medical sciences in beijing , china . a total of 413 patients with non - small cell lung cancer ( nsclc ) performer the stair - climbing test between january 2010 and july 2015 . we excluded 74 patients due to lack of complete clinical data , 53 patients were excluded because they failed to correctly perform the pulmonary function test , 12 patients were excluded as they received lung resection and esophagectomy at the same time , and 103 patients were excluded because they did not receive surgery afterwards . thus , 171 eligible nsclc patients were enrolled in this study , including 41 females and 130 males . none of the patients had received any antineoplastic therapy such as radiotherapy and/or chemotherapy in the 6 months prior to surgery . all patients in the study participated in the symptom - limited stair - climbing test preoperatively by climbing 5 floors from the 1 floor to the 6 floor in our surgical building . there are 20 steps and 1 intermediate landing between each floor , and each step is 0.153 m in height . the patients were instructed before the stair - climbing test and encouraged to climb the stairs as fast as they could without stopping to rest until they reached the highest floor possible . the maximum height ( to the 6 floor ) was 18.4 m. the test was complete when patients reached either the 6 floor or the patients presented an apparent symptom such as severe dyspnea with oxygen desaturation , severe leg fatigue , or chest pain , which indicated exhaustion and they could not climb any more . all patients were accompanied by 2 physicians all the time during the test , who would monitor their symptoms and recorded test results simultaneously . heart rate and capillary oxygen saturation were recorded before the test , at each floor during the test , and after resting for 15 min after completing the test , using a portable pulse - oxy meter with finger probe , called the autobox ( newtech , shenzhen , china ) . the resting oxygen saturation ( osat ) and lowest osat during the test were recorded and used to calculate the desaturation level . for air leak management , single thoracic drainage in the chest was induced into the 2 intercostal space , midclavicular line ( pneumonectomy ) , and the 7 intercostal axillary line ( wedge resection and lobectomy ) . with the exception of pneumonectomy , all the drainage tubes were put into the chest with the front - most end reaching the top of the chest . at the end of the operation , residual lung tissues were carefully examined , and then air - leak tissue was stitched by prolene thread until there was no air leak from the residual lung tissue . spirometry , flow - volume curves , and absolute lung volumes were measured using a pulmonary function test apparatus . the diffusion capacity for carbon monoxide ( dlco ) was determined using a standard single - breath technique . best results were selected in 2 acceptable measuring results at intervals of at least 510 min between 2 measurements . all patients underwent surgical resection for lung cancer by either thoracotomy ( n=129 ) or video - assisted thoracoscopic surgery ( vats , n=42 ) . procedures included pneumonectomy in 22 cases , lobectomy in 122 cases , and pulmonary wedge resection in 27 cases . postoperative treatment was focused on early mobilization , chest physiotherapy and physical rehabilitation , pain control , and antibiotic and anti - thrombotic prophylaxis . postoperative complications and deaths were defined as those occurring 030 days postoperatively , according to the guideline reported by bolliger et al . , statistical analyses were performed using sas software ( sas institute , cary , nc ) . categorical variables of clinical characteristics are shown as percentages and were compared using the chi - square test . continuous variables are presented as means standard deviations and compared by use of the t test . the utility of the complication as a predictive marker for drainage tube length of postoperative stay and hospital stay was investigated using kaplan - meier analysis . logistic regression analysis was used to determine the association of preoperative factors with postoperative complications . factors with a significant difference in univariate analysis ( p value < 0.05 ) were measured again by multivariate analysis to determine their independence . a total of 171 non - small cell lung cancer patients were included in the study . one of these deaths was a 75-year - old male and another was a 59-year - old female , whose exercise oxygen desaturation ( eod ) were both greater than 5 , and both died from respiratory failure 1 and 80.5 , respectively , days postoperatively despite being supported by mechanical ventilation . the third patient was a 64-year - old male who died from postoperative bleeding in the chest after a lobectomy . there was no air leak from the chest of patients for more than 2 days before closing the chest , and no patient had empyema . baseline information and the comparison between the complication group and the complication - free group are shown in table 2 . there were no significant differences in sex ratio , bmi , smoking , lung resection , spirometry measurements ( fev1 , mvv% , dlco and dlco% ) , or stair - climbing test results ( pre - climb sao2 , climbing - end sao2 , and climbing - end heart rate ) between the groups . while comparing those parameters between the complication group and the complication - free group , there were significant differences in age , fev1% , mvv , height of climbing , eod , and heart rate change between the 2 groups . finally , logistic regression analysis showed that eod and heart rate change were independent predictors of complications ( table 3 ) . a model for predicting the probability of postoperative complications based on logistic regression was hypothesized as : probability = e/(1+e ) , =1.136 + ( 0.114eod ) + ( 0.040heart rate change ) , in which e represented the base of natural logarithms and the was the regression coefficient in the logistic regression . the accuracy of the model was evaluated by use of the roc curve , which showed that the area under the roc curve ( auc ) was 0.750 ( 95% ci : 0.6680.831 ) ( figure 1 ) . if the cutoff probability value was larger than 0.193 , the patient was considered to have high risk of suffering from postoperative cardiopulmonary complications , and vice versa . patients without a postoperative cardiopulmonary complication had a median length of drainage time of 4.3 days ( 95% ci , 3.44.5 ) and a median length of hospital stay of 9.4 days ( 95% ci , 8.99.8 ) , as compared to 5.9 days ( 95% ci , 5.36.4 ) ( figure 2 , p<0.001 ) , and 13.1 days ( 95% ci , 10.915.3 ) ( figure 3 , p<0.001 ) for patients with postoperative complications . complications such as chronic obstructive pulmonary disease ( copd ) and coronary artery disease are very common among those patients , with high risk of surgical morbidity and mortality . thus , it is critical to perform preoperative assessment on cardiopulmonary function to predict the risk of postoperative complications and death . use of vital capacity for the prediction of surgical risk was first reported as early as 1955 . since then , lung functional testing has been used to predict the risk of postoperative complications and even mortality . the 2 most commonly used parameters of conventional pulmonary function test are fev1 and dlco , which were confirmed by univariate analysis in our study . however , multivariate analysis showed that fev1% was not an independent predictor of postoperative complications for patients with nsclc . before this study , poor exercise capacity has long been used as the major predictor of postoperative cardiopulmonary complications after lung resection in patients with nsclc . however , nsclc patients with poor exercise capacity not only have high risk of postoperative cardiopulmonary complications , but also have longer hospital stay . in the present study , our results showed that eod and heart rate change were independent predictors of postoperative cardiopulmonary complications for patients with nsclc , confirming the predictions from other studies . brunelli reported that complication rates in patients with and without eod > 4% and < 4% were 36% and 22% , respectively . in addition , a previous study by our team found the rate of postoperative cardiopulmonary complications was significantly higher in patients with eod 5% and heart rate change < 55 beats / min than that in the group with eod < 5% and heart rate change 55 beats / min . therefore , in the present study , we successfully established a model to predict the risk of postoperative cardiopulmonary complication using scts parameters of eod and heart rate change . it is worth noting that , in the present study , the patients without postoperative cardiopulmonary complication had a shorter hospital stay than those with postoperative cardiopulmonary complications . also , the patients without postoperative cardiopulmonary complication had a shorter drainage time than those with postoperative cardiopulmonary complications . the possible explanation is that a patient who has postoperative cardiopulmonary complications creates more pleural effusion than one does not , further confirming that sct may be an effective method to predict postoperative cardiopulmonary complications in nsclc patients . however , well - established formal cardiopulmonary exercise testing ( cpet ) is still be recommended for precise assessment of these patients with high eod or low heart beat change during the stair - climbing test . we provided definitive evidence that eod and heart rate change in sct are independent predictors for postoperative cardiopulmonary complications in nsclc patients . a follow - up study with a much larger patient pool however , based on the information of our study , it is highly recommended for all lung resection candidates with poor conventional function test results to perform a stair - climbing test .

backgroundthere is currently no reliable method to predict major postoperative cardiopulmonary complications for patients with non - small cell lung cancer ( nsclc ) . in this study , we hypothesized that exercise oxygen desaturation ( eod ) and heart rate change results in a stair - climbing test ( sct ) would predict postoperative cardiopulmonary complications for patients with nsclc.material/methodswe examined 171 patients ( 41 females and 130 males ) with nsclc by preoperative sct from january 2010 to july 2015 . among them , 27 underwent wedge resection , 122 underwent lobectomy , and 22 underwent pneumonectomy . the correlation between postoperative cardiopulmonary complications and parameters of sct and pulmonary function test ( pft ) parameters were analyzed retrospectively.resultsthe overall 30-day postoperative morbidity of the patients was 46/171 ( 26.9% ) , with death occurring in 3/171(1.8% ) . the age , fev1% , mvv , height of climbing , eod , and heart rate change were found to be significantly different between the group with postoperative cardiopulmonary complications and those without . binary logistic regression analysis showed that eod and heart rate change were independently correlated with postoperative cardiopulmonary complications . in addition , a model predicting the probability of postoperative cardiopulmonary complication based on logistic regression for multivariable analysis was used to confirm our findings.conclusionsa symptom - limited sct with oxygen saturation monitoring is a safe , simple , and low - cost method to evaluate cardiopulmonary function preoperatively .

a positive family history of prostate cancer ( pca ) is an established risk factor for pca . first - degree relatives of affected men have a 23 fold increased risk of pca . when 3 or more first - degree relatives are affected ( or at least 2 first - degree relatives before the age of 55 years ) , the family is considered a hereditary prostate cancer ( hpc ) family according to the so - called johns hopkins or carter criteria . yet , only a few very rare high - penetrance gene mutations have been identified to cause hpc . in recent years , genome - wide association studies have added approximately 40 low - penetrance genetic polymorphisms that are associated with an increased risk of pca [ 4 , 5 ] . several polymorphisms have also been identified that are associated with serum prostate - specific antigen ( psa ) , the most commonly used marker for early detection of pca [ 6 , 7 ] . an ongoing matter of debate is whether psa testing should be used for population - wide screening . in a population - based setting , the european randomized study of screening for prostate cancer ( erspc ) showed a decrease in pca mortality of 31% in the screening arm after correction for non - attendance and contamination . by contrast , the prostate , lung , colorectal and ovarian cancer screening trial ( plco ) , found no effect of psa screening on mortality , but suffered from methodological problems which severely hamper interpretation of the results . previous studies into the effectiveness of psa sceening in men with an increased risk due to family history have yielded largely inconsistent results . these range from a marked benefit for men in high - risk pca families ( particularly for families with early onset pcas ) to a decreased risk of pca for non - affected men in hpc families [ 13 , 14 ] . the increasing use of psa testing in the general population has also had an important influence on men with a family history of pca . men with a positive family history are relatively more active in pursuing psa testing than men in the general population . this has led to an increased detection of mainly of small localized tumors . to guide the public and physicians in translating the results of the erspc and plco into clinical practice , the dutch association of urology ( nvu ) and the dutch college of general practitioners ( nhg ) concurrently released a policy statement in march 2009 . this statement referred men to a website ( http://www.prostaatwijzer.nl/ ) with information about pca and psa testing and advised them to consult their gp for further counseling . it did , however , not discuss dealing with a family history of pca or hpc . to date , in absence of official internationally accepted guidelines , the advice is to attempt to distinguish genetic predisposition - based families with multiple pca cases from ascertainment - based multiple - case families and offer pca screening only to the former . this study assessed the knowledge of dutch urologists , general practitioners ( gps ) and clinical geneticists ( cgs ) about hpc and pca as a phenotype in hereditary syndromes . furthermore , their post - erspc attitude towards pca testing and the role of family history in clinical decision - making about pca testing were assessed . to our knowledge , this is the first study to investigate this in different professions that counsel men about pca testing . after publication of the results of the erspc and plco and the statements of the nvu and nhg , an online survey was developed . this survey , targeted at dutch urologists , gps and cgs , contained questions about hpc and assessed the participants general attitude towards pca testing . in addition to this , it inquired into the role that family history played in the physician s daily clinical practice regarding pca testing . the survey also included case descriptions of a man requesting to be tested for pca . this fictitious man presented at different ages , in absence of family history and physical complaints . the survey could be completed anonymously . in january 2010 , all dutch urologists ( n = 351 ) , clinical oncogeneticists ( n = 32 ) , cgs in training ( n = 50 ) and genetic counselors ( n = 68 ) were invited by e - mail from their respective professional associations . gps in the region of the comprehensive cancer centre east ( ccce ) who were enlisted to receive the ccce s e - newsletter ( n = 300 ) were invited by e - mail . for statistical analysis because of small numbers , clinical oncogeneticists , cgs in training and genetic counselors were pooled into one stratum ( cgs ) . these were submitted by 109 urologists ( 31% ) , 69 gps ( 23% ) and 46 cgs ( 31% ) . one pediatric urologist and two cgs were excluded from analyses because they never counseled men for pca testing . occasionally , participants did not complete all questions , causing small differences in the subtotals for different questions . of the urologists , 66% ( 71/107 ) counseled men about pca testing at least once a week and 93% ( 100/107 ) did this at least once a month . in comparison , 85% of the gps ( 59/69 ) did this at least once a month , as opposed to only 2% ( 1/44 ) of the cgs . by contrast , the cgs had the most accurate knowledge of the hpc criteria : 72% ( 31/43 ) correctly selected the minimum of three affected first - degree relatives for the criterion that is most frequently fulfilled ( table 1 ) . in comparison , only 36% ( 38/105 ) of the urologists and 18% ( 12/66 ) of the gps correctly selected this criterion ( p < 0.001 ) . cgs were also best informed about the number of affected first - degree relatives with a pca diagnosis before 55 years of age to meet the definition of hpc , although the differences between the groups were smaller . the third definition of hpc , i.e. , three consecutive generations with pca , was known to only a few participants.table 1responses to the question what is the minimum number of relatives with prostate cancer to meet the carter criteria for hereditary prostate cancer ( hpc)?urologistsgpscgsnumber of affected first - degree relatives ( all ages ) two35 ( 33%)9 ( 14%)1 ( 2% ) three38 ( 36%)12 ( 18%)31 ( 72% ) four1 ( 1%)2 ( 3%)2 ( 5% ) > four3 ( 3%)0 ( 0%)1 ( 2% ) do nt know15 ( 14%)39 ( 59%)7 ( 16% ) not a criterion13 ( 12%)4 ( 6%)1 ( 2% ) total1056643number of affected first - degree relatives ( diagnosis < 55 years of age ) two72 ( 68%)33 ( 48%)34 ( 76% ) three6 ( 6%)0 ( 0%)3 ( 7% ) four1 ( 1%)1 ( 1%)0 ( 0% ) > four5 ( 5%)2 ( 3%)0 ( 0% ) do nt know12 ( 11%)32 ( 46%)7 ( 16% ) not a criterion10 ( 9%)1 ( 1%)1 ( 2% ) total1066945number of consecutive generaties with prostate cancer two10 ( 10%)10 ( 15%)9 ( 21% ) three15 ( 15%)6 ( 9%)5 ( 11% ) four3 ( 3%)1 ( 2%)1 ( 2% ) > four2 ( 2%)1 ( 2%)0 ( 0% ) do nt know25 ( 25%)39 ( 58%)10 ( 23% ) not a criterion44 ( 44%)10 ( 15%)19 ( 43% ) total996744p 0.001 for differences between the physician groupscorrect answers are italicized responses to the question what is the minimum number of relatives with prostate cancer to meet the carter criteria for hereditary prostate cancer ( hpc ) ? p 0.001 for differences between the physician groups correct answers are italicized almost all cgs ( 41/42 , 98% ) listed at least one inherited trait with pca as part of the phenotype , compared to only 24% ( 25/103 ) of the urologists and 9% ( 6/66 ) of the gps . the most frequently mentioned traits were the brca2 gene mutation ( n = 60 ) , the brca1 gene mutation ( n = 40 ) and lynch syndrome ( n = 10 ) . the rare hpc1 , hpcx , msr1 , rnasel and hpc2/elac2 mutations were sporadically mentioned . urologists had the least reservations towards pca testing in a man with no physical complaints and no family history of pca : 46% ( 32/69 ) of the gps and 49% ( 22/45 ) of the cgs preferred to refrain from testing unless there were strong reasons to test ( table 2 ) , as compared to 31% ( 33/108 ) of the urologists . for a man presenting at 55 and 75 years of age , cgs were more inclined not to test for pca . at 45 years of age , more physicians in all groups would not test for pca.table 2responses to the question would you test this man for pca ? regarding a man with no physical complaints / no family history of pca , requesting to be tested for pcaage at presentationtest for prostate cancerurologistsgpscgsgeneral attitudewill test , unless 35 ( 32%)13 ( 19%)3 ( 7%)will not test , unless 33 ( 31%)32 ( 46%)22 ( 49%)leave choice to patient37 ( 34%)22 ( 32%)9 ( 20%)other*3 ( 3%)2 ( 3%)11 ( 25%)45 years of ageyes20 ( 19%)5 ( 7%)1 ( 2%)no12 ( 11%)18 ( 26%)28 ( 64%)first discuss pros and cons of prostate cancer testing75 ( 70%)46 ( 67%)15 ( 34%)55 years of ageyes29 ( 27%)16 ( 23%)1 ( 2%)no0 ( 0%)1 ( 1%)16 ( 36%)first discuss pros and cons of prostate cancer testing79 ( 73%)52 ( 75%)27 ( 61%)75 years of ageyes21 ( 19%)10 ( 15%)9 ( 21%)no8 ( 7%)3 ( 4%)14 ( 33%)first discuss pros and cons of prostate cancer testing79 ( 73%)56 ( 81%)20 ( 47% ) * answers under other : most often ( 8/11 ) cgs indicated not to perform this kind of testing themselves , but would refer the man to their gpp < 0.001 for differences between the physician groupsno significant difference between urologists and gps ; p = 0.40no significant difference between urologists and gps ; p = 0.45 responses to the question would you test this man for pca ? regarding a man with no physical complaints / no family history of pca , requesting to be tested for pca * answers under other : most often ( 8/11 ) cgs indicated not to perform this kind of testing themselves , but would refer the man to their gp p < 0.001 for differences between the physician groups no significant difference between urologists and gps ; p = 0.40 no significant difference between urologists and gps ; p = 0.45 age played a role when considering pca testing . of the urologists , 70% reported to use age limits , with 45 years as the mean and median lower age limit . this lower age limit was higher for gps ( 60% reported age limits ) and cgs ( 30% reported age limits ) , with 50 years of age being the median lower age limit . the median maximum age limit was 80 years ( mean 77 ) for urologists and gps , compared to 75 years ( mean 74 ) for cgs . cgs always took family history into consideration when deciding whether or not to test for pca . by contrast , 3540% of urologists and gps answered that family history would not influence the decision whether or not to test for pca ( table 3 ) . this did not vary between physicians with different general attitudes towards psa testing ( p = 0.47 for the urologists and p = 0.78 for the gps ) , as was assessed in a previous question.table 3responses to the question ( a ) does family history play a role in the decision whether or not to test a man for pca ? and the follow - up question ( b ) how extensively do you inquire about the family history?urologistsgpscgsa . does family history play a role ? yes67 ( 62%)44 ( 65%)40 ( 98% ) no41 ( 38%)24 ( 35%)1 ( 2% ) total1086841b only pca29 ( 43%)16 ( 37%)0 ( 0% ) pca and other malignancies38 ( 57%)27 ( 63%)40 ( 100% ) total674340 responses to the question ( a ) does family history play a role in the decision whether or not to test a man for pca ? and the follow - up question ( b ) how extensively do you inquire about the family history ? a majority of the urologists ( 76% ) knew the erspc and plco results , compared to only 14 and 8% of cgs and gps , respectively . ninety - two percent ( 75/82 ) of the urologists who knew the studies found the erspc results more valuable . the statements of nvu and nhg , advising men to visit the website and consult the gp if further counseling was needed , were better known than the results of the trials : 85% ( 92/108 ) of the urologists and 59% ( 41/69 ) of the gps was familiar with the statements . of them , 12% ( 11/91 ) of the urologists and 24% ( 10/41 ) of the gps did not agree with the statements . a positive family history of pca is an important risk factor for pca and the balance between pros and cons of psa testing may be different in men with affected relatives . only one in three urologists and one in five gps is familiar with the criteria for hpc . the brca1 gene mutation was frequently selected as an inherited trait with pca as part of the phenotype . the evidence for an increased risk of pca due to a brca1 gene mutation is quite weak , though . by contrast , for brca2 gene mutations ( selected by 14 urologists and all 42 cgs ) there is fairly solid evidence of familial clustering of aggressive pca [ 18 , 19 ] . recently , an elevated risk of pca for carriers of a mismatch - repair gene mutation was indeed found . this has , however , not been confirmed in other studies , so it remains unclear whether the physicians who selected lynch syndrome are correct . it should be noted that urologists and gps hardly ever counsel patients with an elevated pca risk based on these inherited traits . in general , urologists reported the least reservations towards pca testing and would test at a younger age than gps and cgs . however , the majority of urologists and gps stated to first discuss the risks and benefits of psa testing and only test if a man would still want to be tested . so , even though 41% of gps was not familiar with the statements regarding pca testing and 92% of them did not know the erspc and plco results , they adhered just as well to the guidelines as the urologists . participants who disagreed with the nvu / nhg statements , mostly indicated that the statements lacked attention for patients preferences . in contrast with the urologists and gps , cgs would more often not test for pca . this might be explained by the fact that the cgs mainly have an advisory role and refer their patients to a gp or urologist for pca testing . cgs hardly ever counsel men about pca testing , as there is no frequently occurring genetic defect known to cause pca . this may change , however , when more data become available about the risk of pca among brca2 carriers . one of the most striking observations might be that more than one in three urologists and gps would not take family history into account when deciding whether or not to test a man for pca . intuitively , one would think that men with a positive family history , and thus a higher a priori risk of pca , would benefit more from psa screening . arguing against this is that hpc cases do not seem to differ from sporadic cases with respect to gleason scores and pca - specific survival [ 21 , 22 ] . even more so , screening programs amongst non - affected men in hpc families have shown that the chance of finding pca in non - affected men in hpc families is low . although we did not address this in our study , when a man requesting pca testing does have , e.g. , an affected brother , he will very likely be tested , not in the least for reasons of anxiety management . however , whether this is beneficial , is doubtful . to better guide physicians in this matter , an addendum to pca guidelines should be developed in a multidisciplinary collaborative effort , describing how to deal with pca testing in case of a positive family history and hpc . the conclusion from a previous study to assess the extent and nature of the family history ( predisposition - based vs. ascertainment - based ) might well serve as a starting point for such a guideline . in addition to this , the use of decision aids , e.g. , the swop - pri should be promoted , as they already include the effect of family history in the risk estimates . the results of this study should be interpreted with some caution . although the responder groups were reasonably large in absolute numbers , the response rate was only 31% at best ( for urologists and cgs ) . hence , it is difficult to extrapolate the results to all dutch physicians providing pca counseling . even more so , if physicians with more interest in this topic completed the survey more often , the results regarding knowledge about hpc and adherence to guidelines might be overoptimistic . on the other hand , intuitively physicians who take care of most of the counseling are most eager to complete the survey . it is also important to bear in mind that the results may not easily be extrapolated to other countries as they may be influenced by the health care system . in the netherlands , e.g. , men can not visit a urologist without a referral from their gp . in conclusion , however , these guidelines do not include family history and many physicians indicated not to consider family history . hence , pca counseling might not be optimal for men with a positive family history . we propose that additional guidelines on this topic are developed in a multidisciplinary effort to optimize counseling .

a family history of prostate cancer ( pca ) is an established risk factor for pca . in case of a positive family history , the balance between positive and adverse effects of prostate - specific antigen ( psa ) testing might be different from the general population , for which the european randomized study of screening for prostate cancer ( erspc ) showed a beneficial effect on mortality . this , however , went at the cost of considerable overtreatment . this study assessed dutch physicians knowledge of heredity and pca and their post - erspc attitude towards pca testing , including consideration of family history . in january 2010 , all dutch urologists and clinical geneticists ( cgs ) and 300 general practitioners ( gps ) were invited by email to complete an anonymous online survey , which contained questions about hereditary pca and their attitudes towards pca case - finding and screening . 109 urologists ( 31% ) , 69 gps ( 23% ) and 46 cgs ( 31% ) completed the survey . cgs had the most accurate knowledge of hereditary pca . all but 1 cg mentioned at least one inherited trait with pca , compared to only 25% of urologists and 9% of gps . cgs hardly ever counseled men about pca testing . most urologists and gps discuss possible risks and benefits before testing for pca with psa . remarkably , 3540% of them do not take family history into consideration . knowledge of urologists and gps about heredity and pca is suboptimal . hence , pca counseling might not be optimal for men with a positive family history . multidisciplinary guidelines on this topic should be developed to optimize personalized counseling .

the world health organization considers leishmaniasis as one of the most neglected tropical diseases which has received little attention and resources despite its serious impacts on both the economic developments and quality of life ( who 2012 ) . leishmaniasis is one of the most important vector - borne disease and public health problem in iran that transmitted by sandflies to human and other animals . the main visceral foci are located in ardabil ( northwest ) and fars ( southwest ) . cutaneous leishmaniasis is the main vector - borne disease in the country with an average of more than 22,000 cases in the last decade ( karimi et al . 2014 ) , about 80% of them are zoonotic cutaneous leishmaniasis ( zcl ) , the endemic foci of this type are in rural areas of 17 out of 31 province ( yaghoobi - ershadi 2012 , karimi et al . zcl is a disease that primarily uses animals such as rodents as reservoir hosts ( mirzaei et al . humans are an accidental host that can be involved in the transmission cycle of leishmania parasites ( rouhani et al . geographically , zcl is widely distributed in africa , the middle east , central asia , and the rajasthan area of india ( rouhani et al . 2014 ) . in iran based on animal reservoir host , there are four foci of disease in our country : the first one has been located in central and northeast of iran , where rhombomys opimus and phlebotomus papatasi play important roles as reservoir and vector of the disease . the second focus of zcl is located in the west and southwest of iran , where tatera indica replaced with r. opimus as a reservoir and p. papatasi as a vector . baluchistan province , in the southeast of iran is considered as the third focus of zcl . in this region meriones hurrianae has been approved as a natural reservoir host . from the reported evidences , it is apparent that the most rural areas of provinces in central and southern iran can be considered as the zcl focus where m. libycus is the primary and main reservoir host of the disease , while r. opimus and t. indica were absent and p. papatasi is considered as the proven vector of zcl ( yaghoobi - ershadi et al . golestan province is one of the well - known foci of zcl in iran and two counties of this province are famous : gonbad - e - kavoos and maraveh tapeh that located in north and northeastern of this province . in one study carried out in gonbad - e - kavoos county , 4% of population have acute ulcer and 78% scar and in other study in maraveh tapeh county the prevalence of acute ulcer and scare rate were 3.03% and 63.7% respectively ( sofizadeh 2007 , cherabin et al . many researchers have argued that r. opimus and m. libycus are reservoir hosts of zcl and p. papatasi is the main vector of this disease in golestan province ( rassi et al . the main aim of this study was to determine relation between rodents active burrows and incidence of zcl in golestan province using spatial analysis . a cross - sectional study was carried out in golestan province from february 2013 to march 2014 . golestan province is located ( 3738n and 5458 e ) in northeastern of iran and is bounded by caspian sea and mazandaran province in the west , semnan province in the south , north khorasan province in the east and a borderline with turkmenistan in the north ( fig 1 ) . most parts of golestan province are plain and more than 2/3 of the plain area has arid and semiarid climates and 1/3 of others have a mild climate . the area of the province is 20437.74 km ( 1.3 areas of iran ) with 1823117 population . there are 14 counties , 27 districts , 60 rural districts , 25 cities and 1732 villages in golestan province . maximum and minimum of temperatures were recorded as 40.8 and 02 c respectively and the mean annual relative humidity was recorded as 74% . the total annual rainfall was 772 mm and the minimum precipitation in august and maximum in february . location of study area , golestan province in iran three villages were randomly selected in all rural districts ( 60 ) of golestan province and the number of active rodents burrows were counted in area of one hectare . furthermore , all villages with positive human cases in 2013 were obtained regardless of the number of cases reported from each village maps of the spatial distribution of the disease were prepared using gis software . the number of human positive cases in each rural districts of the province , as well as the incidence of disease were calculated . for calculating , the incidence rate of the disease , we used population at risk in the denominator . the relationship between the number of rodents active burrows with incidence rate and the number of positive cases of disease in rural districts were calculated using spss software version 13 and sperman test . a cross - sectional study was carried out in golestan province from february 2013 to march 2014 . golestan province is located ( 3738n and 5458 e ) in northeastern of iran and is bounded by caspian sea and mazandaran province in the west , semnan province in the south , north khorasan province in the east and a borderline with turkmenistan in the north ( fig 1 ) . most parts of golestan province are plain and more than 2/3 of the plain area has arid and semiarid climates and 1/3 of others have a mild climate . the area of the province is 20437.74 km ( 1.3 areas of iran ) with 1823117 population . there are 14 counties , 27 districts , 60 rural districts , 25 cities and 1732 villages in golestan province . maximum and minimum of temperatures were recorded as 40.8 and 02 c respectively and the mean annual relative humidity was recorded as 74% . the total annual rainfall was 772 mm and the minimum precipitation in august and maximum in february . three villages were randomly selected in all rural districts ( 60 ) of golestan province and the number of active rodents burrows were counted in area of one hectare . furthermore , all villages with positive human cases in 2013 were obtained regardless of the number of cases reported from each village maps of the spatial distribution of the disease were prepared using gis software . the number of human positive cases in each rural districts of the province , as well as the incidence of disease were calculated . for calculating , the incidence rate of the disease , we used population at risk in the denominator . the relationship between the number of rodents active burrows with incidence rate and the number of positive cases of disease in rural districts were calculated using spss software version 13 and sperman test . among all counties of golestan province , the highest incidence rate of the disease were observed in moravehtappeh ( 121.5/100000 ) and gonbad kavoos ( 99.3/100000 ) respectively ( table 1 ) . these two counties were endemic foci of cl in golestan province . in the rest of counties , demographic characters of zcl in golestan province ( 2013 ) the highest ( 53.8% ) and lowest ( 11.5% ) of cl cases were observed in the age groups of 14 and 1 years old respectively ( table . it should be noted that , 60.2 % of patients were male . in the assess the existence of rodent 's active burrows in a radius of 300 meters from the villages , they were found only in the rural districts of gonbad kavoos and moraveh tappeh counties . in the rest of studied districts , there were not rodent 's active burrows , or were located at a distance of one kilometer of the villages . however , the maximum number of rodent 's active burrows was observed in rural district of korand in gonbad kavoos county ( fig . 2 ) . distribution of rodent 's active burrows in golestan province , 2013 the number of positive cases as well as the incidence of disease were calculated 0100/100000 and 035/100000 in the different rural districts of golestan province respectively . it must be mentioned , similar to rodent 's active burrows , the most positive cases and the highest incidence of disease were found in rural district of korand in gonbad kavoos county ( fig . 2 , 3 ) . number of positive cases of zcl in golestan province , 2013 based on the statistical analysis of data , there were significant difference between the number of rodent 's active burrows with the number of positive cases ( 0.465 , p < 0.001 ) as well as , the incidence of disease ( 0.470 , p < 0.001 ) ( table 1 ) . the most positive cases were found in the northern villages of plain areas in october and november ( figs . 4 , 6 ) . incidence of zcl in golestan province , 2013 trend of zcl cases in golestan province ( 2013 ) spatial distribution of zcl cases in golestan province ( 2013 ) there was a significant positive correlation between the number of rodent 's active burrows and incidence of disease as well as the number of positive cases in studied areas ( 0.470 , p < 0.001 ) . according to fig . based on literature review of cutaneous leishmaniasis in golestan province , the causative agent of disease ( zcl ) was l. major and p. papatsi has been reported as the principal vector to human . two rodents of r. opimus and m. lybicus were main and secondary reservoir host of disease respectively ( rassi et al . 2008 , parvizi and hedayati 2010 , roshanghalb and parvizi 2012 , bordbar and parvizi 2014 ) . since the sand flies are often active in a distance of 200 meters from its habitats ( rassi and hanafi bojd 2006 ) , therefore , the presence of rodents burrows , one kilometer from the village , are considered as one of the risk factors of disease transmission . based on the results of our study , the active rodents burrows were only observed in rural districts of maraveh tappeh and gonbad - e kavoos counties . the previous studies have demonstrated that two species of r. opimus and m. libycus , are chosen the plains with soft soil to build their colonies under bushes and along streams ( nadim et al . 2009 ) , so , in the rural district of palizan to centroid qazanqaya village ( 561528.686e 375531.132n ) due to the rocky nature of area as well as , in the rural districts of shalami to centroid arab qari haji village ( 55458.727e 374123.994n ) and golidagh to centroid golidagh village ( 555641.944e 373841.094n ) due to the mountainous nature of the region , no rodent burrows were observed . we found several active burrows with high population of rodents in both rural districts of the korand to centroid korand village ( 55315.842e 37 5751.47n ) and atrak to centroid dashliburun village ( 544845.653e 373757.254n ) located in gonbad - e kavoos county . in the rural districts of agh - abad to centroid agh - abad village ( 551426.891e 371814.929n ) and sultan ali to centroid sultan ali village ( 5537.15e 37144.863n ) in some villages , the rodents were active with low abundance . in the tamran rural district to centroid tamer ghare ghozi village ( 552955.568e 372951.35n ) located at the kalaleh county , there was no rodent burrows at a distance of one kilometer from the villages . also , in the rest counties of golestan province , no rodent 's active burrows were observed nearby villages . based on our study , there were high correlation between the number of rodent 's active burrows with the number of positive cases ( 0.465 , p < 0.001 ) as well as , the incidence of disease ( 0.470 , p < 0.001 ) . this was due to the abundance of active wild rodents as the reservoir of disease and vicinity of their colonies to human settlements . therefore , high number of positive cases as well as high incidence of disease were observed in the rural districts of moravehtappeh ( moravehtappeh county ) and korand and also atrak in gonbad kavoos county . it should be noted that , two rural districts of agh - abad and sultan - ali in gonbad kavoos county were in the second rank of positive human cases . all positive cases in other rural districts of the province were imported cases , due their business trip to endemic areas . evaluation forms for these patients revealed that in most counties , the patients had a history of traveling to endemic areas of disease including rural districts of maraveh tapeh , atrak or korand . some patients had also at least one travel to the provinces of semnan , khorasan and isfahan . for example , the number of 20 cases of cutaneous leishmaniasis had been reported from the district of south fenderesk to centroid dar kalateh village ( 545738.738e 365758.499n ) located at the county of ramian , they were workers of brick kilns , who had traveled to the province of semnan in the early spring and had returned to their homes in the early autumn of 2013 . the highest incidences of the disease were found in the counties of maravehtappeh and gonbad kavoos counties respectively , while the high number of positive cases were observed in the second county . because the most population of the maraveh tappeh county ( 43.1% ) were living in endemic areas and were at greater risk of the disease , while in the county of gonbad - e kavoos county about 15% of the population were living in endemic areas . the hightest ( 53.8% ) and lowest ( 11.5% ) of cl cases were observed in the age groups of > 14 and < 1 respectively ( table 1 ) . whereas most patients ( 53.8% ) were adults over 14 years old , these results were similar to studies conducted in maraveh tapeh and gonbad - e kavoos countis in this province ( cherabin et al . 2012 , sofizadeh et al . 2013 ) and in the rural districts of damghan , and kasahan in the provinces of semnan and isfahan respectively ( doroodgar et al . 2009 , mohammadi azni et al . 2010 ) . the lowest morbidity rate of disease ( 11.5% ) was observed in age group of 1 years old . this age group had the lowest presence in outdoors and considering to hypoendemic of disease in studied areas ( sofizadeh et al . our study showed , the males are infected more than females ( 60.2% ) . because , the males are busy in agriculture tasks outside of home during the nights of summer and attending to active peak of sand flies ( sofizadeh et al . 2009 ) they have received more infected bites . the greatest number of cases were occurred in the months of october and november and coincided with the second peak of sand flies in september ( sofizadeh 2009 ) . due to direct relation between the number of rodent 's burrows and the incidence of disease , we propose to use different methods of reodent control as complementary measure for the control of disease . results of current study indicated the direct relationship between the burrows of rodents and zcl prevalence . therefore the presences of rodents as the reservoir host of disease play an important role on prevalence as well as incidence rate of disease . considering this , control of rodents will have an important role in controlling the disease . the other point that we need to pay attention , do actions that take more than a kilometer distance between human and the rodent 's life places .

background : zoonotic cutaneous leishmaniasis ( zcl ) is one of the most important vector - borne diseases in iran . wild rodents play as a reservoir . the main aim of this study was to determine spatial analyses of the relationship between rodent 's active burrows and incidence of zcl in golestan province , north east of iran.methods:the cross - sectional study was conducted in 59 rural districts in golestan province . spatial distribution of rodent 's active burrows , human cases of zcl and incidence of disease were collected , using geographical information systems ( gis ) . the relationship of them were analyzed by sperman test , spss software version no.13.results:the most number of rodents active burrows , human positive cases ( 100 persons ) and high incidence of disease ( 35/1000 ) were observed in korand rural district of gonbad - e kavoos county . there was significant correlation between the number of rodents active burrows with incidence rate of disease ( 0.470 , p < 0.001 ) as well as the number of cases in each districts ( 0.465 , p < 0.001 ) , there is high correlation between higher incidence rate and human positive cases in districts with number of rodents active burrows.conclusion:vicinity of wild rodents burrows to villages plays an important role in transmission of zcl to humans .

genetic factors significantly influence late - onset alzheimer 's disease ( ad ) though by some estimates 3065% of the genetic variance remains unexplained by the four established ad genes ( tanzi , 2012 ; mahley and rall , 2000 ) . polymorphisms of the apoe gene , specifically the epsilon 4 ( 4 ) allele , account for most of the known heritability of late - onset ad ( tanzi , 2012 ) . a positive family history is a risk factor for late - onset ad ( tanzi , 2012 ; cupples , 2004 ; silverman , 1994 ) , with studies indicating a 24-fold larger risk in first - degree relatives . some of this risk is additive to the known risk conferred by the apoe gene suggesting a missing heritability . no measure of self - reported fh status to evaluate subjects at risk hence , studies examining the effect of fh on biomarker phenotypes may improve the interpretation of biomarker tests and the counseling of at risk subjects . several studies have examined the effect of fh on biomarkers ( lampert , 2013 ; honea , 2011 ; xiong , 2011 ; andrawis , 2012 ; okonkwo , 2012 ) . for example , several cross - sectional studies in mild cognitive impairment or normal subjects report that first - degree relatives have a higher prevalence of abnormal cerebrospinal fluid beta - amyloid and/or tau phenotypes , even after accounting for the known effects of age and apoe4 ( lampert , 2013 ; xiong , 2011 ) . one of these studies also estimated that the unexplained genetic heritability in fh ( for an effect on beta - amyloid in mci ) was about half the size of the apoe4 effect ( lampert , 2013 ) . prior studies have also examined the effect of fh on hippocampal volume ( lampert , 2013 ; honea , 2011 ; andrawis , 2012 ; okonkwo , 2012 ) . in a prior cross - sectional study of normal , mci , and ad subjects , we failed to find an effect of fh on hippocampal volume ( lampert , 2013 ) . however , in another study of normal subjects derived from the ku brain aging project , maternal fh was reported to influence 2-year volume loss in the precuneus , parahippocampal and hippocampus independent of apoe4 ( honea , 2011 ) . supporting this was another 1-year multisite study , which found that maternal ( but not paternal family history ) was associated with increased hippocampal atrophy in mci subjects but not in normal or ad subjects ( andrawis , 2012 ) . however , another 4-year study of middle aged normals , in the wisconsin aging study , found that fh status predicted greater atrophy only within a posterior sub - region of the hippocampus but not in other gray matter regions , and that there was no effect of maternal versus paternal history ( okonkwo , 2012 ) . reasons for discrepancy may be differences in inclusion criteria , sample size , follow - up duration , image analyses , and covariates used . the alzheimer 's disease neuroimaging initiative ( adni ) is a highly successful national longitudinal biomarker research study ( weiner , 2010 ) and as such it is ideal for more definitive testing of preliminary results generated by single site studies . stringent subject selection criteria , serial mri scans using qualified scanners and phantoms , and standardized central mr image analyses are some of the many strengths of adni . the goal of this present analysis was to use adni data to test the effect of fh on longitudinal atrophy rates ( up to 48 months ) of 20 brain regions in subjects with mci . we decided to focus on subjects with mild cognitive impairment for three reasons : adni included twice as many mci subjects as controls ; mci subjects are at greater risk for progression to ad than normal controls ; mci subjects have a greater rate of atrophy than normal controls . hence , the mci group offered greater power for testing our hypotheses and was also more relevant to the type of subject seen in routine clinical practice . our primary hypothesis was that a positive fh would be associated with greater rate of atrophy in brain regions known to degenerate early in ad such as the hippocampus , amygdala , entorhinal cortex , and cortical gray matter . data used in the preparation of this article were obtained from the alzheimer 's disease neuroimaging initiative ( adni-1 ) database ( http://adni.loni.usc.edu ) ( weiner , 2010 ) . the adni was launched in 2003 by the national institute on aging ( nia ) , the national institute of biomedical imaging and bioengineering ( nibib ) , the food and drug administration ( fda ) , private pharmaceutical companies , and nonprofit organizations , as a 5-year public private partnership . the primary goal of adni has been to test whether serial magnetic resonance imaging ( mri ) , positron emission tomography ( pet ) , other biological markers , and clinical and neuropsychological assessment can be combined to measure the progression of mild cognitive impairment and early alzheimer 's disease ( ad ) . determination of sensitive and specific markers of very early ad progression is intended to aid researchers and clinicians to develop new treatments and monitor their effectiveness , as well as lessen the time and cost of clinical trials . the principal investigator of this initiative is michael w. weiner , md , va medical center and university of california san francisco . adni is the result of efforts of many co - investigators from a broad range of academic institutions and private corporations , and subjects have been recruited from over 50 sites across the u.s . and . the initial goal of adni was to recruit 800 subjects but adni has been followed by adni - go and adni-2 . to date these three protocols have recruited over 1500 adults , ages 5590 , to participate in the research , consisting of cognitively normal older individuals , people with early or late mci , and people with early ad . the follow - up duration of each group is specified in the protocols for adni-1 , adni-2 and adni - go . subjects originally recruited for adni-1 and adni - go had the option to be followed in adni-2 . for up - to - date information , for additional details readers can also refer to the adni-1 procedures manual ( adni , 2013 ; alzheimer 's disease neuroimaging initiative , 2013 ) . only subjects with a baseline diagnosis of mci were eligible for this study . additionally , subjects selected for analysis were required to have data for all of the following parameters : baseline age , race , gender , and years of education ; baseline mini - mental state examination ( mmse ) score ; apoe genotyping results ; and family history status of ad . subjects also needed initial visit 1.5 t mr scans analyzed centrally by freesurfer software ( v.4.4 ) to derive cortical thickness and sub - cortical volume data ; and a minimum of two other follow - up mri time points ( 6 , 12 , 18 , 24 , 36 , or 48 months from baseline ) with centrally freesurfer analyzed mri data . to be classified as mci in adni a subject needed an inclusive mmse score between 24 and 30 , subjective memory complaint , objective evidence of impaired memory calculated by scores of the wechsler memory scale logical memory ii adjusted for education , a score of 0.5 on the global cdr , absence of significant confounding conditions such as current major depression , normal or near normal daily activities , and absence of clinical dementia . for a detailed list of all selection criteria readers fh data was collected by an interview with the subject and their study partner regarding the presence of ad in the subject 's parents and siblings . a control typically self - reported while the study partner was the main source of information for memory - impaired subjects . a positive family history ( fh+ ) was characterized as having a parent or sibling , living or deceased , who had been reported as diagnosed with ad . a negative family history ( fh ) meant having no reported parents or siblings with a history of ad . apoe allele genotyping of all subjects was executed using dna extracted from peripheral blood cells with details given elsewhere ( adni , 2013 ) . adni used 1.5 t mp - rage t1-weighted mr images that were later pre - processed and corrected for nonlinearity via gradwarp . the scans were implemented using a standardized adni protocol adjusted for use at each specific collection site and then underwent scaling and vetting to meet quality control criteria . for more detailed information regarding the specific mr acquisition protocols and control methods used we selected 20 brain regions of interest , including 4 regions known to atrophy in early ad ( hippocampus , amygdala , cortical gray matter volume , and entorhinal cortex ) as well as other regions of exploratory interest . specific details about these techniques have been previously described in publications ( sgonne , 2004 ; fischl , 2002 ; sled et al . , 1998 ; fischl et al . , 2001 ; fischl et al . , 1999 ; fischl and dale , 2000 ; han , 2006 ; hostage et al . , 2014 ) . for more information please see http://adni.loni.usc.edu . for each region of the brain , the longitudinal variation in thickness / volume was modeled as a log - linear function of time and other subject information . the response vij(t ) is the thickness / volume in the jth region of the brain , j = 1 , 2 , , 20 for the ith subject , i = 1 , , 184 , measured at the tth time point , t = 0 , 6 , 12 , 18 , 24 , 36 , or 48 months . the terms in the model include j , the baseline thickness volume , measured at time 0 , for a 75 year old male subject with no family history of alzheimer 's . the baseline subject represents the most common subject characteristics in the study , and the reference age represents the first quartile of ages in the study . there is also a subject specific random effect , ai , assumed to have a gaussian distribution with 0 mean and sd a . this term accounts for subject specific variation in the baseline brain volume , due to factors such as difference in intra - cranial brain volume . in addition , we have a linear effect of age ( centered at 75 ) , with coefficient aj , an additional effect for females , fj , as well as an effect of positive family history , fhj . the aforementioned effects are all at t = 0 and specific to region j. next , we model atrophy ( or volume increase ) as a linear function in time , decomposed into effects of the demographic variables : firstly , j is the baseline rate of atrophy , corresponding to the baseline subject described above . there is also a subject specific random rate of atrophy , bi , assumed to have a gaussian distribution with 0 mean and sd b . this term allows the rate of atrophy to vary across subjects , as a proxy for terms not explicitly included in the model , such as genetic and lifestyle factors . we also have an additional atrophy effect for females , fj , as well as an atrophy effect of positive family history , fhj . finally , ijt is a random measurement error , assumed to have a zero mean gaussian distribution with sd . the brain volumes vij(t ) were log transformed to ensure that the distribution of the estimated error terms ( residuals ) of the model better conformed to the assumption of gaussianity . we also used a second model , where additional genetic information was incorporated:(1.2)log(vij(t))=j+aij+ajage+fj+fhj+e4j+jt+bijt+fjt+fhjt++e4jt+ijt . in model ( 1.2 ) , the common terms represent the same things as in model ( 1.1 ) , except that the baseline subject additionally possesses the apoe 3/3 allele . the term e4j denotes the additional baseline effect of the apoe 4 + allele , while e4j denotes the additional atrophy effect of the apoe 4 + allele . both models were fit separately to data from each region of the brain , using restricted maximum likelihood ( reml ) , as implemented in the nmle package [ pinheiro et al . linear and nonlinear mixed effects models . r package version 3.1 - 113 , 2013 ] in the r computing platform ( http://www.r-project.org ) . our a priori primary hypothesis regions included those well known to atrophy in early ad such as the hippocampus , amygdala , entorhinal cortex and cortical gray matter . there were no significant differences by fh for gender , years of education , or mmse score . there was a difference in age ( p = 0.05 ) , and as expected there was an overrepresentation of the apoe4 allele in fh+ subjects ( p = 0.002 ) . total follow - up was up to 48 months , and the mean follow - up time for the fh group ( 27.8 months ) was not significantly different from the fh+ group ( 29.5 months , p < 0.25 ) . as shown in the first three columns in table 2 , most brain regions showed a significant atrophy over time ( i.e. base rate ) . age and gender , subjects with a positive fh had greater atrophy of the cerebral cortex ( p < 0.009 ) , amygdala ( p < 0.01 ) , entorhinal cortex ( p < 0.01 ) and hippocampus ( p < 0.053 ) . table 3 depicts the estimated effects and significance of apoe 4 + and fh on longitudinal atrophy rates . in this model , some 15 regions showed a significant apoe4 + effect including all the predicted primary regions ( such as the hippocampus , amygdala , entorhinal cortex and cerebral cortex ) . however , none of the primary predicted regions showed a significant fh effect in this model and the atrophy rate attributable to fh was considerably smaller than that attributable to apoe4 . 1 compares atrophy rates due to apoe4 + versus those due to fh+ it shows that most fh effects are small relative to apoe4 + effects , as evidenced by the wider spread of apoe4 + effects while most fh+ rates remained close to zero . table 4 summarizes the model when fit to only 3 homozygotes there was no significant fh effect on any predicted region and effect sizes ( cohen 's d ) ranged from 0.05 to 0.22 . for example , the fh effect size on the amygdala atrophy rate was 0.17 ( p < 0.14 ) . exploratory analyses comparing the effect of fh in apoe 4 + versus 3/3 subjects showed that some regions exhibit a slightly greater fh effect on atrophy in the 3/3 group ( e.g. amygdala ) whereas some other regions have a greater fh effect in the 4 + group ( e.g. hippocampus ) but none of these were statistically different . duration of follow - up available for cognition was longer than that available for mri scans and we examined the baseline and last available time point within the 4 year window . duration of follow - up tended to be longer in fh+ subjects ( mean 43.7 months ) than fh subjects ( mean 38.5 months ) ( p = 0.02 ) . fh+ subjects ( 57% ) had numerically higher rate of conversion from mci to dementia than fh subjects ( 45% ) but the difference was not statistically significant . fh+ subjects ( mean 7 points 10 ) had numerically greater decline in cognition ( adas - cog ) than fh subjects ( mean 5.7 points 9 ) but the fh effect was not statistically significant in a multivariate model with age , gender , education , baseline cognition , e4 and time . gender and apoe4 genotype ( p = 0.313 ) were significant in this model . to our knowledge , this is the first national long - term ( up to 48 months ) examination of the effects of fh on atrophy rates of multiple regions in mci subjects at risk for future ad . we found that a positive fh was associated with accelerated atrophy rates for three key structures ( amygdala , hippocampus , and entorhinal cortex ) plus cortical gray volume in mci but that the residual fh effect on atrophy rate , after covarying for apoe e4 , was no longer significant for any brain structure . we also found that in the subset of apoe3 homozygotes , there was no significant fh effect on any key structure . these data suggest that any missing heritability within fh ( other than apoe4 ) for explaining atrophy rate in mci subjects is likely to be quite small and nonsignificant . lastly , given the large size of the main e4 effect relative to the main fh effect in mci subjects , our data suggests that much of the e4 effect may not be contained wholly in the fh effect . there appeared to be a weak interaction between apoe status and fh in that the effect of fh on amygdala atrophy rates seemed larger in e3 homozygotes than e4 carriers , but this too was not significant . prior studies of fh effects on csf [ reviewed in lampert , 2013 ] and fdg - pet [ reviewed in mosconi , 2007 ] biomarkers have generally found consistent changes . however , prior studies of fh effects on longitudinal hippocampal atrophy rates have yielded potentially conflicting findings ( honea , 2011 ; andrawis , 2012 ; okonkwo , 2012 ) . honea ( 2011 ) found a positive effect for maternal fh , independent of e4 status , on 2-year hippocampal atrophy rate in normal controls and proposed that this supported the mitochondrial hypothesis of ad . andrawis ( 2012 ) did not find a significant effect of fh on 1-year atrophy rates in normal controls or ad patients but did find an effect for maternal fh in mci subjects . yet another study of normal middle - aged adults , by okonkwo ( 2012 ) , found a fh effect on atrophy only in a posterior subregion of the hippocampus and only in e4 subjects there was no fh effect in e4 + subjects . in addition , okonkwo et al . found no significant difference between maternal and paternal fh on atrophy rate ( okonkwo , 2012 ) . the follow - up in okonkwo 's study was 4-years and longer than prior studies but their controls were younger . these studies relied on just 2 mri scans ( baseline and follow - up ) to extract atrophy rates . our study extends these data by examining mci subjects over a longer follow - up period ( 3 mri scans and up to 48 month follow - up ) and by examining multiple brain regions . the use of 3 mri scans over this period may have allowed for a more accurate estimate of the slope of change than in prior studies . further , our examination of 20 different brain regions allowed us to examine changes in regions associated with cognitive brain circuits known to be affected early in ad . overall , our data do not support a significant residual ( after covarying for apoe status ) fh effect on rates of atrophy of any key brain region . since we did not examine maternal versus paternal fh a strength of this current study is the use of a relatively large sample of carefully selected mci subjects , data collected in a standardized manner from many sites across the country , multiple imaging time points , and relatively long duration of follow - up . fh status was ascertained via interviews with the subjects and their study partners , so it is possible that there may have been a reporter bias ( for example , some respondents may not know the difference between ad and dementia ) . that said , our findings are still relevant because fh is collected by simple history in most clinics and biomarker research studies . we studied only mci subjects and did not examine interactions of fh with amyloid or tau phenotypes . prior studies have shown that amyloid positivity may be linked to e4 status [ reviewed in lampert , 2013 ] and accelerated atrophy [ reviewed in honea , 2011 ] , and so it is conceivable that there may also be an interaction of amyloid status with fh . we also did not analyze subregions of the hippocampus and hence could not directly test findings that fh might affect only specific subregions ( okonkwo , 2012 ) . we also did not examine the effect of fh on glucose metabolic status , another marker that has been linked to fh ( mosconi , 2007 ) . thus , our findings can not be generalized to other biomarkers or other diagnostic groups . as stated earlier , we did not test maternal versus paternal fh since we did not collect that data . likewise , we were unable to test parental versus sibling fh as there is a vast majority of patients with an affected parent ( n = 51 parental , n = 9 both , n = 18 sibling ; 77% parental ) and therefore not enough power to separate the two . future directions of this study should include further analysis to determine if there are other genetic factors ( e.g. whole genome data ) in addition to apoe4 that predict atrophy rates in brain regions susceptible to ad as well as studies examining the effect of fh on multiple biomarkers ( mri , csf , pet ) over longer periods of time . since fh has been linked to beta - amyloid positivity and amyloid deposition in turn has been linked to atrophy , future studies should examine fh effects in healthy controls who are amyloid positive ( i.e. , preclinical ad ) . only a subset of adni-1 subjects had csf or amyloid pet data . adni2 , where every subject underwent florbetapir pet , will allow for a better test of this theory in the near future . such information may serve to further improve personalized testing and drug development for at - risk patients . experimental regionsabbreviationhippocampus total volumehippocampus.total.volamygdala total volumeamygdala.total.volentorhinal cortexentorhinal.cortex.taparahippocampal taparahippocampal.taposterior cingulate taposterior.cingulate.taprecuneus taprecuneus.tasuperior.parietal tasuperior.parietal.tasuperior.temporal tasuperior.temporal.tatemporal.pole tatemporal.pole.tatransverse.temporal tatransverse.temporal.tacerebral cortex total volcerebral.cortex.total.volfusiform gyrus tafusiform.gyrus.tainferior parietal tainferior.parietal.tainferior temporal tainferior.temporal.tamiddle temporal tamiddle.temporal.tacerebellar cortex ( total volume)cerebellar.cortex.total.volcerebellar white mater ( total volume)cerebellar.wm.total.volumepericalcarine cortexpericalcarine.tapostcentral gyruspostcentral.taprecentral gyrusprecentral.ta

a positive family history ( fh ) raises the risk for late - onset alzheimer 's disease though , other than the known risk conferred by apolipoprotein 4 ( apoe4 ) , much of the genetic variance remains unexplained . we examined the effect of family history on longitudinal regional brain atrophy rates in 184 subjects ( 42% fh+ , mean age 79.9 ) with mild cognitive impairment ( mci ) enrolled in a national biomarker study . an automated image analysis method was applied to t1-weighted mr images to measure atrophy rates for 20 cortical and subcortical regions . mixed - effects linear regression models incorporating repeated - measures to control for within - subject variation over multiple time points tested the effect of fh over a follow - up of up to 48 months . most of the 20 regions showed significant atrophy over time . adjusting for age and gender , subjects with a positive fh had greater atrophy of the amygdala ( p < 0.01 ) , entorhinal cortex ( p < 0.01 ) , hippocampus ( p < 0.053 ) and cortical gray matter ( p < 0.009 ) . however , when e4 genotype was added as a covariate , none of the fh effects remained significant . analyses by apoe genotype showed that the effect of fh on amygdala atrophy rates was numerically greater in 3 homozygotes than in e4 carriers , but this difference was not significant . fh+ subjects had numerically greater 4-year cognitive decline and conversion rates than fh subjects but the difference was not statistically significant after adjusting for apoe and other variables . we conclude that a positive family history of ad may influence cortical and temporal lobe atrophy in subjects with mild cognitive impairment , but it does not have a significant additional effect beyond the known effect of the e4 genotype .

spontaneous pneumothorax ( sp ) comprises the largest group and is classified into primary spontaneous ( psp ) and secondary spontaneous pneumothorax . psp usually occurs in young , tall , thin men , especially smokers , in the absence of an underlying lung disease . although , sp is a relatively common condition , bilateral primary spontaneous pneumothorax ( bpsp ) is a very rare clinical condition with an occurrence ranging from 1.3 to 1.9% of all cases of sp . herein , we present a case of bpsp in an overweight young male patient , which is an exceptionally rare event . an otherwise - healthy , non - smoker , 20-year - old , overweight ( bmi 29.8 kg / m ) greek male patient was presented to the emergency department complaining of low - intensity chest pain and breathlessness of progressive worsening over the preceding 7 days . on admission , blood pressure was 125/70 mmhg , heart rate 100 beats / min , sao2 97% on air and axillary temperature 36.8c . initial plain chest x - ray demonstrated bilateral pneumothoraces ( more prominent on the left side ) and no deviation of the trachea ( fig . 1 ) . plain chest x - rays on admission revealing bilateral pneumothorax . on emergency basis , the patient was managed with bilateral tube thoracostomy through the fifth intercostal spaces . during hospitalization , both lungs were gradually expanded ; the use of continuous aspiration was necessary for a couple of days , in order to achieve complete expansion of the left lung ( fig . 2 ) . next day the plain x - ray revealed recurrence of pneumothorax on the left side ( fig . the tube was removed after 5 days ; no recurrence took place at this time . during the next 3 days the patient remained under observation ; serial physical examinations and chest x - rays were normal and the patient was discharged home . recurrence of pneumothorax on the left side after removing the chest tube . during hospitalization a chest computed tomography ( ct ) was performed and revealed the presence of blebs at the apices of both upper lungs ( fig . the patient was referred to a tertiary center for an elective video - assisted thoracoscopic surgery intervention ( vats ) . three years after , no recurrence of pneumothorax took place and the patient remains healthy . psp usually occurs in the absence of any obvious underlying lung disease ; however , blebs and bullae apparently play a role in the pathogenesis of psp , as they are found on ct or during thoracoscopy or thoracotomy in 4879% of these patients . diagnosis of bilateral pneumothorax presents difficulties ; while unilateral pneumothorax is relatively easily suspected from the patient 's medical history , physical and radiological findings , the diagnosis of bilateral pneumothorax is usually delayed . the presence of equally diminished breath sounds in both sides and the presence of a not deviated trachea often mislead clinicians and delay chest drain insertion which is usually performed after the transaction of chest ct . although sp is a relatively common condition , bpsp is a very rare clinical condition . lee et al . , in their study of the 616 patients with 807 episodes of psp , found that the incidence of bpsp was 1.6% ( 13 patients ) . in univariate regression analysis , patients with psp compared with patients with bpsp had significantly lower body weight , bmi , higher body height / body weight ratio and higher incidence of bilateral blebs / bullae seen in hrct of the lung . however , in multiple regression analysis , only bmi and the presence of bilateral blebs / bullae retained statistically significant importance . huang et al . found that only lower bmi and smoking were significantly associated with the formation of bpsp . in their study , the proportion of bilateral blebs / bullae seen in hrct was higher in the bpsp group than that in the psp group ( 63 vs 53% , respectively ) , but the difference was not statistically significant ( p = 0.724 ) . the present report describes a case of bpsp in an overweight patient showing that psp is an existing condition in patients with bmi value higher than the normal . generally , if an sp affects < 20% of one lung , observation is efficient ; the absorption rate of the air is 1.25% ( 5075 ml / day ) . if pneumothorax affects > 20% of the lung or if it increases during observation , chest tube drainage may be required . air leakage can be diminished in 5 h and in 48 h in 52 and 82% of patients with tube thoracostomies , respectively . in patients with bpsp , one side should always be drained regardless of the extent of pneumothorax , whereas the other side can be managed by simple observation depending on the extent of the air in the pleura space . prolonged air leakage is the most common indication for operation in the first episode of pneumothorax . today compared with open thoracotomy , this procedure provides visualization of the entire thoracic cavity by video and causes less postoperative pain . single - stage bilateral vats procedure for bpsp has been advocated as an elective procedure to avoid subsequent anesthetic and operative procedures and longer hospital stays . if any underlying pulmonary disease is detected during surgical treatment , the existence of pleural communications should be investigated and mediastinal pleura should be examined carefully . in addition , apical pleurodesis further reduces the risk of recurrence . in our case , the patient was successfully managed initially with a bilateral tube thoracostomy and finally with an elective bilateral vats procedure . in conclusion , bpsp is a rare clinical condition and usually develops in patients with low bmi and bilateral blebs / bullae . bpsp in overweight - obese patients is an existing condition and its early diagnosis requires high suspicion index . bpsp needs urgent assessment and management , followed by bilateral single - stage vats treatment as a safe and effective procedure .

bilateral primary spontaneous pneumothorax ( bpsp ) is a rare condition , accounting for 1.6% of spontaneous pneumothoraces . patients with primary spontaneous pneumothorax have typically low body weight . bpsp in overweight / obese patients is an exceptionally rare event . the present report describes a case of an otherwise - healthy 20-year - old male patient with bmi 29.8 kg / m2 presented to our hospital owing to low - intensity chest pain and shortness of breath 7 days after the onset of symptoms . plain chest x - ray revealed the presence of bilateral pneumothorax . chest tubes were inserted on both sides . during hospitalization , a computed tomography demonstrated multiple blebs on the surface of the upper lobes of both lungs . hereupon , the patient referred to a tertiary center and underwent elective single - stage minimally invasive bilateral video - assisted thoracoscopic surgery . the present case report shows that bpsp in overweight / obese patients is an existing condition .

segments of dna with near - identical sequence ( segmental duplications or duplicons ) in the human genome can be hot spots or predisposition sites for the occurrence of non - allelic homologous recombination or unequal crossing - over leading to genomic mutations such as deletion , duplication , inversion or translocation . these structural alterations , in turn , can cause dosage imbalance of genetic material or lead to the generation of new gene products resulting in diseases defined as genomic disorders . previous studies to identify segmental duplications in the human genome have analyzed older versions of the genome assembly , which contained higher amounts of unfinished sequence and incorrectly mapped regions , and have used different computational approaches all performed by the same group [ 6 - 8 ] . with the human genome sequence now nearing completion , we have examined its content for segmental duplications using two distinct computational methods . in the first , we utilized the rapid blast2 algorithms that allow direct chromosomal - wide sequence comparisons to be made . all blast results reported in table formats can be subsequently grouped , parsed and analyzed for the detection of duplicated sequences . in addition , we have shown previously that there is a strong correlation between ambiguously mapped snps ( ambsnps ) , as well as the density of snps , and segmental duplications . ambsnps are snps that were annotated to map to two locations on a particular chromosome in the ncbi dbsnp . a subset of these ambsnps are not true snps but are likely to be computer - generated nucleotide mismatches from paralogous copies of duplicated sequences and should be more appropriately labeled as paralogous sequence variants ( psvs ) . another subset is likely to be false ambsnps of genomic sequences that have been misassigned in genome assemblies . here , we report our analysis of all potential psvs in the human genome and their correlation with segmental duplications as detected by our blast analysis . furthermore , we provide a critical assessment on the three latest human genome assemblies from our analysis of sequence misassignments as identified from this study . on the basis of the june 2002 ( ncbi build 30 ) human genome assembly , a total of 107.4 mb ( 3.53% ) of the human genome content ( 3,043.1 mb ) were found to be involved in recent segmental duplications by our blast analysis criteria ( table 1 ) . this content is composed of more than 1,530 distinct intrachromosomal segmental duplications ( 80.3 mb or 2.64% of the total genome , figure 1 ) and 1,637 distinct interchromosomal duplications ( 43.8 mb or 1.44% of the total genome ) . in addition , 29% of all duplications are located in unfinished regions of the current genome assembly . we have also found that 38% of the duplications ( 52.3 mb ) can be considered as tandem duplications - defined here as two related duplicons separated by less than 200 kb . in this study , we only analyzed large ( size 5 kb ) and recent ( sequence identity 90% ) duplications because we can achieve higher confidence and to prioritize those regions for their potential involvement in diseases . previously , bailey and colleagues reported a total of 5.2% of the human genome involved in recent segmental duplications . the 1.6% discrepancy between our findings could be due to the difference in our detection criteria ( size cutoff of 5 kb used in this study versus 1 kb used in bailey et al . ) . moreover , we have identified 38.9 mb of sequences ( 1.28% of the june 2002 genome assembly ) likely to be artifactual duplications resulting from sequence misassignment errors present in the assembly . by comparing our results with those published previously , we found that 482/2579 clones that we identified to be involved in duplication were novel . the molecular mechanism by which segmental duplications are created is still unclear at the moment . a recent report has suggested that alu repeat clusters had a role as mediators of recurrent chromosomal rearrangements . we have examined whether elevated amounts of repetitive elements could be found in duplicon junctions . we inspected all duplication borders from our results and calculated the occurrence of different repeat types within the 500 bp window outside each duplicon junction . the whole - genome average frequencies were determined by sampling random 500 bp windows across the genome ( excluding gap regions ) . overall , we found that there are significant enrichment ( or relative fold increase ) for the presence of small ribonucleoprotein rna ( srprna ) , satellite , long terminal ( ltr ) and sine / alu repeats ( see additional data file ) . in addition , our data also showed that for some chromosomes the amount of duplicated sequence is higher in the pericentromeric and subtelomeric regions of chromosomes ( figure 1 ) , supporting the hypothesis that these repeat - dense regions have made an important contribution to the evolution of the human genome . regions containing recently occurring segmental duplications can harbor rapidly evolving hominoid - specific genes , as well as novel gene families that are unique to primates . using the national center for biotechnology information refseq annotation of these , 475 genes were fully contained within duplicated regions and were best candidates for recent whole - gene duplication . we have carried out functional analysis of these 475 genes using the gene ontology consortium database and found that there is a significant increase in gene duplications for genes involved in immune defense ( antibodies , blood - group antigens ) and reproduction ( pregnancy , sex differentiation ) ( see additional data file ) . we were aware that in silico detection methods , such as the ones used in this study , would not allow us to distinguish completely true duplications from artifactual duplications arising from misassigned sequences , especially in cases where sequence identity between two detected duplications exceeded 99.5% over a substantial length ( > 5 kb ) in regions composed of draft sequences . although a small proportion of such results ( duplications with > 99.5% identity ) might represent unfinished regions of the genome that contain true duplications that have arisen very recently in the evolution of the human genome ( such as the large and nearly perfect palindromic repeats located in the azfc region on chromosome yq11.223 involved in male infertility ) , we suspect that most of the duplications ( > 99.5% identity and contain draft sequences ) are in fact sequence misassignment errors in the genome assembly . an explanation for such errors would be when two identical sequences belonging to the same genomic location were misassigned to distinct regions in the genome assembly . we have used the ncbi e - pcr to evaluate our results ( potential sequence misassignment errors ) from the june 2002 human genome assembly . using some of the largest interchromosomal misassignment errors detected in our study , we found that none of the sts markers located within these misassigned sequences maps to their incorrectly assigned chromosomes . for example , ac121339 is incorrectly mapped to 3q13.13 in the june 2002 genome assembly , as supported by a consensus number of chromosome x sequence - tagged site ( sts ) markers ( figure 2 , table 2 ) . from this genome assembly , we identified that a total of 38.9 mb of sequences , representing 1.28% of the total sequence content , are involved in such potential errors ( a full list of potentially misassigned sequences can be obtained from ) that would require additional effort and further sequencing to achieve resolutions . we also analyzed an additional two previous versions of the human genome assemblies , december 2001 and april 2002 , and our results showed that there has been a dramatic reduction in potential errors in the latest human genome assembly compared to the two previous genome assemblies ( table 3 ) . furthermore , we examined the distribution of the amount of duplications in five different categories on the basis of their level of sequence identity to each other ( table 3 ) . we observed a large reduction in duplications that fall within the 98 - 100% category , supporting the fact that the genome assemblies continue to improve and correct errors made . in addition , our data showed that there have been major improvements for chromosomes 5 , 6 , 7 , 13 , 14 and 19 over the last three genome assemblies . and for chromosomes that had reached finished status , such as chromosomes 20 , 21 and 22 ambsnps are snps that were annotated to map to two locations on a particular chromosome in the ncbi dbsnp . here we show on a genome scale that ambsnps most specifically correlate with intrachromosomal segmental duplications , suggesting they are paralogous sequence variants ( psvs ) ( figure 1 ) . these psvs were perhaps mistakenly introduced into dbsnp by automated in silico - generated analysis , arising from nucleotide mismatches in paralogous copies of duplicated sequences . overall , a surprisingly high proportion , 8.6% ( 199,965 of 2,327,473 ) , of the refsnps were annotated as ambsnps from dbsnp ( build 108 ) . a significant number of the ambsnps ( 139,974 of 199,965 or 70.0% ) are located within duplicated regions as identified by blast and should be regarded as psvs ( see additional data file ) . the non - identification by blast analysis of regions that contain ambsnps first , the duplicated copy(s ) could have been removed from the sequence assembly or the two have been conflated , that is , mistakenly thought to be the same sequence owing to their high sequence similarity . a second possibility is that the duplication is smaller than 5 kb and was excluded in our blast analysis . a third possibility is that a collection of ambsnps could have been generated from misassigned sequences ( identical sequences but misassigned to two difference locations in the genome ) in older assembly builds due to sequencing errors or true snps ( with high polymorphism rate ) in the sequence . we also observed that the density of ambsnps generally correlates with the size of the putative duplication , although this might be affected by the level of sequence identity between duplications . for example , two duplicated sequences sharing 98% sequence identity compared to 95% over the same length might contain fewer psvs as the number of base - pair mismatches would be fewer in the former . in addition , we observed that regions identified by our blast method but that do not contain ambsnps often correspond to artifactual duplications generated from assembly errors . the size , orientation , and contents of segmental duplications are highly variable and most of them show great organizational complexity . this is perhaps due to successive transposition and rearrangement events leading to the creation of segmental duplications . in many cases , for example , one of the largest segmental duplicons detected in this study was 359 kb in size at the williams - beuren locus on 7q11.23 . in this case , the two duplicons are separated by 1.6 mb of intervening sequence with the telomeric duplicon comprising several separate smaller modules as compared to the primary duplicon . the results presented in our study ( provided in tables available at ) would also allow rapid identification of new duplicons that are potentially responsible for chromosome rearrangements and genomic disorders . for example , the location of the duplicons on chromosomes 9q34/22 q11 that have been suggested to mediate recombination leading to the philadelphia chromosome was identified in our analysis , as were other medically relevant chromosomal regions ( table 4 ) . the characterization of most large segmental duplications is complicated by the fact that many of them ( 29% of all duplications ) are only represented as draft sequences from the current genome assembly . despite the fact that both blast and psvs analyses allowed us to identify most segmental duplications involved in known genomic disorder mutations ( table 4 ) , estimations of the size of rearranged regions were different from those previously reported . in fact , with the exception of several small duplications and the segmental duplications on chromosome 22 , other regions containing duplications involved in genomic disorders were often erroneously assembled and misplaced . furthermore , we have searched the celera human genome c3 ( publicly released version ) and c4 ( subscription - based version ) sequence assemblies for large duplications found on chromosome 7 . we observed that most of them were not represented in large scaffolds , but instead were located in their sequence gaps , or only partially found at ends of scaffolds leading into gaps ( see table 4 ) . this suggests that the whole - genome assembly approach alone might not be able to finish such duplicated regions in mammalian genomes . on the basis of the june 2002 ( ncbi build 30 ) human genome assembly , a total of 107.4 mb ( 3.53% ) of the human genome content ( 3,043.1 mb ) were found to be involved in recent segmental duplications by our blast analysis criteria ( table 1 ) . this content is composed of more than 1,530 distinct intrachromosomal segmental duplications ( 80.3 mb or 2.64% of the total genome , figure 1 ) and 1,637 distinct interchromosomal duplications ( 43.8 mb or 1.44% of the total genome ) . in addition , 29% of all duplications are located in unfinished regions of the current genome assembly . we have also found that 38% of the duplications ( 52.3 mb ) can be considered as tandem duplications - defined here as two related duplicons separated by less than 200 kb . in this study , we only analyzed large ( size 5 kb ) and recent ( sequence identity 90% ) duplications because we can achieve higher confidence and to prioritize those regions for their potential involvement in diseases . previously , bailey and colleagues reported a total of 5.2% of the human genome involved in recent segmental duplications . the 1.6% discrepancy between our findings could be due to the difference in our detection criteria ( size cutoff of 5 kb used in this study versus 1 kb used in bailey et al . ) . moreover , we have identified 38.9 mb of sequences ( 1.28% of the june 2002 genome assembly ) likely to be artifactual duplications resulting from sequence misassignment errors present in the assembly . by comparing our results with those published previously , we found that 482/2579 clones that we identified to be involved in duplication were novel . the molecular mechanism by which segmental duplications are created is still unclear at the moment . a recent report has suggested that alu repeat clusters had a role as mediators of recurrent chromosomal rearrangements . we have examined whether elevated amounts of repetitive elements could be found in duplicon junctions . we inspected all duplication borders from our results and calculated the occurrence of different repeat types within the 500 bp window outside each duplicon junction . the whole - genome average frequencies were determined by sampling random 500 bp windows across the genome ( excluding gap regions ) . overall , we found that there are significant enrichment ( or relative fold increase ) for the presence of small ribonucleoprotein rna ( srprna ) , satellite , long terminal ( ltr ) and sine / alu repeats ( see additional data file ) . in addition , our data also showed that for some chromosomes the amount of duplicated sequence is higher in the pericentromeric and subtelomeric regions of chromosomes ( figure 1 ) , supporting the hypothesis that these repeat - dense regions have made an important contribution to the evolution of the human genome . regions containing recently occurring segmental duplications can harbor rapidly evolving hominoid - specific genes , as well as novel gene families that are unique to primates . using the national center for biotechnology information refseq annotation of these , 475 genes were fully contained within duplicated regions and were best candidates for recent whole - gene duplication . we have carried out functional analysis of these 475 genes using the gene ontology consortium database and found that there is a significant increase in gene duplications for genes involved in immune defense ( antibodies , blood - group antigens ) and reproduction ( pregnancy , sex differentiation ) ( see additional data file ) . we were aware that in silico detection methods , such as the ones used in this study , would not allow us to distinguish completely true duplications from artifactual duplications arising from misassigned sequences , especially in cases where sequence identity between two detected duplications exceeded 99.5% over a substantial length ( > 5 kb ) in regions composed of draft sequences . although a small proportion of such results ( duplications with > 99.5% identity ) might represent unfinished regions of the genome that contain true duplications that have arisen very recently in the evolution of the human genome ( such as the large and nearly perfect palindromic repeats located in the azfc region on chromosome yq11.223 involved in male infertility ) , we suspect that most of the duplications ( > 99.5% identity and contain draft sequences ) are in fact sequence misassignment errors in the genome assembly . an explanation for such errors would be when two identical sequences belonging to the same genomic location were misassigned to distinct regions in the genome assembly . we have used the ncbi e - pcr to evaluate our results ( potential sequence misassignment errors ) from the june 2002 human genome assembly . using some of the largest interchromosomal misassignment errors detected in our study , we found that none of the sts markers located within these misassigned sequences maps to their incorrectly assigned chromosomes . for example , ac121339 is incorrectly mapped to 3q13.13 in the june 2002 genome assembly , as supported by a consensus number of chromosome x sequence - tagged site ( sts ) markers ( figure 2 , table 2 ) . from this genome assembly , we identified that a total of 38.9 mb of sequences , representing 1.28% of the total sequence content , are involved in such potential errors ( a full list of potentially misassigned sequences can be obtained from ) that would require additional effort and further sequencing to achieve resolutions . we also analyzed an additional two previous versions of the human genome assemblies , december 2001 and april 2002 , and our results showed that there has been a dramatic reduction in potential errors in the latest human genome assembly compared to the two previous genome assemblies ( table 3 ) . furthermore , we examined the distribution of the amount of duplications in five different categories on the basis of their level of sequence identity to each other ( table 3 ) . we observed a large reduction in duplications that fall within the 98 - 100% category , supporting the fact that the genome assemblies continue to improve and correct errors made . in addition , our data showed that there have been major improvements for chromosomes 5 , 6 , 7 , 13 , 14 and 19 over the last three genome assemblies . and for chromosomes that had reached finished status , such as chromosomes 20 , 21 and 22 , the number of errors was negligible . ambsnps are snps that were annotated to map to two locations on a particular chromosome in the ncbi dbsnp . here we show on a genome scale that ambsnps most specifically correlate with intrachromosomal segmental duplications , suggesting they are paralogous sequence variants ( psvs ) ( figure 1 ) . these psvs were perhaps mistakenly introduced into dbsnp by automated in silico - generated analysis , arising from nucleotide mismatches in paralogous copies of duplicated sequences . overall , a surprisingly high proportion , 8.6% ( 199,965 of 2,327,473 ) , of the refsnps were annotated as ambsnps from dbsnp ( build 108 ) . a significant number of the ambsnps ( 139,974 of 199,965 or 70.0% ) are located within duplicated regions as identified by blast and should be regarded as psvs ( see additional data file ) . the non - identification by blast analysis of regions that contain ambsnps could be due to one of three possibilities . first , the duplicated copy(s ) could have been removed from the sequence assembly or the two have been conflated , that is , mistakenly thought to be the same sequence owing to their high sequence similarity . a second possibility is that the duplication is smaller than 5 kb and was excluded in our blast analysis . a third possibility is that a collection of ambsnps could have been generated from misassigned sequences ( identical sequences but misassigned to two difference locations in the genome ) in older assembly builds due to sequencing errors or true snps ( with high polymorphism rate ) in the sequence . we also observed that the density of ambsnps generally correlates with the size of the putative duplication , although this might be affected by the level of sequence identity between duplications . for example , two duplicated sequences sharing 98% sequence identity compared to 95% over the same length might contain fewer psvs as the number of base - pair mismatches would be fewer in the former . in addition , we observed that regions identified by our blast method but that do not contain ambsnps often correspond to artifactual duplications generated from assembly errors . the size , orientation , and contents of segmental duplications are highly variable and most of them show great organizational complexity . this is perhaps due to successive transposition and rearrangement events leading to the creation of segmental duplications . in many cases , for example , one of the largest segmental duplicons detected in this study was 359 kb in size at the williams - beuren locus on 7q11.23 . in this case , the two duplicons are separated by 1.6 mb of intervening sequence with the telomeric duplicon comprising several separate smaller modules as compared to the primary duplicon . the results presented in our study ( provided in tables available at ) would also allow rapid identification of new duplicons that are potentially responsible for chromosome rearrangements and genomic disorders . for example , the location of the duplicons on chromosomes 9q34/22 q11 that have been suggested to mediate recombination leading to the philadelphia chromosome was identified in our analysis , as were other medically relevant chromosomal regions ( table 4 ) . the characterization of most large segmental duplications is complicated by the fact that many of them ( 29% of all duplications ) are only represented as draft sequences from the current genome assembly . despite the fact that both blast and psvs analyses allowed us to identify most segmental duplications involved in known genomic disorder mutations ( table 4 ) , estimations of the size of rearranged regions were different from those previously reported . in fact , with the exception of several small duplications and the segmental duplications on chromosome 22 , other regions containing duplications involved in genomic disorders were often erroneously assembled and misplaced . furthermore , we have searched the celera human genome c3 ( publicly released version ) and c4 ( subscription - based version ) sequence assemblies for large duplications found on chromosome 7 . we observed that most of them were not represented in large scaffolds , but instead were located in their sequence gaps , or only partially found at ends of scaffolds leading into gaps ( see table 4 ) . this suggests that the whole - genome assembly approach alone might not be able to finish such duplicated regions in mammalian genomes . we have used two different computational approaches to identify the locations of all recent segmental duplications in the current human genome draft sequence . the fidelity of the results reflects the quality of the assembly examined and the parameters used . in addition , our approach has detected numerous potential sequence misassignment errors in the current genome annotation , allowing rapid error detection in future sequence assemblies . the segmental duplication map of the human genome should serve as a guide for investigation of the role of duplications in genomic disorders , as well as their contributions to normal human genomic variability . it is clear that genomic regions containing segmental duplications present a major challenge to the completion of the human genome sequence by april 2003 . focused efforts including targeted sequencing of allele - specific clones , high - resolution fluorescence in situ hybridization , and expert curation would be required to validate the actual ( or proposed ) organization of these complex regions as well as to complete the human genome reference sequence . we obtained the december 2001 , april 2002 , and june 2002 ( ncbi build 28 , 29 and 30 respectively ) human genome assemblies through the university of california , santa cruz human genome browser . all chromosome sequences were lower - case masked for highly repetitive elements by repeatmasker ( a.f.a . each of the 25 masked chromosome sequences ( including one unmapped chromosome sequence ' chrun ' ) was compared against itself by chromosome - wide blast2 to detect intrachromosomal segmental duplications ( 25 comparisons made ) , as well as pairwise comparisons to each of the other 24 chromosomes to detect interchromosomal segmental duplications ( 600 comparisons made ) . all blast results were subsequently parsed to eliminate low - quality and fragmented alignments under the following criteria : blast results having 90% sequence identity , 80 bp in length , and with expected value 10 . all identical hits ( same coordinate alignments ) , including suboptimal blast alignments recognized by multiple , overlapping alignments , as well as mirror hits ( reverse coordinate alignments ) from the blast results of the intrachromosomal set were removed . contiguous alignments separated by a distance of less than 3 kb , then 5 kb , and subsequently 9 kb were joined ( stepwise ) into modules in order to traverse masked repetitive sequences and to overcome breaks in the blast alignments caused by insertions / deletions and sequence gaps . such contiguous sequence alignment modules represent sequence similarity between the subject and query chromosome sequence in question ( at their respective positional coordinates ) . this pairwise sequence comparison procedure serves as a rapid and robust way to detect duplication relationships . however , because of the use of masked sequences , our method would only yield a poor ( on average 0.1 - 0.5 kb ) resolution for the determination of the precise duplication alignment boundaries . results were classified as either duplications or ' questionable ' results based on sequencing status of the region and the percent sequence similarity between the detected alignments . questionable duplications are results that fall within regions containing draft sequences with > 99.5 % detected sequence identity with another region . we consider these questionable duplications to be involved in potential sequence misassignment errors in the human genome assembly and would require further effort to achieve resolution . detailed information regarding segmental duplications as well as potential sequence misassignment errors identified by our analysis were presented using the generic genome browser . we have also summarized our results in table formats that include information on size of duplications , chromosomal band locations , level of identity between duplicated copies , sequenced clones ( accession numbers ) and their sequencing status , as well as genes mapped to these regions . in addition , we have plotted the size of each intrachromosomal duplication ( y - axis ) against its chromosome position ( x - axis ) along each chromosome to indicate the intrachromosomal segmental duplication content of each chromosome ( figure 1 ) using the publicly available visualization tool genomepixelizer . results generated from the detection of segmental duplications were subsequently converted into coordinate files as input for display using genomepixelizer . each chromosome snp table , containing annotation regarding ambsnps that have appeared twice in a particular chromosome , were extracted and sorted along with their corresponding chromosomal positions . the number of ambsnps was tabulated along a 10-kb window to produce density plots of ambsnps along the length of each chromosome ( figure 1 ) . we obtained the december 2001 , april 2002 , and june 2002 ( ncbi build 28 , 29 and 30 respectively ) human genome assemblies through the university of california , santa cruz human genome browser . all chromosome sequences were lower - case masked for highly repetitive elements by repeatmasker ( a.f.a . each of the 25 masked chromosome sequences ( including one unmapped chromosome sequence ' chrun ' ) was compared against itself by chromosome - wide blast2 to detect intrachromosomal segmental duplications ( 25 comparisons made ) , as well as pairwise comparisons to each of the other 24 chromosomes to detect interchromosomal segmental duplications ( 600 comparisons made ) . all blast results were subsequently parsed to eliminate low - quality and fragmented alignments under the following criteria : blast results having 90% sequence identity , 80 bp in length , and with expected value 10 . each blast report was sorted by chromosomal coordinates . all identical hits ( same coordinate alignments ) , including suboptimal blast alignments recognized by multiple , overlapping alignments , as well as mirror hits ( reverse coordinate alignments ) from the blast results of the intrachromosomal set were removed . contiguous alignments separated by a distance of less than 3 kb , then 5 kb , and subsequently 9 kb were joined ( stepwise ) into modules in order to traverse masked repetitive sequences and to overcome breaks in the blast alignments caused by insertions / deletions and sequence gaps . such contiguous sequence alignment modules represent sequence similarity between the subject and query chromosome sequence in question ( at their respective positional coordinates ) . this pairwise sequence comparison procedure serves as a rapid and robust way to detect duplication relationships . however , because of the use of masked sequences , our method would only yield a poor ( on average 0.1 - 0.5 kb ) resolution for the determination of the precise duplication alignment boundaries . results were classified as either duplications or ' questionable ' results based on sequencing status of the region and the percent sequence similarity between the detected alignments . questionable duplications are results that fall within regions containing draft sequences with > 99.5 % detected sequence identity with another region . we consider these questionable duplications to be involved in potential sequence misassignment errors in the human genome assembly and would require further effort to achieve resolution . detailed information regarding segmental duplications as well as potential sequence misassignment errors identified by our analysis were presented using the generic genome browser . we have also summarized our results in table formats that include information on size of duplications , chromosomal band locations , level of identity between duplicated copies , sequenced clones ( accession numbers ) and their sequencing status , as well as genes mapped to these regions . in addition , we have plotted the size of each intrachromosomal duplication ( y - axis ) against its chromosome position ( x - axis ) along each chromosome to indicate the intrachromosomal segmental duplication content of each chromosome ( figure 1 ) using the publicly available visualization tool genomepixelizer . results generated from the detection of segmental duplications were subsequently converted into coordinate files as input for display using genomepixelizer . each chromosome snp table , containing annotation regarding ambsnps that have appeared twice in a particular chromosome , were extracted and sorted along with their corresponding chromosomal positions . the number of ambsnps was tabulated along a 10-kb window to produce density plots of ambsnps along the length of each chromosome ( figure 1 ) . the following additional data are availiable as a single file with this article : a table describing the relative frequency increase of repeat types at duplicon junctions ( additional data file 1 ) ; a table giving the number of ambiguousgly mapped snps within segmental duplications ( number of psvs ) ( additional data file 1 ) ; a figure ( additional data file 1 ) showing functional profiling of genes involved in recent whole - gene duplication vs human genome average . this work was supported by the canadian institutes of health research ( cihr ) and genome canada to s.w.s . and x.e . is a senior scientist at the centre de regulaci genmica ( crg ) and a visiting scientist of the hospital for sick children research institute . s.w.s . is a scholar of cihr and international scholar of the howard hughes medical institute . left , graphical views of the paralogous relationships between recent segmental duplications ( graphics produced using genomepixelizer ; each line represents a duplicated module ; coloring scheme , red = 99% to 100% sequence identity , purple = 96% to 98% , green = 93% to 95% , and blue = 90% to 92% ) . middle panel : segmental duplications as detected by blast analysis ( size of duplication in kb plotted against the length of chromosome in mb ) . right panel : ambsnps density plot ( number of ambsnps plotted against the length of chromosome in mb ) . ac121339 is incorrectly mapped to 3q13.13 in the june 2002 human genome assembly as shown by a consensus number of chromosome x sts markers . segmental duplication content of the human genome * previous data on segmental duplications distributed by chromosomes as reported in . comparison of duplications and potential sequence misassignment errors in genome assemblies * all numbers shown in the table are 100 kb . * segmental duplications involved in known genomic disorders and chromosome rearrangements identified by blast and ambsnp analyses this table represents a partial list of all known genomic disorders and chromosome rearrangements . * only the start coordinates ( based on june 2002 assembly ) for duplicons are shown . results from blast analysis with chromosome coordinates and size of duplicon . for several genomic mutations ( williams - beuren syndrome , prader - willi syndrome and angelman syndromes , and digeorge syndrome ) the duplicons shown are incomplete , most of which are composed of several duplication modules . the ' - ' sign indicates that the second duplicon is in the inverse orientation . the number of ambsnps ( ambiguously mapped single - nucleotide polymorphisms ) found within the genomic segment . the ambsnp analysis defines regions containing high densities of contiguous ambsnps . for some of the segmental duplications involved in genomic disorders , the contiguous lengths of ambsnps are much larger than those detected by blast . the specific sizes of the segmental duplications have to be resolved by detailed characterization of the different modules . celera representation : s , both copies found in large ( > 500 kb ) sequence scaffolds ; p , partially hit , single copy found , or less than perfect alignments ; m , missing from large sequence scaffolds , hitting numerous fragments .

rapid computational heuristics have been developed based on blast analysis to detect segmental duplications , as well as regions containing potential sequence misassignments in the human genome assemblies . most of the sequences in the human genome that have undergone recent segmental duplications have now been identified .

cryptogenic organizing pneumonia ( cop ) , also known as bronchiolitis obliterans organizing pneumonia ( boop ) , is a relatively rare disorder with distinctive clinical , imaging and pathological features . the pathological findings are characterized by plugs of granulation tissue lying within small airways , alveolar ducts and alveoli and by chronic inflammatory cell infiltration in the alveolar walls . cop is defined as idiopathic and any cause for the development of pneumonia such as infection or underlying tissue disease is excluded . patients with cop manifest rapid clinical and imaging improvement with corticosteroid therapy , but suffer from frequent relapses . through histopathological examination and clinical observation , uveitis is usually classified into two categories , granulomatous and nongranulomatous . hypopyon is the accumulation of white blood cells in the anterior chamber and is often observed in nongranulomatous uveitis . nongranulomatous presentations are mostly idiopathic or due to hla - b27 antigen involvement [ 2 , 3 , 4 ] . it is well known that systemic diseases that can be associated with nongranulomatous uveitis include behet 's disease , ankylosing spondylitis , inflammatory bowel disease and psoriasis . although the cause is not fully disclosed , certain ocular and systemic conditions might be the underlying association with nongranulomatous uveitis . in this report , we describe two cases of cop who developed bilateral anterior uveitis with hypopyon unresponsive to topical corticosteroid therapy but responsive to systemic corticosteroid therapy . in addition , the clinical condition of their cop was concomitant to that of their intraocular inflammation . a 65-year - old woman was referred for evaluation of bilateral decreased vision and glaucoma . she had undergone a series of treatment for breast cancer with surgery , radiation and chemotherapy 14 years earlier . concerning her ocular history , she had been diagnosed as having an epiretinal macular membrane on her right eye and received a vitrectomy and phacoemulsification 3 years prior . the intraocular inflammation was resistant to topical betamethasone administration but decreased spontaneously in 1 year . despite the uveitis being mostly controlled , the intraocular pressure in both eyes had gradually increased and she was referred to our hospital . the initial ophthalmic examination disclosed a best - corrected visual acuity ( bcva ) of 20/50 in the right eye and 20/80 in the left eye . the intraocular pressure was 25 mm hg in the right and 40 mm hg in the left eye . slit - lamp examination demonstrated no sign of uveitis or retinal disease besides pseudophakia in the right eye and a cataract in the left eye . the results of the laboratory investigations of serum including angiotensin - converting enzyme , antinuclear antibodies , rheumatoid factor and antineutrophil cytoplasmic antibodies were unremarkable despite for the presence of elevated c - reactive protein . serological analyses indicated that there was no active infection of syphilis , human t - cell lymphoma virus 1 , herpes simplex virus or varicella zoster virus . the results of human leukocyte antigen type b testing were positive for b-60 and b-61 . after admission , the patient underwent phacoemulsification surgery with intraocular lens implantation and a viscocanalostomy in the left eye for her cataract and glaucoma . her bcva improved to 20/40 in the right eye and 20/32 in the left eye , and both the intraocular inflammation and intraocular pressure were controlled . five months later , the cellular infiltration with hypopyon in the anterior chamber was seen in both eyes ( fig . then , the patient received systemic prednisolone ( psl ) 40 mg / day administered orally to treat the aggravated cop , and both lung symptoms and intraocular inflammation diminished . when psl dosage was reduced to 5 mg / day during tapering , cellular infiltration in the anterior chamber and hypopyon relapsed and bcva of her eyes , particularly of her left eye , declined despite administration of topical dexamethasone . an optical coherence tomography ( oct ) examination showed a macular edema in her left eye ( fig . although a subconjunctival triamcinolone injection was given when her left bcva reached 20/200 , there was little improvement . then , oral psl was increased to 30 mg / day , and the intraocular inflammation subsequently decreased and her bcva gradually recovered ( fig . , yttrium - aluminum - argon ( yag ) laser treatment was performed to treat posterior capsule opacification in her left eye . during the clinical course , there was temporal intraocular pressure elevation , which could be maintained by topical antiglaucoma medication . at the middle of the relapse period , cytological examination of infiltrated cells in the anterior chamber showed neutrophils and a few lymphocytes , but no atypical cell . the genomic dna in aqueous humor was analyzed to screen infectious pathogens including bacteria , parasites , and viruses by comprehensive polymerase chain reaction . , there were no physical findings of skin disease , oral aphtha , pudendal ulcers , joint inflammation , spondylitis or digestive symptoms . a 69-year - old woman presented with blurred vision in both eyes . her ocular history expressed age - related macular degeneration in both eyes 6 years prior . subsequently , she was referred to our hospital . the initial ophthalmic examination disclosed a bcva of 20/25 in the right eye and 20/20 in the left eye . slit - lamp examination demonstrated bilateral fine keratic precipitates , hypopyon and intensive cell infiltration in the anterior chamber . there was a mild cataract but no sign of posterior uveitis or diabetic change in the retina . 3 . the results of the laboratory investigations of serum including angiotensin - converting enzyme , antinuclear antibodies , rheumatoid factor , and antineutrophil cytoplasmic antibodies were unremarkable despite for the presence of elevated c - reactive protein and hba1c ( 7.1% ) . serological analyses showed no active infection of syphilis , human t - cell lymphoma virus 1 , herpes simplex virus or varicella zoster virus . the results of human leukocyte antigens type b testing were positive for b-51 and b-61 . one month later , she was administered systemic psl 40 mg / day orally to treat cop . the intraocular cell infiltration and hypopyon diminished in 1 month . during psl an oct examination showed an epiretinal membrane and macular edema in both eyes ( fig . the psl administration was reduced gradually and then halted , with no recurrence of uveitis . therefore , the cytological examination of infiltrated cells in the anterior chamber or the genomic dna analysis of infectious pathogens was not performed . during observation , there were no physical findings of skin disease , oral aphtha , pudendal ulcers , joint inflammation , spondylitis or digestive symptoms . a 65-year - old woman was referred for evaluation of bilateral decreased vision and glaucoma . she had undergone a series of treatment for breast cancer with surgery , radiation and chemotherapy 14 years earlier . concerning her ocular history , she had been diagnosed as having an epiretinal macular membrane on her right eye and received a vitrectomy and phacoemulsification 3 years prior . the intraocular inflammation was resistant to topical betamethasone administration but decreased spontaneously in 1 year . despite the uveitis being mostly controlled , the intraocular pressure in both eyes had gradually increased and she was referred to our hospital . the initial ophthalmic examination disclosed a best - corrected visual acuity ( bcva ) of 20/50 in the right eye and 20/80 in the left eye . the intraocular pressure was 25 mm hg in the right and 40 mm hg in the left eye . slit - lamp examination demonstrated no sign of uveitis or retinal disease besides pseudophakia in the right eye and a cataract in the left eye . the results of the laboratory investigations of serum including angiotensin - converting enzyme , antinuclear antibodies , rheumatoid factor and antineutrophil cytoplasmic antibodies were unremarkable despite for the presence of elevated c - reactive protein . serological analyses indicated that there was no active infection of syphilis , human t - cell lymphoma virus 1 , herpes simplex virus or varicella zoster virus . the results of human leukocyte antigen type b testing were positive for b-60 and b-61 . after admission , the patient underwent phacoemulsification surgery with intraocular lens implantation and a viscocanalostomy in the left eye for her cataract and glaucoma . her bcva improved to 20/40 in the right eye and 20/32 in the left eye , and both the intraocular inflammation and intraocular pressure were controlled . five months later , the cellular infiltration with hypopyon in the anterior chamber was seen in both eyes ( fig . then , the patient received systemic prednisolone ( psl ) 40 mg / day administered orally to treat the aggravated cop , and both lung symptoms and intraocular inflammation diminished . when psl dosage was reduced to 5 mg / day during tapering , cellular infiltration in the anterior chamber and hypopyon relapsed and bcva of her eyes , particularly of her left eye , declined despite administration of topical dexamethasone . an optical coherence tomography ( oct ) examination showed a macular edema in her left eye ( fig . although a subconjunctival triamcinolone injection was given when her left bcva reached 20/200 , there was little improvement . then , oral psl was increased to 30 mg / day , and the intraocular inflammation subsequently decreased and her bcva gradually recovered ( fig . , yttrium - aluminum - argon ( yag ) laser treatment was performed to treat posterior capsule opacification in her left eye . during the clinical course , there was temporal intraocular pressure elevation , which could be maintained by topical antiglaucoma medication . at the middle of the relapse period , cytological examination of infiltrated cells in the anterior chamber showed neutrophils and a few lymphocytes , but no atypical cell . the genomic dna in aqueous humor was analyzed to screen infectious pathogens including bacteria , parasites , and viruses by comprehensive polymerase chain reaction . , there were no physical findings of skin disease , oral aphtha , pudendal ulcers , joint inflammation , spondylitis or digestive symptoms . she had undergone treatment for ovarian cancer with surgery 8 years earlier . in addition , she had been regularly examined and treated for cop and nephrolithiasis . her ocular history expressed age - related macular degeneration in both eyes 6 years prior . the initial ophthalmic examination disclosed a bcva of 20/25 in the right eye and 20/20 in the left eye . slit - lamp examination demonstrated bilateral fine keratic precipitates , hypopyon and intensive cell infiltration in the anterior chamber . there was a mild cataract but no sign of posterior uveitis or diabetic change in the retina . 3 . the results of the laboratory investigations of serum including angiotensin - converting enzyme , antinuclear antibodies , rheumatoid factor , and antineutrophil cytoplasmic antibodies were unremarkable despite for the presence of elevated c - reactive protein and hba1c ( 7.1% ) . serological analyses showed no active infection of syphilis , human t - cell lymphoma virus 1 , herpes simplex virus or varicella zoster virus . the results of human leukocyte antigens type b testing were positive for b-51 and b-61 . one month later , she was administered systemic psl 40 mg / day orally to treat cop . the intraocular cell infiltration and hypopyon diminished in 1 month . during psl an oct examination showed an epiretinal membrane and macular edema in both eyes ( fig . the psl administration was reduced gradually and then halted , with no recurrence of uveitis . therefore , the cytological examination of infiltrated cells in the anterior chamber or the genomic dna analysis of infectious pathogens was not performed . during observation , there were no physical findings of skin disease , oral aphtha , pudendal ulcers , joint inflammation , spondylitis or digestive symptoms . in the present study , we described two cases that had common points in their phenotype . first , both cases showed bilateral anterior uveitis with hypopyon . their intraocular inflammation was unresponsive to the administration of topical corticosteroids but showed a greater response to systemic corticosteroid administration ( sca ) . furthermore , both patients were receiving treatments for cop , and had medical histories of malignant tumors . however , neither was identified with any systemic disease that could be associated with uveitis . nongranulomatous uveitis is characterized by fine keratic precipitates , and in severe cases , fibrinous clotting or hypopyon in the anterior chamber . the most common form of nongranulomatous anterior uveitis is acute anterior uveitis , which is associated with the hla - b27 allele [ 2 , 3 , 5 ] . the ocular feature of hla - b27-associated acute anterior uveitis is acute onset of unilateral inflammation , and is common in males between the ages of 2040 years . in this series , both cases were hla - b27 negative , and the uveitis was bilateral . other diseases that could be associated with uveitis were screened . by the absence of systemic symptoms such as skin lesion , genital ulcer , oral aphtha and digestive symptoms , the laboratory investigations also eliminated the involvement of ankylosing spondylitis , arthritis , inflammatory bowel disease , sarcoidosis and infection . in addition , several drugs including cidofovir and rifabutin are reported to induce uveitis . in the present study , neither patient had taken those medicines during the period in which they contracted uveitis . it is also known that the malignant neoplasm can introduce hypopyon that resembles intraocular inflammation [ 7 , 8 , 9 ] . since they had not shown any recurrence for several years , it was assumed that their malignancies were controlled . additionally , the cytological analysis of aqueous humor with cell infiltration in case 1 showed a moderate inflammation pattern without neoplastic cell . through the follow - up period of ovarian cancer without anticancer therapy in case 2 , there was no sign of recurrent cancer or metastasis . from these facts , it is unlikely that their malignancy is associated with uveitis in their eyes . cop is diagnosed by its typical pathological findings of organizing pneumonia and the absence of any identified cause . the organizing pneumonia occurs in cases of autoimmune diseases such as rheumatoid arthritis and sjgren 's syndrome . several reports have described how cop accompanies uveitis as a part of behet 's disease [ 12 , 13 ] . in those cases , although the uveitis in our report developed concomitant to cop , there was no clinical indication to suggest a diagnosis of behet 's disease . additionally , the anterior uveitis in our patients was resistant to topical corticosteroid drops , which rarely occurs during anterior uveitis care . this suggests that anterior uveitis in our cases was associated with cop . at present , there is one report that describes the complications of cop and uveitis without any cause . in this case , bilateral nongranulomatous uveitis was unresponsive to topical corticosteroid treatment , but systemic corticosteroid treatment had a more positive effect . the present findings suggest the possibility that there is a novel cause of uveitis that shares the same etiology with cop . further investigation with increased numbers of case studies and statistical analysis may result in the discovery of new symptoms for uveitis and cop .

two female patients with histories of cancer who showed cryptogenic organizing pneumonia ( cop ) complications and bilateral anterior uveitis with hypopyon were examined . both patients had suffered from cop and received intermitted systemic corticosteroid administration ( sca ) . the first patient , a 65-year - old woman with a history of breast cancer , showed bilateral uveitis with hypopyon . the topical corticosteroid treatment was ineffective . after sca for the treatment of cop was started , the hypopyon gradually dissipated . upon termination of sca , uveitis relapses were controlled by renewed sca . the other patient , a 69-year - old woman with a history of ovarian cancer , showed bilateral anterior uveitis with hypopyon . her intraocular outcome did not improve by the topical corticosteroid administration , but sca that was applied to treat cop led to remission of uveitis . imaging examinations , biochemical analysis , symptoms or hla - b27 antigen screenings in either patient did not explain the development of uveitis . bilateral anterior uveitis is commonly related to autoimmune disease or systemic syndrome . we report two cases with cop that developed bilateral anterior uveitis with hypopyon resistant to topical administration but responsive to systemic administration of corticosteroid . these findings suggest that cop can be associated with the etiology of anterior uveitis .

diagnostic lumbar puncture ( dlp ) is a frequent procedure used in the diagnosis of neural system disorders . post - lumbar puncture syndrome is a common complication of dlp occurring in about 340% of patients [ 14 ] . post - lumbar puncture headache ( plph ) is the predominant symptom of this syndrome , probably resulting from the leakage of cerebrospinal fluid into the epidural cavity through the post - puncture opening in the dura mater . the loss of cerebrospinal fluid causes a drop off in intracranial pressure ( icp ) , causing compensative dilatation of cerebral vessels , which is reflected by the headache [ 13 , 5 ] . this theory has one significant weakness , namely that plph do not occur in every patient following dlp . transcranial doppler ultrasonography ( tcd ) enables us to assess blood flow in the intracranial arteries non - invasively . assuming that tcd parameters are influenced both by changes in cerebral vessel diameters and by fluctuations in icp [ 68 ] , we aimed to verify if dlp resulted in significant changes in cerebral blood flow ( cbf ) that could be visualized by tcd . therefore , we identified a group of subjects who developed plph following dlp and compared their pre- and post - puncture blood flow parameters with respective values of plph - free subjects . this study included patients at the department of neurology , who required a scheduled dlp . exclusion criteria from this group included emergency lumbar punctures , inadequate temporal bone windows , stenosis of the middle cerebral arteries , hemodynamically significant stenosis of the common carotid arteries , atrial fibrillation , and patients who were immobilized . all the procedures were approved by the local ethics committee of the ludwik rydygier collegium medicum in bydgoszcz . tcd was performed 24 h before dlp and repeated within 24 h after the procedure . lumbar puncture was done following standard protocol by one of 11 physicians employed at the department . plph was diagnosed according to the international classification of headache disorders ( ich - ii ) guidelines . the presence and severity of plph was evaluated for 5 days , on a daily basis , using a numeric rating scale ( nrs ) . sixty - six patients were enrolled to this study , including 37 men and 29 women . the mean age of these patients was 47.4 16.5 years ( range from 16 to 88 , table 1 ) . patients were subjected to diagnostic procedures due to cranial mononeuropathy ( n = 12 ) , polyneuropathy ( n = 7 ) , multiple sclerosis ( n = 13 ) , vertigo ( n = 7 ) , diffuse injuries of the brain or spinal cord ( n = 9 ) , epilepsy ( n = 5 ) , and other reasons.table 1clinical characteristics of patients who developed post - lumbar puncture headache ( plph ) and in plph - free individualsparameterplph - free ( n = 45)plph ( n = 21)p valueage in years , mean sd49.3 17.843.2 12.50.16women , n ( % ) 13 ( 28.9)16 ( 76.2)0.00031men , n ( % ) 32 ( 71.1)5 ( 23.8)0.00026history of headache , n ( % ) 9 ( 20)14 ( 66.7)0.00021 tension - type headache , n ( % ) 6 ( 13.3)11 ( 52)0.00034 migraine without aura , n ( % ) 3 ( 6.7)2 ( 9.5)0.21 other type of headache , n ( % ) 0 ( 0)1 ( 4.8)arterial hypertension , n ( % ) 12 ( 26.67)3 ( 14.29)0.26diabetes , n ( % ) 9 ( 20)2 ( 9.5)0.29hypercholesterolemia , n ( % ) 15 ( 33.3)4 ( 19.1)0.23hematocrit in % , mean sd41.8 3.339.1 3.90.006hemoglobin in g / l , mean sd14.2 1.313.0 1.50.001 clinical characteristics of patients who developed post - lumbar puncture headache ( plph ) and in plph - free individuals lumbar puncture was performed in the lateral recumbent position , using the intervertebral space between l4 and l5 or between l5 and s1 , with the aid of a 22-gauge needle ( bd spinal needle quincke type point ) . all the procedures were performed between 11 and 12 a.m. following the procedure , the patients were ordered to stay in bed for 8 h. tcd was performed using a pioneer apparatus ( nicolet / eme ) with a 2-mhz probe . following localization of the temporal window , the middle cerebral artery ( mca ) was identified . the probe was placed so as to record the peak possible velocity from the vessel . the measurements included mean velocity ( vmean ) , peak systolic velocity ( vmax ) , and gosling s pulsatility index ( pi ) at 5456 mm , in the left and right mca . all tcds were performed by the same physician being experienced in neurosonology and unaware of plph presence and severity in patients examined . mean values of normally distributed variables were compared with student s t test for independent samples , whereas the variables whose distributions were not normal were compared with the non - parametric mann distributions of discrete variables were compared between the groups with the aid of test . calculations were performed using statistica 7 ( statsoft , poland ) software , and statistical significance was defined as p 0.05 . this study included patients at the department of neurology , who required a scheduled dlp . exclusion criteria from this group included emergency lumbar punctures , inadequate temporal bone windows , stenosis of the middle cerebral arteries , hemodynamically significant stenosis of the common carotid arteries , atrial fibrillation , and patients who were immobilized . all the procedures were approved by the local ethics committee of the ludwik rydygier collegium medicum in bydgoszcz . tcd was performed 24 h before dlp and repeated within 24 h after the procedure . lumbar puncture was done following standard protocol by one of 11 physicians employed at the department . plph was diagnosed according to the international classification of headache disorders ( ich - ii ) guidelines . the presence and severity of plph was evaluated for 5 days , on a daily basis , using a numeric rating scale ( nrs ) . sixty - six patients were enrolled to this study , including 37 men and 29 women . the mean age of these patients was 47.4 16.5 years ( range from 16 to 88 , table 1 ) . patients were subjected to diagnostic procedures due to cranial mononeuropathy ( n = 12 ) , polyneuropathy ( n = 7 ) , multiple sclerosis ( n = 13 ) , vertigo ( n = 7 ) , diffuse injuries of the brain or spinal cord ( n = 9 ) , epilepsy ( n = 5 ) , and other reasons.table 1clinical characteristics of patients who developed post - lumbar puncture headache ( plph ) and in plph - free individualsparameterplph - free ( n = 45)plph ( n = 21)p valueage in years , mean sd49.3 17.843.2 12.50.16women , n ( % ) 13 ( 28.9)16 ( 76.2)0.00031men , n ( % ) 32 ( 71.1)5 ( 23.8)0.00026history of headache , n ( % ) 9 ( 20)14 ( 66.7)0.00021 tension - type headache , n ( % ) 6 ( 13.3)11 ( 52)0.00034 migraine without aura , n ( % ) 3 ( 6.7)2 ( 9.5)0.21 other type of headache , n ( % ) 0 ( 0)1 ( 4.8)arterial hypertension , n ( % ) 12 ( 26.67)3 ( 14.29)0.26diabetes , n ( % ) 9 ( 20)2 ( 9.5)0.29hypercholesterolemia , n ( % ) 15 ( 33.3)4 ( 19.1)0.23hematocrit in % , mean sd41.8 3.339.1 3.90.006hemoglobin in g / l , mean sd14.2 1.313.0 1.50.001 clinical characteristics of patients who developed post - lumbar puncture headache ( plph ) and in plph - free individuals lumbar puncture was performed in the lateral recumbent position , using the intervertebral space between l4 and l5 or between l5 and s1 , with the aid of a 22-gauge needle ( bd spinal needle quincke type point ) . the volumes of collected cerebral fluid ranged from around 610 ml . all the procedures were performed between 11 and 12 a.m. following the procedure , the patients were ordered to stay in bed for 8 h. tcd was performed using a pioneer apparatus ( nicolet / eme ) with a 2-mhz probe . following localization of the temporal window , the middle cerebral artery ( mca ) was identified . the probe was placed so as to record the peak possible velocity from the vessel . the measurements included mean velocity ( vmean ) , peak systolic velocity ( vmax ) , and gosling s pulsatility index ( pi ) at 5456 mm , in the left and right mca . all tcds were performed by the same physician being experienced in neurosonology and unaware of plph presence and severity in patients examined . mean values of normally distributed variables were compared with student s t test for independent samples , whereas the variables whose distributions were not normal were compared with the non - parametric mann distributions of discrete variables were compared between the groups with the aid of test . calculations were performed using statistica 7 ( statsoft , poland ) software , and statistical significance was defined as p 0.05 . post - lumbar puncture headache was observed in 21 out of 66 patients who underwent dlp ( 32% ) , whereas symptoms of post - lumbar puncture syndrome were not noted in the remaining 45 subjects ( 68% ) . in most patients ( 86% ) , the symptoms manifested on the first day following dlp , on average 17.7 10.2 h after the procedure ( range 744 h ) . plph persisted 5.5 1.8 days on average ( range 210 days ) . female patients predominated amongst the plph cases ( 16 , compared to five men , p < 0.0003 ) . most patients who developed plph ( n = 14 ) had a history of headaches , mostly tension headaches . the mean age of patients with plph was lower than for unaffected individuals ( 43.2 vs. 49.3 years ) , but this difference was insignificant ( table 1 ) . in both groups of patients , no significant differences were observed in vmean , vmax and pi between the right and left mcas both before dlp and following this procedure ( table 2).table 2pre- and post - puncture values ( mean sd ) of mean velocity ( vmean ) , peak systolic velocity ( vmax ) , and gosling s pulsatility index ( pi ) in the right ( mca r ) and left ( mca l ) middle cerebral arteries of patients who developed post - lumbar puncture headache ( plph ) and in plph - free individualsplph - free ( n = 45)plph ( n = 21)p valuepre - puncture vmean , mca r ( m / s)55.2 9.771.9 24.90.0002post - puncture vmean , mca r ( m / s)55.5 12.658.4 20.20.47p value0.860.0004pre - puncture vmean , mca l ( m / s)55 12.0169.12 22.560.0015post - puncture vmean , mca l ( m / s)54.2 13.259.4 19.10.19p value0.700.008pre - puncture vmax , mca r ( m / s)89.9 16.2109.3 36.90.004post - puncture vmax , mca r ( m / s)91.1 21.589.8 30.10.84p value0.700.0002pre - puncture vmax , mca l ( m / s)89.8 19.4104.3 32.20.03post - puncture vmax , mca l ( m / s)88.7 20.490.7 28.00.74p value0.740.009pre - puncture pi , mca r0.99 0.200.86 0.140.01post - puncture pi , mca r1.0 0.210.89 0.120.03p value0.460.41pre - puncture pi , mca l0.98 0.220.84 0.130.01post - puncture pi , mca l0.99 0.230.86 0.130.01p value0.430.65 pre- and post - puncture values ( mean sd ) of mean velocity ( vmean ) , peak systolic velocity ( vmax ) , and gosling s pulsatility index ( pi ) in the right ( mca r ) and left ( mca l ) middle cerebral arteries of patients who developed post - lumbar puncture headache ( plph ) and in plph - free individuals in patients who developed plph , bilateral pre - puncture values of vmean and vmax were significantly higher and pi was significantly lower compared to unaffected individuals . no significant differences were observed between these groups in terms of post - puncture vmean and vmax , but the post - puncture pi was still significantly lower in plph cases ( table 2 ) . moreover , hemoglobin concentration and hematocrit values were significantly lower in patients who developed headache compared to unaffected individuals ( table 1 ) . in plph cases , the post - puncture values of vmean and vmax were significantly lower than the respective baseline parameters . significant puncture - related changes in bilateral vmean and vmax were not observed in patients who did not develop plph . after the puncture , a slight increase in pi was observed bilaterally in both groups , but these changes lacked statistical significance ( table 2 ) . when study results were stratified according to patient gender , significant post - puncture changes in vmean and vmax were observed only in women ( p < 0.01 and p < 0.07 in right and left mca , respectively ) . a significant inverse correlation was present between plph severity on nrs and bilateral pre - puncture pi ( fig . 1).fig . 1correlation between post - lumbar puncture headache ( plph ) severity on numeric rating scale and pre - puncture gosling s pulsatility index ( pi ) in the left middle cerebral artery correlation between post - lumbar puncture headache ( plph ) severity on numeric rating scale and pre - puncture gosling s pulsatility index ( pi ) in the left middle cerebral artery post - lumbar puncture headache is believed to result from cerebrospinal fluid leakage through the post - puncture opening , with a subsequent decrease in intracranial pressure and cerebral vessel dilatation [ 13 , 5 ] . this theory was confirmed by our study . following the puncture , we observed significant decreases in vmean and vmax in bilateral mcas of patients who developed plph when compared to their respective pre - puncture values . the drop off in vmean and vmax observed in this group of patients may result from the dilatation of intracranial vessels , a consequence of lowered icp . similar puncture - related decreases in vmax in patients with plph were previously described by gobel et al . , but only affecting the right mca . in our study , significant puncture - related changes in vmean and vmax were not observed in bilateral mcas of plph - free individuals . presumably , the loss of cerebrospinal fluid in these patients was not significant enough to be reflected by compensative dilatation of cerebral vessels . the value of pi illustrates the resistance of a vascular bed ( supplied by a given artery ) when examined by tcd . there is a linear relationship between changes in pi and icp . a decrease in pi may result both from lowered icp and from vascular dilatation [ 7 , 8 ] . assuming that the loss of cerebrospinal fluid with further decrease in icp is the reason for plph , pi should decrease during the early phase of post - lumbar puncture syndrome . reactive dilatation of cerebral vessels due to decreased icp should result in further pi drop off . we did not observe significant puncture - related changes in pi , both in patients who developed plph and in unaffected individuals . presumably , icp fluctuations were slight or transient and therefore we failed to record them . noticeably , baseline ( pre - puncture ) pi values in patients who later developed plph were significantly lower compared to subjects free of this condition . this finding suggests that compensative dilatation of cerebral vessels does not accompany every decrease in icp but is rather characterized by low values of this parameter . there exist linear relationships between pi or vmax changes and changes in icp in cerebral vessels . pi is known to decrease and vmax to increase with lowered icp [ 7 , 8 ] . thus , it is likely that the baseline icp in patients who later developed plph was lower than in unaffected individuals . consequently , further icp drop off due to cerebrospinal fluid loss might result in compensative vascular dilatation in patients with plph . a relatively higher baseline icp in plph - free individuals might in turn protect them against intracranial hypotension and resulting post - puncture headache . this theory was supported by a significant correlation observed in our study , between pre - puncture pi and plph severity : the lower the baseline pi ( and indirectly icp ) the more severe the plph . in cases with plph , baseline vmean and vmax values were higher and pi lower than in unaffected individuals . vmean and vmax are known to decrease with age [ 6 , 11 ] . consequently , higher baseline values of these parameters in plph patients might be associated with the higher age of this group , compared to unaffected individuals . in our study , age differences between these two groups were insignificant , and therefore , unlikely to be responsible for the results found in this experiment . the predominance of women amongst the plph patients might be another potential reason for flow velocity differences observed between affected and unaffected individuals . higher vmean and vmax values were previously demonstrated in females compared to males and these differences were attributed to higher values of cbf in women [ 11 , 12 ] . additionally , our study revealed significantly lower hematocrit and hemoglobin values in plph subjects than in unaffected individuals . a lowered hematocrit is known to enhance flow velocity , and decreases pi due to reduced resistance . consequently , decreased hematocrit values might be responsible for higher values of vmean and vmax and lower pi observed in the plph cases . , hematocrit values do not influence flow velocities unless they are below 35% . in conclusion , the findings of this study partially confirm the theory behind the etiology of plph stated in the introduction , and suggest that higher baseline values of vmean and vmax and low pi in bilateral mcas predispose patients to this condition . additional risk factors of plph were observed to include female gender , history of headaches , and decreased hematocrit and hemoglobin levels . finally , within the 24 h following puncture , significant decreases in vmean and vmax were observed in bilateral mcas of patients who developed plph .

the aim of this study was to verify if diagnostic lumbar puncture ( dlp ) in post - lumbar puncture headache ( plph ) patients is related to significant changes in cerebral blood flow which could be visualized by transcranial doppler ( tcd ) . sixty - six patients were enrolled in this study . tcd was performed 24 h before dlp and repeated within 24 h after the procedure . the measurements included mean velocity ( vmean ) , peak systolic velocity ( vmax ) , and gosling s pulsatility index ( pi ) , in the left and right middle cerebral artery ( mca ) . plph was observed in 21 patients ( 32% ) . no significant differences were noted in vmean , vmax and pi between the right and left mcas both before dlp and following this procedure . in patients who developed plph , bilateral pre - puncture values of vmean and vmax were significantly higher and pi was significantly lower compared to unaffected individuals . no significant differences were observed between these groups in terms of post - puncture vmean and vmax , but the post - puncture pi was still significantly lower in plph cases . in plph cases , the post - puncture values of vmean and vmax were significantly lower than the respective baseline parameters . a significant inverse correlation was present between plph severity and bilateral pre - puncture pi . in conclusion , this study revealed that higher baseline values of vmean and vmax and low pi in bilateral mcas predispose patients to plph .

cardiovascular disease ( cvd ) is the leading cause of mortality in both men and women in developed countries . nonetheless , sex - associated differences regarding the age of cvd onset and its progression are observed worldwide . incidence of cvd in premenopausal women is markedly lower than age - matched men in epidemiological studies ( messerli et al . , 1987 ; bairey merz et al . , 2006 ; shaw et al after menopause , however , the incidence is comparable or even higher in women than in men ( lerner and kannel , 1986 ; eaker et al . , 1993 ) , making cvd the primary cause of death in postmenopausal women ( 55 versus 43% in men ) , exceeding all cancer deaths ( rosamond et al . , 2008 ) . the lower cvd risk among fertile women is often attributed to the protective role of estrogens at the vascular level . according to epidemiological observations and extensive basic research , estrogen and other sex steroids estrogen modulates a myriad of molecular pathways that improve vascular function , whether at the physiological level or when administered as hormone replacement therapy ( hrt ; grodstein et al . , 2000 , 2001 ; miller and duckles , 2008 ) nevertheless , some clinical studies have questioned the protective value of hrt against vascular disease . two randomized clinical trials , the women s health initiative ( whi ; rossouw et al . , 2002 ) and the heart and estrogen / progestin replacement study ( hers i and ii ; gambacciani et al . , 2002 ) , indicate that hrt may increase cvd risk and events in postmenopausal women . the reason for this paradox could be attributable to many patient characteristics , including age . although aging occurs progressively in both men and women , the onset of menopause marks a sudden increase in the appearance of aging - associated signs in women , and more specifically in the progress of vascular aging . information about the role of age and menopause in the development of cvd in women is scarce . this review of clinical and experimental data on the effects of aging , estrogens , and hrt on vascular function of females aims to clarify how menopause and aging contribute jointly to vascular aging and how estrogen modulates vascular response at different ages . the numerous vascular effects of estrogens are triggered by complex genomic and non - genomic mechanisms . they include modulation of vascular function and inflammatory response as well as metabolic and hemodynamic effects . estradiol , the most abundant and potent estrogen in humans , mainly binds and activates estrogen receptors ( ers ) . vascular estrogen signaling involves at least three ers identified in both vascular smooth muscle and endothelium , reinforcing the idea that estrogen has a key role in controlling vascular function . the classical subtypes er ( soloff and szego , 1969 ) and er ( kuiper et al . , 1996 ) vary not only in their tissue distributions , but also in their agonist / antagonist profile with respect to several compounds ( cano and hermenegildo , 2000 ) . these er subtypes belong to the intracellular receptors classically defined as nuclear ligand - activated transcription factors . activation of these receptors by the corresponding hormones affects gene expression by acting on estrogen - response elements in the target genes and modulating transcriptional events ( beato et al . , 1995 ) . estrogen binding to er and er regulates gene expression in a time- and tissue - dependent manner , generating controversy about the type of receptor involved in vascular protection ( murphy , 2011 ) . in the cardiovascular system , both er and er have been identified in the endothelium , smooth muscle cells , adventitia , and adrenergic nerve endings of arteries from various territories and several species , including humans ( karas et al . , 1994 ; kim - schulze et al . , 1996 ; although it has been reported that cultured endothelial cells do not express er ( toth et al . , 2009 ) , other investigators have demonstrated the presence of both er and er mrna in endothelium ( wagner et al . , 2001 ) and data from our group demonstrate the protein expression of both er and er in huvec ( sobrino et al . , 2009 , 2010 ) . in addition to their classic nuclear location , er can also target the plasma membrane , enabling estrogen activation of several signaling pathways , including those involved in calcium mobilization ( zhang et al . prakash et al . , 1999 ) and the phosphatidylinositol-3-kinase ( pi3k ) pathway ( hisamoto et al . , a third type of er , g - protein coupled , and mainly located in the plasma membrane , was initially named gpr30 ( takada et al . , 1997 ) , then renamed gper by the international union of basic and clinical pharmacology , iuphar ( alexander et al . , 2008 ) . gper is expressed in both endothelial and smooth muscle cells of human arteries and veins ( haas et al . , gper activates rapid signaling cascades such as extracellular signal - related kinase and pi3k ( meyer et al . , 2011 ) . several rapid and non - genomic estrogen effects formerly attributed to er have now been described as gper - mediated ( prossnitz and barton , 2011 ) . the vascular protection conferred by estrogen may be mediated indirectly by its influence on the metabolism of lipoproteins or by a direct action on the modulation of molecular pathways in the vessel wall , and more specifically on endothelial cells ( hermenegildo et al . , 2002 ) . vascular endothelium not only regulates vascular tone through flow - mediated mechanisms , but also confers antithrombotic and antiinflammatory properties to the blood vessel . nitric oxide ( no ) , the primary endothelial - derived mediator , is involved in many physiological processes , including vasodilation and inhibition of thrombosis , cell migration , and proliferation ( dudzinski and michel , 2007 ; lamas et al . estrogen is known to increase no bioavailability by mechanisms that either directly increase no generation ( figure 1 ) or decrease superoxide anion o2 concentration , thereby attenuating o2-mediated no inactivation . mechanisms involved in estrogen - induced increases in no availability include : ( 1 ) transcriptional stimulation of endothelial no synthase ( enos ) gene expression ( huang et al . , 1997 ; sumi and ignarro , 2003 ) ; ( 2 ) non - genomic activation of enzyme activity via a pi3k / phosphokinase b ( pkb / akt)-mediated signaling pathway ( hisamoto et al . , 2001 ) ; ( 3 ) increased intracellular free ca concentration ( [ ca]i ) in endothelial cells ( rubio - gayosso et al . , 2000 ) ; ( 4 ) decreased production of asymmetric dimethylarginine ( adma ) , the enos endogenous inhibitor ( monsalve et al . , 2007 ) ; and ( 5 ) attenuated o2 concentrations ( wassmann et al . estradiol effects on enos - mediated nitric oxide ( no ) production include both genomic and non - genomic effects . genomic effects include the classical intracellular estrogen receptors ( er ) , which after binding of e2 interact with estrogen - response element ( ere ) in dna , resulting in an increased enos expression . moreover , e2 binding to gper leads to activation of different transcriptional factors such as camp response element ( cre ) which also induces enos expression . among non - genomic effects , er and gper regulate the e2-induced enos activity ( modified from sobrino , 2011 ) . estrogens such as 17-estradiol , estrone , and estriol have been described to act as reactive oxygen species ( ros ) scavengers by virtue of the hydrogen - donating capacity of their phenolic molecular structure ( halliwell and grootveld , 1987 ; dubey and jackson , 2001 ) . however , in these studies the direct effect of estrogens as scavengers can only be observed at concentrations above 1 m ( arnal et al . , 1996 ; kim et al . , 1996 ) . considering that plasma concentrations of estrogen in physiological conditions are within the nanomolar range , it is likely that direct scavenger action is not estrogen s main antioxidant mechanism . estrogen modulates ros concentration through a mechanism that involves interaction with its estrogenic nuclear receptors to decrease oxidative proteins and/or increase antioxidant enzymes expression . many studies have associated changes in estrogen levels with altered levels of antioxidant enzymes including glutathione peroxidase , catalase , and superoxide dismutase ( capel et al . , 1981 ; robb and stuart , 2011 ; sivritas et al . , 2011 ) . moreover , estrogen modulates nadh / nadph oxidases and at1 receptor gene expression , both of which are major sources of o2 production ( wassmann et al . , 2001 ; dantas et al . , estrogen also has a modulating effect on constrictive factors and positively upregulates the production of endothelium - derived relaxing factors such as pgi2 ( sobrino et al . , 2009 , 2010 ) and the endothelium - derived hyperpolarizing factors ( golding and kepler , 2001 ) , both of which are important mediators of vascular relaxation in resistance - sized arteries . the beneficial effects of estrogen on the endothelium can be partially explained by an inhibitory effect on the production or action of the cyclooxygenase ( cox)-derived vasoconstrictor agents prostaglandin h2 , pgh2 , and thromboxane a2 , txa2 ( davidge and zhang , 1998 ; dantas et al . , 1999 ; novella et al . , 2010 ) , and of endothelin-1 ( et-1 ; david et al . , 2001 ) . it regulates contractile responses by a direct modulation of ca mobilization into the vascular smooth muscle cells . direct interaction of estradiol with voltage - gated maxi - k channel subunit beta , which confers higher ca sensitivity , may modulate vascular smooth muscle ( valverde et al . , 1999 ) . estrogen does not inhibit ca release from the intracellular stores ( crews and khalil , 1999 ; murphy and khalil , 1999 ) . however , supraphysiological concentrations of estrogen impede ca influx from the extracellular space ( han et al . , 1995 ; crews and khalil , 1999 ; murphy and khalil , 1999 ) by inhibiting ca entry through voltage - gated ca channels ( freay et al . , 1997 ; kitazawa et al . , 1997 ; crews and khalil , 1999 ; murphy and khalil , 1999 ) . expression of the l - type ca channels in cardiac muscle is substantially increased in er - deficient mice ( johnson et al . , 1997 ) , suggesting er - mediated regulation of ca mobilization . estrogen also exerts direct modulation on the components of the renin - angiotensin system ( ras ) , a key regulator of blood pressure and smooth muscle cell growth . production of angiotensin ii ( ang ii ) , the active hormone of the ras , is reduced by estrogen inhibition of angiotensin - converting enzyme ( ace ) expression . in animal models of menopause and in postmenopausal women , chronic estrogen replacement reduces ace activity in the circulation and in tissues including the kidney and aorta ( brosnihan et al . , 1999 ; seely et al . , 2004 ) . furthermore , estrogen attenuates expression of and tissue response to type 1 angiotensin receptor ( at1 ) in the aorta , heart , and kidney ( silva - antonialli et al . vascular aging is associated with endothelial dysfunction , arterial stiffening and remodeling , impaired angiogenesis , defective vascular repair , and an increasing prevalence of atherosclerosis ( lakatta and levy , 2003 ; erusalimsky , 2009 ) . aging - associated changes in structure and function of large elastic arteries are seen even in the absence of clinical cvd ( moreau et al . , 2003 ) . although aging per se has detrimental effects in the vasculature , the lack of estrogen due to menopause may add an aggravating cvd risk factor in women , compared to arterial aging in men . in middle - aged females , aging - associated vascular dysfunction is potentiated by lack of estrogen due to menopause or ovariectomy and improves with estrogen replacement ( harman , 2004 ; stice et al . , 2009 ; novella et al . , 2010 unfortunately , the onset of menopause coincides with a time when aging - associated damage may be noted , making it particularly difficult to distinguish between the contributions of aging and the lack of estrogen . vascular aging is a natural phenomenon that could be simply described as a consequence of physical stress , beginning early in life . arteries are elastic tissues , susceptible to fatigue , and fracture over time as a consequence of extension - relaxation cycles during heartbeats ( avolio et al . , 1983 ) . in cross - sectional studies , postmenopausal females taking hrt have less arterial stiffness than their non - treated peers ( moreau et al . , 2003 ; radial artery distensibility fluctuates in accordance with estrogen levels during menstrual cycles ( giannattasio et al . , 1999 ) . basic research using animal models of estrogen withdrawal and aging suggests a modulatory role for estrogen in the molecular mechanisms to prevent arterial stiffening ( zhang et al . , 2000 ) a recent study reported that hrt improves arterial compliance , an effect related in part to estrogen actions in the control of endothelial - dependent vasodilatory tone ( moreau et al . , 2012 ) . collagen and elastin content of arterial walls is a key factor in arterial thickening and stiffening . it is mostly regulated by matrix metalloproteinases ( mmp ) , enzymes capable of degrading components of the extracellular matrix . during estrogen replacement in ovariectomized rats increases mmp activity and restores aged arteries to structural properties similar to those of younger animals studied ( zhang et al . , 2000 ) . dysfunction of both endothelial and smooth muscle molecular signaling appears during the aging process and favors vasospasm , thrombosis , inflammation , and abnormal cell migration and proliferation ( lakatta and levy , 2003 ; briones et al . endothelial dysfunction in the elderly has been associated with malfunctioning of vascular tissue , resulting in atherosclerosis , hypertension , and coronary artery disease ( lakatta and levy , 2003 ; herrera et al . , 2010 ) , renal dysfunction ( schmidt et al . , 2001 ; erdely et al . , 2003 ) , and alzheimer disease ( price et al . , 2004 ) . in women , a slight age - related decrease in endothelium - dependent relaxation persists until middle age ( around 50 years ) . after that , the declining response to the endothelium - dependent vasodilator hastens , even exceeding the rate experienced by men ( taddei et al . , the mechanisms for age - associated endothelial dysfunction are multiple , although most are associated with decreased no bioavailability ( hayashi et al . , 2008 ; santhanam et al . , 2008 ; erusalimsky , 2009 ; kim et al . , reduced endothelium - dependent and no - mediated vasodilation has been described in both human and animal models of aging ( kim et al . , 2009 ; virdis et al . lower no production in the elderly may be based on decreased no synthesis or increased no degradation . suggested mechanisms to explain reduced no production include : ( 1 ) decreased expression of enos ( briones et al . , 2005 ; yoon et al . , 2010 ) ; ( 2 ) a lack of no precursor ( l - arginine ; santhanam et al . , 2008 ) and enos cofactor tetrahydrobiopterin ( bh4 ; yoshida et al . , 2000 ; eskurza et al . , 2005 ; meyer et al . , 2011 ) ; and ( 3 ) increased endogenous enos inhibitor adma ( xiong et al . , 2001 ; kielstein et al . , on the other hand , strong evidences support the hypothesis that age - associated increase in oxidative stress and consequent production of o2 is a potent contributor to lower no bioavailability and increased endothelial dysfunction ( jacobson et al . , 2007 ; rodriguez - manas et al . , there is little information to correlate the progression of aging with the production / degradation of no in women . although several studies have described decreased expression of enos in senile female rats and mice ( wynne et al . , 2004 ; novensa et al . , 2011 ) , aging - associated effects on enos in women can be easily confounded with the effects of lack of estrogen , since most of these studies grouped women into just two time - points : premenopausal and menopausal groups . even though the decline in no bioavailability could sufficiently explain most of the changes in the functioning of vascular cells , other molecules crucial to control of vascular function are also modified by aging . in the regulation of vascular tone , cox - derived factors are particularly important as they can induce both vascular relaxation ( pgi2 ) and contraction ( txa2 and pgh2 ) . some studies have reported a prevalence in the production of relaxing cox factors in the vasculature of young and healthy individuals ( tang and vanhoutte , 2008 ) . with aging , cox - dependent vasoconstrictors production becomes evident , leading to increased vascular contraction ( taddei et al . however , the cox isoform involved in the generation of contractile prostanoids remains unclear . in functional studies developed in femoral arteries of aged rats , oxygen free radicals participate in the augmented endothelium - dependent contractions mediated by cox - derived prostanoids . both the constitutive and inducible isoforms of cox contribute to this endothelial dysfunction ( shi et al . molecular studies performed in endothelial cells from aged rats showed an increase in mrna levels of cox-1 , cox-2 , and other enzymes involved in the synthesis of prostanoids ( tang and vanhoutte , 2008 ) , demonstrating the importance of the arachidonic acid cox cascade in the endothelial and vascular dysfunction associated with aging . moreover , functional studies have demonstrated an interaction between no and prostanoids pathways . in aorta from aged female mice , no bioavailability increases when the cox pathway is inhibited ; both gene and protein expression of cox-1 are increased ( novella et al . , 2011 ) . furthermore , activation of inflammatory pathways in the vascular wall plays a central role in the process of vascular aging . even in the absence of traditional risk factors for atherosclerosis , an age - associated shift to a proinflammatory gene expression profile , known as endothelial activation , induces upregulation of cellular adhesion molecules and cytokines , which increases endothelial leukocyte interactions and permeability , mechanisms considered crucial to initial steps in the development of atherosclerosis ( herrera et al . , 2010 ; seals et al . , 2011 ) . accordingly , a sex - associated difference in inflammatory responses during aging has been proposed . inflammatory atherosclerosis and associated acute coronary heart disease develop earlier in life in men than in women ( roger et al . , 2011 ) and are associated with earlier death , although men and women present the same overall plaque burden ( frink , 2009 ) . in animal models of atherosclerosis , male sex contributes to a faster and more severe progression of lipid deposition , remodeling , and aortic lesions ( pereira et al . with the wide - ranging data from experimental research , estrogens might appear to promise protection against the progression of vascular aging and cvd in women . epidemiological observational studies also suggest that postmenopausal women on hrt are less likely to develop cvd than non - users at the same age ( grodstein et al . , 2000 , 2001 ) nevertheless , these studies contrast with the large prospective clinical trials , hers and whi , which failed to show reduced cardiovascular events in postmenopausal women on hrt . in fact , whi suggested that hrt was associated with increased risk to the cardiovascular system ( rossouw et al . , 2002 ) . possible reasons for this discrepancy have been extensively discussed and include the average age of women entering most hrt clinical trials , 65 years and older , which results in a study population with some degree of aging - associated vascular damage . in addition , participants had been estrogen - deficient for an average of 10 years before starting hrt , a relatively late start that could modify the status of ers and molecular signaling so as to attenuate the benefits of estrogen . for instance , during aging ers can undergo posttranslational modifications such as methylation , which decreases their expression and activity . we recently reported that aging contributes to increased dna methylation in female mice aorta , which could be associated with the decrease in the modulatory effects of estrogen ( novensa et al . , 2011 ) . a few clinical studies also provide evidence for aging - associated dysregulation of er methylation and suggest that focal epigenetic changes in er could contribute to decreased estrogen activity and to the development of atherosclerosis in elderly women ( post et al . , 1999 ; detailed examination of whi data reveals that early initiation of estrogen replacement produces more favorable results than the later average initiation employed in the whi studies overall ( grodstein et al . , 2006 ; these findings , together with observational studies , have led scientists to create the so - called timing hypothesis that estrogen - mediated benefits to prevent cvd only occur when treatment is initiated before the detrimental effects of aging are established on vascular walls ( harman , 2006 ) . these effects include endothelial dysfunction and pathophysiological actions , such as increased vascular calcification and generalized stiffening of the arterial tree that increase the prevalence of hypertension and atherosclerosis ( lakatta and levy , 2003 ; erusalimsky , 2009 ; kovacic et al . , 2011 ) . little information is available on whether and how vascular effects of estrogen are modified with aging in females . aging has been associated with significant reductions in the direct estrogen - mediated mechanisms of vascular relaxation ( wynne et al . , 2004 ; the lack of estrogen responses in these animal studies was not related to age - associated changes in the plasma levels of estrogen or activity of er , but rather to possible age - related changes in estrogen - mediated signaling pathways in the vasculature . modifications in the ratio between er and er in older female mice are associated with the lack of protective effects of estrogen on no production and with a reversal in its antioxidant effect to a pro - oxidant profile ( novensa et al . , 2011 ) . moreover , clinical studies have revealed that cvd risk factors in postmenopausal women were lower among women aged 5059 years at hrt initiation ( manson et al . these studies clearly establish the complexity of estrogen effects , which may be influenced by pathophysiological conditions including aging and subclinical cvd . despite convincing arguments by the followers of the timing hypothesis the potential extrapolation of the protective effects of estrogen replacement , well described in young females , to older women remains controversial . the field still lacks detailed experimental and clinical research on the long - term effects of estrogen and how it modulates cardiovascular function during aging . our society is aging progressively , and increased life expectancy enhances the risks for diseases associated with the natural fatigue of the body , including cvd . despite this undeniable reality , there is evidence that vascular aging in women does not follow the same chronology as in men . the vascular protective effects exerted by estrogens have been proposed as the major reason for reduced signs of vascular aging and cvd risk in premenopausal women , compared to men . when natural estrogen withdrawal occurs and a woman enters her climacteric stage , effects of sudden vascular aging become evident , leading to vascular dysfunction and increased risk of a cardiovascular event . the lack of crucial information from clinical trials and the discrepancies in the available data on the regulation of the female cardiovascular system can lead to inappropriate diagnosis and treatment of cvd in older women . women have been treated like men , despite the notable sex - associated differences in the elements of aging and disease processes . much research effort is still needed to understand age- and sex - related differences in cardiovascular control , establish the impact of the menstrual cycle and hrt on vascular function , and propose new therapeutic strategies to improve cvd diagnosis and treatment and the overall management of vascular senescence in women . 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 .

aging is associated with structural and functional changes in the vasculature , including endothelial dysfunction , arterial stiffening and remodeling , impaired angiogenesis , and defective vascular repair , and with increased prevalence of atherosclerosis . cardiovascular risk is similar for older men and women , but lower in women during their fertile years . this age- and sex - related difference points to estrogen as a protective factor because menopause is marked by the loss of endogenous estrogen production . experimental and some clinical studies have attributed most of the protective effects of estrogen to its modulatory action on vascular endothelium . estrogen promotes endothelial - derived no production through increased expression and activity of endothelial nitric oxide synthase , and modulates prostacyclin and thromboxane a2 release . the thromboxane a2 pathway is key to regulating vascular tone in females . despite all the experimental evidence , some clinical trials have reported no cardiovascular benefit from estrogen replacement therapy in older postmenopausal women . the timing hypothesis , which states that estrogen - mediated vascular benefits occur only before the detrimental effects of aging are established in the vasculature , offers a possible explanation for these discrepancies . nevertheless , a gap remains in current knowledge of cardiovascular aging mechanisms in women . this review comprises clinical and experimental data on the effects of aging , estrogens , and hormone replacement therapy on vascular function of females . we aim to clarify how menopause and aging contribute jointly to vascular aging and how estrogen modulates vascular response at different ages .

sixty - four patients in nepal that met us department of defense enrollment criteria ( 9 ) for influenzalike illness were evaluated by using onsite rapid influenza tests ( optical immunoassay rapid diagnostic tests , thermo electron corp . throat swab specimens were collected within the first 72 hours of onset of symptoms , routed through the armed forces research institute for medical sciences in bangkok , thailand , and shipped on dry ice to brooks city base in san antonio , texas , for clinical characterization and diagnosis using traditional culturing techniques and monoclonal antibody staining ( 10 ) . antigenic analysis of select isolates was performed at the centers for disease control and prevention ( cdc ) in atlanta , georgia , by using the hemagglutination inhibition ( hi ) assay and postinfection ferret antisera ( 11 ) . rna was extracted from 48-hour shell vial cultures ( 10 ) by using the magnapure lx ( roche molecular , mannheim , germany ) and rna isolation kit ii ( roche molecular ) according to the manufacturer 's protocols . for reverse transcription - polymerase chain reaction ( rt - pcr ) amplification , 5 l rna was added to a 50-l master mixture containing 1 reaction buffer , 1.6 mmol / l mgso4 , 1 enzyme mixture , and 400 nmol / l primers ( h3-f7 , 5-act - atc - att - gct - ttg - agc-3 and h3r-1184 , 5-atg - gct - gct - tga - gtg - ctt-3 ) by using the superscript iii one - step rt - pcr system ( invitrogen , carlsbad , ca , usa ) . pcr thermocycling consisted of an rt step at 50c for 30 min , hot start activation at 95c for 3 min , followed by 40 amplification cycles of 95c for 30 s , 52c for 15 s , and 68c for 1 min , with a final extension cycle at 68c for 7 min . all pcr products were visualized after electrophoresis in 2% precast gels stained with ethidium bromide ( invitrogen ) under uv illumination . the ha1 amplicon ( 1177 bp ) was sequenced by using the h3-f7 and h3r-1184 pcr primers ( described above ) and 2 additional internal oligonucleotides , h3r-466 ( 5-ggt - gca - acc - aat - tca - atc-3 ) and h3f-282 ( 5-cag - caa - ctg - tta - ccc-3 ) . unincorporated fluorescent nucleotides were removed by using a dye ex 96-well plate kit ( qiagen ) according to the manufacturer 's recommendations . nucleotide sequencing was performed by using the big dye terminator v3.1 kit and analyzed by using an abi 3100 genetic analyzer ( both from applied biosystems , foster city , ca , usa ) according to the manufacturer 's specifications . multiple sequence alignments , protein translation , and phylogenetic analysis were performed with the dnastar ( dnastar inc . , three - dimensional ha protein structures were generated by using molmol ( 12 ) and the swiss - pdb viewer programs ( 13 ) . ha nucleotide sequences for all 26 nepal isolates depicted in the phylogenetic analysis are available from genbank under accession nos . sixty - four patients in nepal that met us department of defense enrollment criteria ( 9 ) for influenzalike illness were evaluated by using onsite rapid influenza tests ( optical immunoassay rapid diagnostic tests , thermo electron corp . throat swab specimens were collected within the first 72 hours of onset of symptoms , routed through the armed forces research institute for medical sciences in bangkok , thailand , and shipped on dry ice to brooks city base in san antonio , texas , for clinical characterization and diagnosis using traditional culturing techniques and monoclonal antibody staining ( 10 ) . antigenic analysis of select isolates was performed at the centers for disease control and prevention ( cdc ) in atlanta , georgia , by using the hemagglutination inhibition ( hi ) assay and postinfection ferret antisera ( 11 ) . rna was extracted from 48-hour shell vial cultures ( 10 ) by using the magnapure lx ( roche molecular , mannheim , germany ) and rna isolation kit ii ( roche molecular ) according to the manufacturer 's protocols . for reverse transcription - polymerase chain reaction ( rt - pcr ) amplification , 5 l rna was added to a 50-l master mixture containing 1 reaction buffer , 1.6 mmol / l mgso4 , 1 enzyme mixture , and 400 nmol / l primers ( h3-f7 , 5-act - atc - att - gct - ttg - agc-3 and h3r-1184 , 5-atg - gct - gct - tga - gtg - ctt-3 ) by using the superscript iii one - step rt - pcr system ( invitrogen , carlsbad , ca , usa ) . pcr thermocycling consisted of an rt step at 50c for 30 min , hot start activation at 95c for 3 min , followed by 40 amplification cycles of 95c for 30 s , 52c for 15 s , and 68c for 1 min , with a final extension cycle at 68c for 7 min . all pcr products were visualized after electrophoresis in 2% precast gels stained with ethidium bromide ( invitrogen ) under uv illumination . the ha1 amplicon ( 1177 bp ) was sequenced by using the h3-f7 and h3r-1184 pcr primers ( described above ) and 2 additional internal oligonucleotides , h3r-466 ( 5-ggt - gca - acc - aat - tca - atc-3 ) and h3f-282 ( 5-cag - caa - ctg - tta - ccc-3 ) . unincorporated fluorescent nucleotides were removed by using a dye ex 96-well plate kit ( qiagen ) according to the manufacturer 's recommendations . nucleotide sequencing was performed by using the big dye terminator v3.1 kit and analyzed by using an abi 3100 genetic analyzer ( both from applied biosystems , foster city , ca , usa ) according to the manufacturer 's specifications . multiple sequence alignments , protein translation , and phylogenetic analysis were performed with the dnastar ( dnastar inc . , madison , wi , usa ) software package . three - dimensional ha protein structures were generated by using molmol ( 12 ) and the swiss - pdb viewer programs ( 13 ) . ha nucleotide sequences for all 26 nepal isolates depicted in the phylogenetic analysis are available from genbank under accession nos . clinical evaluations and throat specimens were obtained from 64 patients from 3 refugee camps in southeastern nepal ( figure 1 ) . of the 64 patients , 61 were refugees from bhutan , 1 was a foreign aid worker from japan , and 2 were nepalese nationals . most of the patients were < 10 years of age ; 36 were male and 28 were female . none had previously been vaccinated against influenza and of the 64 specimens collected , 42 ( 66% ) tested positive for influenza a by culture . the green circle shows the location of 3 bhutan refugee camps where the outbreak occurred in early july 2004 . ( map courtesy of http://www.maps.com ) hi was performed by using postinfection ferret antisera with reference antigens that included the 20042005 h3n2 vaccine seed strain ( a / wyoming/03/2003 ) and the 20052006 southern hemisphere h3n2 vaccine strain ( a / wellington/1/2004 ) . when compared with a / wyoming/03/2003 , 4 of 9 nepal isolates showed 4-fold lower titers ( 1:320 versus 1:1,280 hi units ) than that allowed for homologous titer of the reference antisera . six of 9 nepal isolates were antigenically distinct when compared with the a / wellington/1/2004 strain and showed a 4-fold ( 1:160 versus 1:640 ) reduction in titer to ferret antisera ( table 1 ) . * test antigens are considered antigenically different from the reference strain if hi titers show a 4-fold difference . a rt - pcr - based molecular subtyping showed that all 42 specimens were the h3n2 influenza subtype . twenty - six of the 42 influenza a positive samples were randomly selected for molecular characterization using direct nucleotide sequencing of the ha gene . the 26 nepal isolates exhibited 99.8% nucleotide sequence identity and contained the fujian - like amino acid substitutions at positions 155 ( h155 t ) and 156 ( q156h ) in the ha protein ( table 2 ) . alignment of the 329amino acid ha protein from 26 isolates obtained from this outbreak with the 2004/05 a / wyoming/3/03 vaccine strain and previous h3n2 vaccine strains indicated 4 evident amino acid changes present in most of the isolates ( table 2 ) . all 4 amino acid changes observed within most of these outbreak isolates are present within a / california/7/04 , a variant strain selected as the h3n2 vaccine strain for the 20052006 influenza season . * n , asparagine ; t , threonine ; h , histidine ; i , isoleucine ; p , proline ; k , lysine ; s , serine ; v , valine ; q , glutamine . consensus sequence derived from a multiple sequence protein alignment of 26 ha1 hemagglutinin sequences from nepal . of the 26 nepal strains examined , 24 exhibited a novel lysine - to - asparagine substitution at position 145 in the ha protein ( k145n ) . this substitution is noteworthy because most strains characterized in 20032004 , including the fujian/411/2002 vaccine strain , contained a lysine ( k ) at this position . prior to this outbreak , the us department of defense had only observed k145n substitutions in 6 strains obtained from ramstein , germany , ( data not shown ) in june 2004 . additionally , all 26 nepal sequences exhibited a serine - to - asparagine substitution at position 189 ( s189n ) that had also been observed in the 6 isolates from germany , as well as in a few isolates from asia characterized at the end of the 20032004 influenza season . two other substitution mutations in the ha1 hemagglutinin , i.e. , valine to isoleucine at position 226 ( v226i ) and serine to proline at position 227 ( s227p ) , were also observed in 24 ( 92% ) and 26 of 26 of the nepal isolates , respectively . both substitutions differ from most influenza a h3n2 field isolates collected in 20032004 , including the fujian and wyoming vaccine strain for 20042005 ( table 2 ) . the phylogeny of h3n2 ha proteins indicates a drifting of the nepal isolates from the a / fujian/411/03 and a / wyoming/03/03 vaccine strains and shows that these outbreak isolates have a higher genetic homology to a / wellington/1/04 , a prototype strain selected as the 20052006 southern hemisphere h3 vaccine strain ( figure 2 ) . the a / wellington/1/04 strain contains 2 of the 4 amino acid changes ( s227p and s189n ) observed in the nepal isolates , but does not contain the k145n and v226i substitutions . unrooted phylogenetic analysis of ha1 hemagglutinin nucleotide sequences from 26 nepal isolates and h3n2 vaccine and reference strains . the nepal isolates have drifted from the 20042005 a / fujian/411/03 vaccine strain ( and a / wyoming/03/03 vaccine seed strain ) and are genetically equivalent to a / california/7/04 , the 20052006 northern hemisphere vaccine strain . a k145n substitution ( branch point indicated by the arrow ) was observed in 24 of 26 nepal isolates and represents a genetic marker for the dominant lineage of h3n2 viruses during the 20042005 season . nucleotide and amino acid sequences for all nepal isolates are available from genbank under accession no . the asterisk indicates isolates from table 2 that were antigenically distinct from a / wyoming/303 . three - dimensional views of influenza ha proteins highlighting amino acid changes in a representative nepal isolate and the a / wyoming/3/03 vaccine strains are shown in figure 3a and b , respectively . the mutation at position 145 ( shown in yellow ) , which is located adjacent to antibody - binding site a and within a known glycosylation site , introduces an asparagine - for - lysine substitution . this substitution results in a more accessible receptor - binding cleft located directly above residue 145 ( comparing panels a and b ) . located above the receptor - binding pocket is a serine - to - asparagine change ( shown in green ) that possibly alters the regional surface topography at position 189 within antibody - binding site b. a serine - to - proline mutation at position 227 ( shown in magenta ) appears to marginally affect the ha surface features . this substitution resides within antibody - binding site d , which corresponds to residues 225228 , which make up the left side of the receptor - binding pocket ( 14 ) . interestingly , this proline residue is located within a barrel ( a protein motif consisting of an antiparallel sheet domain ) and does not appreciably alter the predicted protein structure , as shown by the absence of any substantial changes in the computer - modeled , 3-dimensional structure compared with the ha1 of a / wyoming/3/2003 . three - dimensional top view of the ha1 hemagglutinin structures for a ) a representative a / nepal/1648/04 virus and b ) vaccine strain a / wyoming/3/03 . most ( 24/26 ) of the nepal isolates contain a lysine to asparagine substitution ( shown in yellow ) at position 145 ( k145n ) . magenta , residues 226 and 227 ; orange , residue 189 ; green , residues 155 and 156 ; yellow , residue 145 . hemagglutinin molecules were generated by using the respective amino acid sequences with molmol ( 12 ) . clinical evaluations and throat specimens were obtained from 64 patients from 3 refugee camps in southeastern nepal ( figure 1 ) . of the 64 patients , 61 were refugees from bhutan , 1 was a foreign aid worker from japan , and 2 were nepalese nationals . most of the patients were < 10 years of age ; 36 were male and 28 were female . none had previously been vaccinated against influenza and of the 64 specimens collected , 42 ( 66% ) tested positive for influenza a by culture . the green circle shows the location of 3 bhutan refugee camps where the outbreak occurred in early july 2004 . hi was performed by using postinfection ferret antisera with reference antigens that included the 20042005 h3n2 vaccine seed strain ( a / wyoming/03/2003 ) and the 20052006 southern hemisphere h3n2 vaccine strain ( a / wellington/1/2004 ) . when compared with a / wyoming/03/2003 , 4 of 9 nepal isolates showed 4-fold lower titers ( 1:320 versus 1:1,280 hi units ) than that allowed for homologous titer of the reference antisera . six of 9 nepal isolates were antigenically distinct when compared with the a / wellington/1/2004 strain and showed a 4-fold ( 1:160 versus 1:640 ) reduction in titer to ferret antisera ( table 1 ) . * test antigens are considered antigenically different from the reference strain if hi titers show a 4-fold difference . a rt - pcr - based molecular subtyping showed that all 42 specimens were the h3n2 influenza subtype . twenty - six of the 42 influenza a positive samples were randomly selected for molecular characterization using direct nucleotide sequencing of the ha gene . the 26 nepal isolates exhibited 99.8% nucleotide sequence identity and contained the fujian - like amino acid substitutions at positions 155 ( h155 t ) and 156 ( q156h ) in the ha protein ( table 2 ) . alignment of the 329amino acid ha protein from 26 isolates obtained from this outbreak with the 2004/05 a / wyoming/3/03 vaccine strain and previous h3n2 vaccine strains indicated 4 evident amino acid changes present in most of the isolates ( table 2 ) . all 4 amino acid changes observed within most of these outbreak isolates are present within a / california/7/04 , a variant strain selected as the h3n2 vaccine strain for the 20052006 influenza season . * n , asparagine ; t , threonine ; h , histidine ; i , isoleucine ; p , proline ; k , lysine ; s , serine ; v , valine ; q , glutamine . consensus sequence derived from a multiple sequence protein alignment of 26 ha1 hemagglutinin sequences from nepal . of the 26 nepal strains examined , 24 exhibited a novel lysine - to - asparagine substitution at position 145 in the ha protein ( k145n ) . this substitution is noteworthy because most strains characterized in 20032004 , including the fujian/411/2002 vaccine strain , contained a lysine ( k ) at this position . prior to this outbreak , the us department of defense had only observed k145n substitutions in 6 strains obtained from ramstein , germany , ( data not shown ) in june 2004 . additionally , all 26 nepal sequences exhibited a serine - to - asparagine substitution at position 189 ( s189n ) that had also been observed in the 6 isolates from germany , as well as in a few isolates from asia characterized at the end of the 20032004 influenza season . two other substitution mutations in the ha1 hemagglutinin , i.e. , valine to isoleucine at position 226 ( v226i ) and serine to proline at position 227 ( s227p ) , were also observed in 24 ( 92% ) and 26 of 26 of the nepal isolates , respectively . both substitutions differ from most influenza a h3n2 field isolates collected in 20032004 , including the fujian and wyoming vaccine strain for 20042005 ( table 2 ) . the phylogeny of h3n2 ha proteins indicates a drifting of the nepal isolates from the a / fujian/411/03 and a / wyoming/03/03 vaccine strains and shows that these outbreak isolates have a higher genetic homology to a / wellington/1/04 , a prototype strain selected as the 20052006 southern hemisphere h3 vaccine strain ( figure 2 ) . the a / wellington/1/04 strain contains 2 of the 4 amino acid changes ( s227p and s189n ) observed in the nepal isolates , but does not contain the k145n and v226i substitutions . unrooted phylogenetic analysis of ha1 hemagglutinin nucleotide sequences from 26 nepal isolates and h3n2 vaccine and reference strains . the nepal isolates have drifted from the 20042005 a / fujian/411/03 vaccine strain ( and a / wyoming/03/03 vaccine seed strain ) and are genetically equivalent to a / california/7/04 , the 20052006 northern hemisphere vaccine strain . a k145n substitution ( branch point indicated by the arrow ) was observed in 24 of 26 nepal isolates and represents a genetic marker for the dominant lineage of h3n2 viruses during the 20042005 season . nucleotide and amino acid sequences for all nepal isolates are available from genbank under accession no . the asterisk indicates isolates from table 2 that were antigenically distinct from a / wyoming/303 . three - dimensional views of influenza ha proteins highlighting amino acid changes in a representative nepal isolate and the a / wyoming/3/03 vaccine strains are shown in figure 3a and b , respectively . the mutation at position 145 ( shown in yellow ) , which is located adjacent to antibody - binding site a and within a known glycosylation site , introduces an asparagine - for - lysine substitution . this substitution results in a more accessible receptor - binding cleft located directly above residue 145 ( comparing panels a and b ) . located above the receptor - binding pocket is a serine - to - asparagine change ( shown in green ) that possibly alters the regional surface topography at position 189 within antibody - binding site b. a serine - to - proline mutation at position 227 ( shown in magenta ) appears to marginally affect the ha surface features . this substitution resides within antibody - binding site d , which corresponds to residues 225228 , which make up the left side of the receptor - binding pocket ( 14 ) . interestingly , this proline residue is located within a barrel ( a protein motif consisting of an antiparallel sheet domain ) and does not appreciably alter the predicted protein structure , as shown by the absence of any substantial changes in the computer - modeled , 3-dimensional structure compared with the ha1 of a / wyoming/3/2003 . three - dimensional top view of the ha1 hemagglutinin structures for a ) a representative a / nepal/1648/04 virus and b ) vaccine strain a / wyoming/3/03 . most ( 24/26 ) of the nepal isolates contain a lysine to asparagine substitution ( shown in yellow ) at position 145 ( k145n ) . magenta , residues 226 and 227 ; orange , residue 189 ; green , residues 155 and 156 ; yellow , residue 145 . hemagglutinin molecules were generated by using the respective amino acid sequences with molmol ( 12 ) . the 4 substitutions described represent a growing lineage of influenza a ( h3n2 ) viruses characterized since july 2004 . three amino acid changes are confined within known antibody - binding sites , i.e. , the s189n change within antibody - binding site b ( 4,5 ) and the v226i and s227p changes residing in antibody - binding site d ( 4,5 ) . because of rotational restrictions , a proline substitution at position 227 ( s227p ) would typically give rise to considerable conformation change ; however , this particular substitution is located within a barrel motif and therefore has little effect on regional protein conformation . cumulatively , field isolates characterized subsequent to this outbreak continue to exhibit these 4 changes , and they appear to constitute a distinct branch in the phylogeny of ha sequences when compared with h3n2 isolates from the 20032004 season . . this change may affect protein - protein interactions since it is immediately adjacent to antibody - binding site a , where neutralizing antibodies have been shown to bind ( 4,5 ) . furthermore , since the k145n substitution is located within a glycosylation site , the charge alteration may affect glycosyl transferase activity , which results in altered glycosylation . differences in glycosylation have been shown to contribute to antigenic variation by preventing antibody binding to antigenic sites ( 15 ) . additionally , 3-dimensional analysis suggests this amino acid substitution may also promote enhanced receptor binding since the asparagine r group is shorter , which may make binding requirements less stringent and the receptor cleft more accessible . the 3-dimensional depiction provides a unique regional residue perspective , demonstrating how the rapidly evolving ha surface antigens in the vaccine strain differ at the molecular level . collectively , the clinical isolates obtained from this outbreak in nepal can not be considered antigenically distinct from the a / wyoming/3/03 vaccine strain because only 4 of 9 isolates evaluated exhibited 4-fold lower titers by hi ( table 1 ) . furthermore , the varying reactivity noted in several isolates from this outbreak having identical ha1 sequences is suggestive that other viral antigens aside from the ha1 protein may have contributed to the antigenic variability observed in the hi panel . with the exception of a / nepal/1670/2004 and a / nepal/1672/2004 , all isolates evaluated by hi ( table 1 ) exhibited identical ha1 amino acid sequences and varying antigenicity profiles to a / wyoming/03/2003 reference antisera . one explanation for this observation is that genetic differences in other influenza surface proteins contribute to the observed immunoreactivity . alternative viral surface protein candidates include the neuraminidase , ha2 , and m2 glycoproteins , which have been shown to exhibit antigenic properties ( 1619 ) . in this report , we describe the genetic analysis of the ha proteins from viruses obtained from an early season outbreak and compare them to current vaccine strains . three amino acids changes ( s189n , i226v , and s227p ) were noted in known ( 4,5 ) antibody - binding sites ( table 2 ) . the fourth change ( k145n ) , which was located within a glycosylation site , may enhance viral binding since the smaller asparagine r group is located close to the ha receptor - binding cleft ( figure 3 ) . phylogenetic analyses show that the nepal isolates make up a distinct branch in the evolution of h3n2 viruses when they are compared with vaccine and reference strains ( figure 2 ) . however , antigenic data appear more ambiguous , suggesting a multigenic effect that can not solely be attributed to properties of the influenza ha ( table 1 ) . studies are in progress to characterize the neuraminidase , m2 , and ha2 proteins to determine the molecular basis responsible for antigenicity differences observed within isolates from this outbreak . the k145n substitution change has become a marker for an increasingly large subset of the fujian - like viruses . cdc and the us department of defense have recently characterized viruses with the k145n change in singapore , taiwan , china , australia , canada , and the united states . in february 2005 , who reported the emergence of a new influenza h3n2 strain in the united states . the a / california/7/2004 strain , which was first identified in the united states in september 2004 , contains all 4 changes observed in isolates from this nepalese outbreak . the a / california/7/2004 strain differs by only 1 amino acid in ha1 ( which is of no immunologic importance ) from most isolates from the outbreak in nepal . all viruses characterized ( 150 globally isolated strains ) subsequent to the preparation of this report ( march 2005 ) by the us department of defense are genetically similar in amino acid sequence to these nepalese strains ( and the a / california strain ) . most of the isolates ( 80% ) analyzed by cdc since october 2004 are antigenically related to a / california ( 20 ) , which indicates that this strain has emerged as the dominant influenza a h3n2 strain . these data indicate that these viruses may persist as the dominant strain at the onset of the 20052006 influenza season . in february 2005 , who recommended inclusion of an a / california/7/2004-like strain in the 20052006 trivalent influenza vaccine to afford immunologic protection from this variant h3n2 virus . our findings emphasize the importance of continued molecular surveillance for characterizing emerging influenza drift variants .

worldwide emergence of variant viruses has prompted a change in the 20052006 h3n2 influenza a vaccine strain .