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mesenteric pseudocyst is a term used to describe abdominal cystic mass without the origin of abdominal organ.(1 ) this has been classified according to embryologic , ehiologic , histologic , and ther data , causing considerable confusion . it was considered the term mesenteric cyst as merely descriptive , and apply a histologic classification such as lymphangioma , pseudocyst , enteric duplication cyst , enteric cyst , and mesothelial cyst.(2 ) we presented a case of mesenteric pseudocyst of the small bowel in a 70-year - old man . a 70-year - old man was referred to our hospital for operation of gastric cancer with a 1-month history of progressively worsening epigastric and intermittent peri - umbilical discomfort . he had no specific previous medical or surgical history including cancer . on physical examination , esophago - gastro - duodenoscopy ( egd ) showed a 3.5 cm sized excavated lesion on the posterior wall of angle . endocopic biopsy confirmed a histologic diagnosis of poorly differentiated adenocarcinoma including signet ring cell component . endoscopic ultrasonography revealed invasion of caner to the proper muscle layer . abdominal computed tomography ( ct ) scan showed a focal mucosal enhancement in posterior wall of angle of stomach , a 2.4 cm sized enhancing mass on distal small bowel loop without distant metastases or ascites in rectovesical pouch , and multiple gallbladder stones ( fig . 1 ) . these physical , laboratory , and radiological findings prompted us to diagnose early gastric cancer , and gastrointestinal stromal tumor of small bowel . laboratory testing revealed alfa - fetoprotein level of 2.88 ( normal range , 0 to 9 ng / ml ) , carcino - embryonic antigen level of 1.45 ng / ml ( normal range , 0 to 5 ng / ml ) , carbohydrate antigen ( ca ) 19 - 9 level of 6.5 u / ml ( normal range , 1 to 35 u / ml ) , and ca 72 - 4 level of 4.8 u / ml ( normal range , 0 to 4 u / ml ) . other laboratory test results were within normal limit . the patient underwent subtotal gastrectomy with gastroduodenostomy , segmental resection of small bowel , and cholecystectomy . mesenteric mass was adhered severely with greater omentum at the mesenteric side of small bowel , and mesenteric fat tissues . small bowel , mesentery , and mesenteric mass were resected en - bloc methods , and end to end anastomosis was performed . after fixation of the surgical specimen , macroscopic examination revealed a uni - locular cyst measuring 332 cm in size . pathological examination revealed 3 cm sized fibrous cystic wall without endothelial or epithelial lining and foam cell collection ( fig . 2 , 3 ) . pathologic stage of gastric cancer was t1bn1m0 ( 6th international union against cancer tnm staging system ) ; invasion to submusosa , metastases to 4 perigastric lymph nodes out of 16 retrieved nodes , and negative resection margin . mesenteric pseudocysts are very rare intraabdominal mass with an incidence of about 1 case per 100,000 hospital admissions.(3 ) ros et al.(2 ) first used the term " pseudocyst " in the classification of mesenteric cyst . mesenteric pseudocyst could be located in the small bowel , large bowel mesentery and even retroperitoneum.(1,4 ) most reports were pseudocyst of large bowel or retroperitoneum.(1 ) although most mesenteric pseudocysts are asymptomatic , symptomatic mesenteric cysts could be associated with cyst size , cyst location , and complications , including infection , rupture , hemorrhage , and intestinal obstruction.(5 ) in our patient , there was no specific symptom associated with mesenteric pseudocyst except for intermittent vague periumbilical discomfort . if egd and ct scan were not performed in this patient presenting non - specific abdominal pain , the diagnosis of mesenteric pseudocyst would be delayed . to the best of our knowledge , this is the first case report describing incidentally detected mesenteric pseudocyst of small bowel in gastric cancer patients . when clinician performed staging work up for gastric cancer , should be aware the possibility of associated intraabdominal lesions .

mesenteric pseudocyst is rare . this term is used to describe the abdominal cystic mass , without the origin of abdominal organ . we presented a case of mesenteric pseudocyst of the small bowel in a 70-year - old man . esophago - gastro - duodenoscopy showed a 3.5 cm sized excavated lesion on the posterior wall of angle . endocopic biopsy confirmed a histologic diagnosis of the poorly differentiated adenocarcinoma , which includes the signet ring cell component . abdominal computed tomography scan showed a focal mucosal enhancement in the posterior wall of angle of the stomach , a 2.4 cm sized enhancing mass on the distal small bowel loop , without distant metastases or ascites in rectal shelf , and multiple gallbladder stones . the patient underwent subtotal gastrectomy with gastroduodenostomy , segmental resection of the small bowel , and cholecystectomy . the final pathological diagnosis was mesenteric pseudocyst . this is the first case report describing incidentally detected mesenteric pseudocyst of the small bowel in gastric cancer patients .

Introduction Case Report Discussion

hepatitis e virus ( hev ) is a nonenveloped , single stranded rna virus which belongs to the hepeviridae family . hev is a causative agent for acute hepatitis in one - third of the world 's population and fulminant hepatitis in pregnant women . the virion is relatively resistant to environmental conditions and remains infectious even in rough situation such as sewage . therefore the major route of hev transmission is the ingestion of the fecal contaminated water ; however , hev can be spread zoonotically and by blood transfusion especially in industrialized countries . although there is an inclusive debate on the parental route of transmission , available evidence seems to prove the ability of the virus to cause congenital infections . hev infection is a significant public health concern especially in developing countries , where large outbreaks as a result of poor sanitation and lack of sewage infrastructures have been reported . there is also a growing support for the claims that seroprevalence of hev infection in industrialized countries is increasing . patients with chronic liver disease , travelers to endemic areas , and people working with animals like pigs , cows , sheep , and goats are at high risk of hev infection [ 5 , 810 ] . pregnant women infected in third semester develop fulminant hepatic failure particularly in the endemic areas of hev infection [ 11 , 12 ] . iran is an endemic country for hepatitis e infection [ 7 , 13 ] , but hev prevalence has not been determined among general population in all parts of this country . most conducted studies in iran have reported the hev prevalence in specific groups and studies on hev prevalence in general populations are limited . hev prevalence information in general population can be a better indicator of the public health and hygiene . ahvaz is a large city in the south - west of iran with a population of about 1.18 million inhabitants that consists of two ethnic groups : arab and farsi . ahvaz is located in the banks of the karun river , which is the main river in this area . no data is available so far on the prevalence of hev among general population of ahvaz city ; therefore this study was conducted to determine the prevalence of hev among adults in south - west of iran . this cross - sectional study was approved by the ethical committee of ahvaz jundishapur university of medical science with research project number 91112 . to estimate the prevalence of anti - hev igg and igm antibodies in the general population of ahvaz city , 510 blood samples from the adult population of ahvaz city were collected randomly using the multistage cluster sampling method from february to july 2014 . ahvaz is a large city in the south - west of iran that consists of 8 districts and has 94 public health centers . in the first stage , 4 public health centers were selected randomly from each district . in the next stage , the family registry code was used to randomly select 16 households within each public health center . from each family , one subject the trained interviewers visited the subjects in their homes and completed a questionnaire containing information of age , gender , and race / ethnicity for each individual . the subjects who refused to participate in the study were replaced with the next random participants . the serum samples were tested in duplicate for anti - hev igg and igm antibodies by using dia.pro hev ab elisa kit and hev igm elisa kit ( dia.pro , italy ) according to the manufacturer 's instructions . statistical analyses were performed using spss 17 package program ( spss inc . , chicago , il , usa ) and p values of less than 0.05 were considered statistically significant . data were analyzed and compared by descriptive statistics and chi - square test or fisher 's exact test . out of 510 study subjects , 206 ( 40.4% ) were male and 304 ( 59.6% ) were female . the average age of participants was varying from 18 to 81 years while the mean age sd was 45.89 14.63 years . the subjects were classified into six age groups : 1830 , 3140 , 4150 , 5160 , 6170 , and over 71 years . 70 ( 13.7% ) subjects were between 18 and 30 years old , while 135 ( 26.5% ) were between 31 and 40 years old , 135 ( 26.5% ) were between 41 and 50 years old , 80 ( 15.7% ) were between 51 and 60 years old , 55 ( 10.8% ) were between 61 and 70 years old , and 35 ( 6.9% ) were older than 71 years . based on race / ethnicity , 53.7% ( 274 ) of cases were arab and 46.3% ( 236 ) were farsi . of the 510 subjects , 235 ( 46.1% ) are shown to be positive for anti - hev igg antibody by dia.pro hev ab elisa kit , while 275 ( 53.9% ) were negative . with regard to gender and race , 86/206 ( 41.7% ) in the male group and 149/304 ( 49% ) in the female group were positive for anti - hev igg antibodies . 134/274 ( 48.9% ) in the arab group and 101/236 ( 42.8% ) in the farsi group are shown to be positive for anti - hev igg antibody . however , the seroprevalence was higher among arab and female groups ; hev seropositivity was not statistically associated with gender ( p = 0.106 ) and race ( p = 0.168 ) . meanwhile , there was statistical difference in anti - hev igg seroprevalence rate between the subjects grouped according to age ( p < 0.001 ) , so that seroprevalence of hev increased with age from 14.3% ( 10/70 ) in subjects aged 1830 years to 71.4% ( 25/35 ) in persons over 71 years old , with a peak among 6170 year - olds ( 90.9% , 50/55 ) . the highest rate of anti - hev seroprevalence was seen in subjects aged 6170 years ( table 1 ) . when we evaluated anti - hev igm antibody seroprevalence rate in the gender and race groups , no significant differences were observed between the subjects regarding gender ( 1% in females and 1.9% in males , p = 0.448 ) and race ( 2.2% in arab and 0.4% in farsi , p = 0.130 ) . however , with regard to age , 4/70 ( 5.7% ) in the age group 1830 years and 3/135 ( 2.2% ) in the age group 3140 years were positive for anti - hev igm antibodies . there was a significant difference between the age groups regarding hev seropositivity ( p = 0.012 ) . the highest rate of anti - hev seroprevalence was observed in subjects aged 1830 years ( table 2 ) . overall , 7 blood samples ( 1.4% ) are shown to be positive for hev - specific - igm antibodies , while 503 samples ( 98.6% ) were negative . hepatitis e infection is a worldwide public health concern , which causes large outbreaks of acute hepatitis in developing countries especially asia , middle east , and africa and also sporadic cases of the infection in developed countries such as south america and europe . although hev is mainly transmitted via the fecal - oral route especially contaminated water in endemic areas , transmission via the blood transfusion has also been suggested according to the high prevalence of anti - hev igg among blood donors [ 4 , 13 , 14 ] . epidemiological studies in different parts of the world show the wide variation in hev prevalence patterns , though the hev seroprevalence rates are higher among less developed countries . high prevalence rates are often reported from south asia , egypt in the middle east , and the far east except japan , and low rates are often found in europe and the americas . iran is an endemic country for hepatitis e infection [ 7 , 13 ] , since hev seroprevalence in general population is above 5% . ataei et al . in 2005 reported hev seroprevalence rate of 3.8% among general population in isfahan province , iran . assarehzadegan et al . in 2005 reported hev prevalence rate of 11.5% among blood donors in khuzestan province . study , hev prevalence was 9.3% in general population of tehran . in another study by nazer et al . , the prevalence of hev was reported to be 7.8% in khorramabad city in 2009 . regarding hev prevalence among the general population of other countries , the overall hev prevalence rate was reported to be 22.5% among general population in bangladesh by labrique et al . , about 3.20% in french blood donors by boutrouille et al . , 13% in the general population in england by ijaz et al . , 1.9% in the general population in netherlands , and 5.3% in the general population of japan . in the present study we investigated the hev seroprevalence among adult population in ahvaz city and found that anti - hev igg and igm seroprevalence were 46.1% and 1.4% , respectively . the result of the current study is considerably higher than that reported among adults in other parts of iran : 9.3% in nahavand , 8.1% in isfahan , 7.8% in western iran , 7.3% in sari , and 7.915% in tehran ; it is also higher than that reported among adult population of some other countries : 3.9% in united kingdom , 16.8% in germany , 7.3% in spain , about 20% in korea , 23% in thailand , 3942% in usa , and 5.9% in turkey ; however , it is lower than that reported among rural population older than four years in egypt ( 5178% ) [ 16 , 34 ] , pregnant women in nile delta , egypt ( 84% ) [ 16 , 35 ] , general population older than 11 years in central malaysia ( 5067% ) , tribes population ( 50100% ) and adult population ( 1677% ) in andaman islands , india , and homeless children in cochabamba city , bolivia ( 66% ) . however , a part of this difference may be due to differences in the used elisa detection kits , the time of sampling , and the demographics and size of studied population . overall , our results compared with the previous studies from iran indicate that the geographic distribution of hev infection is different even within a specific country , which most likely reflects different levels of exposure to infection over time due to different living conditions in different regions and fecal - oral transmission of hev . in the current study , the hev seroprevalence rate significantly increased with age from 14.3% in people aged below 31 years to 90.9% in persons aged 6170 years . improvement of public health and hygiene results in decreased exposure to the virus over time . however , exposure to hev increases with age . this is consistent with most studies which reported a significant association between age and higher anti - hev positive values , since the prevalence of the disease increases with age [ 26 , 29 , 31 , 36 ] . similarly high seroprevalence was found among adult population older than 60 years in china ( 7080% ) , adult population older than 80 years in bangladesh ( 67% ) , and adult population older than 80 years in hong kong ( 5260% ) . similar to the results of previous studies [ 5 , 13 , 17 , 29 , 37 ] , our results show that the presence of anti - hev igg and igm antibodies is not associated with gender ; also we did not find any association between race / ethnicity and hev seropositivity . our data showed that the anti - hev igg prevalence rate among adult population in ahvaz is 46.1% , the highest rate reported in different parts of iran . the implication is that ahvaz city is a highly endemic area for hev and the main route of hev transmission in this city is most likely karun river . evidence for this claim is that the drinking water source of the city is supplied from karun river and this river is commonly used for swimming , fishing , and other household needs by inhabitants . since the major transmission route of hev is most often the fecal contaminated drinking water and also this virus is relatively resistant to environmental conditions and remains infectious in sewage , the river can be considered as the water source for hev infection . however more studies are required to confirm this hypothesis . therefore , type e hepatitis is more common among adult population of ahvaz city compared with other parts of iran and this finding should be considered in the differential diagnosis of hepatitis infections and also prediction of possible outbreaks . in conclusion , high anti - hev igg seroprevalence of 46.1% was observed among the adults population living in ahvaz city of iran . determination of hev prevalence in different regions can be used for the purpose of hev epidemiology by developing a prevalence map on the base of hev geographical distribution . in addition to epidemiological purposes , hev prevalence information is important in evaluating the public health and hygiene and in identifying the major route of hev transmission in iran .

background . knowledge regarding prevalence of hev in general population can be an indicator of the public health and hygiene . therefore , this study was conducted to evaluate the prevalence of hev among adults in south - west of iran . methods . blood samples were taken from 510 participants , 206 ( 40.4% ) males and 304 ( 59.6% ) females from february to july 2014 . detection of anti - hev igg and igm antibodies was carried out by elisa test . results . the overall anti - hev igg and igm prevalence rates were 46.1% and 1.4% , respectively . anti - hev igg and igm seropositivity were not statistically associated with gender and race / ethnicity . meanwhile , there were significant differences between the age groups regarding hev igg and igm seropositivity . hev igg seroprevalence increased with age from 14.3% in subjects aged 1830 years to 71.4% in persons over 71 years old , and considerably individuals aged 61 to 70 years had the highest hev prevalence ( 90.9% ) . also , 5.7% in the age group 1830 years and 2.2% in the age group 3140 years were positive for anti - hev igm antibodies and the highest rate was observed in subjects aged 1830 years . conclusion . in conclusion , high hev igg seroprevalence of 46.1% was observed among adults in south - west of iran .

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

early diagnosis of rib fractures can rapidly indicate the source of thoracic pain and be helpful in pain management of trauma patients . other than pain , rib fractures have been reported to be associated with morbidity and mortality in elderly patients ( 3 - 5 ) . in most emergency departments , ultrasonography is considered as one of the most useful screening tools for rapid evaluation of trauma patients ( 6 ) . application of ultrasonography for assessment of chest wall injuries has been reported from 1980s ( 7 ) . since then the technology of ultrasound devices has significantly improved so that images with higher resolutions are obtained . in light of these improvements , the diagnostic value of this modality has been considerably enhanced ( 9 ) . in this regard , studies have illustrated a considerably high diagnostic value of ultrasonography in detection of thoracic fractures , even higher than that of chest radiography ( 8 , 10 - 12 ) . for instance in his narrative review , chan referred to ultrasonography as a reliable diagnostic tool for detection of thoracic bone fractures ( 13 ) . nevertheless , still no comprehensive review has been carried out comparing the diagnostic values of chest ultrasonography and radiography in detection of thoracic fractures . one solution is to perform a meta - analysis on the available evidence ( 15 , 16 ) . accordingly , the present systematic review and meta - analysis aimed to determine the diagnostic values of chest ultrasonography and radiography in detection of thoracic bone fractures . search strategy and selection criteria the study protocol was established based on the guidelines of meta - analysis of observational studies in epidemiology statement ( moose ) ( 19 ) . after selection of keywords from medical subject heading ( mesh ) terms and emtree , two reviewers ( m.y , p.g ) independently carried out an extended systematic search in databases of medline ( via pubmed ) , embase ( via ovidsp ) , isi web of knowledge , scopus , cochrane library , and proquest without any time or language limitations . sonography or ultrasound or radiography or chest film or chest radiograph combined with rib fractures or chest wall fracture or additionally , the bibliographies of original and review articles as well as google scholar were also searched . all the studies evaluating the diagnostic accuracy of ultrasonography or chest radiography in detection of chest wall fractures were assessed . review and editorial articles , case reports and studies with sample populations of less than 10 patients were excluded . two reviewers ( m.y , p.g ) independently worked on summarizing the data regarding assessing quality of studies , baseline characteristics of patients ( age , gender , the number of patients with and without hemothorax , the etiology of hemothorax ) , the characteristics of ultrasonography device ( transducer , frequency ) , physicians in charge of imaging interpretation , blinding status , sampling method ( consecutive , convenience ) , study design ( retrospective , prospective ) . finally the number of true positive ( tp ) , true negative ( tn ) , false positive ( fp ) , and false negative ( fn ) cases were extracteded . disagreements were discussed with the third reviewer ( m.h ) and a solution was proposed . in cases of data inaccessibility , data presented as charts were extracted via the method proposed by sistrom and mergo ( 28 ) . in cases where only the sensitivity and specificity were presented , reliable web - based programs were used to calculate the number of tp , tn , fp , and fn cases . charecteristics of included studies 1 , ( + / - ) : number of patient with fracture / number of patient without fracture ; 2 , number are presented as mean standard deviation or ( range ) . ct : computed tomography ; cxr : chest radiography ; ep : emergency physician ; na : not applicable ; nr : not reported ; us : ultrasonography quality assessment quality of the studies were assessed based on the guidelines of 14-item quality assessment of diagnostic accuracy studies ( quadas2 ) tool ( 29 ) . the quality assessment were performed based on following items : acceptable reference tests , accounting for indeterminate results , avoiding differential verification bias , disease progression bias , incorporation bias and verification bias , blind index test interpretation , blind interpretation of reference test , explained withdrawal , relevant clinical data available , and representative spectrum . a total grading of poor , fair , and good was attributed to each survey and only the fair and good studies were included in the meta - analysis . statistical analysis analysis was performed using stata 11.0 statistical software via midas module . to evaluate the screening performance characteristics of ultrasonography and radiography in detection of chest wall fractures , summary receiver operative curves ( srocs ) were drawn and pooled sensitivity , specificity , positive likelihood ratio and negative likelihood ratio with 95% confidence interval ( 95% ci ) were calculated . due the high heterogeneity between the included studies , mixed - effects binary regression model was used . heterogeneity was evaluated through calculations of i2 and 2 tests and a p value of less than 0.1 together with an i2 greater than 50% were considered as positive heterogeneity ( 30 ) . deek s asymmetry funnel plot was used to search for publication bias . in all the analyses , p value of less than 0.05 study characteristics 17 out of 3894 studies found in the comprehensive search were included in the systematic review and meta - analysis ( 1 , 2 , 8 , 10 - 12 , 14 , 17 , 18 , 20 - 27 ) . 5 studies had assessed the diagnostic accuracy of ultrasonography in detection of thoracic bone fractures ( 1 , 2 , 18 , 20 , 21 ) , 8 diagnostic value of chest radiography ( 14 , 17 , 22 - 27 ) , and 4 diagnostic values of ultrasonography and radiography simultaneously ( 8 , 10 - 12 ) . 1667 cases ( 807 with and 860 without fractures ) were extracted from the 17 mentioned articles , whose age ranged from 0 to 92 years old . figure 1 shows the inclusion process of articles and table 1 summarizes the characteristics of included studies . the results of the analyses are presented as srocs and funnel plots in figures 3 to 5 . the area under the curve of sroc for ultrasonography and radiography in detection of chest wall fractures were found to be 0.99 ( 95% ci : 0.97 - 0.99 ) and 0.97 ( 95% ci : 0.96 - 0.99 ) , respectively ( figure 3 ) . pooled sensitivity and specificity of ultrasonography in detection of thoracic bone fractures were 0.97 ( 95% ci : 0.90 - 0.99 ; i2= 88.88 , p<0.001 ) and 0.94 ( 95% ci : 0.86 - 0.97 ; i2= 71.97 , p<0.001 ) , respectively ( figure 4-a ) . these characteristics for radiography were found to be 0.77 ( 95% ci : 0.56 - 0.90 ; i2= 97.76 , p<0.001 ) and 1.0 ( 95% ci : 0.91 - 1.00 ; i2= 97.24 , p<0.001 ) , respectively ( figure 5-a ) . in addition , pooled positive and negative likelihood ratios of ultrasonography were 16.26 ( 95% ci : 6.26 - 38.87 ; i2= 59.14 , p<0.001 ) and 0.03 ( 95% ci : 0.01 - 0.11 ; i2= 86.76 , p<0.001 ) , respectively ( figure 4-b ) , while these measures for radiography were reported to be 774.63 ( 95% ci : 7.0 - 8573.0 ; i2= 96.62 , p<0.001 ) and 0.23 ( 95% ci : 0.11 - 0.48 ; i2= 96.94 , p<0.001 ) , respectively ( figure 5-b ) . there were significant heterogeneity between the articles ( figure 4 and 5 ) . specificity of ultrasonography in detection of thoracic bone fractures was directly correlated with frequency of transducer ( 90% vs. 95% ) . the sensitivity of this modality was found to be higher in detection of rib fractures rather than fractures of clavicle or sternum ( 97% vs. 91% ) . moreover it was found that the sensitivity would be higher if the procedure is performed by a radiologist ( 96% ) compared to an emergency medicine specialist ( 90% ) . studies with sample sizes of greater than 100 patients reported higher diagnostic accuracies for ultrasonography in detection of thoracic bone fractures ( 97% vs. 91% ) . as can be seen in table 2 , the most important factor affecting sensitivity of chest radiography is the interpreting physician . the sensitivity was found to be 66% when the radiogram was interpreted by an emergency medicine specialist while it was 80% when interpreted by a radiologist . furthermore , consecutive sampling method compared with convenience ( 80% vs. 73% ) and sample size of more than 100 patients ( 82% vs. 73% ) were also found to be sources of heterogeneity . base on the results of the present meta - analysis sensitivity of chest ultrasonography in detection of thoracic bone fractures following trauma was prominently higher than radiography ( 97% vs. 77% ) . yet , the specificity of radiography was found to be significantly higher than ultrasonography in this regard ( 100% vs. 94% ) . on this basis and according to calculated likelihood ratios , a negative result of ultrasonography in detection of thoracic fracture is more reliable than radiography ( negative likelihood ratio=0.03 ) , while a positive result of chest radiography is more reliable than ultrasonography ( positive likelihood ratio=774.63 ) . subgroup analysis of diagnostic accuracy for chest radiography and ultrasonography in detection of thoracic bone fractures , p value < 0.1 was considered as significant for heterogeneity ; ci : confidence interval . ultrasonography had a higher sensitivity in diagnosis of rib fractures rather than other chest wall bones , while the type of fracture had no effect on diagnostic value of radiography . this finding can be ascribed to the higher attention that physicians pay to rib fractures rather than other chest wall bones such as scapula and sternum . fractures are diagnosed via ultrasonography based on observation of cortical bone disruption . in cases of small fractures , detection of this sign in sonogram and distinguishing it from other findings is highly dependent on the skills of the operator . the role of operator s skills in detection of injuries via ultrasonography was verified in the present study as well ( 31 - 34 ) . the present study found that the specificity of this modality increased with frequencies of higher than 10mhz which might be due to the higher resolution obtained with higher frequencies ( 35 ) , making it easier to detect the signs of fracture . some narrative review articles and qualitative systematic reviews are indicative of the potential benefit of ultrasonography in detection of chest wall fractures . in this regard , chan , in his systematic review conducted on studies indexed in medline , declares that ultrasonography has a higher sensitivity in detection of thoracic bone fractures compared to radiography ( 13 ) . finding the diagnostic accuracy of ultrasonography to be two times the ability of radiography in fracture diagnosis , dietrich et al . also referred to ultrasonography as a useful diagnostic tool for detection of rib fractures ( 36 ) . presence of considerable heterogeneity between the included articles and simultaneous inclusion of retrospective and prospective studies in the meta - analysis were major limitations of this study . ess : effective sample sizes summary receiver operative curves ( sroc ) for ultrasound ( a ) and chest radiography ( b ) in detection of thoracic bone fractures . auc : area under the curve ; sens : sensitivity ; spec : specificity forest plot of screening performance characteristics of chest ultrasonography in detection of thoracic bone fractures . sensitivity and specificity ( a ) ; diagnostic likelihood ratio ( dlr ) ( b ) . ci : confidence interval forest plot of screening performance characteristics of chest radiography in detection of thoracic bone fractures . sensitivity and specificity ( a ) ; diagnostic likelihood ratio ( dlr ) ( b ) . base on the findings of the present meta - analysis , screening performance characteristic of ultrasonography in detection of thoracic bone fractures was found to be higher than radiography . however , these characteristics were more prominent in detection of rib fractures and in cases where was performed by a radiologist . this research has been supported by tehran university of medical sciences & health services grant number : 93 - 02 - 38 - 25618 . all authors passed four criteria for authorship contribution based on recommendations of the international committee of medical journal editors .

introduction : the potential benefit of ultrasonography for detection of thoracic bone fractures has been proven in various surveys but no comprehensive conclusion has been drawn yet ; therefore , the present study aimed to conduct a thorough meta - analytic systematic review on this subject . methods : two reviewers independently carried out a comprehensive systematic search in medline , embase , isi web of knowledge , scopus , cochrane library , and proquest databases . data were summarized as true positive , false positive , true negative and false negative and were analyzed via stata 11.0 software using a mixed - effects binary regression model . sources of heterogeneity were further assessed through subgroup analysis . results : data on 1667 patients ( 807 subjects with and 860 cases without thoracic fractures ) , whose age ranged from 0 to 92 years , were extracted from 17 surveys . pooled sensitivity and specificity of ultrasonography in detection of thoracic bone fractures were 0.97 ( 95% ci : 0.90 - 0.99 ; i2= 88.88 , p<0.001 ) and 0.94 ( 95% ci : 0.86 - 0.97 ; i2= 71.97 , p<0.001 ) , respectively . the same measures for chest radiography were found to be 0.77 ( 95% ci : 0.56 - 0.90 ; i2= 97.76 , p<0.001 ) and 1.0 ( 95% ci : 0.91 - 1.00 ; i2= 97.24 , p<0.001 ) , respectively . the sensitivity of ultrasonography was higher in detection of rib fractures , compared to fractures of sternum or clavicle ( 97% vs. 91% ) . moreover , the sensitivity was found to be higher when the procedure was carried out by a radiologist in comparison to an emergency medicine specialist ( 96% vs. 90% ) . conclusion : base on the findings of the present meta - analysis , screening performance characteristic of ultrasonography in detection of thoracic bone fractures was found to be higher than radiography . however , these characteristics were more prominent in detection of rib fractures and in cases where was performed by a radiologist .

Introduction: Methods: Results: Discussion: Conclusion: Conflict of interest: Funding support: Authors contributions:

aortic dissection following coronary angiography and angioplasty is a rare , but potentially fatal complication with an overall reported incidence of 0.01 - 0.04% [ 1 - 3 ] . the incidence is significantly higher during percutaneous coronary interventions ( 0.12% ) than during elective diagnostic procedures . moreover , in the setting of acute myocardial infarction ( ami ) , the reported incidence is 0.19% , significantly higher than the 0.01% reported in the absence of ami . management and outcomes have varied considerably in the literature . while up to 50% mortality was reported in one series following surgical repair of the dissections , other studies reported more favorable outcomes with conservative management . we report a case of an elderly woman who developed type a aortic dissection with moderate to severe aortic regurgitation and heart failure , diagnosed approximately 1 month following a diagnostic coronary angiography . she underwent emergency surgical repair of the dissection ; however , she could not be successfully weaned off of cardiopulmonary bypass despite maximum pharmacological interventions and support and died in the operating room . a 63-year - old white female with known hypertension , hyperlipidemia , and prior pacemaker insertion for atrial fibrillation with symptomatic bradycardia , was admitted to our hospital with recent onset substernal chest pressure radiating to her jaw , with diaphoresis . her vital signs were stable , and her physical examination was non - revealing , but was limited by her morbid obesity . her electrocardiogram revealed atrial fibrillation with demand ventricular pacing and her cardiac biomarkers were negative for acute coronary syndrome . echocardiogram revealed normal left ventricular systolic function and normal aortic root diameter , otherwise was technically difficult ( fig . coronary angiography revealed 60 - 70% eccentric lesion in the second obtuse marginal , otherwise mild atherosclerotic disease . ( a ) baseline two - dimensional ( 2d ) parasternal long axis echocardiographic image obtained prior to index cardiac catheterization showing normal size aortic root and ascending aorta ( ao ) . ( b ) corresponding 2d parasternal echocardiographic image 1 month following cardiac catheterization showing massive dilatation of the aortic root and ascending aorta ( ao ) with a dissection flap . lv : left ventricle ; rv : right ventricle ; la : left atrium . she was readmitted 1 month later with recurring episodes of chest pain , shortness of breath , orthopnea and reduced exercise tolerance which started ever since her recent discharge , for which she had two emergency room visits and a clinic follow - up visit . blood pressure ( bp ) was 152/93 mm hg , and heart rate was 105 bpm . she had an elevated bnp of 1,400 pg / ml , but otherwise negative cardiac biomarkers , and was therefore treated with diuretics , which resulted in a significant decrease of her bp to 81/51 mm hg . repeat echocardiogram was performed , which revealed normal left ventricular systolic function , and severely dilated aortic root , measuring 6.7 cm , with aortic dissection flap noted ( fig . ct angiogram revealed aortic dissection extending proximally to the aortic root above the coronary ostia ( fig . ( a ) axial ct angiographic image showing the dissection starting at the aortic root and sparing the origins of the coronary arteries , with massive dilatation of the aortic root . ( b ) sagittal ct angiographic image showing the extension of the dissection to the origin of the brachiocephalic artery . the patient was taken emergently to surgery and despite extensive surgical repair and pharmacologic support , she could not be weaned off of cardiopulmonary bypass and expired in the operating room . type a aortic dissection is a lethal condition with an overall surgical mortality of up to 34% in unstable patients , as reported in 526 cases by the international registry of acute aortic dissection ( irad ) . the irad registry reported 28 cases ( 5.3% ) of iatrogenic type a aortic dissection ( itaad ) as a complication of cardiac surgery or cardiac catheterization , nine of which ( 32% ) died . the german registry for acute aortic dissection type a ( geraada ) reported a comparable incidence of 100 cases ( 4.7% ) of itaad out of a total of 2,137 cases of type a aortic dissection , but with a lower 30-day mortality of 16% . leontyev et al reported 48 cases over a 15-year follow - up , undergoing surgery for itaad , with a frequency of 0.06% ( 36 cases ) of open heart surgeries , and 0.01% ( 12 cases ) of cardiac catheterizations ; early surgical mortality was 50% for itaads associated with coronary angiography . dunning et al reported nine cases ( 0.02% ) of coronary artery - aortic dissections out of over 40,000 cardiac catheterizations performed over a 6-year period ; these were significantly more prevalent in the setting of ami ( 0.19% ) , compared with non - ami ( 0.01% ) . while outcomes were favorable for the less severe dissections treated conservatively , two of the reported cases had dissection extending into the arch , and both died following surgery . they proposed a classification scheme based on the extent of aortic dissection beyond the involved coronary cusp ( fig . 3 ) , and concluded that the best treatment in class 1 and 2 dissections is stenting of the intra - coronary entry point when possible and close clinical follow - up , while class 3 dissections usually require surgical intervention . gomez - moreno et al reported 17 cases ( 0.04% incidence ) of itaad associated with cardiac catheterizations over a 10-year follow - up period , with a significantly higher incidence after interventional procedures ( 0.12% ) than after diagnostic procedures ( 0.01% ) . patients were treated conservatively with either stenting to seal the entry door or expectant management ; no patients died during hospitalization or follow - up . nunez - gil et al reported 14 cases ( 0.02% incidence ) of iatrogenic dissection of the descending aorta / arch without coronary involvement over a 15-year follow - up treated conservatively , with only one hospital death . tanasie et al reported eight cases over a 5-year follow - up of itaads referred for multi - detector coronary tomography ( mdct ) evaluation ; one patient required surgery and died , while all others treated conservatively with either stenting or expectant management survived . dunning classification of itaad . several cases of conservatively treated itaad have been reported in the literature with favorable outcomes . sakakura et al reported a 79-year - old male with itaad following stenting of proximal right coronary artery ( rca ) for angina who was treated with intravascular ultrasound ( ivus)-guided stenting of the entry point with good outcome . fiddler et al reported a 65-year - old female who underwent rca stenting for ami resulting in guidewire - associated itaad with evidence of tamponade ; there was spontaneous hemodynamic stabilization and subsequent ct demonstrating no intimal dissection flap , prompting expectant management with favorable outcome . kerut et al reported a 79-year - old male undergoing coronary angiography for angina , who developed subintimal hematoma , without dissection flap , in the left coronary sinus and 4 cm upwards as demonstrated by ct and transesophageal echocardiography ( tee ) ; these findings resolved spontaneously and she was treated conservatively with favorable outcome . ghaffari and pourafkari reported itaad proximal to the origin of the right brachiocephalic artery in a 30-year - old male with bicuspid aortic valve undergoing aortography in preparation for aortic coarctation stenting . the dissection spontaneously sealed , and they attributed this to curved catheter position in the aortic arch and high - pressure contrast jet through the catheter side holes in the setting of aortopathy . gorog et al reported a 56-year - old female undergoing coronary angiography who developed aortic dissection starting at the femoral artery extending into the aortic arch , treated successfully with a self expanding metallic stent into the iliac artery via the contralateral femoral artery approach . shah et al reported two cases of itaad , the first was a 56-year - old male receiving rca stent for inferior ami who developed itaad starting at the rca cusp , successfully treated with stenting of the proximal rca to cover the entry point . the second case was a 68-year - old male who developed left main coronary dissection with retrograde extension into the coronary cusp during stenting of the left circumflex , treated successfully with left main stenting ; he subsequently underwent an uneventful elective coronary artery bypass grafting ( cabg ) surgery . welch et al reported a 65-year - old female treated with multiple rca stents for inferior ami who developed an intramural hematoma extending from the right sinus of valsalva to the origin of the brachiocephalic artery , with an entry point . lambelin et al reported a 75-year - old female who developed cardiac tamponade and cardiogenic shock due to intimal tear in the ascending thoracic aorta , not involving the coronaries , during angiography for aortic insufficiency ; she was successfully treated with immediate surgery . noguchi et al reported a 66-year - old male with inferior ami undergoing circumflex stenting , who developed cardiac tamponade and hypotension due to itaad originating at the brachiocephalic artery , without involvement of the coronaries ; he underwent successful emergency surgical repair . yilik et al reported two cases of itaad treated surgically , the first was a 65-year - old female who developed retrograde aortic dissection during angioplasty of the left anterior descending ( lad ) for unstable angina ; due to inability to localize the tear , she underwent uneventful surgical repair . the second case was a 73-year - old female who developed itaad during angioplasty to the rca , the guidewire could not be introduced into the true lumen and therefore she underwent successful surgical repair . tochii et al reported a 69-year - old male who developed aortic dissection during balloon angioplasty to the left subclavian which extended retrogradely to the ascending aorta and required surgical intervention with good outcome . the above case reports , summarized in table 1 [ 8 - 18 ] , demonstrate the heterogeneity of itaad with regard to patient characteristics and presentations , cause and location of the dissection , and treatment options . it is obvious that severe dissections are associated with high early mortality and therefore warrant emergency surgery , which in itself carries very high risk of mortality . our patient is unique in that her presentation was delayed at 1 month from her index cardiac catheterization , with progressive symptoms , likely causing extensive aortic root dilatation and extension of dissection ( dunning class iii ) . negative outcomes may in fact be more prevalent than our discussion implies , but may tend to be under - reported in the literature . associated risk factors reported in several studies may be related to patient characteristics versus procedural variables , some of which may be modifiable ( table 2 ) . prompt recognition and management is crucial to avoid the extensive dissection and dilatation of the aorta , as we report in our patient , and reduce mortality . low risk dissections appear to respond well to conservative medical and localized interventional treatments , with serial hemodynamic monitoring , imaging and follow - up . high risk dissections overall appear to require surgical management , despite the increased surgical risk . associated risk factors reported in several studies may be related to patient characteristics versus procedural variables , some of which may be modifiable ( table 2 ) . prompt recognition and management is crucial to avoid the extensive dissection and dilatation of the aorta , as we report in our patient , and reduce mortality . low risk dissections appear to respond well to conservative medical and localized interventional treatments , with serial hemodynamic monitoring , imaging and follow - up . high risk dissections overall appear to require surgical management , despite the increased surgical risk .

we report a 63-year - old female with hypertension , hyperlipidemia , and prior pacemaker insertion for atrial fibrillation with symptomatic bradycardia , who was admitted with substernal chest pressure and diaphoresis . her electrocardiogram revealed atrial fibrillation with demand ventricular pacing and her cardiac biomarkers were negative for acute coronary syndrome . echocardiogram revealed normal left ventricular systolic function and normal aortic root diameter . coronary angiography revealed 60 - 70% obtuse marginal lesion , otherwise mild disease . she was treated medically and discharged in stable condition . she was readmitted 1 month later with recurring chest pain , and shortness of breath which started shortly after her most recent discharge . blood pressure was 152/93 mm hg , and heart rate was 105 bpm . bnp was elevated at 1,400 pg / ml , and other cardiac biomarkers were negative . she was treated with diuretics , which resulted in decrease of her blood pressure to 81/51 mm hg . repeat echocardiogram revealed severely dilated aortic root , measuring 6.7 cm , with aortic dissection flap and moderate to severe aortic regurgitation . ct angiogram revealed aortic dissection extending proximally to the aortic root above the coronary ostia and distally to the left subclavian artery takeoff . she underwent surgery ; she , however , could not be weaned off from cardiopulmonary bypass and died in the operating room . this case illustrates the importance of having a high index of suspicion for iatrogenic aortic dissection following cardiac catheterization as a cause of recurrence of cardiac symptoms , as early detection may help avert a catastrophic outcome , as we report in our patient .

Introduction Case Report Discussion Conclusion Disclosures

a significant percentage of overweight and obese individuals show signs of insulin resistance , up to and including the metabolic syndrome . overweight individuals with insulin resistance are in turn at increased risk of type 2 diabetes and cardiovascular disease ( cvd ) . cvd has been the leading cause of morbidity and mortality in the united states for more than 80 years . impaired fasting glucose , hypertension , atherogenic dyslipidemia , obesity , and endothelial dysfunction have each been shown to increase the risk of type 2 diabetes mellitus and cvd . in the epidream cohort study , a 1-mmol / l increase in fasting plasma glucose was associated with a 17% increase in the risk of future cardiovascular events and death . in the idea study , the frequency of cvd and type 2 diabetes mellitus increased proportionally with waist circumference for both genders . in the framingham heart study , the risk of major coronary heart disease events increased by nearly 25% for every 5-mg / dl decrease in high - density lipoprotein cholesterol ( hdl - c ) below the median values . therapeutic lifestyle change , with an emphasis on diet , is recommended as a first - line treatment for the prevention of diabetes and cvd in at - risk individuals . nuts in particular are gaining increasing attention in this area , with a recent meta - analysis indicating reduced risk of diabetes among those consuming more nuts and less red meat . emerging clinical trial and epidemiological evidence indicates that nuts can favorably alter levels of oxidative stress , inflammation , and lipids and can improve glucose metabolism and vascular reactivity [ 1215 ] . walnuts are a uniquely rich source of -linolenic acid ( ala ) , and epidemiological studies suggest that plant - derived ala may confer particular cardiovascular benefits . a meta - analysis investigating the impact of walnut consumption on blood lipids showed that walnut - enriched diets significantly decreased total cholesterol and low - density lipoprotein cholesterol ( ldl - c ) when compared with control diets for the duration of the short - term trials . furthermore , walnuts are also rich in tocopherol , phenolic antioxidants , folic acid , and magnesium , nutrients that have been shown to impact endothelial function favorably . previously , we reported that a walnut - enriched ad libitum diet improves endothelium - dependent vasodilatation in type 2 diabetic individuals , suggesting a potential reduction in overall cardiac risk in this population . to our knowledge , the effects of walnuts on endothelial function in adults with visceral adiposity and free of diabetes have not been examined further . we therefore conducted a randomized controlled crossover trial to investigate the effects of daily walnut ingestion in overweight adults with central obesity on endothelial function as well as body mass index ( bmi ) , weight , waist circumference , lipid panel , insulin sensitivity , and blood pressure . forty - six participants ( 18 men and 28 women ) were recruited from the lower naugatuck valley in connecticut through flyers and newspaper advertisements . participants were required to be nonsmoking adults aged 3075 years with a bmi greater than 25 and a waist circumference of more than 40 inches for men or more than 35 inches for women . all included participants also exhibited 1 or more additional risk factors for metabolic syndrome : blood pressure 130/85mmhg or taking antihypertensive medication , fasting serum glucose ( fpg ) 100 mg / dl , fasting serum triglyceride ( tg ) level 150 mg / dl , or fasting hdl - c 40 mg / dl in men or 50 mg / dl in women . participants were excluded if they were pregnant or had been diagnosed with atherosclerotic vascular disease , diabetes , severe hypertension , sleep apnea , tuberculosis , acquired immune deficiency syndrome , cancer , psychotic disorder , and/or eating disorder . participants were also excluded if they had a prior history of substance abuse , consumed restricted diets by choice ( i.e. , vegan , carbohydrate - restricted , etc . ) , were allergic to walnuts or any other nuts , or were unwilling to refrain from taking medication for 12 hours prior to assessment . individuals who regularly used nonsteroidal anti - inflammatory drugs or vasoactive medication , fiber supplements , aspirin , lipid - lowering medications , or antihypertensive medications and had been taking them at a stable dosage for less than 3 months were also excluded from participation . those passing the telephone screening ( n = 92 ) underwent a clinical screening examination consisting of height , weight , bmi , blood pressure measurements , and laboratory testing including fasting serum lipids and fpg . this study was a randomized , controlled , single - blind , crossover clinical trial . all participants first took part in a dietary group session that was led by a registered dietitian . this group session was the initiation of the 4-week run - in period to allow for diet and weight stabilization . the 3-day diet records were done once during the original run - in period , once during each 8-week dietary period , and once during the 4-week washout period . at the end of the run - in period , participants ( n = 46 ) were randomly assigned to one of two possible treatment sequences consisting of an 8-week walnut - enriched ad libitum diet and an ad libitum diet without walnuts for 8 weeks . treatment assignments were separated by a 4-week washout period . during the walnut - enriched diet treatment , participants were given an 8-week supply of walnuts . they were instructed to consume 56 g of shelled , unroasted english walnuts per day as a snack or with a meal . a consumption log sheet was provided for participants to keep a record of their walnut consumption . participants were instructed to maintain their baseline medication and supplement use and physical activity level throughout the study . to ensure that weight remained stable during the walnut - enriched phase , participants were counseled by a registered dietitian during the group session about strategies for equivalently substituting calories from walnuts for calories from other foods in the diet but were instructed to otherwise continue with their usual dietary patterns . compliance with treatment and dietary patterns was assessed from subjects diet records and consumption log sheets . during each visit height , weight , blood pressure , fasting serum lipids , fpg , and fasting serum insulin were measured . endothelial function was measured noninvasively in the right brachial artery by a high - frequency ultrasound scanning machine ( sonos 4500 , phillips medical systems , andover , ma ) in accordance with published guidelines and our previous endothelial function studies [ 20 , 2224 ] . subjects were required to rest in a quiet , temperature - controlled , softly lit room for 15 minutes before scanning was initiated . the right brachial artery was imaged longitudinally , 25 cm above the antecubital fossa , by an experienced registered vascular technologist ( rvt ) who was blinded to the treatment assignments . a resting scan was performed , and arterial flow velocity was measured . an occluding cuff placed on the upper arm was inflated to a pressure of 250 mmhg for 5 minutes and rapidly deflated to induce reactive hyperemia . brachial artery scans were acquired on magnetic optical disk continuously between 30 and 180 seconds after cuff deflation , including a repeated flow - velocity measurement during the first 15 seconds after cuff release . brachial artery diameters were analyzed by commercially available software ( brachial analyzer , medical imaging application , iowa city , ia ) . dilatation from baseline was measured at 5080 seconds after cuff deflation to assess endothelium - dependent vasodilatation . to test intraobserver reliability , a random sample of 20 brachial artery reactivity studies was re - read by the same rvt to ensure consistency of measurement and interpretation . endothelial function was measured as flow - mediated vasodilation ( fmd ) , the percentage change of brachial artery diameter from before cuff inflation to 60 seconds after cuff release . in addition to brachial diameter at 60 seconds after cuff release , flow after cuff deflation within the first 15 seconds was used as an indicator of stimulus strength , hyperemic flow being the stimulus for endothelial reactivity . to account for potential variability in stimulus strength , fmd was divided by flow at 15 seconds after cuff deflation to create a stimulus - adjusted response measure . fasting serum lipids ( consisting of total cholesterol , hdl - c , ldl - c , tgs , and total cholesterol : hdl ratio ) , fpg , and fasting serum insulin were measured at the griffin hospital laboratory using standard procedures at each visit . insulin resistance ( homa - ir ) values were generated from fpg and fasting serum insulin levels ( homa calculator version 2.2.1 ) to gauge the degree of insulin resistance . height , weight , bmi , and waist circumference were measured at each visit . to measure weight , participants were asked to remove their heavy outer garments ( jacket , coat , etc . ) and shoes and stand in the center of the platform with weight distributed evenly on both feet . waist circumference was measured at the umbilicus with the measurement tape surrounding the abdomen horizontal to the floor . blood pressure was determined with the use of the datascope accutorr plus automatic digital blood pressure device ( data - scope corp , mahwah , nj ) with the subject supine after a 5-minute period of rest . both systolic and diastolic diet records were analyzed using the food processor ii , esha research 's basic nutrition and diet analysis software ( version 7.0 , esha research , salem , or ) . the study protocol and consent form were approved by the griffin hospital ( derby , ct ) institutional review board . signed informed consent was obtained from all study participants , and all participants received monetary compensation for their participation . repeated - measures analysis of variance ( anova ) was used to assess differences in intraindividual responses across treatments . paired t tests were also used to compare baseline mean values of all outcome measures among participants by group assignment . the combined effect of independent variables ( age , race , bmi , hypertensive status , dyslipidemia , and treatment sequence ) and treatment assignment was assessed with respect to all outcome measures using multivariable anova models . the sample size was determined to allow for 20% attrition and noncompliance and to provide > 80% power to detect a minimal difference of 3% in fmd between treatments ( 21 ) with maximum allowable type i error of 5% . the sample size was calculated using the formula n = [ ( z + z)sd]/d , where n = the number of participants needed , z = 1.96 for 2 sided at 5% , z = 0.84 at 80% power , sd = 6.5% , and d = 3% ( minimal detectable difference in change in fmd between intervention and control assignments ) ; n = [ ( 1.96 + 0.84 ) 6.5]/3 = 37 participants are needed . allowing for 20% withdrawal and loss to follow - up , 46 participants were needed for the trial . forty - six participants ( 18 men and 28 women ) were recruited from the lower naugatuck valley in connecticut through flyers and newspaper advertisements . participants were required to be nonsmoking adults aged 3075 years with a bmi greater than 25 and a waist circumference of more than 40 inches for men or more than 35 inches for women . all included participants also exhibited 1 or more additional risk factors for metabolic syndrome : blood pressure 130/85mmhg or taking antihypertensive medication , fasting serum glucose ( fpg ) 100 mg / dl , fasting serum triglyceride ( tg ) level 150 mg / dl , or fasting hdl - c 40 mg / dl in men or 50 mg / dl in women . participants were excluded if they were pregnant or had been diagnosed with atherosclerotic vascular disease , diabetes , severe hypertension , sleep apnea , tuberculosis , acquired immune deficiency syndrome , cancer , psychotic disorder , and/or eating disorder . participants were also excluded if they had a prior history of substance abuse , consumed restricted diets by choice ( i.e. , vegan , carbohydrate - restricted , etc . ) , were allergic to walnuts or any other nuts , or were unwilling to refrain from taking medication for 12 hours prior to assessment . individuals who regularly used nonsteroidal anti - inflammatory drugs or vasoactive medication , fiber supplements , aspirin , lipid - lowering medications , or antihypertensive medications and had been taking them at a stable dosage for less than 3 months were also excluded from participation . those passing the telephone screening ( n = 92 ) underwent a clinical screening examination consisting of height , weight , bmi , blood pressure measurements , and laboratory testing including fasting serum lipids and fpg . this study was a randomized , controlled , single - blind , crossover clinical trial . all participants first took part in a dietary group session that was led by a registered dietitian . this group session was the initiation of the 4-week run - in period to allow for diet and weight stabilization . the 3-day diet records were done once during the original run - in period , once during each 8-week dietary period , and once during the 4-week washout period . at the end of the run - in period , participants ( n = 46 ) were randomly assigned to one of two possible treatment sequences consisting of an 8-week walnut - enriched ad libitum diet and an ad libitum diet without walnuts for 8 weeks . treatment assignments were separated by a 4-week washout period . during the walnut - enriched diet treatment , participants were given an 8-week supply of walnuts . they were instructed to consume 56 g of shelled , unroasted english walnuts per day as a snack or with a meal . a consumption log sheet was provided for participants to keep a record of their walnut consumption . participants were instructed to maintain their baseline medication and supplement use and physical activity level throughout the study . to ensure that weight remained stable during the walnut - enriched phase , participants were counseled by a registered dietitian during the group session about strategies for equivalently substituting calories from walnuts for calories from other foods in the diet but were instructed to otherwise continue with their usual dietary patterns . compliance with treatment and dietary patterns was assessed from subjects diet records and consumption log sheets . during each visit height , weight , blood pressure , fasting serum lipids , fpg , and fasting serum insulin were measured . endothelial function was measured noninvasively in the right brachial artery by a high - frequency ultrasound scanning machine ( sonos 4500 , phillips medical systems , andover , ma ) in accordance with published guidelines and our previous endothelial function studies [ 20 , 2224 ] . subjects were required to rest in a quiet , temperature - controlled , softly lit room for 15 minutes before scanning was initiated . the right brachial artery was imaged longitudinally , 25 cm above the antecubital fossa , by an experienced registered vascular technologist ( rvt ) who was blinded to the treatment assignments . an occluding cuff placed on the upper arm was inflated to a pressure of 250 mmhg for 5 minutes and rapidly deflated to induce reactive hyperemia . brachial artery scans were acquired on magnetic optical disk continuously between 30 and 180 seconds after cuff deflation , including a repeated flow - velocity measurement during the first 15 seconds after cuff release . brachial artery diameters were analyzed by commercially available software ( brachial analyzer , medical imaging application , iowa city , ia ) . dilatation from baseline was measured at 5080 seconds after cuff deflation to assess endothelium - dependent vasodilatation . to test intraobserver reliability , a random sample of 20 brachial artery reactivity studies was re - read by the same rvt to ensure consistency of measurement and interpretation . endothelial function was measured as flow - mediated vasodilation ( fmd ) , the percentage change of brachial artery diameter from before cuff inflation to 60 seconds after cuff release . in addition to brachial diameter at 60 seconds after cuff release , flow after cuff deflation within the first 15 seconds was used as an indicator of stimulus strength , hyperemic flow being the stimulus for endothelial reactivity . to account for potential variability in stimulus strength , fmd was divided by flow at 15 seconds after cuff deflation to create a stimulus - adjusted response measure . fasting serum lipids ( consisting of total cholesterol , hdl - c , ldl - c , tgs , and total cholesterol : hdl ratio ) , fpg , and fasting serum insulin were measured at the griffin hospital laboratory using standard procedures at each visit . insulin resistance ( homa - ir ) values were generated from fpg and fasting serum insulin levels ( homa calculator version 2.2.1 ) to gauge the degree of insulin resistance . height , weight , bmi , and waist circumference were measured at each visit . to measure weight , participants were asked to remove their heavy outer garments ( jacket , coat , etc . ) and shoes and stand in the center of the platform with weight distributed evenly on both feet . waist circumference was measured at the umbilicus with the measurement tape surrounding the abdomen horizontal to the floor . blood pressure was determined with the use of the datascope accutorr plus automatic digital blood pressure device ( data - scope corp , mahwah , nj ) with the subject supine after a 5-minute period of rest . both systolic and diastolic pressures were calculated as the mean value of 2 readings 5 minutes apart . diet records were analyzed using the food processor ii , esha research 's basic nutrition and diet analysis software ( version 7.0 , esha research , salem , or ) . the study protocol and consent form were approved by the griffin hospital ( derby , ct ) institutional review board . signed informed consent was obtained from all study participants , and all participants received monetary compensation for their participation . repeated - measures analysis of variance ( anova ) was used to assess differences in intraindividual responses across treatments . paired t tests were also used to compare baseline mean values of all outcome measures among participants by group assignment . the combined effect of independent variables ( age , race , bmi , hypertensive status , dyslipidemia , and treatment sequence ) and treatment assignment was assessed with respect to all outcome measures using multivariable anova models . the sample size was determined to allow for 20% attrition and noncompliance and to provide > 80% power to detect a minimal difference of 3% in fmd between treatments ( 21 ) with maximum allowable type i error of 5% . the sample size was calculated using the formula n = [ ( z + z)sd]/d , where n = the number of participants needed , z = 1.96 for 2 sided at 5% , z = 0.84 at 80% power , sd = 6.5% , and d = 3% ( minimal detectable difference in change in fmd between intervention and control assignments ) ; n = [ ( 1.96 + 0.84 ) 6.5]/3 = 37 participants are needed . allowing for 20% withdrawal and loss to follow - up , 46 participants were needed for the trial . forty - six overweight adults participated in the study . sixty - one percent of the participants were female . three subjects dropped out of the study due to changes in medication , 1 dropped out due to inability to comply with the protocol , and 2 dropped out due to schedule conflicts . hdl = high - density lipoprotein , ldl = low - density lipoprotein , homa - ir = homeostasis model assessment insulin resistance . dietary intake . dietary intakes of polyunsaturated fatty acids ( pufas ) , n-3 fatty acids , n-6 fatty acids , and fat increased significantly during the walnut - enriched diet treatment compared with the control diet ( pufas , p < 0.01 ; n-3 fatty acids , p < 0.01 ; n-6 fatty acids , p < 0.01 ; and fat , p = 0.01 ) . selected nutrient intake values are mean sd ; p values were obtained from 1-way analysis of variance . endothelial function . in this overweight population with at least 1 risk factor for metabolic syndrome , consumption of a walnut - enriched diet for 8 weeks improved fmd significantly from baseline as compared with the control diet ( p = 0.019 ; see table 3 ) . change in outcome measures values are mean sd . hdl = high - density lipoprotein , ldl = low - density lipoprotein , homa - ir , homeostasis model assessment insulin resistance . significant ( p < 0.05 ) from baseline ; p values were obtained from repeated - measures analysis of variance. p < 0.05 from paired student t test . daily consumption of the walnut - enriched diet for an 8-week period did not change anthropometric measures from baseline ( bmi : p = 0.481 ; weight : p = 0.439 ; waist circumference : p = 0.344 ) . the control diet was associated with a reduction in bmi ( p = 0.016 ) and body weight ( p = 0.019 ) relative to the walnut - enriched diet . lipid panel . after consumption of a walnut - enriched diet for 8 weeks , participants mean measures of total cholesterol , ldl - c , hdl - c , and tg did not differ significantly from baseline as compared with the mean measures of control diet ( total cholesterol : p = 0.69 ; ldl - c : p = 0.98 ; hdl - c : p = 0.89 ; tg : p = 0.32 ) . blood pressure . after consumption of a walnut - enriched diet for 8 weeks , participants blood pressure values decreased nonsignificantly from baseline as compared with the control diet without walnut supplementation ( systolic : p = 0.07 ; diastolic : p = 0.352 ) . fasting plasma glucose , fasting insulin , and homa - ir . fasting plasma glucose and fasting insulin did not change significantly in participants consuming a walnut - enriched diet from baseline as compared with control group participants . our findings are unchanged , controlling for age , race , gender , bmi , hypertensive status , dyslipidemia , and treatment sequence , using multivariable anova models . forty - six overweight adults participated in the study . sixty - one percent of the participants were female . three subjects dropped out of the study due to changes in medication , 1 dropped out due to inability to comply with the protocol , and 2 dropped out due to schedule conflicts . hdl = high - density lipoprotein , ldl = low - density lipoprotein , homa - ir = homeostasis model assessment insulin resistance . dietary intake . dietary intakes of polyunsaturated fatty acids ( pufas ) , n-3 fatty acids , n-6 fatty acids , and fat increased significantly during the walnut - enriched diet treatment compared with the control diet ( pufas , p < 0.01 ; n-3 fatty acids , p < 0.01 ; n-6 fatty acids , p < 0.01 ; and fat , p = 0.01 ) . selected nutrient intake values are mean sd ; p values were obtained from 1-way analysis of variance . endothelial function . in this overweight population with at least 1 risk factor for metabolic syndrome , consumption of a walnut - enriched diet for 8 weeks improved fmd significantly from baseline as compared with the control diet ( p = 0.019 ; see table 3 ) . hdl = high - density lipoprotein , ldl = low - density lipoprotein , homa - ir , homeostasis model assessment insulin resistance . significant ( p < 0.05 ) from baseline ; p values were obtained from repeated - measures analysis of variance. p < 0.05 from paired student t test . daily consumption of the walnut - enriched diet for an 8-week period did not change anthropometric measures from baseline ( bmi : p = 0.481 ; weight : p = 0.439 ; waist circumference : p = 0.344 ) . the control diet was associated with a reduction in bmi ( p = 0.016 ) and body weight ( p = 0.019 ) relative to the walnut - enriched diet . lipid panel . after consumption of a walnut - enriched diet for 8 weeks , participants mean measures of total cholesterol , ldl - c , hdl - c , and tg did not differ significantly from baseline as compared with the mean measures of control diet ( total cholesterol : p = 0.69 ; ldl - c : p = 0.98 ; hdl - c : p = 0.89 ; tg : p = 0.32 ) . blood pressure . after consumption of a walnut - enriched diet for 8 weeks , participants blood pressure values decreased nonsignificantly from baseline as compared with the control diet without walnut supplementation ( systolic : p = 0.07 ; diastolic : p = 0.352 ) . fasting plasma glucose , fasting insulin , and homa - ir . fasting plasma glucose and fasting insulin did not change significantly in participants consuming a walnut - enriched diet from baseline as compared with control group participants . our findings are unchanged , controlling for age , race , gender , bmi , hypertensive status , dyslipidemia , and treatment sequence , using multivariable anova models . the daily addition of 56 g of walnuts to the diet for 8 weeks significantly improved endothelial function in overweight adults with visceral obesity as compared with an ad libitum diet not supplemented with walnuts . a beneficial trend in systolic blood pressure reduction was observed that did not quite reach statistical significance . despite the walnut dose representing more than 350 kcal , weight gain was not observed in the walnut treatment arm of the study , and the addition of walnuts was even associated with a decline in waist circumference . lipid panel measures , fasting insulin , insulin sensitivity , and fasting glucose levels did not change . one of the most important observations in this study involves the improved brachial artery vasoactivity results . in general , these findings confirm and extend the results of most previous walnut consumption studies [ 20 , 2528 ] , respective of the methodological differences that exist among these trials . . also showed improvement in endothelial function after nut consumption by assessing by peripheral artery tonometry in metabolic syndrome participants . it has been hypothesized that one mechanism through which nut consumption improves endothelial function occurs via improvements in lipid panel indices . however , in this study , the lipid panel measures were unaffected by walnut intake . the overall evidence for potential cardioprotective effects of walnut intake is robust . in a pooled analysis of 25 nut intervention trials spanning 7 countries , sabate et al . showed that nuts produce favorable lipid - regulating effects in individuals with bmi < 30 . it has been argued that this effect could be due to overweight individuals displaying reduced lipid responsiveness . the results of this trial are in agreement with those observed by sabate et al . the average bmi of participants in the present trial is 33.2 4.4 , and lipid - regulating effects were not observed as a result of a walnut - supplemented ad libitum diet . the lipid panel is only one traditional cvd risk factor albeit an important one among a cluster of traditional and nontraditional risk factors within the current models of cvd etiology . endothelial function is increasingly being viewed as a prognostic marker of susceptibility to future cardiac events , and changes in risk factors other than serum lipids may account for part of the strong inverse association between nut consumption and cvd risk . we observed a beneficial trend in blood pressure reduction at the end of the walnut - supplemented ad libitum diet phase when compared with the ad libitum diet alone . our observations correspond with other nut and walnut consumption trials that have observed either a positive or a neutral influence on blood pressure responsiveness with increased consumption . mechanisms through which walnuts may elicit a blood pressure - lowering response could involve their high content of monounsaturated fatty acids , pufas , magnesium , and fiber and their low levels of sodium and saturated fatty acids . participants anthropometric measures did not change significantly from baseline during the walnut - enriched diet phase of the intervention . given walnuts rich fatty acid composition and high metabolic energy profile , there was initial apprehension that walnut consumption ( and nut consumption in general ) would lead to undesired weight gain . such concerns have been attenuated by evidence from epidemiological trials and randomized clinical trials , which have shown that walnut consumption does not tend to elevate weight , waist circumference , or bmi . given that participants physical activity levels remained unaltered throughout the study , the reduction in weight seen here could be due to a placebo effect , with participation in the trial encouraging subjects to eat more healthfully and/or to consume less food . the lack of weight increase seen during the walnut - enriched phase of the trial could be explained by participants partially substituting walnuts for other foods in the diet and by walnuts ability to increase energy expenditures while providing a high satiety and low metabolizable energy source . of note , waist circumference declined during the walnut study phase , despite a higher calorie intake . in a study by salas - salvado et al . , the prevalence of metabolic syndrome was reduced as a result of decreased waist circumference after a mediterranean diet supplemented with mixed nuts . the effects of walnuts on appetite , satiety , weight , and body composition clearly warrant further study . lastly , this trial showed that a walnut - enriched ad libitum diet does not change fasting plasma glucose , fasting insulin , and homa - ir levels when compared with the control diet . overall , nut consumption studies provide varied results when it comes to glucose and insulin homeostasis . such findings are difficult to interpret given that these studies employ participants of varied health status , nuts of various sorts and differing amounts , and variable intervention durations and number of participants . longer - term effects have generally been associated with observational studies , whereas intervention ( feeding ) studies have typically been of short duration , as is true in this case . we acknowledge that our study has several limitations , the most important being the absence of a prescribed standardized diet , which would then allow us to attribute the findings to the only independent variable under investigation , walnut consumption . nevertheless , the ad libitum crossover design employed here compensates for this concern and strengthens the real - life applicability of our findings . the practical amount of walnuts consumed by the participants in this trial ( 56 g ) adds further real - world applicability of our findings . the external validity of our trial is limited by the racial and gender homogeneity of the participating population . compliance assessment in this trial was carried out via 3-day food records and walnut consumption log sheets , which may not provide a perfectly accurate record of subject compliance . even so , given the positive results in the primary outcome measure , endothelial function , such concerns are of reduced importance . since the present study was a trial of short length , it is uncertain whether our findings could be extrapolated to trials of longer duration . we acknowledge that our study has several limitations , the most important being the absence of a prescribed standardized diet , which would then allow us to attribute the findings to the only independent variable under investigation , walnut consumption . nevertheless , the ad libitum crossover design employed here compensates for this concern and strengthens the real - life applicability of our findings . the practical amount of walnuts consumed by the participants in this trial ( 56 g ) adds further real - world applicability of our findings . the external validity of our trial is limited by the racial and gender homogeneity of the participating population . compliance assessment in this trial was carried out via 3-day food records and walnut consumption log sheets , which may not provide a perfectly accurate record of subject compliance . even so , given the positive results in the primary outcome measure , endothelial function , such concerns are of reduced importance . since the present study was a trial of short length , daily intake of 56 g of walnuts improves endothelial function in overweight adults with at least 1 sign of metabolic syndrome . despite adding calories to an ad libitum diet , walnut intake was not associated with weight gain and was actually associated with a nonsignificant decline in waist circumference . this study provides suggestive evidence of a role for walnuts in protecting against diabetes and heart disease in at - risk individuals . the study also indicates that walnuts may be added to an ad libitum diet without weight gain , at least in the short term .

objectivesmetabolic syndrome is a precursor of diabetes and cardiovascular disease ( cvd ) . walnut ingestion has been shown to reduce cvd risk indices in diabetes . this randomized controlled crossover trial was performed to investigate the effects of daily walnut consumption on endothelial function and other biomarkers of cardiac risk in a population of overweight individuals with visceral adiposity.methodsforty-six overweight adults ( average age , 57.4 years ; 28 women , 18 men ) with elevated waist circumference and 1 or more additional signs of metabolic syndrome were randomly assigned to two 8-week sequences of walnut - enriched ad libitum diet and ad libitum diet without walnuts , which were separated by a 4-week washout period . the primary outcome measure was the change in flow - mediated vasodilation ( fmd ) of the brachial artery . secondary measures included serum lipid panel , fasting glucose and insulin , homeostasis model assessment insulin resistance values , blood pressure , and anthropometric measures.resultsfmd improved significantly from baseline when subjects consumed a walnut - enriched diet as compared with the control diet ( 1.4% 2.4% versus 0.3% 1.5% ; p = 0.019 ) . beneficial trends in systolic blood pressure reduction were seen , and maintenance of the baseline anthropometric values was also observed . other measures were unaltered.conclusiondaily ingestion of 56 g of walnuts improves endothelial function in overweight adults with visceral adiposity . the addition of walnuts to the diet does not lead to weight gain . further study of the potential role of walnut intake in diabetes and cvd prevention is warranted .

INTRODUCTION MATERIALS AND METHODS Participants Study Design Vascular Reactivity Testing: Brachial Artery Reactivity Studies Outcome Measures Diet Record Analysis Ethics Statistical Analysis RESULTS Demographic and Baseline Primary Endpoint Secondary Endpoints DISCUSSION Limitations CONCLUSION

we used the lrn as a conduit to maintain the confidentiality and anonymity of the variola testing sites . a convenience sample of 45 laboratory workers completed an online survey developed by researchers at the university of nebraska medical center ( omaha , ne , usa ) . nonidentifying demographic information was collected , in addition to any adverse effects after vaccination and perceived barriers to revaccination . to determine a significant difference existed regarding the success ( presence or absence of a take after vaccination ) of the vaccine based on intervals between vaccines , we measured the mean interval ( in years ) between vaccinations . respondents mean age was 46 years ; they had worked a mean of 20.5 years in the laboratory setting . eighty - four percent of respondents reported that the only adverse events from vaccination were related to the skin irritation caused by the occlusive dressings worn over the vaccination lesion . sixty - seven percent listed a medical condition in themselves or a close household contact as the barrier to revaccination . the mean interval from first to second vaccination was 4.8 years for vaccinees who had a successful vaccine and 6.0 for those who did not . statistical analysis demonstrated no significant difference ( p = 0.149 ) between the number of years between first and second vaccinations and the take rates . sixty - two percent of respondents indicated they did not work with non highly attenuated orthopoxviruses . ( i.e. , developed lesions ) regardless of number of years since previous vaccination , suggesting that immunity might have waned . therefore , our data do not provide evidence to suggest that the acip recommended interval for revaccination be prolonged . although most respondents reported having no adverse effects from the vaccine , for some this vaccination caused discomfort . many reported symptoms related to the occlusive dressing worn as a precautionary measure to ensure that the lesion site was properly covered during work hours . other measures to ensure the lesion is covered appropriately , such as nonocclusive dressings and long sleeves , may be considered given that laboratory workers do not have direct contact with patients . although the lrn asks this small group of laboratory workers to comply with the acip recommendations , the question remains whether this requirement should include laboratory workers who do not handle orthopoxviruses . revaccination of most laboratory workers at variola testing sites every 3 years would be expected to be sufficient to provide an initial immunologic response , whereas laboratory workers who do not handle orthopoxviruses could be vaccinated in the same fashion as other health care and public health workers who have at least 1 recent ( since 2003 ) documented successful vaccination ( 5 ) . this recommendation is based on the same premise as using the vaccine as prophylaxis for documented exposure to a smallpox - infected person . this practice was used regularly during the smallpox eradication program . because the average incubation period for vaccinia is 34 days shorter than the incubation period for smallpox , a person exposed to smallpox would have a 34 day window in which to be vaccinated with and immunologically respond to vaccinia , which also confers immunity to smallpox ( 6 ) compromised immune systems or cardiac risk factors that make vaccinees ineligible for vaccination are more likely to develop as they age ( 7 ) . most barriers to revaccination were related to medical conditions ( compromised immunity and/or exfoliative skin disorders ) that place vaccinees at high risk for adverse events to the currently licensed smallpox vaccine . the conditions are an added challenge for the aging pool of laboratory workers assigned to national variola testing sites ( 8) . currently unlicensed third - generation smallpox vaccines may be considered ( pending licensure ) as replacements to acam2000 ( sanofi pasteur biologics , lyon , france ) , the currently licensed vaccinia vaccine , for laboratory workers at national variola testing sites or perhaps an even broader population of laboratory workers throughout the united states . third - generation vaccines are nonreplicating and safer in populations that might have contraindications to traditional vaccines ( 911 ) . the risk to the us population from a release of smallpox this reduced risk stems not from a lower threat from terrorism but from the existence of a stockpile of the new acam2000 smallpox vaccine , in addition to a cadre of health care and public health professionals who could be revaccinated quickly and mobilized accordingly ( 12 ) . more research on the immunogenicity of smallpox vaccine is needed but is challenged by the absence of smallpox disease to test the efficacy of vaccination . researchers now appreciate that the complex mechanism of the immune response to vaccinia and/or smallpox infection might lead to better treatment options for infectious and autoimmune diseases ( 7 ) . future opportunities may arise to challenge the vaccine with the actual virus to measure vaccine efficacy and provide sound recommendations to protect all public health and health care responders against smallpox ( 13 ) . in the meantime , ensuring that recommendations created to protect some populations are properly interpreted and applied is important to protecting the most vulnerable persons without exposing others to unnecessary harm .

to evaluate the need to revaccinate laboratory workers against smallpox , we assessed regular revaccination at the us laboratory response network s variola testing sites by examining barriers to revaccination and the potential for persistence of immunity . our data do not provide evidence to suggest prolonging the recommended interval for revaccination .

The Study Conclusions

a 57yearold woman who had initially been implanted with a double chamber pacemaker for complete atrioventricular block was upgraded to crtp at another center after a year due to left ventricular dysfunction and dyspnea . she was referred to us 3 months after the upgrade due to wound infection by pseudomonas sp caused by gossybypoma ( retained compress ) . preoperative transthoracic ( tte ) and transesophageal ( toe ) echocardiography did not reveal any signs of endocarditis , and the patient was afebrile with negative blood cultures . a patent foramen ovale ( pfo ) with a small left to right shunt traction with locking stylets allowed complete extraction of the activefixation right atrial and right ventricular leads , and the coronary sinus lead was explanted by simple traction . the following morning she developed sudden dysarthria and left facial palsy , and emergent cerebral angiography revealed an occlusion of the right frontal branch of the middle cerebral artery ( fig . mechanical thrombectomy with a solitaire fr revascularization device ( covidien , plymouth , mn , usa ) retrieved a thrombus attached to dense fibrinous tissue ( fig . normal vessel flow was recovered ( fig . 2c and d ) , and neurological recovery was complete within 60 min of symptom onset . oral antibiotics were continued for a further 2 weeks , and followup at 6 months was uneventful . transesophageal echocardiography of the bicaval view in 2 dimensions ( a ) and using color doppler ( b ) , showing a mobile interatrial septum ( asterisk ) with a patent foramen ovale ( solid arrow ) . ( a and b ) occlusion of frontal branch of right middle cerebral artery ( arrow ) with hypoperfusion of a large territory ( asterisk ) . ( c and d ) restored flow after mechanical thrombectomy . ( a ) the solitaire fr revascularization device ( covidien ) used for mechanical thrombectomy . herein , we describe the case of a patient suffering acute ischemic stroke due to paradoxical embolization of dense fibrous material through a pfo following transvenous lead extraction ( tle ) . endocardial pacemaker leads cause fibrin deposits on their surface which may form sheaths in over 85% of patients 1 or adhere with the vessel walls , tricuspid valves , and endocardium . thrombi can be detected on the leads in around 30% of patients by toe and intracardiac ultrasound 2 , 3 , and findings of asymptomatic pulmonary emboli in up to 15% of patients as well as increased pulmonary pressure point toward a high rate of subclinical embolism in patients with cardiovascular implantable electronic devices ( cieds ) 1 , 3 , 4 . one particular concern is the risk of systemic thromboembolism in patients with intracardiac shunts and cieds ; in all shunts other than pfos , the risk of systemic thromboembolism is doubled 5 and such conditions are at present considered a contraindication for endocardial lead placement 6 . in the case of pfos , the risk is less well documented , but retrospective data points toward a hazard ratio of > 3 for stroke 7 , although study bias may have exaggerated this figure . tle is a valuable technique for management of device infection and lead failure , and its safety has improved thanks to technological advances and increasing experience . preliminary data from the electra registry 8 , the only prospective european registry of tle , show a relatively low rate of major complications ( 2.53.9% depending on the procedural volume of centers ) . one complication of particular concern when extracting leads is the risk of systemic embolism . indeed , residual fibrous deposits persisting after tle have been described 9 , sometimes large enough as to be described as our case illustrates that these residual deposits may not always be visible with toe , probably because they may be confined to the vessels ( e.g. , innominate or subclavian vein ) but may embolize later . although the rate of stroke seems to be low ( 0.1% ) during tle , our case highlights the potentially dramatic consequences of systemic embolization , even in the absence of visible vegetations on the leads . moreover , current guidelines offer no clear recommendations to help lower the risk of embolization during tle 10 . routine anticoagulation , used by some centers mainly to lower the rate of venous occlusion , is probably of little use due to the fibrinous nature of the tissue . the use of distal embolization protection devices during tle in patients with vegetations has been reported in several cases , whether in the pulmonary 11 or systemic circulation 12 . however , the efficacy of these devices seems limited by the risk of delayed embolism as demonstrated in our case , and because residual dense fibrinous tissue probably persists for several days or weeks . based on our case and these considerations , we believe paradoxical embolism causing stroke is a real concern in patients with pfos undergoing tle . although its incidence seems to be low , the consequences can be catastrophic and the costs linked with the morbidity of stroke are important . although this may raise the concern of additional cost , most patients undergoing tle have a preoperative echocardiographic examination , either tte or toe , in order to evaluate preoperative tricuspid valve regurgitation or presence of vegetations . the additional cost and time linked to evaluate for a pfo is negligible . in case of pfos with large shunts , we suggest considering percutaneous pfoclosure before extraction in noninfected patients or surgical extraction and pfoclosure in patients with endocarditis . in cases with small pfos , we recommend strict avoidance of maneuvres increasing righttoleft shunting ( e.g. , coughing , valsalva ) and 2448 h neurological surveillance . the effectiveness of mechanical thrombectomy in cases of embolization after lead extraction is also highlighted in this case . because of the dense fibrous nature of embolus ( or in cases where the embolus is of foreign body material ) , pharmacological thrombolysis is likely to be ineffective , as well as increasing the bleeding risk considerably in the postoperative period . therefore , we strongly suggest performing tle in centers with neuroradiological standby , as rapid action may prevent irreversible sequellae .

key clinical messagesystemic embolization is a dreaded complication of transvenous lead extraction ( tle ) , even without visible vegetations . preoperative patent foramen ovale evaluation is important , justifying neurological surveillance or consideration of surgical extraction in selected cases . in case of stroke after tle , mechanical thrombectomy is a successful therapy , and should be readily available .

Case Report Case Discussion Conflicts of interest Funding

stroke and other cerebrovascular diseases caused by cerebral vascular blockage or bleeding cause loss of normal blood circulation in the affected area and anoxia , resulting in irreversible brain damage which manifests as functional impairments in consciousness , movement , senses , recognition , comprehension , and language . common mobility impairments include : lateral muscle weakness , abnormal lateral muscle tension , abnormal postural control , abnormal coordination , abnormal movement sequencing and loss of joint coordination . these are all factors which contribute to problems with postural control , thereby affecting standing balance ability1 . postural control evaluation and training are important contributors to the rehabilitation of balance , walking ability and other daily living activities performed by stroke patients3 , 4 . the modern theory of movement control and movement learning holds that in humans all intentional movements are task - specific5 , i.e. the response is specific to the environment or situation . however , rehabilitation therapies involving task - specific actions often have better effects than traditional non - task - specific rehabilitation therapies6 . according to barclay - goddard , stevenson , poluha , et al.7 , through traditional treatment , stroke patients can increase standing symmetry when using a force plate for treatment . van peppen , kortsmit , lindeman , et al.8 concluded that stroke patients using visual feedback with traditional treatments showed greater improvements than patients receiving standard treatment only in standing symmetry , postural sway movement with the eyes open and closed , and walking speed9 . dynamic balance ability is the ability to change postural without losing one s balance10 . limits of stability ( los : the maximum center of gravity displacement possible without loss of balance ) are also reduced in stroke patients . damage to the central nervous system function directly affects posture , movement , and thus , balance ability . reduced muscle strength and mobility the hemiparesis that often results from stroke tends to force patients to complete movements using a compensation strategy , e.g. fixing the affected part and using the healthy part to complete the target movement . this behavior will decrease the free movement factors in the central nervous system and reduce the complexity of movement choice , making it easier to complete the intended action . however , this behavior does not require postural control , so nervous impulses to the affected side reduce , which further hinders functional recovery of the affected side in rehabilitation12,13,14 . this means that techniques which oblige stroke patients to practice centralized postural and balance control on the affected side will have considerable impact on the stroke patients recovery of balance , walking and other mobility - related functions . in this study , a device to enable stroke patients to learn better movement control during rehabilitation than relying on traditional rehabilitation therapies was tested . the device uses two force plates to measure right and left side centers of foot pressure ( cop ) , and visual feedback with different side reaction ratios to prompt movements of the affected side while the non - affected support base remains in a fixed position . a schematic diagram of the device and task used in this study is shown in fig . 1fig . patients stand on the signal capture device ( dual force plate ) , and are prompted with a task from the interactive game through a monitor . the patient then changes the body center to move an object as prompted by a task shown on the monitor . all of these physical actions are used to execute balance training and task assessment . system schematic diagram this study used a cop capture device consisting of a load cell , signal transmitter and an analog / digital converter with a universal serial bus ( usb ) interface . weight distribution data collected from the foot plantar stress center are displayed in a two - dimensional plane and entered into the game for coordination . figure 1a shows the cop capture device which consists of two plates ( 30 245 10 mm , 304 stainless steel ) , one for each foot . the bottom displays an equilateral triangle ( width 165 mm ) consisting of three load cells acting as a unit ( fig . the center of this equilateral triangle is the geometric center of the stepping zone . the user needs to step on the center of the stepping zone to align the geometric center of the plantar area and the center of the load cell as closely as possible . figure 1c and 1d show how movements are captured when the cop capture device has been set up . the load cell retrieves signals from the right and left feet of the user , and the movements of foot pressure center are post - processed to construct the center distribution figure . if the region d is located in the two - dimensional coordinate system , which is the density function (x , y ) , region d is further divided into left and right sides , sub - regions dl and dr . the overall weight of the system is the subject s weight on both feet , which is calculated using the formula : the center of gravity of the system is calculated using the formula : the three vertices of a subject s plantar force on the region dl(dr ) , have force and moment arm values li ( ri ) and xli , yli ( xri , yri ) ; for i = 13 , respectively . figure 2fig . 2.the relationship between the force and the moment arm shows the relationship between the force and the moment arm diagram , and the center of gravity is be derived as follows : the relationship between the force and the moment arm figure 3fig . a signal transmitter is used to provide the operational voltage to the load cell and amplify the signal for transmission to the analog / digital converter . converted data is saved in a buffer zone and transmitted when the software requests it . after acquisition by the analysis software , data is processed by a finite impulse response filter ( to a low - pass filter , 10 hz ) and organized in chronological sequence . the software was written in microsoft visual c # , for ( 1 ) setting the function of the analog / digital converter with the usb interface ; ( 2 ) processing load cell data tables ; ( 3 ) processing vector parameters ; ( 4 ) center distribution plots after vector processing ; ( 5 ) a graphic user interface to display raw data and center distribution plots after vector processing ; and ( 6 ) a graphic user interface to set vector processing and system parameters . schematic diagram of the capture device the game engine was developed using the microsoft .net framework . microsoft visual c # with xna game studio was used to build this engine with ( 1 ) an event processing queue ( 2 ) a graphic engine ( 3 ) a user interface ( 4 ) a database interface ( fig . 4.architecture of the game engine ) . architecture of the game engine the cop capture device of this system used the proportion , single load cell value : whole load cell value = single foot center point distribution value : single foot center point reflect plane boundary , to reflect the collected weight distribution in the virtual plane . the six distribution values are then merged to become the pressure centers of the right and left feet and the whole cop in the virtual plane of the game engine using this rule : healthy side : affected side = 1 : n ( n is decided by the medical staff based on the patient s diagnosed medical condition : n=1.5 for case a , and 4 for case b in this study ) . if the cop values are over the boundary the display is locked to that boundary . in this study the task for patients was to use cop to aim an arrow and to shoot it at a target , when the sum value of the healthy side load cell is less than 20% of the value for the load cell unit as a whole , red warnings were displayed on the monitor to tell the patient to move their cop to the other side . the formula controlling movement of the target is given by : horizontal boundary bh= vertical boundary bv=1,000 ( unit)moving velocity v : 1v10 ( unit / fps ) moving velocity changing rate n : 10n100 ( fps ) the target moved randomly throughout the game . the user was considered to have located the target when the angle formed by the target and the monitor center ( the location of the camera ) and the difference was less than 5. at this point , the arrow was shot at the target . the time taken to locate the target the experiments in this study were approved by the human ethics committee , taipei medical university hospital , taiwan . inclusion criteria were stroke with hemiplegia , and an age between 25 to 60 years . this study reports the results of two subjects who used the device for movement control training of the affected side . training sessions consisted of following the randomly moving target as described above , with one five - minute break in the middle of the session . baseline performance was measured before the start of training , and performance was measured again at the end of the training period . the tools used to evaluate performance were : the berg balance scale ( bbs)15 , 16 , 14 balance tasks scored on a five - point ( 04 ) scale with 0 representing the poorest performance ; the motor assessment scale ( mas)17 which evaluates motor function on a seven - point ( 06 ) scale , in this study , only items relevant to standing balance were used ; and the newly - developed device was used to evaluate the cop distribution . the evaluation included the cop motion distribution of the left and right feet and whole body while standing with the eyes open for one minute , and standing with the eyes closed for one minute . the effect on balance performance training using the device was assessed using two patients . the side ratio n was 1.5 for case a ( the reaction in the game was 60% on the affected side and 40% on the non - affect - side ) , and 4 for case b ( affected - side 80% , non - affected - side 20% ) . table 1table 1.basic information the two cases in this studysubjectabgendermalemaleage ( years)4939height ( cm)167168weight ( kg)8272bmi29.425.51main symptomsstrokestrokeonset period6 months11 monthsdominant side / affected sideright/ rightright / rightother illnessesasthma , hypertension , aphasianohabits and customsstopped drinking and smoking ( about a year)exercise at least one hour a daystopped smoking ( 8 months)exercise one to two hours a day summarizes patient data . 5.the cop distribution of case a shows the cop distribution for patient a : before training in the eyes open condition ( fig . . 6d ) . the cop numerical distribution is shown in fig . 7fig . the cop distribution of case a the cop distribution of case b cop distribution before and after training baseline and post - training balance assessments using the bbs showed that neither patient a nor patient b regressed on the items for which performance at baseline was already at a ceiling . the bbs improvement of case a was 19.44% and that of case b was 11.9% , showing that the interactive game used in this study can help stroke patients improve their balancing ability on the stroke affected side . detailed results are presented in table 2table 2.balance ability assessment with bbssubjectbbs sub taskbefore trainingafter trainingprogressasit to standwith hand assistancewithout hand assistancesignificantstanding unsupported with eyes closedunstablemore stablesignificantstand unsupported with feet togetherwith hand assistancewithout hand assistance and longer standing timeaveragereaching forward with outstretched armsome reachlonger reachaveragepick up object from the floor from a standing positionno changenonturn to look behind over left and right shoulders while standingcant look behind over affected sidecan look behind over affected sidesignificantturn 360low speedfaster speed ( but the score not enough to next level)weakplace alternate foot on bench or stool while standing unsupportedcould not step on a stoolstepped on the stool twice without any supportsignificantstand unsupported with one foot in frontnot able to standunsupported with one foot in front for 8 ssignificantstanding on one legcould not stand on the affected side , short term on the healthy sidestill could not stand on the affected side , but more time on the healthy sideaveragebstand unsupported with feet togetherstand for a long time , but need assistance to drew the feetdrew the feet by himselfsignificantplace alternate foot on bench or stool while standing unsupportedtook 24 sonly took 12 ssignificantstand unsupported with one foot in frontnot able to standstood for 20 ssignificantreaching forward with outstretched arm18 cm19 cmweakpick up object from the floor from a standing positionno changenonturn to look behind over left and right shoulders while standingno changenonturn 360took 13 stook 12 sweakbbs scorecase acase bbefore / after / rate of progress36 point / 43point /19.44%42 point / 47 point /11.9% . table 2 shows that the progress of patient a was not sufficient on some of the bbs items to elicit an improvement in score , although there was some evidence of progress . we speculate that additional training time would have elicited larger gains in performance . on items such as reaching forward with an outstretched arm , and picking up an object from the floor from a standing position , there were no obvious changes in performance , presumably because these items involve upper limb movement . patient b was tested after a longer recovery period and was in better physical condition , and his baseline balance performance was better than that of patient a. among the items which were worst at baseline , items involving upper limb movement showed little change after the training , but items related to standing balance showed obvious improvement . table 3table 3.balance ability assessment with massubjectmas sub taskbefore trainingafter trainingprogressabalance sittingcould maintain sitting , but the weight of two body side was unevenimproved . could look around and touch the side floor but not the front floor.averagesitting to standingcould complete independ- ently , but the weight was uneven.the uneven situation was improved but the speed was rather slow.averagewalkinghad to have assistance all the time for walking.could walk steadily without assistance device but still slow.averagebbalance sittingnormal performancesome progressweaksitting to standinggood performancebetter performancesignificantwalkinggood performancewalking speed was obviously fastersignificantmas scorecase acase bbefore / after / rate of progress6 point/12 point /100%16 point /17 point /6.25 the test program included seated balance and transitions from sitting to standing and walking . the cop distribution data is shown in table 4table 4.cop distributionsubjectcenter point average positionaverage value of the center pointdrifting position and the center point of the average distanceoe ( before / after)ce ( before / after)oe ( before / after)ce ( before / after)a(16.20 , 1.97 ) / ( 2.55 , 0.36)(13.80 , 3.35 ) / ( 13.48 , 22.41)13.39 / 4.9214.78 / 10.39cop distribution change : position moved to right side ( affected side ) from left side ( healthy side).progress : oe : 63.25% , ce : 29.70% drift area of center point ( before / after ) : oe : 1,284.90 / 543.50 ( progress 57.70% ) ce : 1,673.30 / 1,238.10 ( progress 26%)b(27.10 , 93.99)/ ( 22.89 , 56.71)(12.23 , 76.06 ) / ( 23.42 , 59.19)6.92 / 5.1214.03 / 14.82cop distribution change : moved from the upper side to the center ( the virtual center of the center point on mapping plane)progress : oe : 26.01% , ce : 5.63% drift area of center point ( before / after ) : oe : 767.20 / 574.30 ( progress 25.14% ) ce : 1599.90 / 873.20 ( progress 45.42% ) . after training , the cop distribution of patient a shifted towards the right ( affected side ) ; presumably because the patient habitually supported standing with the healthy side . the affected side showed smaller effort during the game , so the patients were forced to use their healthy side to finish the game . over time they became used to having their affected side absorb the weight when standing on the force plate . the data of the center point drift and the average value of the center point of the average distance showed that there was considerable improvement in the eyes open condition . this may be because the patients knew they could find a datum point and maintain that datum point . in fact , the average value for the center position distance in the eyes closed condition indicated a slight deterioration in performance . patient a showed a 19.44% improvement in performance on the bbs and 100% improvement in performance on the mas following training using the device and interactive game . although patient a used his healthy side to support his body weight in a standing position , under certain circumstances he could switch to using the affected side . patient a showed considerable improvement in static standing balance control with his eyes open . patient b showed much smaller improvements in performance : an 11.9% improvement in performance on the bbs , and a 6.25% performance on the mas . in center point plane performance , there was no significant change in the center point position ; and there was no detectable change in average drift extent , even though the center point drift area was smaller . it is our opinion , that the difference in the effectiveness of the training for the two patients was due to differences in the length of recovery . case b was tested after an 11 month recovery period and had already achieved considerable gains in mobility , most of which were persistent . this persistence was derived from functional use of the movements in the course of daily life . the training produced a considerable improvement in static center point drift but no obvious improvement in center point position . it is our opinion , that patient a made significant progress in all aspects of balance because testing occurred after a shorter recovery period , and patient a had not recovered as much mobility as patient b , leading to larger training effects . in summary , an interactive game with dual force and different side ratio developed for this study can help stroke patients train their balance on their stroke affected side , but this method is only effective for patients who have recently experienced stroke and offers little benefit to patients who have recovered a significant degree of mobility . we were unable to determine the level of baseline mobility at which training ceases to be effective as this study assessed only two cases . future research should investigate the effects of our rehabilitation protocol in stroke patients with hemiplegia with varying recovery periods , e.g. 6 to 11 months , bbs score below a certain threshold , e.g. 42 , or mas scores below a certain threshold , e.g. 16 to provide further evidence regarding its efficacy .

[ purpose ] stroke and other cerebrovascular diseases are major causes of adult mobility problems . because stroke immobilizes the affected body part , balance training uses the healthy body part to complete the target movement . the muscle utilization rate on the stroke affected side is often reduced which further hinders affected side functional recovery in rehabilitation . [ subjects and methods ] this study tested a newly - developed interactive device with two force plates to measuring right and left side centers of pressure , to establish its efficacy in the improvement of the static standing ability of patients with hemiplegia . an interactive virtual reality game with different side reaction ratios was used to improve patient balance . the feasibility of the proposed approach was experimentally demonstrated . [ results ] although the non - affected - side is usually used to support the body weight in the standing position , under certain circumstances the patients could switch to using the affected side . a dramatic improvement in static standing balance control was achieved in the eyes open condition . [ conclusion ] the proposed dual force plate technique used in this study separately measured the affected and non - affected - side centers of pressure . based on this approach , different side ratio integration was achieved using an interactive game that helped stroke patients improve balance on the affected side . only the patient who had suffered stroke relatively recently benefited significantly . the proposed technique is of little benefit for patients whose mobility has stagnated to a certain level .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

nine human hereditary neurodegenerative disorders , including huntington 's disease ( hd ) and several spinocerebellar ataxias ( scas ) , are caused by the abnormal expansion of cag repeats located in the translated sequences of different genes ( fig . 1 ) . for example , hd is triggered by the expression of at least 36 cag repeats located in exon 1 of the htt gene , which encodes the large and multifunctional huntingtin protein ( htt ) . sca1 is caused by at least 39 cag repeats present in exon 8 of the atxn1 gene , which codes for the ataxin-1 protein involved in rna metabolism and transcriptional regulation , and sca3 ( also known as machado joseph disease ) is induced by at least 60 cag repeats that occur in exon 10 of the atxn3 gene encoding a protein with deubiquitinating activity . the cag repeats are translated into polyglutamine tracts in protein products , and the entire group of resulting disorders is known as the polyglutamine ( polyq ) diseases . each polyq disorder primarily affects a different population of neurons , although the causative genes are widely expressed in the central nervous system and peripheral tissues . the expanded repeats are thought to exert their pathogenic effects at the protein level , mainly through a gain of toxic function by the mutant protein . for comparison , myotonic dystrophy type 1 ( dm1 ) , which is another example of a triplet repeat expansion disease ( tred ) , is caused by 503,000 copies of ctg tandem repeats located in the 3 untranslated sequence of the dystrophia myotonica protein kinase gene ( dmpk ) . in dm1 , the gain of toxic function by mutant transcript containing cug repeats 1comparison of the lengths of the cag repeat tracts that occur in polyq disease - related transcripts and the cug repeats in the transcript responsible for dm1 . the normal repeat range is marked in green , and the mutant repeat range is marked in red and specified . the starting threshold for dm1 mutation ( 50 repeats ) is denoted by a hatched line comparison of the lengths of the cag repeat tracts that occur in polyq disease - related transcripts and the cug repeats in the transcript responsible for dm1 . the normal repeat range is marked in green , and the mutant repeat range is marked in red and specified . the starting threshold for dm1 mutation ( 50 repeats ) is denoted by a hatched line the pathogenic number of cag repeats in genes implicated in polyq diseases is , in most cases , lower than the number of cug repeats in the dmpk gene of dm1 patients , and cag expansions typically span a much narrower range of repeat lengths ( fig . 1 ) . nevertheless , a considerable fraction of hd and sca patients harbor mutant cag repeats of a length corresponding to the lower range of cug repeat lengths found in dm1 . the expression levels of individual polyq disease genes and the dmpk gene differ within and between tissues ; however , they all belong to the moderate or low expression category [ 8 , 9 ] . the structural features of the cag repeats are similar to the cug repeats , forming hairpin structures in transcripts when the repeat tracts are long enough [ 1016 ] . the repeats of normal length form small and semistable hairpins , and mutant repeats form long hairpins that are more stable . according to the results of crystallographic studies , the cag repeat duplexes and cug repeat duplexes show a high degree of similarity [ 18 , 19 ] , suggesting that the stem portion of these hairpins may share some protein - binding properties [ 20 , 21 ] . in this review , we first discuss the main cellular and molecular hallmarks of the mutant protein and mutant rna gain - of - function mechanisms from studies of polyq diseases and dm1 pathogenesis . we then present recent findings indicating that transcripts containing expanded cag repeats might also be toxic and contribute to the pathogenesis of polyq disorders . finally , we focus on the experimental models used to demonstrate rna toxicity in polyq diseases . we discuss the features of these models and propose generating new cellular and animal models dedicated to elucidating the toxic effects triggered by mutant rna and protein in polyq diseases . the protein products of genes that undergo mutations leading to polyq diseases differ in their cellular functions and cover a wide range of molecular weights . a common feature of these proteins is the presence of a polyq tract that is expanded as the result of the mutation . however , the exact nature of protein gain - of - function toxicity in polyq diseases remains a subject of debate [ 22 , 23 ] , and two mechanisms have been considered : the gain of a new toxic function by the mutant protein and the enhancement of the normal protein function to toxic levels . in either case , the key to understanding the details of such pathogenic mechanisms is the identification and characterization of new toxic protein protein interactions and imbalanced normal interactions . a systematic search for proteins associated with normal and expanded the data showed that the proteins that preferentially interacted with mutant htt are enriched for intrinsically disordered proteins and proteins involved in key cellular functions , such as energy production , protein trafficking , rna modification and translation , mitochondrial functions , cellular stress , and cell death . these results confirmed and expanded earlier findings regarding the cellular processes and pathways that are altered in hd [ 2527 ] . the pathogenesis of polyq diseases is believed to be initiated by the expanded polyq tract , which is thought to acquire a -sheet conformation and cause the mutant protein to misfold . alternatively , the expanded polyq stretch may stabilize one of several conformations characteristic of the wild - type protein that may normally exist in equilibrium ; such an effect enhances some intermolecular interactions of the mutant protein at the cost of other interactions . in addition to the polyq tract , the context of other protein domains may also contribute to pathogenesis , as demonstrated in animal models of spinal bulbar muscular atrophy , sca1 , and hd . in several instances ( reviewed in [ 3234 ] ) , single amino acid substitutions in functional domains of relevant proteins resulted in dramatic changes of disease phenotypes pointing to the critical role of protein toxicity in polyq diseases . the cellular hallmarks of polyq diseases are nuclear and cytoplasmic amyloid - like aggregates formed by mutant proteins , their proteolytic fragments [ 35 , 36 ] , or by fragments translated from aberrantly spliced shorter mrnas that sequester other proteins , predominantly those containing unstructured regions [ 24 , 28 , 38 ] . the formation of amyloid - like deposits is also observed in parkinson and alzheimer diseases , with which the protein toxicity of polyq diseases is often compared . however , in efforts aimed at demonstrating rna toxicity in polyq diseases , reference to dm1 and other rna - triggered treds , e.g. , sca8 and hdl2 ( huntington 's disease - like 2 ) [ 40 , 41 ] , is both appropriate and justified [ 20 , 21 , 4244 ] . dm1 is the prototype of treds caused by toxic rna [ 6 , 7 ] . terms such as toxic rna and rna toxicity are used to reflect the fact that the disease - causing mutation is passed from the gene to the transcript only , while the protein structure is unaffected by the mutation . the hallmarks of cells expressing expanded cug repeats are nuclear rna foci , which are toxic to cells because they sequester important cellular proteins , reducing their normal , functional levels ( reviewed in [ 46 , 47 ] ) . one such protein trapped in mutant rna foci is muscleblind - like ( mbnl1 ) alternative splicing factor ; together with the concomitant upregulation of the antagonistic splicing factor cug - bp1 , mbnl1 deficiency results in the misregulation of the developmentally regulated alternative splicing of numerous mbnl1-regulated genes . the compromised functions of some of these genes have been linked to the clinical symptoms of dm1 , e.g. , the altered splicing of insulin receptor in insulin resistance , chloride channel 1 in myotonia , sarcoplasmic / endoplasmic reticulum ca atpase 1 and 2 in muscle wasting , cardiac troponin t in cardiac abnormalities , and tau ( mapt ) in cognitive deficits . the binding of the multifunctional helicases p68 and p72 to mutant cug repeats facilitates the binding of mbnl1 to the repeats and likely links the dm1 pathogenesis pathway to the nuclear step of microrna biogenesis , a process in which these helicases are involved as cofactors of the ribonuclease drosha complex . direct evidence was provided for the crosstalk between the dm1 pathogenesis pathway and the cytoplasmic ribonuclease dicer step of microrna biogenesis , in which the mbnl1 protein is also implicated . it was demonstrated that mbnl1 sequestered by mutant cug repeats can not effectively function in its newly recognized role as a pre - mirna cleavage regulator , resulting in the compromised expression and function of some mirnas , including mir-1 , an effect linked to dm1-related heart defects . the expanded cug repeats were also shown to trigger immune responses in dm1 tissue in which the pkr , oas , tlr3 , and rig i genes were activated . the protein products of genes that undergo mutations leading to polyq diseases differ in their cellular functions and cover a wide range of molecular weights . a common feature of these proteins is the presence of a polyq tract that is expanded as the result of the mutation . however , the exact nature of protein gain - of - function toxicity in polyq diseases remains a subject of debate [ 22 , 23 ] , and two mechanisms have been considered : the gain of a new toxic function by the mutant protein and the enhancement of the normal protein function to toxic levels . in either case , the key to understanding the details of such pathogenic mechanisms is the identification and characterization of new toxic protein protein interactions and imbalanced normal interactions . a systematic search for proteins associated with normal and expanded the data showed that the proteins that preferentially interacted with mutant htt are enriched for intrinsically disordered proteins and proteins involved in key cellular functions , such as energy production , protein trafficking , rna modification and translation , mitochondrial functions , cellular stress , and cell death . these results confirmed and expanded earlier findings regarding the cellular processes and pathways that are altered in hd [ 2527 ] . the pathogenesis of polyq diseases is believed to be initiated by the expanded polyq tract , which is thought to acquire a -sheet conformation and cause the mutant protein to misfold . alternatively , the expanded polyq stretch may stabilize one of several conformations characteristic of the wild - type protein that may normally exist in equilibrium ; such an effect enhances some intermolecular interactions of the mutant protein at the cost of other interactions . in addition to the polyq tract , the context of other protein domains may also contribute to pathogenesis , as demonstrated in animal models of spinal bulbar muscular atrophy , sca1 , and hd . in several instances ( reviewed in [ 3234 ] ) , single amino acid substitutions in functional domains of relevant proteins resulted in dramatic changes of disease phenotypes pointing to the critical role of protein toxicity in polyq diseases . the cellular hallmarks of polyq diseases are nuclear and cytoplasmic amyloid - like aggregates formed by mutant proteins , their proteolytic fragments [ 35 , 36 ] , or by fragments translated from aberrantly spliced shorter mrnas that sequester other proteins , predominantly those containing unstructured regions [ 24 , 28 , 38 ] . the formation of amyloid - like deposits is also observed in parkinson and alzheimer diseases , with which the protein toxicity of polyq diseases is often compared . however , in efforts aimed at demonstrating rna toxicity in polyq diseases , reference to dm1 and other rna - triggered treds , e.g. , sca8 and hdl2 ( huntington 's disease - like 2 ) [ 40 , 41 ] , is both appropriate and justified [ 20 , 21 , 4244 ] . dm1 is the prototype of treds caused by toxic rna [ 6 , 7 ] . terms such as toxic rna and rna toxicity are used to reflect the fact that the disease - causing mutation is passed from the gene to the transcript only , while the protein structure is unaffected by the mutation . the hallmarks of cells expressing expanded cug repeats are nuclear rna foci , which are toxic to cells because they sequester important cellular proteins , reducing their normal , functional levels ( reviewed in [ 46 , 47 ] ) . one such protein trapped in mutant rna foci is muscleblind - like ( mbnl1 ) alternative splicing factor ; together with the concomitant upregulation of the antagonistic splicing factor cug - bp1 , mbnl1 deficiency results in the misregulation of the developmentally regulated alternative splicing of numerous mbnl1-regulated genes . the compromised functions of some of these genes have been linked to the clinical symptoms of dm1 , e.g. , the altered splicing of insulin receptor in insulin resistance , chloride channel 1 in myotonia , sarcoplasmic / endoplasmic reticulum ca atpase 1 and 2 in muscle wasting , cardiac troponin t in cardiac abnormalities , and tau ( mapt ) in cognitive deficits . the binding of the multifunctional helicases p68 and p72 to mutant cug repeats facilitates the binding of mbnl1 to the repeats and likely links the dm1 pathogenesis pathway to the nuclear step of microrna biogenesis , a process in which these helicases are involved as cofactors of the ribonuclease drosha complex . direct evidence was provided for the crosstalk between the dm1 pathogenesis pathway and the cytoplasmic ribonuclease dicer step of microrna biogenesis , in which the mbnl1 protein is also implicated . it was demonstrated that mbnl1 sequestered by mutant cug repeats can not effectively function in its newly recognized role as a pre - mirna cleavage regulator , resulting in the compromised expression and function of some mirnas , including mir-1 , an effect linked to dm1-related heart defects . the expanded cug repeats were also shown to trigger immune responses in dm1 tissue in which the pkr , oas , tlr3 , and rig i genes were activated . although the contribution of rna toxicity to the pathogenesis of polyq diseases was previously postulated based on the structural similarities between the cag and cug repeats in transcripts [ 10 , 17 ] , the first compelling experimental evidence for such an involvement was provided in 2008 by bonini and colleagues . using a drosophila melanogaster model system , the researchers demonstrated that the expression of both translated and untranslated rna molecules containing long hairpin - forming cag repeat tracts causes neurodegeneration , whereas the expression of rna containing caa - interrupted cag repeats resulted in considerably less pronounced neurodegenerative features . caa triplets also encode glutamine but are unable to form a hairpin structure , and their occurrence within cag repeat tracts [ 60 , 61 ] destabilizes cag repeat hairpins . other studies that were performed in nonhuman model systems provided further evidence for the toxicity of cag repeats . hsiao and colleagues showed that in transgenic caenorhabditis elegans , similar to cug repeats , cag repeats of toxic length formed nuclear foci that colocalized with cembl , which is the worm homolog of mbnl1 . worms expressing the toxic cag repeats showed shortened lifespan and reduced motility rates , which is consistent with the phenotypes of worms expressing toxic cug repeats . as shown by pan and colleagues , the expression of 200 cag repeats from the 3utr of egfp was toxic in transgenic mouse muscle and testis tissues in which the expression of the transgene was directed . importantly , ribonuclear foci and abnormal phenotypes were observed in the absence of a polyq product expression . the formation of cag repeat rna foci that colocalize with mbnl1 protein was , for the first time , demonstrated by cooper and colleagues through the expression of very long exogenous cag repeats in monkey cosm6 cells . a question that was addressed using human cell lines was whether less cag repeats could also form nuclear foci and cause alternative splicing aberrations similar to those known for dm1 . the results showed that these effects are triggered in both hela and neuroblastoma sk - n - mc cells transiently expressing mutant cag repeats . in addition , we demonstrated that the expanded cag repeats present in htt and atxn3 transcripts also occur within intranuclear mbnl1-positive rna foci in human hd and sca3 fibroblasts . although the focus formation was accompanied by the aberrant alternative splicing of several endogenous mbnl1-dependent transcripts , this only occurred in cells expressing approximately 70 cag repeats and not a lower number of approximately 45 cags . very recently , bates and colleagues have demonstrated the cag repeat length - dependent aberrant splicing of the htt exon 1 giving rise to short polyadenylated exon 1-intron 1 mrna which is translated to the toxic htt exon 1 protein . the aberrant splicing is triggered by the increased binding of the alternative splicing factor srsf6 to expanded cag repeats . thus , it is likely that the pathogenesis of polyq disorders involves , at least to some extent , the primary elements of the rna gain - of - function mechanism that is well characterized in dm1 . consistent with the above nuclear effects , a protein that is responsible for the nuclear export of transcripts harboring expanded cag repeats has been identified by chan and colleagues as the small nuclear rna u2-associated factor u2af65 . this factor binds directly to expanded cag repeats and forms a complex with the nuclear export receptor nxf1 and its reduction in symptomatic tissues is linked to accumulation of mutant rna in the nucleus . an rnai mechanism has been shown to be involved in pathogenesis in several in vivo and cellular models of polyq diseases . as demonstrated by bonini and colleagues and richards and colleagues in two independent studies , the co - expression of exogenous expanded cag and cug repeats in fly cells resulted in a neurodegenerative phenotype . the complementary repeats likely formed a double - stranded rna that was cleaved by dicer-2 into 21 nt cag / cug repeat sirnas , which were active in silencing in an ago2-dependent process . margolis and colleagues discovered a natural antisense transcript , httas , at the hd repeat locus that contains the cug repeat tract . this transcript was shown to regulate sense htt transcript expression in a process dependent on the repeat length and partly dependent on dicer and the risc pathway . the involvement of an rnai mechanism was also demonstrated by marti and colleagues in human cell lines expressing mutant cag repeats flanked by the htt exon 1 sequence . the mutant cag repeats , both translated and untranslated , gave rise to a toxic small rna species ( scag ) in a dicer - dependent manner and caused a downstream silencing effect in an ago2-dependent manner . again , the cytotoxic effects were triggered only by cag repeats and not by caa repeats . the involvement of the mirna biogenesis pathway in the pathogenesis of polyq diseases is only circumstantial , based on the ability of the mbnl1 [ 16 , 64 , 65 ] and p68 proteins to bind to both cug repeats and , though less efficiently , cag repeats . the recent study by chan and colleagues revealed that cag repeat expansion induces nucleolar stress in polyglutamine diseases , demonstrating that the expanded cag repeat rna interacts directly with nucleolin and that this interaction triggers a number of aberrant downstream cellular processes , eventually leading to cell apoptosis . these downstream effects include hypermethylation of the upstream control element of the rrna promoter and the inhibition of rrna transcription . the reduced level of rrna results in the accumulation of free ribosomal proteins , and the interaction of these proteins with mdm2 e3 ubiquitin ligase causes mitochondrial p53 accumulation . the interaction between p53 and the antiapoptotic protein bcl - xl causes the oligomerization of the proapoptotic protein bak at the mitochondrial membrane and allows for cytochrome c release to the cytosol , an event that activates the caspase cascade and induces apoptosis . collectively , several mechanisms of rna toxicity have been revealed that may likely participate in the pathogenesis of polyq diseases ; however , the entire scale of toxic rna contribution to the disorders and the range of cellular processes and pathways altered by mutant transcripts remain unknown . various genetic models have been used to demonstrate the first examples of cag repeat rna toxicity described in the previous section . the characteristics of these model systems and the main results obtained from their application are compiled in table 1 . it is apparent that this type of rna toxicity has thus far been modeled in lower invertebrates , nematode worms , and fruit flies in addition to mammalian systems , including mice and monkey and human cell lines . however , a common issue in modeling human diseases in lower organisms is the relevance of the results obtained with simpler model systems of human disease . indeed , relevance is hard to prove by merely a sufficient similarity of the genetic pathways operating in model organisms and humans . for polyq diseases and treds in general , this task is more difficult , as these late - onset diseases only occur naturally in humans and not in shorter living animals in which the experimental induction of similar effects requires stronger triggers , i.e. , longer repeat tracts and in some cases higher expression levels . in most studies , the expression of transgene was determined but the level not referred to the expression of endogene ( homologue of human polyq gene ) or any other gene . most authors related the expression of a single construct to the expression of other transgene variants . in case of mouse models r6/2 , bachd , and yac128 , the expression level of transgene did not differ much from that of homologous endogene [ 71 , 72 ] . in case of constructs tested in mammalian systems , containing cmv promoters , the expression level of transgene was reported as very high [ 64 , 65 , 73 ] . the worm and fly systems satisfactorily fulfilled the requirement of genetic similarity , and both systems were capable of demonstrating some molecular and phenotypic hallmarks of rna toxicity . although more relevant and potentially more informative than the fly systems , transgenic mouse models have less frequently been used , and human cellular models have been used for this purpose only twice.table 1characteristics of model systems used for the investigation of rna - mediated toxicity caused by expanded cag repeats . the table is organized according to the species of model organism and includes human cell lines . note that some constructs contained either pure cag or caa repeats , and some contained other caa - interrupted cag repeats . the cag repeats interrupted with caa or pure caa tracts are presented in boldconstruct characteristicsmain conclusionsreferencerepeat tractsequence contextpromoter / expressioncaenorhabditis elegans(cag ) 83gfp 3utrmyo-3 ( muscle)cag repeats , similar to cug repeats , are toxic at the rna level in a length - dependent manner(cag ) 125(cag ) 200(ctg ) 125drosophila melanogaster(cag ) 52 , 99myc flaggmr - gal4 ( eye)the expression of expanded and translated cag or caa repeats causes neurodegeneration(caa ) 94(cag ) 933utruntranslated cag repeats do not induce pathologysca3trq78(cag)atxn3 truncatedgmr - gal4 ( eye ) or elav - gal4 ( neurons)untranslated cag repeats induce progressive neural dysfunctionsca3trq78(caa / g)sca3nq84(cag)atxn3 full lengthlong , untranslated caa - interrupted cag repeats are not toxicsca3nq81(caa / g)(cag ) 100 or 250dsred2 3utr(caa / g ) 105 or 262(ctg ) 250dsred2 3utrgmr - gal4 ( eye ) , 24b - gal4 ( muscle ) , or hs - gal4 ( ubiquitous by heat - shock)simultaneously expressed expanded cag and cug repeats are toxic , and rnai is involved in toxicity(cag ) 250rcag 100cheerio transcript or uas drivengmr - gal4 ( eye ) , elav - gal4 ( neurons ) or da - gal4 ( ubiquitous)the expression of cag and cug repeats leads to rnai - dependent neurodegenerationrctg 100sca3trq78(cag)atxn3 truncatedgmr - gal4 ( eye)the nxf1/u2af65-mediated rna export pathway is involved in expanded cag repeat - mediated toxicitysca3trq78(caa / g)the interaction of cag repeats with nucleolin triggers numerous downstream toxic effects(cag ) 100 or 250dsred2 3utrelav - gal4 ( neurons)the cag100-expressing flies show aberrant expression of numerous brain genes(caa / g ) 1054x rcag 100short sequence or gfp 5utrda - gal4 or actin5c - gal4 ( ubiquitous)the expression of cug or cag ( but not caa ) repeat - containing transcripts causes morphological defects4x rctg 1004x rcaa 100mus musculus(cag ) 44htt cdna full - lengthcmv ( ubiquitous)/construct described to be highly expressed in particular transgenic lineno observed neuropathological changes or behavioral abnormalities in 1-year - old mice overexpressing mutant htt transcript(cag ) 200egfp 3utrgsg ( skeletal muscle)transgenic mice expressing egfp transcripts with long cag repeats in the 3utr develop pathogenic featuresr6/2 hd micehtt exon 1htt promoter ( ubiquitous)/average tissue expression : 75 % relative to endogene nxf1/u2af65 rna export pathway associates with expanded cag rna - mediated toxicity(cag)115 - 150expanded repeat cag rnas interact with nucleolin and trigger nucleolar stress and induce apoptosis via the p53 pathwaybachd 97q ( 48 cag , 49 caa)yac128 125q ( 116 cag , 9 caa)htt full lengthhtt promoter ( ubiquitous)/expression in brain close to endogenous httdistinct caa interruption patterns may be implicated in the observed phenotypic differences in two hd modelsmammalian cultured cells(ctg ) 960dmpk exons 1115cmv cosm6 monkey cells /expression higher than endogenousmrnas containing cag repeats form nuclear rna foci that colocalize with mbnl1(cag ) 960(cag ) 74egfp 3utr fusion construct egfp - mutatxn3 ( 69 cag)cmv , hela , and sk - n - mc human cell lines / expression higher than endogenousexogenous transcripts expressing long untranslated cug or cag repeats trigger similar splicing aberrations in model cells(cag ) 200(ctg ) 74(ctg ) 20040 caghtt exon 1hmec , hpde , hela , and sh - sy5y human cell linesthe expression of expanded htt exon-1 transcript with cag repeats gives rise to small cag - repeated rnas generated by rnai and induces cell death80 cag80 caa - prot80 cag - rnathe repeated pattern was cag cag caa cag caa caaevery 20 repeats were interrupted with ctcgaevery 20 repeats were interrupted with tcgag characteristics of model systems used for the investigation of rna - mediated toxicity caused by expanded cag repeats . the table is organized according to the species of model organism and includes human cell lines . note that some constructs contained either pure cag or caa repeats , and some contained other caa - interrupted cag repeats . the cag repeats interrupted with caa or pure caa tracts are presented in bold the repeated pattern was cag cag caa cag caa caa every 20 repeats were interrupted with ctcga every 20 repeats were interrupted with tcgag the genetic systems shown in table 1 differ considerably with regard to their molecular design . most models are transgenic and express exogenous expanded repeats that have the potential to trigger pathogenesis by a variety of mechanisms involving aberrant interactions with host proteins . of importance in these systems is the intragenic localization of repeated sequences . in some experimental systems , the repeats are located within the open reading frame ( orf ) and are typically transcribed and translated ; the expressed mutant repeats are present within the sequence context of either the full - length human disease transcript or only a fragment of this transcript . alternatively , the repeats are placed within the 3utr sequence of a heterologous gene and are only transcribed . the genes hosting mutant repeats are usually a marker gene , such as gfp or dsred . in addition , the length and purity of the cag repeat tracts are important factors in the design of a model system ; the repeated sequence typically falls into the upper range of repeat lengths found in human subjects suffering from the disease and is often modified to contain caa repeats . the ideal model of a polyq disease would be one that closely recapitulates the human disease with regard to most of its aspects . thus , human cell lines are well suited to study the details of the molecular mechanisms that are implicated in triggering pathogenesis . the rapid development of induced pluripotent stem ( ips ) cell technology enables somatic cells to be transformed into ips cells and then differentiated to neuronal cell lines . moreover , the advent of gene - editing technologies has begun to enable the precise engineering of human disease - related endogens . the expression of mutant exogenous full - length cdna using the well - established technologies of transgenesis may also be considered as an option for the construction of in vitro models of polyq diseases . a good transgenic mouse model of polyq disease should express the cag repeat mutation within the sequence context of the entire human gene , with sufficient upstream and downstream regulatory sequences to ensure the proper spatial and temporal expression of the human gene at natural levels [ 72 , 76 ] . in general , model systems expressing a full - length human mrna , despite showing milder disease phenotypes , are better suited for studies aimed at discovering new mechanisms of rna toxicity than systems expressing only shorter or longer mrna fragments . in the full - length rna models , the mutant transcript has a chance to go through all steps of mrna cellular journey from its birth in the nucleus to death in the cytoplasm ; therefore , all steps altered by the mutation may be identified . a serious difficulty in identifying the involvement of rna toxicity in polyq diseases is the overlap with the polyq toxicity in models expressing cag repeats from the orf , and placing the repeat in the untranslated sequence of a heterologous gene is frequently used to overcome this difficulty and trace the effects of mutant rna . however , preventing transcript translation by the mutation of the start codon in a polyq disease gene or its cdna resolves this problem while preserving the natural sequence context and expression level for the repeats . based on the assumption that the non - aug - initiated translation of repeated sequences is not a very common phenomenon , the start codon mutation may indeed be considered a better solution . by mutating the start codon also in constructs expressing caa repeats or caa - interrupted cag repeats , the four model types depicted in fig . 2 may be generated . with these in vitro and in vivo models critical experiments using both transcriptome - wide and more focused candidate approaches have to be done to obtain clear - cut answers regarding the role of mutant transcripts in the pathogenesis of polyq diseases.fig . 2four types of models that can be used to separate rna toxicity and protein toxicity in the investigation of the pathogenesis of cag repeat diseases . the depicted models express one of two types of repeated sequences , either cag or caa . both sequences code for glutamine , but only the cag repeat forms a hairpin structure in transcripts . in these types of models , both repeats are expressed in their translated or untranslated forms , depending on the absence or presence of an aug start codon mutation . potentially toxic entities , an expanded cag repeat - containing transcript and a polyq - containing protein , are marked in red ; a transcript containing expanded caa repeats , presumed to be nontoxic , is marked in blue four types of models that can be used to separate rna toxicity and protein toxicity in the investigation of the pathogenesis of cag repeat diseases . the depicted models express one of two types of repeated sequences , either cag or caa . both sequences code for glutamine , but only the cag repeat forms a hairpin structure in transcripts . in these types of models , both repeats are expressed in their translated or untranslated forms , depending on the absence or presence of an aug start codon mutation . potentially toxic entities , an expanded cag repeat - containing transcript and a polyq - containing protein , are marked in red ; a transcript containing expanded caa repeats , presumed to be nontoxic , is marked in blue research on rna toxicity in polyq diseases is still in its infancy . at this stage , there is a marked disproportion between the ability of a model organism to closely reproduce human disease and the frequency with which the model system has been used in the search for rna toxicity hallmarks . nevertheless , most of the genetic models provided in table 1 have succeeded in demonstrating the first examples of rna toxicity caused by expanded cag repeats . future studies along this line will require the more extensive use of dedicated mouse models and the next generation of human cellular models . the studies performed thus far have revealed the involvement of several cellular processes and pathways in cag repeat rna toxicity . these altered processes include aberrant alternative splicing , transcript nuclear transport and export , rna interference , and nucleolar stress resulting in apoptosis . the proteins that participate in direct or indirect interactions with mutant cag repeat transcripts need to be identified in further studies , and their role in pathogenesis needs to be elucidated . such efforts may help to determine the contribution of rna toxicity to the overall toxicity caused by both toxic factors : the protein and the rna . this endeavor may also provide the basis to answer the question of whether the rna and protein toxicities interact with each other to enhance pathogenesis or whether these toxic processes are parallel and independent . another issue to be clarified is whether the rna and protein toxicities require the same or different repeat length thresholds to become operative . the latter alternative may likely be the case , and a higher number of repeats may be required to initiate the rna - mediated toxic processes . finally , depending on the scale and repeat length thresholds for the various pathogenic mechanisms triggered by mutant rna , these factors may impact the causative therapeutic approaches to combat polyq diseases to a higher or lower extent . should rna toxicity be proven to contribute significantly to disease development and progression , both the inhibition of mutant protein synthesis and the blockage or degradation of mutant transcript will be required .

in huntington 's disease and other polyglutamine ( polyq ) disorders , mutant proteins containing a long polyq stretch are well documented as the trigger of numerous aberrant cellular processes that primarily lead to degeneration and , ultimately , the death of neuronal cells . however , mutant transcripts containing expanded cag repeats may also be toxic and contribute to cellular dysfunction . the exact nature and importance of rna toxicity in polyq diseases are only beginning to be recognized , and the first insights have mainly resulted from studies using simple model systems . in this review , we briefly present the basic mechanisms of protein toxicity in polyq disorders and rna toxicity in myotonic dystrophy type 1 and discuss recent results suggesting that the pathogenesis of polyq diseases may also be mediated by mutant transcripts . this review is focused on the experimental systems used thus far to demonstrate rna toxicity in polyq disorders and the design of new systems that will be more relevant to the human disease situation and capable of separating rna toxicity from protein toxicity .

Introduction Mechanisms of protein toxicity in polyQ diseases and RNA toxicity in DM1 PolyQ toxicity CUG repeat toxicity Accumulating evidence for RNA toxicity in polyQ diseases Experimental models used to trace RNA toxicity in polyQ diseases Concluding remarks

with the announcement on 26 october 2011 that eli lilly and company ( indianapolis , in , usa ) was withdrawing drotrecogin alfa ( activated ) from the worldwide market , we witnessed the end of the only drug specifically approved for sepsis . the move was prompted by the failure of prowess - shock , a large international study , to confirm the benefit reported from the original trial , prowess ( protein c worldwide evaluation in severe sepsis ) , 10 years ago . in the aftermath , many questions will be raised . here , , i should declare that i led the long - term follow - up and cost - effectiveness studies accompanying prowess and served on the data safety and monitoring board of prowess - shock ( see acknowledgments for full disclosure ) . drotrecogin was approved largely on the basis of a single phase 3 trial , which was stopped early for efficacy . most drugs are approved after two positive phase 3 trials , but exceptions are made when there are impressive supporting data , there is a compelling unmet clinical need , or the trial results are particularly impressive . although an external us food and drug administration ( fda ) advisory panel was split on whether to approve , the fda nevertheless felt that these conditions were met . it seems , therefore , that the decision to approve , though based on only one phase 3 trial , was consistent with the procedures and habits of regulatory bodies around the world . for all new drugs typically , the drug has been administered to only a few thousand patients , in highly controlled situations , with limited long - term follow - up . second , a longer time to generate more evidence raises the drug development costs for pharmaceutical companies while shortening the post - approval patent life ( when a company recoups its investment ) , thus worsening the risk - reward ratio and potentially choking overall investment in drug development . because approval occurs while uncertainty persists , a variety of post - approval surveillance activities are performed in case the decision must be reversed . in the five years leading up to drotrecogin 's approval in 2001 , the fda approved 597 new therapies . in other words , there is a low , but non - zero , rate of drug withdrawal . a lower rate would be preferable , but without major changes to patent laws or to the science and costs of drug development , the chilling effect of a more stringent approval process on dwindling drug pipelines would likely be considered intolerable . so , while we might lament that a sepsis drug was one of the unlucky ones , the fundamental drug approval process that led to drotrecogin approval does not seem too lenient , wrong , or unreasonable . that said , it is a shame that prowess was stopped early , something outside the control of the fda , as early stopping biases toward an overestimate of treatment effect . and it is a shame that the costs and logistics of running two concurrent phase 3 trials in critical care seem to be insurmountable obstacles in the drug development process . cheaper and easier trials could allow us to generate greater certainty without compromising drug pipeline . the usual reason for withdrawal is determination of a previously unknown yet highly undesired side effect . these studies generally reported mortality benefits similar to that seen in prowess [ 6 - 12 ] . the studies also provided greater information about bleeding risks , which led to further label restrictions . however , somewhat unusually , the withdrawal in this instance was a voluntary decision based not on safety but on failure to confirm efficacy . numerous human and animal studies suggest that it modulates coagulation and inflammatory pathways and interacts with endothelial function in the midst of intense innate immune responses to challenges such as sepsis . a previous simulation exercise of theoretical anti - tumor necrosis factor ( anti - tnf ) anti - body trials in sepsis demonstrated that modest differences in the distribution of unmeasured variables such as host genotype and pathogen characteristics could lead to trials in which the same drug produces opposing results , even when patients meet the same clinical criteria . prowess - shock attempted to enroll patients ideally suited for drotrecogin . in the original prowess study , the largest reduction in absolute mortality was noted in patients who appeared to be sicker ( for example , those presenting with septic shock ) . a subsequent trial - administration of drotrecogin alfa ( activated ) in early stage severe sepsis , or address - targeted patients with lower severity of illness and could not demonstrate efficacy . thus , we now have an original trial suggesting benefit in both low and high severity risk , with a constant relative risk reduction but variable absolute reduction , and two subsequent trials mimicking the low and high ends of the original trial but failed to repeat the positive findings . provided that all trials were conducted well , the results of prowess - shock and address raise doubt about the prowess results , but , equally , the prowess results raise doubt about those of prowess - shock and address . when data from multiple trials are synthesized , such as in a meta - analysis , a negative study does not trump a positive study of similar quality . rather , the results from both trials are combined to give an overall estimate of treatment effect . given the potential for differences in important but unmeasured variables at baseline in sepsis to change the result , such an approach seems wise . however , it seems possible that the result will be a point estimate in favor of drotrecogin over placebo but of a magnitude far smaller than in the original trial and probably no longer statistically significant . if the combined estimate from the literature is , say , a non - significant 2% to 3% reduction in mortality , would we be interested in confirming whether such an effect was significant ? that would normally depend on the cost of one more ( much larger ) confirmatory trial , our desire to tolerate non - fatal bleeding side effects , and the resulting financial impact ( both per - patient cost - effectiveness and global increase in spending ) of adopting the drug should the benefit be confirmed . such quandaries could be formally estimated in a value - of - information analysis - how valuable is it to know the answer with greater certainty ? however , the decision was taken out of our hands . eli lilly and company no doubt considered the potentially very large cost of another trial , the fact that regulatory agencies and the field of critical care might be so skeptical as to threaten the ability to conduct another trial or adopt its results into practice or both , and the likelihood that the trial would be negative . taking these factors together , the company presumably decided that to go forward was just too risky . whether the action of eli lilly and company has left a drug that could cut sepsis mortality by 2% to 3% ' on the table ' is something we will now likely never know . we will all see the details of prowess -shock published in the coming weeks or months . first , should this story reinvigorate our drive to complement clinical enrollment criteria with biomarkers that better select patients likely to benefit from a given immunomodulating agent ? second , should we ask of ourselves what we as a community might have done differently ? certainly , greater engagement in clinical trials would seem to be an obvious first step - a huge part of the costs of clinical trials is that they take a long time and enroll only a tiny fraction of all patients with severe sepsis . our ability to make wiser choices about drugs would be enhanced if we could conduct larger trials more rapidly . much time , energy , and emotion were devoted to forming opinions about prowess and about drotrecogin , often with strongly voiced opinions about both the drug and eli lilly and company . one can hope that decisions about future drugs will be made in environments richer in data and poorer in opinion . if we believe that there is a role for pharmacomanipulation of critical illness , our partnership with the pharmaceutical industry is a prerequisite . thus , we must think about whether the partnership works properly in terms of open , efficient , rapid , and rigorous science for the optimal benefit of all concerned , especially our patients . address : administration of drotrecogin alfa ( activated ) in early stage severe sepsis ; fda : us food and drug administration ; prowess : protein c worldwide evaluation in severe sepsis . the author declares that he was principal investigator on grants received by the university of pittsburgh from eli lilly and company for the conduct of the long - term follow - up and cost - effectiveness studies accompanying prowess . he also received consulting fees and speaking honoraria from eli lilly and company between 1996 and 2004 . he received compensation from eli lilly and company for serving on the data safety and monitoring board for prowess - shock . ( salt lake city , ut , usa ) , which are also engaged in sepsis research . the author gratefully acknowledges shamly austin for her assistance generating data on fda approval and withdrawal rates discussed in this article .

following the failure of prowess - shock to demonstrate efficacy , eli lilly and company withdrew drotrecogin alfa ( activated ) from the worldwide market . drotrecogin was initially approved after the original trial , prowess , was stopped early for overwhelming efficacy . these events prompt consideration of both the initial approval decision and the later decision to withdraw . it is regrettable that the initial decision was made largely on a single trial that was stopped early . however , the decision to approve was within the bounds of normal regulatory practice and was made by many approval bodies around the world . furthermore , the overall withdrawal rate of approved drugs remains very low . the decision to withdraw was a voluntary decision by eli lilly and company and likely reflected key business considerations . drotrecogin does have important biologic effects , and it is probable that we do not know how best to select patients who would benefit . overall , there may still be a small advantage to drotrecogin alfa , even used non - selectively , but the costs of determining such an effect with adequate certainty are likely prohibitive , and the point is now moot . in the future , we should consider ways to make clinical trials easier and quicker so that more information can be available in a timely manner when considering regulatory approval . at the same time , more sophisticated selection of patients seems key if we are to most wisely test agents designed to manipulate the septic host response .

None First, was the initial decision to approve drotrecogin wrong? Second, was the decision to withdraw drotrecogin too hasty? Next steps Abbreviations Competing interests Acknowledgements

mouse luecs were immortalized with polyoma middle t antigen and cultured in mcdb131 medium ( gibco ; life technologies ) supplemented with 20% fbs ( invitrogen ) , 2 mm l - glutamine ( lonza ) , 1 mm na - pyruvate ( gibco ; life technologies ) , 100 g / ml heparin ( sigma - aldrich ) , and 50 g / ml ec growth supplement ( ecgs ) obtained from calf brain . ecs were seeded on plates coated with glutaraldehyde - crosslinked gelatin and cultured in complete medium for 4 days to reach confluence . total rna was extracted from ~ 5 10 cells using the rneasy mini kit ( qiagen ) following manufacturer 's instruction . quality control of the rna samples was performed using an agilent bioanalyzer 2100 ( agilent technologies ) . the rna from three independent extractions was processed for each of the cell line under analysis . a total of 150 ng of rna from each sample was used for rna quality check , labeling and hybridization on a mouse gene 1.0 st genechip array according to the manufacturer 's instructions ( affymetrix ) . three independent biological replicates were performed for each condition ( l1-transfected vs. mock - transfected luecs ) . we used the robust multi - array average ( rma ) to normalized data . a total of 35,512 probesets were loaded and 819,041 pm probes were used for analysis . next , we uploaded normalized data in brb arraytools ( http://linus.nci.nih.gov/brb-arraytools.html ) to run sam analysis ( http://www-stat.stanford.edu/~tibs/sam/ ) in order to identify differentially expressed genes upon l1 overexpression in luec cells . the sam parameters of analysis were the following : number of classes : 2number of probesets : 35,512target proportion of false discoveries ( q - value ) : 0.05delta value used to identify significant probesets : 1.24701fudge factor for standard deviation computed : 0.04825 . number of probesets : 35,512 target proportion of false discoveries ( q - value ) : 0.05 delta value used to identify significant probesets : 1.24701 fudge factor for standard deviation computed : 0.04825 . under these conditions , we identified a total of 3409 significant probesets corresponding to 2684 unique annotated genes ( fig . 1 ) . we then selected probesets having 1.5 fold change difference ( l1 vs. ctr ) that resulted in a set of 496 upregulated and 743 downregulated probesets annotated , corresponding to 361 upregulated and 580 downregulated unique genes , referred to as the l1-ecs signature. next , we uploaded the l1-ecs signature ( probeset level ) in the ingenuity pathway analysis software ( ipa , http://www.ingenuity.com ) to identify biological functions / pathways putatively regulated by l1 . the mouse gene 1.0 st array reference set present in the ipa database was used for the ipa core analysis . only direct relationships in mammals ( i.e. , human , mouse , and rat ) were considered , including endogenous chemicals for gene network analysis . the p - values for biofunction enrichment were corrected for multiple testing using the benjamini - hochberg correction . with these settings we identified 23 bio - functions significantly enriched ( p - value < 0.05 ; benjiamini - hochberg correction ) in l1-overexpressing cells compared to mock - transfected cells . considering the massive changes in transcriptional activity and the predicted effect on a variety of biological functions upon l1 overexpression , we then asked whether l1 could activate / inhibit transcription factor(s ) ( tfs ) . to address this question such an analysis allows the identification of transcriptional regulators activated / inhibited under specific experimental conditions , thus accounting for the observed gene expression changes . strikingly , we identified a total of 18 and 11 tfs predicted to be activated or inhibited by l1 , respectively . of note , 5 tfs were differentially regulated in l1-overexpressing vs. mock - transfected cells and , in particular , four of the activated tfs were upregulated in l1 overexpressing cells ( stat1 , stat2 , irf7 and atf4 ) , while one of the inhibited tfs was downregulated ( foxm1 ) . we then performed the mechanistic networks analysis to further explore the contribution of the tfs in regulating gene networks . we found that stat1 , stat2 , irf7 , atf4 and stat3 ( that was among the 18 tfs predicted to be activated ) interact with each other ( fig . 2 ) , thus exerting a coordinated control on a directional network of 105 genes regulated by l1 ( i.e. 11% of all the l1-regulated genes ) . most of these genes were consistently up- or downregulated with the expression change of their upstream tfs . these findings were supported by functional studies which implicated the jak / stat signaling pathway in the biological response of luecs to l1 overexpression . mouse luecs were immortalized with polyoma middle t antigen and cultured in mcdb131 medium ( gibco ; life technologies ) supplemented with 20% fbs ( invitrogen ) , 2 mm l - glutamine ( lonza ) , 1 mm na - pyruvate ( gibco ; life technologies ) , 100 g / ml heparin ( sigma - aldrich ) , and 50 g / ml ec growth supplement ( ecgs ) obtained from calf brain . ecs were seeded on plates coated with glutaraldehyde - crosslinked gelatin and cultured in complete medium for 4 days to reach confluence . total rna was extracted from ~ 5 10 cells using the rneasy mini kit ( qiagen ) following manufacturer 's instruction . quality control of the rna samples was performed using an agilent bioanalyzer 2100 ( agilent technologies ) . the rna from three independent extractions was processed for each of the cell line under analysis . a total of 150 ng of rna from each sample was used for rna quality check , labeling and hybridization on a mouse gene 1.0 st genechip array according to the manufacturer 's instructions ( affymetrix ) . three independent biological replicates were performed for each condition ( l1-transfected vs. mock - transfected luecs ) . we used the robust multi - array average ( rma ) to normalized data . a total of 35,512 probesets were loaded and 819,041 pm probes were used for analysis . next , we uploaded normalized data in brb arraytools ( http://linus.nci.nih.gov/brb-arraytools.html ) to run sam analysis ( http://www-stat.stanford.edu/~tibs/sam/ ) in order to identify differentially expressed genes upon l1 overexpression in luec cells . the sam parameters of analysis were the following : number of classes : 2number of probesets : 35,512target proportion of false discoveries ( q - value ) : 0.05delta value used to identify significant probesets : 1.24701fudge factor for standard deviation computed : 0.04825 . number of probesets : 35,512 target proportion of false discoveries ( q - value ) : 0.05 delta value used to identify significant probesets : 1.24701 fudge factor for standard deviation computed : 0.04825 . under these conditions , we identified a total of 3409 significant probesets corresponding to 2684 unique annotated genes ( fig . 1 ) . we then selected probesets having 1.5 fold change difference ( l1 vs. ctr ) that resulted in a set of 496 upregulated and 743 downregulated probesets annotated , corresponding to 361 upregulated and 580 downregulated unique genes , referred to as the l1-ecs signature. next , we uploaded the l1-ecs signature ( probeset level ) in the ingenuity pathway analysis software ( ipa , http://www.ingenuity.com ) to identify biological functions / pathways putatively regulated by l1 . the mouse gene 1.0 st array reference set present in the ipa database was used for the ipa core analysis . only direct relationships in mammals ( i.e. , human , mouse , and rat ) the p - values for biofunction enrichment were corrected for multiple testing using the benjamini - hochberg correction . with these settings we identified 23 bio - functions significantly enriched ( p - value < 0.05 ; benjiamini - hochberg correction ) in l1-overexpressing cells compared to mock - transfected cells . considering the massive changes in transcriptional activity and the predicted effect on a variety of biological functions upon l1 overexpression , we then asked whether l1 could activate / inhibit transcription factor(s ) ( tfs ) . to address this question such an analysis allows the identification of transcriptional regulators activated / inhibited under specific experimental conditions , thus accounting for the observed gene expression changes . strikingly , we identified a total of 18 and 11 tfs predicted to be activated or inhibited by l1 , respectively . of note , 5 tfs were differentially regulated in l1-overexpressing vs. mock - transfected cells and , in particular , four of the activated tfs were upregulated in l1 overexpressing cells ( stat1 , stat2 , irf7 and atf4 ) , while one of the inhibited tfs was downregulated ( foxm1 ) . we then performed the mechanistic networks analysis to further explore the contribution of the tfs in regulating gene networks . we found that stat1 , stat2 , irf7 , atf4 and stat3 ( that was among the 18 tfs predicted to be activated ) interact with each other ( fig . 2 ) , thus exerting a coordinated control on a directional network of 105 genes regulated by l1 ( i.e. 11% of all the l1-regulated genes ) . most of these genes were consistently up- or downregulated with the expression change of their upstream tfs . these findings were supported by functional studies which implicated the jak / stat signaling pathway in the biological response of luecs to l1 overexpression . here we describe the methods of analysis that were used to define and characterize the gene expression profile induced by l1 in murine lung endothelial cells ( luecs ) . we used the affymetrix mouse gene 1.0 st arrays , which enabled us to produce gene expression data of a total of 28,869 genes ( 764,885 distinct probes ) . through the combined use of the significant analysis of microarrays ( sam ) and the ingenuity pathway analysis ( ipa ) , we identified a set of 941 genes and 23 bio - functions significantly regulated in l1-overexpressing vs. control luecs . we reasoned that these massive transcriptional changes could be the result of the l1-mediated regulation of tfs . indeed , by using the upstream modulator analysis in ipa we found that 29 tfs were predicted to be activated / inhibited and , among these , 5 tfs were transcriptionally regulated in our profiling experiment . among the 29 tfs identified , many are involved in the inflammatory and/or angiogenic cascades ( stat1 , stat2 , stat3 , irf1 , irf3 , irf7 , rela / p65 , nfkb1 and atf4 ) . of note , while l1 has been causally linked to nf-b signaling in cancer cells , we are the first to report a crosstalk between l1 and stats , irfs and atf4 . our data , therefore , point to l1 as a novel orchestrator of the cancer - associated response that entails the regulation of gene networks and biochemical cascades which , in turn , impact on the pathophysiology of cancer cells as well as tumor microenvironment . importantly , the results of our in silico analysis have been successfully validated experimentally , thus lending further support to their biological relevance .

we recently identified a novel role for the l1 transmembrane glycoprotein ( also known as l1cam or cd171 ) in the regulation of tumor angiogenesis and vessels stabilization . l1 overexpression in cultured endothelial cells of the lung ( luecs ) exerted a pleiotropic effect in that it regulated proliferation , migration , tubulogenesis , vascular permeability , and endothelial - to - mesenchymal transition ( endmt ) . in addition , we provided strong evidence that antibody - mediated targeting of l1 may be an effective strategy for vessel normalization with the potential to increase efficacy of chemotherapeutic agents.high-throughput microarray expression profile revealed that l1 modulates the expression of hundreds of genes mainly involved in cell cycle regulation , dna replication , cellular assembly , migration , development and organization . by using a pathway - oriented analysis strategy we were able to identify a network of 105 genes modulated by l1 through the predicted activation of five transcription factors : stat1 , stat2 , stat3 , irf7 , and atf4 . indeed , l1 overexpression resulted in the strong induction of stat3 phosphorylation which was abolished by antibody - mediated neutralization of il-6r. these results indicated that l1 promoted stat3 activation via the il-6/il-6r axis .

Direct link to deposited data Experimental design, materials and methods Cell culture RNA preparation Gene expression profiling Discussion Conflict of interest

transorbital intracranial injury is rare and may cause serious brain damage and is associated with a high mortality rate . this type of injury , accounting for 4.5% of all orbital pathology,[35 ] represents 0.04% of all head trauma , 24% of penetrating head trauma in adults , and 45% in children . penetrating orbital injury may lead to severe brain injury when the foreign object enters the cranium , leading to both orbital and cerebral complications . a foreign body can penetrate the brain through the orbit in 3 ways : via the orbital roof , the superior orbital fissure , and the optic canal . the diagnosis is confirmed if an entire or partial foreign object is found in the wound , and is difficult to make in the case of trivial trauma . we report a case of a transorbital injury caused by penetration of a metal bar which entered the cerebrum through the orbit . a 13-year - old male sustained an injury to his right orbit , resulting from falling while holding a metal bar . an iron bar [ 3 cm 18 cm ] penetrated his right orbit via the medial canthus and right infraorbital region and was driven into the skull . on admission , he was unconscious , but with normal vital signs . his right eye protruded outside the orbital cavity and was severely swollen and bruised . on neurological examination , he was in a deep coma with a glasgow coma score of 4/15 ( e1m2v1 ) . plain skull radiographs confirmed that the bar crossed the skull from the right orbit to the left occipital bone [ figure 1 ] . computed tomography ( ct ) demonstrated the bar passing through the right orbit and superior orbital fissure and emerging from the left occipital bone , which was fractured by direct compaction . in association with the bar , there was a trajectory of intracerebral hemorrhage , specifically subarachnoid hemorrhage in the basal cisterns with severe cerebral edema [ figure 2 ] . the bar penetrated the third ventricle and suprasellar cistern ; adjacent to the right carotid artery and branches , the dense hemorrhage observed within the suprasellar cistern was likely the result of injury to the right internal carotid artery . angiography could not be performed because the patient rapidly worsened neurologically ( gcs 3 : e1m1v1 ) . lateral skull radiograph shows the bar penetrating the orbit and emerging from the occipital bone . computed tomography demonstrates the bar 's trajectory into the sellar and suprasellar region via the superior orbital fissure . the right internal carotid artery was found to be lacerated , and the right optic nerve and optic chiasm transected near the superior orbital fissure . the bar penetrated the superior orbital fissure , anterior clinoid process , suprasellar cistern , third ventricle , and cerebrum . incidentally , if a metal bar is fixed at the superior orbital fissure , it can usually be mobilized and removed , retracting it carefully from the entry site under direct visualization and avoiding resistance or leverage . severe bleeding from the right internal carotid artery occurred despite this maneuver ; the carotid artery required ligation . the patient died 10 days postoperatively due to ischemic brain injury , diencephalic injury , and intractable increased intracranial pressure with brainstem herniation . . most reports are by pens , knives , or chopsticks . due to its thin wall , the orbit is the most vulnerable structure in the cranium ; penetrating orbital injuries are often associated with traumatic brain injury . the cranium can be violated via the transorbital route through the superior orbital fissure and optic canal . the degree of neurologic damage is related to orbital bone anatomy , as well as the size , shape , and trajectory of the object . intracranial penetration may occur through the orbital roof , superior orbital fissure or optic canal . therefore , medial or canthal injuries , associated with severe visual loss , strongly suggest optic canal damage . the most frequent site of penetration is through the orbital roof because the superior orbital plate of the frontal bone is thin and fragile . this frequently leads to frontal lobe contusion , the prognosis of which is fairly good . objects passing through the superior orbital fissure tend to follow a trajectory , close to the internal carotid artery and are generally associated with optic nerve and other orbital injuries . the orbit is pyramid - like in volume , and as a result , penetrating objects are directed towards the apex , most of which pass through the superior orbital fissure and through optic canal . our case is an example of this type of injury . penetrating injuries through the superior orbital fissure may affect cranial nerves iii , iv , v , vi , the arteries of the circle of willis and the carotid artery . penetrating injuries caused by high speed objects , following a trajectory perpendicular to the orbital wall , result in direct bone fractures . vertically directed objects may pierce the orbital roof and cause damage in the frontal lobe . horizontally penetrating objects may cause ethmoid bone or posterior orbital wall fractures . with sufficient force , the midline may be violated and can damage contralateral structures . penetrating objects , directed medially through the superior orbital fissure , the optic canal , and / or sphenoid wing , may damage the temporal lobe , cavernous sinus , and brain stem . the most frequent pattern of injury ( 68% ) involves the cavernous sinus , temporal lobe , and brainstem . in low velocity injuries , when the penetrating object hits the orbit at a small angle , the object follows a path along the wall of the orbit . penetrating objects , entering the orbit close to the horizontal plane , tend to follow the orbital funnel towards the apex . this mechanism differs from that seen in orbital roof penetration , in which objects traverse the superior orbital fissure and pass laterally along the cavernous sinus toward the temporal lobe . in our case , the iron bar penetrated the orbit at the medial canthus and followed a posterior , medial , and superior trajectory . objects directed superiomedially within the orbit usually affect the superior orbital fissure and / or optic canal , sphenoid wing , and sella turcica , passing across the midline ; then , they enter the suprasellar cistern and third ventricle , causing serious damage to neurovascular structures . these types of injuries are associated with the most severe brain damage , due to laceration of central diencephalic neural structures and major vessels ; therefore , they are associated with the highest morbidity and mortality rates . the injury described in this report usually has a fatal prognosis . in penetrating orbitocranial injuries , the severity of brain damage and outcome depends on the velocity , trajectory , and shape of the object , rather than the type of material . however , porous objects that are prone to fragmentation provide a good medium for infection . immediate complications of transorbital penetrating trauma include intracerebral hematoma , cerebral contusion , intraventricular hemorrhage , pneumocephalus , cranial nerve damage , severe permanent neurological damage , brain stem injury and cerebrovascular injury . delayed complications include cerebrospinal fluid fistula , pneumocephalus , orbital cellulitis , carotid - cavernous sinus fistula , central nervous system infections , traumatic aneurysm and delayed intracranial hemorrhage . retained foreign objects cause severe infections , such as meningitis or brain abscess ( 50% ) . the prognosis is good in absence of direct injury to the brainstem or laceration of major intradural vessels . in our patient , ct is necessary to evaluate the trajectory of the object , the intracranial structures involved and to predict possible complications . however , wooden objects are not always detectable on plain radiographs , making accurate evaluation more difficult . if the injury is caused by a chopstick , and if chopstick is removed before the patient is evaluated by a physician , potentially fatal complications can occur . magnetic resonance imaging ( mri ) is superior to ct for detection of small fragments of wooden foreign bodies . therefore , cerebral angiography should be performed 2 - 3 weeks after an insult to rule out traumatic aneurysms and to identify major vascular injury in a timely fashion . the transorbital or transcranial approach can be chosen depending on the location of the foreign body . removal of the foreign object , debridement and resection of all involved skull bones , hematoma evacuation , careful hemostasis along the trajectory , and meticulous dural closure to reduce cerebrospinal fluid leak are mandatory to prevent potentially fatal infectious complications . vigorous debridement is not necessary and is associated with an increased disability and mortality without clear advantage . antiepileptic medications are recommended in the early stages after injury to prevent seizures . in conclusion , transorbital penetrating cranial injury by a metal bar is rare and may result in potentially life - threatening complications . failure to detect intracranial complications may lead to serious neurological morbidity and is associated with a high mortality rate .

transorbital intracranial injury is uncommon , representing 0.04% of penetrating head trauma with a high mortality rate . orbital penetrating injuries may cause severe brain injury if the cranium is entered , typically via the orbital roof , the superior orbital fissure , or the optic canal . a 13-year - old male sustained a severe brain injury due to penetration of the right orbit with an iron bar . the bar entered the inferiomedial aspect of the orbit and emerged from the left occipital bone . neurological examination revealed deep coma ( gcs : e1m2v1 ) with fixed , dilated , and non - reactive pupils . the bar followed an intracranial trajectory , through the third ventricle and suprasellar cistern . the patient underwent an immediate exploration with removal of the bar . unfortunately , he died 10 days postoperatively due to severe diencephalic injury with brainstem herniation . in this case report , we discuss the radiologic diagnosis and surgical management of transorbital orbitocranial injury by foreign body penetration .

Introduction Case Report Discussion

all study subjects were recruited in a consecutive manner from our glaucoma clinic ; the patients were examined between february and april 2010 at the asan medical center , seoul , korea . on initial evaluation , all subjects underwent a complete ophthalmologic examination including the recording of medical , ocular , and family histories ; visual acuity ( va ) testing ; a humphrey field analyzer ( hfa ) swedish interactive threshold algorithm 24 - 2 full threshold test ( carl zeiss meditec ) ; multiple intraocular pressure measurements using goldmann applanation tonometry ; stereoscopic optic nerve photography ; and cirrus oct . all participants had previously experienced several instances of hfa testing . to minimize any learning effect , only the final hfa test results were used for analysis in the current study . for inclusion , all participants had to meet the following criteria : best - corrected visual acuity ( bcva ) of 20 / 30 or better , with a spherical equivalent within 5 diopters ( d ) and cylinder correction within + 3 d ; presence of a normal anterior chamber and open angle on slit - lamp and gonioscopic examination ; and reliable hfa test results with a false - positive error < 15% , a false - negative error < 15% , and a fixation loss < 20% . any patient with any ophthalmic disease that could result in hfa defects or with a history of diabetes mellitus was excluded . all participants had glaucomatous optic discs , as confirmed and agreed upon by two glaucoma experts ( krs and jhn ) , and when glaucomatous visual field ( vf ) defects meeting at least two of the following criteria were present : 1 ) a cluster of three points with a probability of less than 5% on a pattern deviation map in at least one hemifield , including at least one point with a probability of less than 1% , or a cluster of two points with a probability of less than 1% ; 2 ) glaucoma hemifield test results outside normal limits ; and 3 ) a pattern standard deviation less than 5% . cataract severity was graded as 0 , 1 , or 2 according to the extent of nuclear , cortical , and subcapsular opacity . all procedures conformed to the declaration of helsinki and the study was approved by the institutional review board of the asan medical center at the university of ulsan , seoul , korea . 3.0.0.50 ) uses spectral - domain technology ; an optic disc cube is obtained from a three - dimensional dataset composed of 200 a - scans derived from 200 b - scans that cover a 6 6 mm area centered on the optic disc . after creation of an rnfl thickness map from the cube dataset , the software automatically determines the center of the disc and then extracts a circumpapillary circle ( 1.73 mm in radius ) from the dataset to perform rnfl thickness measurements . maculae were scanned using the macular cube mode ; the data were obtained from a three - dimensional dataset composed of 512 a - scans derived from 128 b - scans that cover a 6 6 mm area centered on the fovea . the ss values of macular and optic disc mode images were compared using the paired t - test . the relationship between macular and optic disc mode image ss was assessed by pearson correlation analysis . clinical characteristics were compared between high- and low - ss patients imaged in each mode , using the mann - whitney u - test . individual factors were subjected to univariate logistic regression analysis and subsequently were included in the multivariate analysis in a backward stepwise manner if the p - value was less than 0.20 . all p - values were two - sided , and p < 0.05 was considered significant . all statistical analyses were performed using spss ver . 15.0 ( spss inc . , 3.0.0.50 ) uses spectral - domain technology ; an optic disc cube is obtained from a three - dimensional dataset composed of 200 a - scans derived from 200 b - scans that cover a 6 6 mm area centered on the optic disc . after creation of an rnfl thickness map from the cube dataset , the software automatically determines the center of the disc and then extracts a circumpapillary circle ( 1.73 mm in radius ) from the dataset to perform rnfl thickness measurements . maculae were scanned using the macular cube mode ; the data were obtained from a three - dimensional dataset composed of 512 a - scans derived from 128 b - scans that cover a 6 6 mm area centered on the fovea . the ss values of macular and optic disc mode images were compared using the paired t - test . the relationship between macular and optic disc mode image ss was assessed by pearson correlation analysis . clinical characteristics were compared between high- and low - ss patients imaged in each mode , using the mann - whitney u - test . individual factors were subjected to univariate logistic regression analysis and subsequently were included in the multivariate analysis in a backward stepwise manner if the p - value was less than 0.20 . all p - values were two - sided , and p < 0.05 was considered significant . ninety - two eyes of 92 patients ( male 32 and female 60 ) were enrolled . the mean age of the study participants was 54.0 13.8 years and the average md value yielded by vf testing was -6.7 6.7 db ( table 1 ) . ss was significantly higher in images obtained by the macular cube mode compared to the optic disc cube mode ( 7.8 1.3 vs. 6.9 1.1 , respectively ; p = 0.001 ) . a comparison of the ss values yielded by the two modes showed a statistically significant positive correlation with mode ( r = 0.581 , p < 0.001 ) ( fig . , 31 images were classified as being of low ss ( ss < 7 ) , whereas 61 were of high ss ( ss 7 ) . however , when the macular cube mode was employed , 16 and 76 eyes were classified into the low- and high - ss groups , respectively . more patients were classified as high - ss subjects when the macular cube mode was employed compared to those so designated when the optic disc cube mode was used ( 82.6% vs. 66.3% , respectively ; p = 0.002 ) . age and va differed significantly between the high- and low - ss groups when either the optic disc or macular cube mode was employed ( optic disc mode : age , p = 0.027 , va , p = 0.012 ; macular cube mode : age , p = 0.046 , va , p = 0.014 ) . with use of either mode , patients in the high - ss group were younger and had better va than those in the low - ss group . however , the extent of cataracts and the vf md did not significantly differ between groups ( tables 2 and 3 ) . upon univariate logistic regression analysis , the extent of cataracts was associated with ss when the optic disc cube mode was used . however , no significant factor emerged upon multivariate analysis ( table 4 ) . in the macular cube mode , no analyzed factor was associated with ss upon either univariate or multivariate analysis ( table 5 ) . oct is now recognized as an important diagnostic tool for providing quantitative data on rnfl and macular thicknesses . a critical feature of oct imaging used in glaucoma diagnosis is the reliability of test results . cheung et al . emphasized the importance of a high ss when rnfl is measured using oct . also , it has been suggested that an ss value of over 7 is essential to guarantee reproducibility of results from the stratus oct . in the present study , we investigated factors associated with image ss obtained by cirrus oct . moreno - montanes et al . reported that cirrus oct afforded a higher ss than did stratus oct . although simple ss comparisons between different oct instruments is not appropriate , because oct versions might vary in their approaches toward image processing and data analysis , it remains possible that cirrus oct may provide higher - quality imaging when image acquisition is performed under the same conditions using different instruments . this is because cirrus oct uses a line - scanning ophthalmoscope fitted with a 750-nm superluminescent diode as a light source , which facilitates image alignment . in the present study , images obtained in the optic cube mode were of lower ss compared to those obtained employing the macular cube mode . the macula is aligned along the visual axis , whereas the optic disc is located in a position nasal to this axis . thus , macular images can be obtained by central fixation , whereas optic disc image acquisition requires eccentric fixation . it follows that if the two structures are tested under the same conditions , it would be easier to retain central fixation , rather than defer to eccentric fixation , during image acquisition . moreover , if the direction of disc attachment with respect to the orbit is unusual , as may be observed with a tilted disc , this can lead to a reduction in ss because reflectance is inconsistent . subjects in the high - ss group , evaluated by either oct mode , were younger and had better va . imaging data obtained using the stratus oct are known to be influenced by the presence of cataracts . the results of the present work indicate that the extent of cataracts did not differ between the high- and low - ss patient groups . in addition , the patients of the present study were relatively young , with a mean age of 54 years , and most cataracts ( 90.2% ) were mild , being of grade 0 or 1 . however , logistic regression analysis showed that the extent of cataracts was significantly associated with ss when the optic disc cube mode was employed , and this suggests that scan quality may vary with cataract density when the cirrus oct is used , as is the case when the stratus oct is employed . meanwhile no meaningful between - group difference was noted when the macular cube mode was used . pupil size can affect both the reproducibility and the quality of images obtained by oct . to exclude any confounding effects of pupil size , we obtained cirrus oct images after pupil dilation in all patients , as mentioned above . all images were obtained by a single experienced examiner ; this may not reflect the real clinical situation . however , we found that it was more difficult to obtain high - quality images when the optic disc cube mode was employed , compared to the macular cube mode used under similar conditions . further , we found that age , va , and extent of cataracts seemed to affect acquisition of high - ss images by the cirrus oct . further work with a larger number of patients is needed to verify the effects of the extent and type of cataracts on image quality , and the impact of ss on glaucoma diagnosis .

purposethe aim of this study was to investigate factors associated with the signal strengths ( ss , image quality scores ) of optic disc and macular images obtained using cirrus spectral domain optical coherence tomography ( oct).methodsninety - two glaucomatous eyes were imaged using the cirrus oct macular and optic disc cube modes after pupil dilation . the influences of patient age , spherical equivalent , cataract presence , and cataract and glaucoma severity ( visual field mean deviation ) , on the ss of images obtained using the two cube modes were compared between patients whose images showed high ss ( ss 7 ) and low ss ( ss < 7).resultsthe signal strength was significantly higher in images obtained using the macular cube compared to the optic disc cube mode ( 7.8 1.3 vs. 6.9 1.1 , respectively ; p = 0.001 ) . age and visual acuity of patients differed significantly between the high- and low - ss groups when data acquired using the optic disc ( p = 0.027 and 0.012 , respectively ) and macular cube modes ( p = 0.046 and 0.014 , respectively ) were analyzed . when the optic disc cube mode was employed , the extent of cataracts was significantly related to ss , whereas when the macular cube mode was used , none of the factors analyzed was significantly associated with ss.conclusionsage , visual acuity , and the extent of cataracts were significantly associated with images of higher ss when the cirrus oct optic disc cube mode was employed .

Materials and Methods Optical coherence tomography imaging Analysis Results Discussion

some studies have shown that colonization of denture inner surfaces by yeasts causes oral and systemic diseases11,23 , particularly in aged and immunodepressed patients and those with reduced salivary flow11,26 . the mechanical removal of debris with the use of toothbrush , dentifrice and water is a popular cleansing technique used by great part of denture wearers7 . although it is a simple , inexpensive and effective method10,25 , a major disadvantage is that the abrasive action could result in wear of the denture base and relining materials10,12 . abrasion caused by brushing of acrylic resin denture bases polymerized by different methods may result in mass loss , surface roughness , loss of surface polishing , problems on denture adaptation due the loss of surface details and difficulty on the maintenance of denture hygiene7,21,22,27 . the critical threshold surface roughness for bacterial adhesion is 0.2 m30 and the acceptable mass loss of acrylic resins is still unknown . the main components are : water , detergent , thickening agent and specific coloring , flavoring and abrasive agents13,15 . most in vitro studies employ motor - driven brushing machines , in order to standardize time , speed , frequency of brush strokes , applied load and amount of dentifrice1928 . the methods used for measurement of abrasion include mass loss , luster changes , surface roughness , microscopic examination and radiometric technique . investigating the abrasion resistance of different denture resins together with the abrasiveness of different dentifrices is of clinical interest to help selecting the most appropriate materials and methods for denture cleansing with no significant damage to the denture base . the aim of this study was to evaluate the mass loss of four commercially available heat - polymerized acrylic resins after simulated toothbrushing with three different dentifrices . the tested null hypothesis was that different denture resins and dentifrices produce different mass loss after toothbrushing . the acrylic resins and dentifrices used in this study are presented in tables 1 and 2 , respectively . plexiglas patterns ( 90x30x3 mm ) were included into investment flasks ( no . 6 jon , jon , so paulo , sp , brazil ) to allow reproduction of specimens by conventional dental procedures . the acrylic resins were mixed following the manufacturer 's instructions and inserted into the mold . after polymerization , the specimens were bench cooled at room temperature before being removed from the mold and were thereafter carefully finished , polished and immersed in water at 37c for 7 days until use17 . the specimens were designed to fit on the custom - made metal plate of the brushing machine . the specimens of all acrylic resins were allocated to 4 groups ( n=4 ) . in group 1 , ( control ) , the specimens were brushed with distilled water ; in group 2 , the specimens were brushed with a dentifrice indicated for natural teeth ( colgate ) ; in groups 3 and 4 , the specimens were brushed with dentifrices specific for complete dentures ( bonyplus and dentu - creme , respectively ) . prior to simulated toothbrushing , the specimens were removed from the water bath , rinsed with tap water , cleaned for 1 min in an ultrasonic bath with deionized water containing 1% of detergent ( limpol neutral , bombril s / a , so bernardo do campo , brazil ) , dried with absorbent paper and weighed in an analytical balance accurate to 0.1 mg after 1 min ( metler toledo gmbh , laboratory & weighing technologies , greifensee , switzerland ) . the specimens were positioned in the specimen holder containing the slurry bath in the mechanical cross - brushing machine ( precision shop , university of so paulo , ribeiro preto , so paulo , brazil ) equipped with 6 soft nylon - bristled toothbrushes ( tek , soft , johnson & johnson ) , in such a way that 6 specimens could be brushed simultaneously . the specimens were subjected to a linear toothbrush abrasion movement with a rate of 356 brush strokes ( forth and back ) per minute , totalizing 35,600 brush strokes for each specimen , which is representative of 2 years of denture cleansing13 . the machine was set to provide 200 g vertical load over each specimen and a 3.8 cm toothbrush trail . brushing was carried out in the presence of a dentifrice / distilled water slurry ( 2:1 ; w / w ) , which was placed into the slurry bath17 . a stainless steel agitating fin was fastened to the end of the brush to ensure adequate mixing , so that settling of the abrasive material would be minimized during brushing . after brushing , the specimens were removed from the specimen holder , rinsed thoroughly and blot dried with soft absorbent paper . mass loss due to wear was calculated as the difference between the mass of each specimen before and after brushing . the results of mass loss were subjected to 2-way anova and tukey 's test ( =0.05 ) to examine the influence of materials and groups . figure 1 shows the means and standard deviation ( sd ) of the mass loss ( mg ) of the tested acrylic resins after toothbrushing . the results of anova are presented on table 3 . according to the tukey 's test for mass loss ( mg ) of acrylic resins and dentifrices , the resins qc-20 and lucitone 550 showed greater mass reduction , which indicated lower abrasion resistance as compared to the other resins . colgate was the most abrasive dentifrice , followed by dentu - creme and bonyplus ( tables 4 and 5 ) . d : dentifrices ; r : acrylic resins different letters indicate statistically significant difference ( p<0.05 ) . since dentifrice and toothbrush association is one of the most common methods for oral hygiene , it should promote good cleansing with minimal damage to teeth , gingival tissues and restorative and prosthetic materials . it is thus important to evaluate the abrasion resistance to brushing of heat - polymerized acrylic resins used for fabrication of denture bases . the acrylic resin hardness , the type of abrasive agent and its concentration , the dimension and form of abrasive particles , the toothbrush and the load applied are some of the different factors that influence the abrasion of acrylic resin by brushing4,7,12 . colgate and dentu - creme dentifrices use calcium carbonate as abrasive particles . in the present study , the results showed that colgate was significantly more abrasive than dentu - creme ( table 4 ) , as reported by freitas and paranhos ( 2006)10 . this previous study10 also showed , by microscopic analysis , that colgate 's abrasive particles presented an irregular spherical form , irregular size and heterogeneous distribution , while dentu - creme 's abrasive particles presented a regular form , small size and homogeneous distribution . this confirms the importance of the abrasive agent 's particles form , size and distribution on the abrasive capacity of dentifrices4,12 . many variables must be considered in the fabrication of dentifrice for cleansing and polishing : acrylic porosity , hardness , size and surface configuration of the cleansing agent , the type of compounds for polishing used along with the cleansing agent , and the hardness of the cleansing and polishing agent20 . some studies have demonstrated the importance of dentifrice abrasiveness on promoting an efficient cleansing because brushing with water alone does not remove stains and organic deposits from dentures13,14 . furthermore , low - abrasion dentifrices do not remove stains from smoker 's dentures either22 . a soft - bristle toothbrush was used in the present study because it is cheap and has good quality , being therefore accessible to most patients . brushing with distilled water caused minimum mass reduction , confirming the results from other studies28 . bonyplus has no abrasive particles and its results were statistically similar to the control group . each specimen was subjected to 35,600 brush strokes , and the load applied on each brush head was 200 g , which was estimated to be equivalent to 2 years of manual brushing28 . artificial brushing is vigorous and may be more abrasive than manual brushing19 , but some works have shown similar results between laboratory and clinical experiments20,21 . resin characteristics , such as monomer / polymer ratio , presence of cross - linking agents , mixture uniformity , polymerization cycle , cooling rate , specimen thickness and surface finishing may influence the interaction between specimens and dentifrices . mass reduction is approved by the iso / tc 106 ( 1996)17 specification as an abrasion indicator and it is the simplest method of producing values for abrasion of acrylic resins25 . due to the experimental conditions of this study sexson and phillips27 showed that mass reduction obtained from brushing rotating acrylic resin specimens was similar to that produced when specimens were allowed to remain stationary , although microscopic examination revealed that the rotating specimens had different surface topography . hence , surface roughness and brightness loss would not provide results valid for this study , in which the specimens remained stationary . during the brushing procedure , the specimens were kept immersed in dentifrice slurry for 100 min and some water sorption could occur . for this reason , the specimens were stored in water at 37c for 7 days before the test in order to balance water sorption17 . when submitted to cycles at higher temperature , heat - polymerized acrylic resins have been shown to produce specimens with higher abrasion resistance2528 . haselden , et al.12 verified that the effects of colgate and dentu - creme dentifrices differed according to the resins used and ranking order was unpredictable . in this study , hence , the ranking order was the same for all acrylic resin brands . qc-20 and lucitone 550 resins showed greater mass reduction as compared to the other resins when submitted to brushing associated to dentifrice ( table 5 ) . an important question concerns the clinical relevance of the abrasion produced by dentifrices on dentures . facq and volpe8 concluded that the abrasion of dentifrices on veneer crowns was clinically insignificant . murray , et al.22 stated that it is possible to estimate that twice daily brushing for 2 min with a dentifrice containing calcium carbonate would result in the removal of 25 m of resin surface per year . therefore , further studies similar to the present one are required to determine the impact of the wear produced by dentifrices on dentures . in vitro experiments are usually helpful to compare the relative effectiveness of denture cleansers and to develop an understanding of the acting mode of each denture cleanser23 . in addition , the present study provides more details concerning the performance of some acrylic resins under abrasive load . based on the obtained results and within the limitations of an in vitro study , it may be concluded that differences exist among heat - polymerized acrylic resins concerning abrasion resistance and that dentifrices specific for denture cleansing tend to cause less damage to acrylic resin surface .

the association between a toothbrush and a dentifrice is the most used denture cleaning method . the purpose of this study was to evaluate the abrasiveness of specific and non - specific denture cleaning dentifrices on different heat - polymerized acrylic resins . sixteen specimens ( 90x30x3 mm ) of each acrylic resin ( qc-20 , lucitone 550 , clssico , vipi - cril ) were prepared and randomly assigned to 4 groups : 1 : control ( distilled water ) , 2 : colgate , 3 : bonyplus and 4 : dentu - creme . the specimens were subjected to simulated toothbrushing in an automatic brushing machine using 35,600 brush strokes for each specimen . brushing abrasion run at a 200-g load with the specimens immersed in 2:1 dentifrice / water slurry . specimens were reconditioned to constant mass and the mass loss ( mg ) was evaluated . data were analyzed by 2-way anova and tukey 's test ( =0.05 ) . analysis of dentifrices ' abrasive particles was made by scanning electron microscopy . colgate produced the greatest mass reduction ( 42.44 mg , p<0.05 ) , followed by dentu - creme ( 33.60 mg ) . bonyplus was the less abrasive ( 19.91 mg ) , similar to the control group ( 19.69 mg ) ( p>0.05 ) . the mass loss values indicated that qc-20 ( 33.13 mg ) and lucitone 550 ( 33.05 mg ) resins were less ( p<0.05 ) resistant to abrasion than clssico ( 26.04 mg ) and vipi - cril ( 23.43 mg ) . in conclusion , colgate produced the greatest abrasion . specific dentifrices for dentures tend to cause less damage to acrylic resins .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

dkk1 is a 29 kda secreted protein belonging to the dickkopf ( dkk ) family , which comprises four main glycoproteins in vertebrates ( dkk1 - 4 ) . dkk1 has been identified as a potent inhibitor of the canonical wnt signaling due to its ability to bind to the wnt coreceptor lrp5/6 , thus blocking the canonical wnt/-catenin pathway . canonical wnt pathway activation is initiated by the direct binding of the wnt glycoprotein to frizzled ( fz ) membrane receptor and to the lrp5/6 coreceptor [ 46 ] . in the absence of wnt wnt - mediated assembly of the activated fz - lrp5/6 receptor complex is followed by the recruitment of the axin - gsk3 to the plasma membrane , resulting in the reduction of the phosphorylation and degradation of -catenin [ 9 , 10 ] . stabilized -catenin accumulates in the cytoplasm and translocates to the nucleus , where it interacts with dna - bound tcf - lef proteins and activates the transcription of target genes . the wnt pathway is involved in many stages of invertebrate and vertebrate development and in adult tissue homeostasis [ 8 , 12 ] . dysfunction within the wnt/-catenin signaling cascade has been associated with many human pathologies [ 8 , 13 ] , such as cancer [ 1417 ] and bone disease [ 18 , 19 ] . lrp5-activating mutations are mainly associated with high - bone mass , while loss - of - function mutations on lrp5 are linked to bone degeneration and osteoporosis [ 20 , 21 ] . the inhibition of wnt signaling by dkk1 has been related to bone degeneration processes and reduced bone mass . in the central nervous system , dkk1 has been associated with the pathophysiology of neuronal degeneration in alzheimer disease ( ad ) [ 2326 ] . dkk1 expression is increased in cortical neurons exposed to -amyloid peptide ( -ap ) , where its aberrant expression is responsible for hyperphosphorylation of tau in neurons challenged with -ap . moreover , increased dkk1 expression was also observed in degenerating neurons in brain samples of ad patients . dkk1 was shown to be neurotoxic when locally infused into brain regions where neurodegenerative processes associated with brain ischemia or ad normally take place . moreover , dkk1 is also associated with neuronal death in cellular and animal models of excitotoxic / ischemic neuronal death , and the treatment of ischemic animals with dkk1 antisense oligonucleotides protects hippocampal neurons against ischemic damage and cultured cortical neurons against nmda toxicity . for these reasons , the dkk1-lrp6 interaction can be considered as a potentially interesting therapeutic intervention point and dkk1 a potential drug target for the treatment of bone and neurodegenerative disorders . a small molecule ( nci8642 ) has been described as an inhibitor of the interaction between dkk1 and one of its receptors ( lrp5 ) , as well as an inhibitor of dkk1 activity in reducing wnt/-catenin signaling activation . in this study , we sought to further characterise nci8642 activity , using biochemical and biophysical approaches , and to extend its characterization in relation to lrp6 , given the prominent expression in brain of lrp6 and its relevance to the neurodegenerative diseases field . human embryonic kidney cells ( hek293 ) were obtained from the german collection of microorganisms and cell cultures . wnt3a all cell lines were cultured in dmem supplemented with fbs and glutamax ( gibco ) . the monoclonal antibody against -catenin was obtained from bd biosciences , the polyclonal goat anti - dkk1 antibody from r&d , while the fluorescently conjugated secondary antibodies were all from invitrogen . the dna encoding the full - length human lrp6 sequence was cloned into the pcdna3.1/zeo(+ ) expression vector ( invitrogen ) . the dna encoding the human dkk1 sequence was cloned into pcdna6.2/clumio - dest ( invitrogen ) . the sequence encoding the secreted alkaline phosphatase from clontech was amplified by pcr and inserted at the c - terminus of dkk1 sequence into the pcdna6.2/clumio - dest - dkk1 plasmid . the luciferase reporter plasmid p4tcf - luc comprises four copies of a tcf - responsive element upstream of a tata element luciferase coding sequence transcriptional unit . all cell lines were cultured in dmem supplied with 10% fbs , 2 mm glutamax , 100 units / ml penicillin , 100 g / ml streptomycin , and the appropriate selection antibiotic ( 50 and 100 g / ml zeocine for pc12-tcfluc and hek293-lrp6 , resp . , 10 g / ml blasticidine for hek293-dkk1 , 0.4 mg / ml g418 for wnt3a l - cells , 20 g / ml hek293-ap - dkk1 ) . for the generation of the stable cell lines , hek293 or pc12 cells were transfected with fugene 6 ( roche ) and subjected to antibiotic selection 24 hours after transfection . clones were selected using immunofluorescence ( hek293-dkk1 , hek293-lrp6 ) , ap enzymatic assay on the culture medium ( hek293-ap - dkk1 ) , or measurement of luciferase activity upon stimulation with wnt3a cm ( pc12-tcf - luc ) . for the production of dkk1 and ap - dkk1 conditioned media ( cm ) , hek293 cells stably expressing the appropriate construct were seeded into a 150-cm flasks at half confluence . the day after media were collected 96 h after seeding , sterile filtered , and stored at 20c in aliquots . the control conditioned optimem was obtained in the same way using the parental hek293 cells . for the functional studies ( -catenin translocation and tcf - luc reporter assay ) , wnt3a cm was used at a 1 : 2 dilution . each batch of dkk1 cm was functionally titrated in order to determine the volume of cm that gives about 50% inhibition of wnt signaling . from dot blot analysis ( data not shown ) , the concentration of dkk1 in the dkk1 cm was estimated to be about 1 g / ml . for the quantitative ap - dkk1 binding assay , ap - dkk1 cm was diluted to obtain a phosphatase activity of 20 mu / ml . for the immunofluorescence detection of cell - bound dkk1 , lrp6 - 293 cells were seeded onto poly - d - lysine - coated coverslips 24 hours before the experiment and incubated with dkk1 or ap - dkk1 cm diluted in cell culture medium for 2 hours at 4c . when indicated , 100 m nci8642 ( or 1% dmso ) was added alongside dkk1 cm . after washing , the cells were fixed in 3% paraformaldehyde ( 15 min , room temperature ) , blocked with pbs/0.1% bsa , and incubated with the primary goat - anti - dkk1 antibody , and fluorescent secondary antibody diluted into blocking solution . images were collected using a zeiss lsm 510 meta confocal microscope . for the quantitative ap - dkk1 binding assay , the ap activity in the cell lysate was measured with attophos ap fluorescent substrate , according to the manufacturer 's instructions . spr was performed using a biacore t100 ( ge healthcare ) . where not specified , experiments were conducted at 25c . kinetic constants were calculated by nonlinear fitting to the association and dissociation curves according to the manufacturer 's instructions . apparent equilibrium dissociation constants ( kd ) were then calculated as the ratio of kd / ka . all immobilization steps were performed at a flow rate of 5 l / min using hbs - ep+ buffer ( 10 mm hepes buffer , ph 7.4 , containing 0.15 m nacl , 3 mm edta , and 0.05% ( v / v ) p20 surfactant ) . full - length human lrp6-fc tagged ( r&d systems ) was immobilized via amine coupling on a flow cell of a cm3 sensor chip . 3000 resonance units ( rus ) were measured at the end of the immobilization procedure . for the indirect capture of lrp6 , protein a was covalently coupled via amine immobilization on cm3 chip . lrp6-fc capture on protein a coated surface was performed by injecting the protein for 180 sec at the concentration of 0.5 g / ml in hbs - ep+ with a flow rate of 10 l / min . control sensorgrams , obtained on an empty flow cell where the coupling reaction had been conducted in the presence of coupling buffer alone , were always subtracted from binding responses . this allowed subtraction of nonspecific binding , which might be generated by residual charged negative groups , which had not been neutralized by the coupling procedure . bovine serum albumine ( bsa ) was immobilized via standard amine coupling procedures on a flow cell of a cm3 sensor chip to obtain the nonrelated control flow cell . for binding and kinetic assays , dkk1 ( r&d systems ) , diluted in hbs - ep+ at concentration ranging from 3 to 54 nm , was injected for 180 sec at the flow rate of 20 l / min over lrp6 either directly immobilized or captured via protein a as described before . for nci8642 binding and kinetic assays , the compound was diluted in hbs - ep+ buffer at concentration ranging from 3 to 50 m and injected over lrp6 surface for 60 sec at the flow rate of 30 l / min . 0.2% dmso was added to hbs - ep+ when performing the experiments with nci8642 compound , and dmso correction module of the software was used to compensate for refractive index change induced by dmso . for inhibition analysis , dkk1 ( 54 nm , diluted in hbs - ep+ buffer ph 7.4 ) was incubated with nci8642 at concentration from 5 to 20 m . after 1 hour at 25c , dkk1 was injected over immobilized lrp6 for 240 s at the flow rate of 10 l / min . pc12-tcf - luc cells were seeded at the density of 100,000 cells / well into black - walled clear - bottom 96-well plates . after 24 hours , culture medium was replaced with 50 l dkk1 cm diluted as appropriate in optimem . after one - hour incubation ( 37c ) , the cells were stimulated by adding 50 l of wnt3a cm ( 50% of the final volume ) and supplemented with either dmso or nci8642 at the indicated final concentration ( final dmso concentration 1% ) . after 24 hours of incubation , the luciferase activity was measured with steady lite substrate ( perkin - elmer ) , according to the manufacturer 's instructions . for each condition 3 , l - cells were seeded into black - walled clear - bottom 96-well plates at a density of 10,000 cells / well . after 24 hours , culture medium was replaced with 50 l dkk1 cm diluted as appropriate in optimem . after one - hour incubation ( 37c ) , the cells were stimulated by adding 50 l wnt3a cm supplemented with either dmso or nci8642 at the indicated final concentration ( final dmso concentration 1% ) . after two - hour stimulation ( 37c ) , the cells were fixed in 3% paraformaldehyde ( 10 min , room temperature ) , permeabilized ( 0.2% triton x-100 in pbs , 10 min , room temperature ) , and blocked with 0.1% bsa in pbs . the cells were then incubated for 2 hours at room temperature with the primary anti--catenin antibody ( 1 : 500 ) , followed by the secondary alexafluor 555 goat - anti - mouse antibody ( 1 : 1000 ) . the ratio of the nuclear and cytoplasmic intensity of -catenin staining was calculated for each cell and averaged within the well . nci8642 was originally characterized as an inhibitor of the lrp5/dkk1 interaction . in order to determine if nci8642 was also able to disrupt the binding of dkk1 to lrp6 , a recombinant hek293 cell line stably expressing lrp6 was generated ( hek293-lrp6 ) . an additional recombinant hek293 cell line was established for the production of human recombinant dkk1 fused to the v5 tag at the c - terminus ( hek293-dkk1 ) . when conditioned medium ( cm ) containing dkk1 was incubated on cells expressing lrp6 and the cell - bound dkk1 was detected by immunofluorescence , a strong membrane signal was observed in hek293-lrp6 cells ( figure 1(a ) , panel b ) , while it was almost absent on the parental hek293 cells ( figure 1(a ) , panel a ) . when dkk1 cm was supplemented with 100 m nci8642 , the binding of dkk1 ( figure 1(a ) , panel d ) was reduced compared to the dmso - treated control ( figure 1(a ) , panel c ) . in order to obtain a quantitative measure of the dkk1/lrp6 interaction , a construct coding for human dkk1 fused to a secreted domain of alkaline phosphatase was generated ( ap - dkk1 ) and used to establish a stable hek293 cell line secreting ap - dkk1 in the culture medium . the ap tag at the c - terminus does not impair dkk1 binding to lrp6 , as shown by immunofluorescence of cell - bound ap - dkk1 on hek293-lrp6 ( figure 1(a ) , panels e and f ) . the binding of ap - dkk1 to hek293-lrp6 , quantified by enzymatic ap assay , was reduced when ap - dkk1 was incubated in the presence of increasing concentrations of nci8642 . the binding of dkk1 was reduced in a concentration - dependent manner compared to dmso sample ( figure 1(b ) ) . the calculated ic50 was 14.6 m ( pic50 = 4.8 , stdev 0.2 , n = 10 independent experiments ) . when flowed over the surface of the sensor chip coated with recombinant lrp6-fc , dkk1 bound to immobilized receptor both specifically and in a concentration - dependent manner ( figure 2(a ) , kon 5.3 10 m s , koff 1.8 10 s ) . indirect capturing of lrp6-fc on recombinant protein a and direct lrp6-fc covalent coupling via amine groups produced similar interaction with recombinant human dkk1 ( kd of 8.4 and 3.5 10 m , resp . ) , demonstrating that the covalent coupling of the receptor to the chip surface does not influence its binding site . the interaction constants obtained are in agreement with the literature data [ 1 , 34 , 35 ] . the lrp6-dkk1 interaction was also verified using recombinant dkk1 cm instead of commercial recombinant dkk1 ( figure 2(b ) ) . when we investigated whether nci8642 was able to bind to the lrp6 receptor , we observed a consistent and effective binding signal that is not present in the control flow cell . the kinetic analysis of the interaction by injection of an nci8642 dilution series ( from 3 m to 50 m ; figure 2(c ) ) allowed the determination of a kd of 4.7 10 m for nci8642 on lrp6 . we also analyzed the effect of nci8642 on the binding of dkk1 to lrp6 , to determine whether the compound could interfere with the interaction between dkk1 and lrp6 . we injected dkk1 and nci8642 over lrp6 previously immobilized . when dkk1 was injected together with nci8642 after 1-hour incubation at 25c , we observed a consistent reduction of dkk1 binding on lrp6 with 5 m and 10 m compound , and even a complete binding inhibition at the concentration of 20 m ( figures 2(d ) and 2(e)-upper panel ) . on the contrary , sequential injections of 20 m nci8642 and 54 nm dkk1 did not inhibit the dkk1-lrp6 interaction . nci8642 was not able to displace dkk1 when injected after it and dkk1 could still bind to lrp6 when injected after nci8642 at the concentration of 20 m ( figure 2(e)-middle and lower panels ) . to investigate the functional activity of nci8642 , we tested the compound in a tcf reporter - based assay in pc12 cells ( neural crest - derived rat pheochromocytoma cells ) . tcf , similarly to lef-1 , is a downstream target transcription factor of the canonical wnt - signaling pathway . a stable pc12 cell line expressing a tcf - dependent luciferase reporter was established ( pc12-tcf - luc ) . wnt3a - mediated response was reverted by dkk1 cm in concentration - dependent manner ( figure 3(a ) ) . for the purpose of compound testing , the dilution factor of dkk1 cm was chosen in order to have about 50% inhibition of wnt signaling ( figure 3(b ) , open symbols ) . in the presence of exogenously added dkk1 the effect was concentration - dependent and was reproducibly and significantly different from dmso treatment at 25 m and 50 m , although higher concentrations of the compound were toxic to the cells and an ic50 value could not , therefore , be calculated . nci8642 was also tested in an assay measuring the modulation of -catenin intracellular accumulation and nuclear translocation . the assay was performed in l - cells , an established model system for studying wnt signaling . upon treatment of l - cells with wnt3a cm , the intracellular amount of -catenin increases , with an enrichment in the nuclear compartment and a concomitant increase in the nuclear / cytoplasmic ratio of -catenin intensity . in the sample treated with control cm , -catenin is hardly detectable , with a nuclear / cytoplasmic ratio close to one . the wnt3a - mediated effect is reverted by dkk1 cm in a concentration - dependent manner ( figures 4(a ) and 4(b ) ) . when nci8642 was added to the cells together with dkk1 , and then the cells were stimulated with wnt3a the measured ic50 of the compound is 12.6 m ( pic50 = 4.9 stdev 0.2 , n = 5 independent measurements ) , in good agreement with the binding data and the reporter assay ( figures 5(a ) and 5(b ) ) . nci8642 is a small molecule that has been recently identified as a specific inhibitor of lrp5-dkk1 interaction . given the role of the wnt signaling in many pathophysiological contexts , modulation of the wnt pathway has become a valuable target in drug discovery . the modulation of the pathway at the level of the cell surface poses the problem of targeting a protein - protein interaction , which might be challenging . recently , the lrp5-dkk1 interaction has been targeted with monoclonal anti - dkk1 antibodies [ 39 , 40 ] . these antibodies were effective in increasing bone density in vivo , both in nave normal growing female mice and in a model of postmenopausal osteoporosis . however , the lack of blood - brain penetration of the antibodies limits their application to peripheral indications such as osteoporosis . on the contrary , a small molecule blocking dkk1 inhibition would have the advantage of being potentially useful also for the treatment of cns pathologies , such as neurodegeneration . our study confirmed that nci8642 acts as a dkk1 inhibitor , and we extended the finding to the interaction of dkk1 to lrp6 . in fact , the small molecule caused the displacement of dkk1 from lrp6 overexpressed in hek293 cells , as shown both by immunofluorescence and quantitative enzymatic assay . the displacement was also demonstrated in a cell - free context by using spr technology , where nci8642 blocks the binding of dkk1 to the immobilized lrp6 . interestingly , the compound was able to block dkk1 binding only when injected together with dkk1 , and not when injected sequentially after dkk1 , which is probably due to the high affinity of dkk1 for lrp6 compared with the micromolar affinity of nci8642 for lpr6 ( figures 2(c ) and 2(e ) ) . spr technology also showed that nci8642 interacts with the immobilized lrp6 with a reproducible and concentration - dependent binding in the absence of dkk1 ( kd 4.7 10 m ) . the interaction between nci8642-lrp6 is highly specific , because no significant binding was observed on two control flow cells . when immobilized on the sensor chip , dkk1 lost the ability to bind lrp6 , suggesting that the immobilization caused a modification of dkk1 structure - function , thus the direct interaction of nci8642 to dkk1 was not measured . nci8642 is also functionally active in the inhibition of dkk1 activity on wnt signaling mediated by lrp5/6 as demonstrated by the tcf - luc and -catenin assays in two different cellular backgrounds . the compound ic50 measured in the tcf - luc and -catenin assays , where lrp5/6 is expressed at endogenous , physiological levels , is comparable with the one obtained in the binding assay on the overexpressed lrp6 . in the tcf - luc assay , we observed a slight increase of the wnt3a - mediated activation of the reporter gene , even in the absence of dkk1 . the hypothesis that it could be due to the presence of endogenous dkk1 in these cells was not confirmed , since we could not detect it by western blot or by quantitative rt - pcr ( data not shown ) . nevertheless , this was not observed in the -catenin translocation assay ( data not shown ) , suggesting that it could be an artifact of the reporter assay , or a cell - line - specific effect . although nci8642 has been reported as a dkk1/lrp5 inhibitor , our data suggest that it is also effective on dkk1/lrp6 interaction . in fact , nci8642 blocked dkk1 binding on lrp6 , both in lrp6 overexpressing cells and in spr analysis . this was also confirmed by functional studies , since only lrp6 , but not lrp5 , is expressed in pc12-tcf - luc cell line , and lrp6 mrna concentration is about 7 times that of lrp5 in l - cells , as assessed by qpcr ( data not shown ) . our results suggest that it is possible to antagonize the dkk1-lrp6 interaction with a small molecule , and this leads to an enhancement of the wnt signaling pathway . although nci8642 micromolar potency might be too low for its application in in vivo experiments , this compound can be considered as a reference small molecule for further drug development aiming at the inhibition of this therapeutically important protein - protein interaction .

background . dkk1 antagonizes canonical wnt signalling through high - affinity binding to lrp5/6 , an essential component of the wnt receptor complex responsible for mediating downstream canonical wnt signalling . dkk1 overexpression is known for its pathological implications in osteoporosis , cancer , and neurodegeneration , suggesting the interaction with lrp5/6 as a potential therapeutic target . results . we show that the small - molecule nci8642 can efficiently displace dkk1 from lrp6 and block dkk1 inhibitory activity on canonical wnt signalling , as shown in binding and cellular assays , respectively . we further characterize nci8642 binding activity on lrp6 by surface plasmon resonance ( spr ) technology . conclusions . this study demonstrates that the dkk1-lrp6 interaction can be the target of small molecules and unlocks the possibility of new therapeutic tools for diseases associated with dkk1 dysregulation .

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

it typically improves in the weeks and months after stroke , yet about 50% of patients are left with long - term residual deficits . few studies have documented the possibility of treatment - induced improvements in post - stroke language functions after 12 months . traditionally , aphasia interventions utilize compensatory communication strategies to assist the participant in immediate communication needs . multi - modal strategies include gesturing , writing , drawing , and augmentative low- and high - technology systems , with a common expectation that use of the alternative communication techniques will decrease naturally as the language capabilities increase . these interventions improve overall communication abilities , but questions have been asked whether they contribute to the recovery of language function , or whether they actually contribute to a learned non - use phenomenon . to counteract the possibility of learned non - use , therapies utilizing restraint have been developed to mirror constraint - induced motor therapies . while it is relatively easy to restrain an unaffected extremity in motor therapies , restraining attempts to communicate non - verbally can be more difficult . constraint - induced aphasia therapy ( ciat ) encourages intensive verbal practice with supported verbal cuing while excluding the use of previously habitual compensatory strategies . in ciat , the theoretical model of taub s motor system for use - dependent cortical re - organization has been applied to a language - based program . in this model , it has been postulated that the behavior of attempting to speak without success leads to communication frustration . this then results in fewer speaking attempts , more reliance on compensatory strategies , and less cortical stimulation in the language areas . the ciat framework provides a structured supportive environment , with clinician guidance and shaping , positive reinforcement from group members , and social interaction opportunities . the theory is that the supportive environment and speaking opportunities will encourage more verbal attempts and stimulate cortical reorganization . thus , the goal of the present study was to provide evidence for the potential efficacy of ciat when compared to no - intervention in patients with chronic ( > 1 year ) post - stroke aphasia . more specifically , this randomized , controlled , blinded pilot study was conducted in order to estimate effect sizes , allowing the design of an appropriately powered trial . subjects were recruited into this institutional review board - approved study by word of mouth from among the stroke and aphasia clinics at the university of cincinnati and university of alabama at birmingham , and from local aphasia support groups . we also listed the study on www.clinicaltrials.gov ( registered nct00843427 ; pi : szaflarski ) , and several contacts were received directly from patients . after providing consent for screening , the inclusion criteria were chronic aphasia related to a single ischemic stroke in the left middle cerebral artery ( lmca ) distribution ( i.e. , diagnosis of a single lmca stroke was confirmed by medical record review including admission notes for the incident stroke and the results of brain imaging obtained prior to enrollment ) , token test in an impaired range ( score 40 ) , and pre - stroke fluency in english . the exclusion criteria were history of degenerative ( e.g. , dementia or parkinson s disease ) or metabolic disorder ( e.g. , encephalopathy ) or supervening illness ( e.g. , brain tumor or other cancer ) , history of severe depression or other mental illness , and positive pregnancy test in women of childbearing age . five potential participants were excluded after interview . the format and the goals of the ciat program all patients indicated their understanding of the goals of the program prior to signing the informed consent ; they also understood that they may be randomized to a no - intervention group , and that the follow - up testing will need to be performed . of the 27 who were offered participation , 3 were excluded before randomization : 2 had a normal token test and 1 was hospitalized for reasons unrelated to the study . fourteen subjects were randomized to receive ciat ( 34 participants per group ; 4 groups were assembled ) and 10 to receive no - intervention ( figure 1 ) . demographic and clinical data of the participants are provided in table 1 . after obtaining the informed consent , we initially screened the patients for the presence of aphasia with token test ( tt ) and categorized the severity of aphasia as mild ( tt=40 - 37 ) , moderate ( tt=36 - 17 ) or severe ( tt=16 - 0 ) . responders were defined as patients with at least a 20% relative improvement in at least 2 of the 5 primary scores in the 2-week period . retainers were those patients whose 12-week score was not less than the 2-week score on at least 2 of the 5 outcome measures . the 2-week ciat protocol was a closely monitored , individualized program embedded within a larger group activity . to ensure consistency of the intervention training ( approximately 46 hours ) was conducted by alb prior to initiating any intervention session including basic theory of learned non - use , and the procedures for the treatment . clinicians also viewed sample videos that exemplified the nature and set - up of the ciat intervention , and they were taught a hierarchy of cues that ranged from most to least supportive ( e.g. , imitation to verbal reminder ) . during the first day of intervention , clinicians had no information on participants abilities as , by design , they did not participate in the initial evaluations . the measures were administered by coordinators who were blinded to group assignment ( figure 1 ) . to enhance the chance for successful implementation of cues during the ciat sessions , clinicians maintained a cuing tracking form that described the interaction between another clinician and their participant partners . the types of cues provided were tracked with a binary notation of whether the cuing resulted in a successful communication . this technique of behaviorally monitoring another clinician rather than self - monitoring was established in our previous study . clinicians and participants were rotated during each day s session in order to provide a balance in communication partners . at the end of day 1 , alb studied the cuing tracking forms and the videos to create an individual treatment plan for the next day s session . the treatment plan included identifying individual baseline linguistic strengths , suggested hierarchy of most beneficial cues , behaviors to constrain , and specific linguistic targets or goals . the treatment plan was reviewed with the clinicians before the second day of therapy . from the second day onwards , clinicians promoted individualized support with cues at a linguistic level suitable for each person and continued data tracking . each day , clinicians reviewed the individual program and levels and made adjustments as needed . at the beginning of each session day , the clinicians reminded each participant of the goals and constraints that had been established for them . for example , a participant who was strong in producing nouns , but limited in verbs , had a goal to add a verb to create a 2-word phrase . if a participant demonstrated a milder aphasia , then the d - level hierarchy of sentences was implemented . the treatment program for each of the 34 participant groups lasted 2 weeks , with direct therapy for about 4 hours per day for 10 consecutive weekdays . the sessions were organized into 4 45-minute periods , with a 1015 minute break in between each session . socialization between clients and clinicians was encouraged throughout the program , even during the break periods . at the beginning of each session , participants were dealt cards and they were instructed to play a go - fish type game . the cards provided the opportunity for the participants to interact with each other during an ongoing game that engaged participants visual , attention , and memory skills , although these were not specifically targeted . the cards provided visual stimuli of line drawings of nouns ( singular , plural to elicit numbers , and with colors ) and photos of action verbs . clinicians were encouraged to use redirecting phrases such as try again and are you sure ? rather than using negative responses . if the other participants responded positively and the communicative exchange was successful , clinicians were instructed not to correct ; communicative success is more important than the sentence accuracy . whenever clinicians were observed to manage cards for the participant , this was discouraged ; clinicians were trained to be supportive while encouraging independence . consistent with the current standards of care , the no - intervention group did not receive any specific treatment and the participants were asked to continue all previous activities as usual . all participants were asked not to take part in any other intervention during their involvement in the study and all complied . patients were randomized by the study statistician ( cjl ) after the patients received all pre - requisite activities ( consenting , clinical record review , neuropsychological aphasia testing ( nat ) ) . patients were assigned to receive either 2 weeks of ciat , or no - intervention , and then to undergo nat within one week and 3 months of ciat completion ( figure 2 consort diagram ) . we used a simple scheme that randomized each block of patients to either ciat or control . randomization occurred after consent , and with the statistician blinded to participant performance on screening and baseline testing . we did not replace subjects who do not complete the full 2 weeks of therapy . after randomization , sealed study charts containing all pre - intervention testing results ( nat ) were funneled through the study biostatistician ( cjl ) to the therapists . therapists set up the intervention groups so that the coordinators ( cb , anm ) who collected all nat data throughout the study remained blinded to group assignment . participants were asked not to reveal their group assignment to coordinators during the post - treatment interactions . all participants received nat which included : ( 1 ) the boston naming test ( bnt ) , ( 2 ) the controlled oral word association test , ( 3 ) the semantic fluency test ( sft ) , ( 4 ) the complex ideation subtest from the boston diagnostic aphasia examination ( bdae ) , ( 5 ) the peabody picture vocabulary test iii ( ppvt iii ) , and ( 6 ) the mini - communicative activities log ( mini - cal ) which is a subjective measure of communicative abilities . the study coordinators , who were extensively trained in the use of these measures and blinded to group assignment , administered the nat measures within 1 week prior to ciat and again during both the first week the twelfth week following ciat completion . all data were entered into redcap ( research electronic data capture ) for subsequent analysis . one patient did not complete the 12 week visit or testing , and 7 additional patients were missing at least 1 nat score . the primary analysis used independent samples t - tests to examine the differences in nat scores at each time point between the intervention and control groups , and effect sizes and 95% confidence intervals were calculated . the secondary analysis was an ad hoc analysis comparing the characteristics of patients who demonstrated a response or change in nat scores to patients who did not . independent t - tests , the mann - whitney u test , and fisher s exact test were used to compare patient characteristics between patients who demonstrated a response or change and patients who did not . all statistical analyses were conducted using spss 22.0 ( ibm corporation , armonk , ny ) and r 2.15.3 ( base package ) . subjects were recruited into this institutional review board - approved study by word of mouth from among the stroke and aphasia clinics at the university of cincinnati and university of alabama at birmingham , and from local aphasia support groups . we also listed the study on www.clinicaltrials.gov ( registered nct00843427 ; pi : szaflarski ) , and several contacts were received directly from patients . after providing consent for screening , the inclusion criteria were chronic aphasia related to a single ischemic stroke in the left middle cerebral artery ( lmca ) distribution ( i.e. , diagnosis of a single lmca stroke was confirmed by medical record review including admission notes for the incident stroke and the results of brain imaging obtained prior to enrollment ) , token test in an impaired range ( score 40 ) , and pre - stroke fluency in english . the exclusion criteria were history of degenerative ( e.g. , dementia or parkinson s disease ) or metabolic disorder ( e.g. , encephalopathy ) or supervening illness ( e.g. , brain tumor or other cancer ) , history of severe depression or other mental illness , and positive pregnancy test in women of childbearing age . five potential participants were excluded after interview . the format and the goals of the ciat program all patients indicated their understanding of the goals of the program prior to signing the informed consent ; they also understood that they may be randomized to a no - intervention group , and that the follow - up testing will need to be performed . of the 27 who were offered participation , 3 were excluded before randomization : 2 had a normal token test and 1 was hospitalized for reasons unrelated to the study . fourteen subjects were randomized to receive ciat ( 34 participants per group ; 4 groups were assembled ) and 10 to receive no - intervention ( figure 1 ) . after obtaining the informed consent , we initially screened the patients for the presence of aphasia with token test ( tt ) and categorized the severity of aphasia as mild ( tt=40 - 37 ) , moderate ( tt=36 - 17 ) or severe ( tt=16 - 0 ) . responders were defined as patients with at least a 20% relative improvement in at least 2 of the 5 primary scores in the 2-week period . retainers were those patients whose 12-week score was not less than the 2-week score on at least 2 of the 5 outcome measures . the 2-week ciat protocol was a closely monitored , individualized program embedded within a larger group activity . to ensure consistency of the intervention , all clinicians completed a training program . training ( approximately 46 hours ) was conducted by alb prior to initiating any intervention session including basic theory of learned non - use , and the procedures for the treatment . clinicians also viewed sample videos that exemplified the nature and set - up of the ciat intervention , and they were taught a hierarchy of cues that ranged from most to least supportive ( e.g. , imitation to verbal reminder ) . during the first day of intervention , clinicians had no information on participants abilities as , by design , they did not participate in the initial evaluations . the measures were administered by coordinators who were blinded to group assignment ( figure 1 ) . to enhance the chance for successful implementation of cues during the ciat sessions , clinicians maintained a cuing tracking form that described the interaction between another clinician and their participant partners . the types of cues provided were tracked with a binary notation of whether the cuing resulted in a successful communication . this technique of behaviorally monitoring another clinician rather than self - monitoring was established in our previous study . clinicians and participants were rotated during each day s session in order to provide a balance in communication partners . at the end of day 1 , alb studied the cuing tracking forms and the videos to create an individual treatment plan for the next day s session . the treatment plan included identifying individual baseline linguistic strengths , suggested hierarchy of most beneficial cues , behaviors to constrain , and specific linguistic targets or goals . the treatment plan was reviewed with the clinicians before the second day of therapy . from the second day onwards , clinicians promoted individualized support with cues at a linguistic level suitable for each person and continued data tracking . the tracking provided constant reminders to only provide cues that resulted in successful communication . each day , clinicians reviewed the individual program and levels and made adjustments as needed . at the beginning of each session day , the clinicians reminded each participant of the goals and constraints that had been established for them . for example , a participant who was strong in producing nouns , but limited in verbs , had a goal to add a verb to create a 2-word phrase . if a participant demonstrated a milder aphasia , then the d - level hierarchy of sentences was implemented . the treatment program for each of the 34 participant groups lasted 2 weeks , with direct therapy for about 4 hours per day for 10 consecutive weekdays . the sessions were organized into 4 45-minute periods , with a 1015 minute break in between each session . socialization between clients and clinicians was encouraged throughout the program , even during the break periods . at the beginning of each session , participants were dealt cards and they were instructed to play a go - fish type game . the cards provided the opportunity for the participants to interact with each other during an ongoing game that engaged participants visual , attention , and memory skills , although these were not specifically targeted . the cards provided visual stimuli of line drawings of nouns ( singular , plural to elicit numbers , and with colors ) and photos of action verbs . clinicians were encouraged to use redirecting phrases such as try again and are you sure ? rather than using negative responses . if the other participants responded positively and the communicative exchange was successful , clinicians were instructed not to correct ; communicative success is more important than the sentence accuracy . whenever clinicians were observed to manage cards for the participant , this consistent with the current standards of care , the no - intervention group did not receive any specific treatment and the participants were asked to continue all previous activities as usual . all participants were asked not to take part in any other intervention during their involvement in the study and all complied . patients were randomized by the study statistician ( cjl ) after the patients received all pre - requisite activities ( consenting , clinical record review , neuropsychological aphasia testing ( nat ) ) . patients were assigned to receive either 2 weeks of ciat , or no - intervention , and then to undergo nat within one week and 3 months of ciat completion ( figure 2 consort diagram ) . we used a simple scheme that randomized each block of patients to either ciat or control . randomization occurred after consent , and with the statistician blinded to participant performance on screening and baseline testing . we did not replace subjects who do not complete the full 2 weeks of therapy . after randomization , sealed study charts containing all pre - intervention testing results ( nat ) were funneled through the study biostatistician ( cjl ) to the therapists . therapists set up the intervention groups so that the coordinators ( cb , anm ) who collected all nat data throughout the study remained blinded to group assignment . participants were asked not to reveal their group assignment to coordinators during the post - treatment interactions . all participants received nat which included : ( 1 ) the boston naming test ( bnt ) , ( 2 ) the controlled oral word association test , ( 3 ) the semantic fluency test ( sft ) , ( 4 ) the complex ideation subtest from the boston diagnostic aphasia examination ( bdae ) , ( 5 ) the peabody picture vocabulary test iii ( ppvt iii ) , and ( 6 ) the mini - communicative activities log ( mini - cal ) which is a subjective measure of communicative abilities . the study coordinators , who were extensively trained in the use of these measures and blinded to group assignment , administered the nat measures within 1 week prior to ciat and again during both the first week the twelfth week following ciat completion . all data were entered into redcap ( research electronic data capture ) for subsequent analysis . one patient did not complete the 12 week visit or testing , and 7 additional patients were missing at least 1 nat score . missing scores were left missing , and are excluded from analysis . the primary analysis used independent samples t - tests to examine the differences in nat scores at each time point between the intervention and control groups , and effect sizes and 95% confidence intervals the secondary analysis was an ad hoc analysis comparing the characteristics of patients who demonstrated a response or change in nat scores to patients who did not . independent t - tests , the mann - whitney u test , and fisher s exact test were used to compare patient characteristics between patients who demonstrated a response or change and patients who did not . all statistical analyses were conducted using spss 22.0 ( ibm corporation , armonk , ny ) and r 2.15.3 ( base package ) . there were 24 patients enrolled in the study , 14 in the intervention group , and 10 in the control group ( see consort diagram ) . two patients in the intervention group did not complete the study , 1 due to lack of transportation and 1 due to hospitalization with an illness unrelated to the study . overall , there were no significant differences in demographic characteristics and past medical history between the 2 groups ( table 1 ) . the mean age was 57 ( sd11 ) years in the intervention group and 51 ( sd13 ) years in the control group . most patients were caucasian , 10/14 ( 71% ) in the intervention group and 9/10 ( 90% ) in the control group . there were few statistically significant differences in nat scores between the intervention and control groups ( table 2 ) . specifically , differences were observed in subjective communication abilities assessed with the mini - cal . patients who received ciat scored higher on the mini - cal at 12 weeks after intervention compared to the control group ( mean 31 vs. 23 ; difference=8 , 95% ci 1.3 to 13.5 , p=0.019 ) . the sft was marginally higher 2 weeks after intervention in ciat patients than controls ( mean 21 vs. 12 ; difference 9 , 95% ci 0.3 to 17.8 , p=0.058 ) . overall , 5/24 ( 21% ) of participants could be classified as a responders , as defined in the methods section . responders were less likely to have a history of hypertension 0/5 ( 0% ) , compared to non - responders 10/14 ( 53% ) , p=0.053 . of the 5 patients who demonstrated a change in nat scores during the course of the study , only 1 ( 20% ) retained the change at the twelve - week visit . there were 24 patients enrolled in the study , 14 in the intervention group , and 10 in the control group ( see consort diagram ) . two patients in the intervention group did not complete the study , 1 due to lack of transportation and 1 due to hospitalization with an illness unrelated to the study . overall , there were no significant differences in demographic characteristics and past medical history between the 2 groups ( table 1 ) . the mean age was 57 ( sd11 ) years in the intervention group and 51 ( sd13 ) years in the control group . most patients were caucasian , 10/14 ( 71% ) in the intervention group and 9/10 ( 90% ) in the control group . there were few statistically significant differences in nat scores between the intervention and control groups ( table 2 ) . specifically , differences were observed in subjective communication abilities assessed with the mini - cal . patients who received ciat scored higher on the mini - cal at 12 weeks after intervention compared to the control group ( mean 31 vs. 23 ; difference=8 , 95% ci 1.3 to 13.5 , p=0.019 ) . the sft was marginally higher 2 weeks after intervention in ciat patients than controls ( mean 21 vs. 12 ; difference 9 , 95% ci 0.3 to 17.8 , p=0.058 ) . responders were less likely to have a history of hypertension 0/5 ( 0% ) , compared to non - responders 10/14 ( 53% ) , p=0.053 . of the 5 patients who demonstrated a change in nat scores during the course of the study , only 1 ( 20% ) retained the change at the twelve - week visit . in this prospective , preliminary , randomized , blinded study of patients with chronic post - stroke aphasia , we estimated the effect of ciat on linguistic performance . while the results are largely not statistically significant , a few points need to be discussed . first , the study included patients with highly variable levels of post - stroke aphasia , making the comparisons between the groups somewhat difficult . second , the observed effects are consistent with improvement in the ciat group beyond that in the control group , with trends towards statistical significance for some of the variables even in this small sample . defining a responder as a participant with 20% improvement on at least 2 of the 5 tests , then the number needed to show significant between - group differences would be 62 and allows for the design of a more comprehensive trial . finally , a significant improvement was noted in the mini - cal results for the ciat group compared to controls . this indicates that , at least subjectively , the patients in ciat group perceived more improvement over time than participants in the control group . reasons for the participants perception of improvement , as measured by the mini - cal , may be related to the social activity of the therapy sessions . in order to isolate the variable of social interaction from ciat theory , future studies should consider including a study arm involving exposure to social interaction with the same duration and frequency as the ciat group , but without the ciat intervention . social factors may be related to both clinicians and other participants in the group . to date , few reports on the potential efficacy of ciat have been published . five studies in patients with chronic aphasia have included both a treatment group and a control group [ 8,2225 ] . only in the original study were participants randomized to treatment group ; however , unlike the current study , the personnel collecting linguistic data were not blinded to treatment assignment in any of these studies . in the original study , in the other 4 studies ciat was compared to other treatment approaches either model - based aphasia therapy ( training based on functional deficit ) , ciat plus written module , pace , or model - oriented aphasia therapy ( moat ) . in addition to the above studies , a recent study compared modified ciat to conventional aphasia therapy in patients with less than 4 months ( subacute ) since the incident ischemic or hemorrhagic stroke . all these studies showed that while participants receiving treatment improved linguistic performance , between - group differences were minimal , if any . thus , when comparing the results of the original study by pulvermuller et al . and the results of the subsequent studies , including ours , the questions of training intensity and social interactions between participants , and participants and clinicians need to be considered ; both can contribute to improved communicative abilities via increased verbal communication practice . we did not show any statistically significant improvement in objective tests of linguistic performance from before to after treatment . one of the reasons for this lack of statistical significance in our small sample size is the high variability in language scores observed in both groups ( figure 3 ) . a future study enrolling a highly - variable group of participants would require a sample size of only 62 per treatment group to show a significant advantage of ciat over passive observation . but , the effects observed by us are in line with the results of the original study by pulvermuller et al . we identified important considerations for study design that will minimize unnecessary variability and maximize the potential for observing treatment benefit . treatment fidelity needs to be monitored , either via independent reviewers performing video or by direct review of the conducted sessions . a transfer package similar to that offered in motor rehabilitation studies will need to be developed and applied to this group . also , the outcome measures should include measures of pre- and post - intervention discourse and assess changes in the severity of aphasia using , e.g. , the western aphasia battery - revised ; assessing linguistic complexity using mean length of utterances ; or indices of syntactic form . in addition to the above , stratification by severity , education , and socioeconomic status might emphasize the magnitude of improvement relative to the degree of aphasia . finally , other factors , including the effects of lesion size and location on changes in aphasia diagnosis ( type ) during the therapy session , should be assessed . modest improvements were noted in the ciat group on the collected objective tests that were not noted in the control group , forming the basis for estimating effect sizes needed for a comprehensive study . moreover , the ciat group reported significant subjective improvement that confirms the importance of this approach . larger randomized controlled trials that adopt the measures suggested to reduce variability and maximize observable benefit are warranted .

backgroundfew studies have documented the possibility of treatment - induced improvements in language functions 12 months or longer after stroke . the purpose of the current study was to provide a preliminary estimate of efficacy of constraint - induced aphasia therapy ( ciat ) when compared to no - intervention in patients with chronic ( > 1 year ) post - stroke aphasia in order to provide the data needed to design an appropriately powered trial.material/methodsthis was a randomized , controlled , single - blinded , pilot trial . we identified 32 patients with chronic post - stroke aphasia . of these , 27 were offered participation , and 24 were randomized ( consort diagram ) : 14 to ciat and to 10 to no - intervention . ciat groups received up to 4 hours / day of intervention for 10 consecutive business days ( 40 hours of therapy ) . outcomes were assessed within 1 week of intervention and at 1 and 12 weeks after intervention and included several linguistic measures and a measure of overall subjective communication abilities ( mini - communicative abilities log ( mini - cal ) ) . to maintain blinding , clinicians treating patients ( ciat group ) did not communicate with other team members and the testing team members were blinded to treatment group assignment.resultsoverall , the results of this pilot trial support the results of previous observational studies that ciat may lead to improvements in linguistic abilities . at 12 weeks , the treatment group reported better subjective communication abilities ( mini - cal ) than the no - intervention group ( p=0.019 ) . other measures trended towards better performance in the ciat group.conclusionsin this randomized , controlled , and blinded pilot study , intensive language therapy ( ciat ) led to an improvement in subjective language abilities . the effects demonstrated allow the design of a definitive trial of ciat in patients with a variety of post - stroke aphasia types . in addition , our experiences have identified important considerations for designing subsequent trial(s ) of ciat or other interventions for post - stroke aphasia .

Background Material and Methods Subjects Definitions Description of the intervention Randomization Blinding Measures Data analysis Results Subjects Primary analysis Secondary analysis Discussion Conclusions

this case is more unusual , as the patient was a known case of recurrent patellar dislocation and presented with an atraumatic locked and vertically rotated patellar dislocation . this type of presentation has never been reported in literature to the best of our knowledge . a 14-year - old healthy male child with previous history of recurrent lateral dislocation of patella presented to accident & emergency department with complaints of inability to walk or bear weight on his left lower limb after he spontaneously dislocated his patella while running on uneven ground . radiographs revealed a laterally displaced and vertically rotated patella along its long axis with the medial patellar edge locked and dipping into the lateral gutter . open reduction was performed along with lateral patellar retinacular release with medial patellar retinaculum plication , to achieve satisfactory patellar stability and patellofemoral tracking . we would recommend that in the settings of patella being vertically dislocated and locked , open reduction would be the management of choice , as these types of dislocations are difficult to relocate by closed reduction . open reduction not only yields better outcomes but also allows the surgeon to perform patellar realignment procedures in order to prevent further patellar dislocations in cases of prior patellar instability . a case of locked patellar dislocation with vertical axis rotation was first described in literature by cooper in 1844 . since then only a handful of cases about the condition intra articular patellar dislocations can be superior or inferior or can be horizontal or vertical . locked vertical dislocation of patella is a rare entity and poses a therapeutic challenge in which closed reduction often requires a general anaesthetic or , may require an open procedure . there have been no published reports on recurrent patellar instability culminating in a vertically rotated and a locked patellar dislocation . we report an unusual case of lateral recurrent dislocation of patella in a 14-year - old healthy child who presented with vertically rotated and locked patella without any history of trauma , which required an open reduction along with proximal extensor apparatus realignment procedure . a 14-year - old healthy male child presented to accident & emergency department with complaints of inability to walk or bear weight on his left lower limb after he spontaneously dislocated his patella while running on uneven ground . upon further inquiry , the patient gave a history of similar episode 10 months back following a fall . at that time , his mother pushed the patella back after which he was taken to the hospital where radiographic images showed that patella was reduced with no associated fracture of patella , tibial tuberosity or femoral condyles . he was given a full length leg cast for 1 month after which he was able to resume his daily activities without any difficulty . previous hospital records revealed that he had history of anterior knee pain with a positive apprehension sign indicating lateral patellar instability . on examination , the knee was markedly swollen with obvious deformity in the lateral aspect in the form of tenting of the skin and soft tissue by underlying patella without any contusion or bruise . the knee was locked in 15 degrees of flexion and patient was unable to perform any movements at the knee joint . standard anteroposterior and lateral plain radiographs were taken which showed a laterally displaced and vertically rotated patella along its long axis with the medial patellar edge locked and dipping into the lateral gutter ( fig . 1 ) . there was no evidence of associated fracture or signs of osteochondral damage . clinical signs of genu valgum , patella alta , tibial torsion or trochlear dysplasia were absent and the quadriceps ( q ) angle on the normal limb was within normal range ( 11 degrees ) . pre - operative radiographs ; antero - posterior and lateral radiographs of the knee joint of the patient showing vertically rotated and locked patellar dislocation in the lateral femoral gutter . an attempt to closed reduction was made under conscious sedation but was not successful . a decision for open reduction the patella was found to be locked with its articular surface facing laterally and the medial edge of patella locked past the lateral femoral condyle and wedged into the lateral gutter . the medial patellar retinaculum was found stretched and attenuated but no tear or defect was noticed . a small incision was given along the lateral retinaculum , the index finger inserted was through it and the wedged medial patellar edge was freed from the lateral femoral condyle ; and at the same time lifting the patella out of the lateral gutter , it was relocated to its anatomical position . the stability and tracking of the patella was checked and it showed a tight lateral retinaculum along with a lax medial retinaculum . release of the lateral retinaculum was done along with medial patellar retinaculum plication using multiple interrupted prolene sutures ( fig . intra - operative image ; image obtained intraoperatively showing reduction of patella with lateral retinaculum release and medial retinaculum plication . post - operative radiographs ; anter - oposterior and lateral radiographs of the knee joint of the patient showing relocated patella to its anatomical position . quadriceps setting exercises and active straight leg raising were started in the second post operative week . progressive active and passive range of motion exercises were started after 3 weeks and full range of motion was achieved by 7 weeks . patient resumed his sporting activities by 6 months post operative with no recurrence or any symptoms at the time of final follow up of 18 months . lateral dislocation of patella is a common entity in adolescents , whereas rotational dislocation of patella which involves rotation of patella along the horizontal or vertical axis is quite rare . in vertical dislocation , the patella rotates along its long axis with the articular surface of the patella facing laterally and gets locked into the lateral gutter . vertical patellar dislocations pose a therapeutic challenge to the orthopaedic surgeons and are difficult to reduce by closed methods . repeated attempts at manipulation can lead to osteochondral fractures , which necessitates open methods to reduce the vertical dislocations . most of the patellar dislocations are seen with direct blow to the medial aspect of patella with knee in near extension or due to indirect force causing sudden contraction of quadriceps muscles while the knee is stretched in valgus and the tight quadriceps act as a bow string and prevents relocation . pre - disposing factors are anatomical variations such as shallow inter condylar groove , patellar hypermotility , patella alta and tibial torsion . in this report , we review a case of lateral dislocation of patella which is of interest ; in that it was vertically rotated locked dislocation of patella which was atraumatic i.e with no direct trauma to the patella in an individual with a history of recurrent dislocation of patella . there was no clinical evidence of medial patella - femoral ligament tear or any radiological evidence of trochlear dysplasia , patella alta , or shallow inter condylar groove . similar cases have been reported previously in the literature but with a concomitant history of trauma . hackl et al reported a locked dislocation with bony avulsion of medial structures after fall . corso et al reported a lateral dislocation with vertical rotation by laterally directed blow to patella during wrestling . gidden and bell reported a vertically rotated irreducible patella due to medial border force in femur as a result of motor bike accident . only a handful of cases have been reported in literature in whom there was no history of direct trauma . gann and nalty reported a vertical patellar dislocation in a 10 year old girl without any trauma . michels et al reported a locked patellar dislocation in a 16 year old girl while she was dancing . this case report is rare in the sense that there was no history of direct trauma preceding the dislocation and the mode of dislocation was running over uneven surface . although from literature review it is apparent that these types of dislocations are mainly traumatic and result from direct blow to medial side of knee , it is to be assumed naturally that the medial attachments of the knee joint would be torn in such circumstances ; but nevertheless , a locked dislocated patella may occur in atraumatic patients with prior history of dislocation without any damage to attachments along the medial border which provides the major support in preventing lateral dislocations . during surgery also , we found that the medial retinaculum to be stretched and lax with no defect or apparent tear , the lateral retinaculum was found to be tight and that the intra operative tracking of patella was abnormal . following the proximal patellar realignment procedure in the form of medial retinacular plication and lateral retinacular release , the patella was found to be tracking well in the trochlear groove . the tight lateral retinaculum with lax medial patellofemoral ligament could be related to previous episodes of recurrent patellar instability and one episode of lateral dislocation of patella in our case . hence , we would recommend that in the settings of patella being vertically dislocated and locked , open reduction would be the management of choice as these types of dislocations are difficult to relocate by closed reduction . open reduction not only yields better outcomes but also allows the surgeon to perform patellar realignment procedures in order to prevent further patellar dislocations in a setting of prior patellar instability . open reduction avoids osteochondral damage associated with repeated attempts of closed reduction and allows the surgeon to perform patellar realignment procedures in order to prevent further patellar dislocations in a setting of prior patellar instability .

introduction : locked vertical patellar dislocations are rare and pose a therapeutic challenge . this case is more unusual , as the patient was a known case of recurrent patellar dislocation and presented with an atraumatic locked and vertically rotated patellar dislocation . this type of presentation has never been reported in literature to the best of our knowledge.case presentation : a 14-year - old healthy male child with previous history of recurrent lateral dislocation of patella presented to accident & emergency department with complaints of inability to walk or bear weight on his left lower limb after he spontaneously dislocated his patella while running on uneven ground . radiographs revealed a laterally displaced and vertically rotated patella along its long axis with the medial patellar edge locked and dipping into the lateral gutter . open reduction was performed along with lateral patellar retinacular release with medial patellar retinaculum plication , to achieve satisfactory patellar stability and patellofemoral tracking.conclusion:we would recommend that in the settings of patella being vertically dislocated and locked , open reduction would be the management of choice , as these types of dislocations are difficult to relocate by closed reduction . repeated attempts of closed reduction may cause osteochondral damage . open reduction not only yields better outcomes but also allows the surgeon to perform patellar realignment procedures in order to prevent further patellar dislocations in cases of prior patellar instability .

Introduction: Case presentation: Conclusion: Introduction Case Presentation Discussion Conclusion

the use of the internet has expanded incredibly across the world over the last few years . the internet provides remote access to others and abundant information in all areas of interest . however , maladaptive use of the internet has resulted in impairment of the individual 's psychological well - being , academic failure , and reduced work performance and , especially leaded to internet addiction disorder ( iad ) [ 14 ] . iad was first raised in 1990s and according to the beard 's definition of iad , an individual is addicted when an individual 's psychological state , which includes both mental and emotional states , as well as their scholastic , occupational , and social interactions , is impaired by the overuse of the medium . in recent years , iad has become more prevalent worldwide ; the recognition of its devastating impact on the users and society has rapidly increased . importantly , recent studies have found dysfunctions of iad are similar to other types of addictive disorders , such as substance abuse disorders and pathological gambling [ 710 ] . people experiencing iad showed clinical features such as craving , withdrawal and tolerance [ 7 , 8 ] , increased impulsiveness , and impaired cognitive performance in tasks involving risky decision - making . as similar with the abnormalities in the dopaminergic neural system in individuals with substance - related addiction , the role of dopaminergic neural system in iad also has been elucidated in a few researches [ 1214 ] . in a recent study , people with iad were found to have altered resting - state glucose metabolism in several brain regions including the major dopamine projection areas such as the striatum and orbitofrontal region . moreover , another study found that adolescents with increased genetic polymorphisms in genes coding for the dopamine d2 receptor and dopamine degradation enzyme were more susceptible to excessive internet gaming compared with an age - matched cohort of controls . in a positron emission tomography ( pet ) imaging study , reduced levels of dopamine d2 receptor in subdivisions of the striatum including the bilateral dorsal caudate and right putamen were found in the individuals with iad . taken together , these findings suggest that iad may also be partly due to impaired dopaminergic neural systems similar to substance - related addiction . dopamine transporter ( dat ) is a protein situated in the presynaptic terminal and striatal dat is responsible for the active dopamine reuptake into the presynaptic neuron and plays a critical role in the regulation of striatal synaptic dopamine levels [ 1618 ] . altered dat concentration in the striatum following chronic substance administration has been reported previously [ 1924 ] . however , whether the abnormality of dat also exists in iad has not been illustrated before . in recent years , imaging of dat has been used as an important tool in clinical settings to display changes in the brain structure of patients with substance - related addiction [ 2124 ] . in addition , the radiotracer tc - trodat-1 , a technetium-99 m ( tc ) labeled tropane derivative ( technetium,2-[[2-[[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo [ 3 , 2 , 1]oct-2-yl]-methyl](2-mercaptoethyl)amino]ethyl]amino]ethanethiolato(3-)]-oxo-[1r-(exo - exo)]- ) , is regarded as a safe and suitable imaging agent for monitoring dat status for human imaging studies [ 21 , 25 , 26 ] . in the present study , we used single photon emission tomography ( spect ) with the radiotracer tc - trodat-1 to investigate striatal dat density to identify potential presynaptic abnormalities in iad subjects compared to age - matched healthy controls . this study aims to test the hypothesis that the altered availability of dat is associated with the pathogenesis of iad . iad was assessed using young 's internet addiction diagnostic questionnaire ( iaddq ) and goldberg 's internet addictive disorder diagnostic criteria ( iaddc ) . all of the questions of iaddq and iaddc were translated into chinese . to be eligible , participants in the iad group responses to the eight questions of iaddq and to satisfy three or more of iaddc ( i.e. , tolerance , withdrawal , craving and unplanned use , failure to reduce use , excessive use , sacrificing social activities to use , and physical and psychological problems associated with use ) . five men ( mean sd , 20.40 2.30 years old ) with iad were randomly selected from the patients seeking treatment at the peking university shenzhen hospital . the iad subjects used the internet almost everyday , and spend more than 8 hours ( mean sd , 10.20 1.48 hours ) everyday in front of the monitor , mostly for chatting with cyber friends , playing online games , and watching online pornographies or adult movies . these subjects were initially familiar with internet mostly at the early stage of their adolescence ( mean age sd , 12.80 1.92 years old ) and had the indications of iad for more than 6 years ( mean sd , 7.60 1.52 years ) . nine age - matched controls ( mean sd , 20.44 1.13 years old ) recruited through advertisement participated in this study . no statistical difference was found for the ages of the participants between the two groups ( p = 0.96 ) . the participants in the control group used internet occasionally or frequently but spent no more than 5 hours a day on the line ( mean sd , 3.81 0.76 hours ) and did not satisfy the diagnosed criteria of iad [ 4 , 27 ] . all the recruited participants were native chinese speakers , never used illegal substances ( sometimes , a few of the participants smoked or drink alcohol , but none of them meet the diagnosis criteria of substance - related disorders ) , had no history of significant medical , neurological or psychiatric diseases , and were right - handed . all the participants gave written informed consent before participation after the nature of the procedure was fully explained , including possible risks and side effects . all procedures for this study were approved by the ethics committee of the peking university shenzhen hospital . trodat-1 ligand ( liquid ) was supplied by the department of chemistry , beijing normal university ( beijing , china ) . the radiotracer tc - trodat-1 , 740 mbq ( 20 mci ) with purity > 90% was synthesized as described previously . and spect studies with tc - trodat-1 were carried out using a siemens diacam / e.cam / icon double detector spect with lower - energy all - purpose collimator ( siemens , erlangen , germany ) . subjects were injected intravenously with 740 mbq ( 20 mci ) of tc - trodat-1 . the acquisition parameters included 64 views over 18 s per view and a 128 128 matrix over 360 with a rotation in 5.6 increments . a butterworth filter was then applied with an order of 15 and a cutoff of 0.33 nyquist frequency . photon attenuation correction was performed using chang 's first order correction method using an attenuation coefficient of 0.15 cm . regions of interest ( rois ) were drawn on 12 transverse images , the pixels were extracted and counts of whole brain and bilateral corpus striatum were carried out . the volume ( v ) and weight ( w ) of bilateral corpus striatum as well as the ratio of corpus striatum / the whole brain ( ra ) was calculated using the mathematical models as described in the previous paper [ 21 , 31 ] . data in the present paper are presented as means standard ( mean sd ) . statistical program for the social sciences for windows , version 11 ( spss 13.0 , spss inc , chicago , usa ) was used to analyze the data . differences between the groups were assessed by student 's t - test . for all of the tests conducted , the criterion for significance was set at p < 0.05 . the dat images of the bilateral corpus striatum in the control group showed a panda - eye shape and dats were distributed uniformly and symmetrically in the corpus striatum . the bilateral corpus striatum was situated on 812 layers , as shown in figure 1(b ) . however , the dat images of the iad subjects displayed different levels of abnormity , in which the corpus stratums were much smaller and showed different shapes , dumbbell , thin strip , lunate shape , or sporadic spot ( figure 1(a ) ) . as shown in figure 1 and table 1 , dat expression level of striatum was significantly decreased in iad subjects . briefly , in comparison with the controls , there were significantly lower values of v ( cm ) , w ( g ) and ra of corpus striatum in the iad group , suggesting that decreased tc - trodat-1 bound to dat or impairment as well as dysfunction of corpus striatum occurred . no statistical difference was found as comparing v or w of the bilateral corpus striatum ( left side and right side ) either in the iad group ( p = 0.67 and p = 0.68 resp . ) or in the health control group ( p = 0.10 and p = 0.11 resp . ) . iad resulted in impaired individual psychological well - being , academic failure , and reduced work performance , especially among adolescents [ 14 ] . however , there is currently no standardized treatment targeted for iad . to develop effective methods for intervention and treatment of iad , it will first require establishing a clear understanding of the underlying neurobiological mechanisms . in the present study , we assessed the dat expression level in iad subjects and healthy controls using tc - trodat-1 spect . we found that dat expression level of striatum was significantly decreased and the values of v , w , and ra of the corpus striatum in iad subjects were greatly reduced . the imaging results provided the direct - viewing proof of altered availability of dat in brain of people with iad . dats play a critical role in the regulation of striatal synaptic dopamine levels [ 1618 ] and have been used as markers of the dopamine terminals . a reduced number of cell membrane dats may possibly reflect pronounced striatal dopamine terminal loss or the brain dopaminergic function impairment which has been found in substance - related addiction [ 2123 ] . pet imaging studies have found increased release of dopamine in the striatum during the video game . patients with pathological gambling also demonstrated high level of dopamine in the ventral striatum during gambling . because increased extracellular dopamine in the striatum is associated with subjective descriptors of reward ( high , euphoria ) [ 11 , 35 ] , individuals with iad may also experience euphoria as the extracellular dopamine in the striatum increases . however , long - time and high concentrations of dopamine have been shown to cause a selective lesion of dopamine terminals [ 32 , 36 ] and decreased size of dopaminergic cell bodies . taken together , the reduced dats found in our study may indicate the neuropathologic damage to the dopaminergic neural system caused by iad . according to our knowledge , this is the first imaging study to examine the abnormality of dat in the brain of iad subjects . furthermore , the imaging results of the present study provide the objective proofs that long - term maladaptive use internet might cause serious problems . however , for complete interpretation of the results of the present study , some limitations should be noted . firstly , the small sample size of our study may limit the generalizability of our results . those positive associations in our study might have been due to chance or a stratification effect in the sample collection , and further studies in independent samples or a larger population are required . secondly , the iad subjects in the present study reported different desired activities when they are sitting in front of the monitor ( including chatting with cyber friends , playing online games , watching online pornographies or adult movies , etc . ) . our study can not determine whether the different types of internet behaviors may cause different brain dat changes . therefore , the present study can only be recognized as exploratory and primary , and more research work should be done before we get the most definitive conclusion . the results from this study provide evidence that iad may induce significant dat losses in the brain and these findings suggest that iad is associated with dysfunctions in the dopaminergic brain systems and are consistent with previous reports in various types of addictions either with or without substances [ 2123 , 37 ] . our findings support the claim that iad may share similar neurobiological abnormalities with other addictive disorders .

in recent years , internet addiction disorder ( iad ) has become more prevalent worldwide and the recognition of its devastating impact on the users and society has rapidly increased . however , the neurobiological mechanism of iad has not bee fully expressed . the present study was designed to determine if the striatal dopamine transporter ( dat ) levels measured by 99mtc - trodat-1 single photon emission computed tomography ( spect ) brain scans were altered in individuals with iad . spect brain scans were acquired on 5 male iad subjects and 9 healthy age - matched controls . the volume ( v ) and weight ( w ) of bilateral corpus striatum as well as the 99mtc - trodat-1 uptake ratio of corpus striatum / the whole brain ( ra ) were calculated using mathematical models . it was displayed that dat expression level of striatum was significantly decreased and the v , w , and ra were greatly reduced in the individuals with iad compared to controls . taken together , these results suggest that iad may cause serious damages to the brain and the neuroimaging findings further illustrate iad is associated with dysfunctions in the dopaminergic brain systems . our findings also support the claim that iad may share similar neurobiological abnormalities with other addictive disorders .

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

compromised renal functions and previous central nervous system ( cns ) disease have been shown to predispose to this neurotoxicity . we describe a case of acute transient encephalopathy in a patient treated with ceftriaxonefor enteric fever infection . the present case illustrates the diagnostic challenges and management of this rare but potentially severe side effect of one of the most commonly prescribed parenteral antibiotics . an eight - year - old male child presented with a history of diarrhea and high - grade fever . the child was conscious , cooperative , well oriented to time , place and persons . the patient was hospitalized and started on ceftriaxone ( 1 g iv daily ) and intravenous fluids . after three days of treatment with iv ceftriaxone , child became afebrile but showed altered mental status with progressive apathy and somnolence . the patient was referred to the dyanand medical college , ludhiana ( punjab ) . in the emergency department , the patient was not in acute distress , had no fever , was hemodynamically stable , but dehydrated . hb 12 g / dl [ normal range 12 to 15 g / dl ] , hct 38% [ normal range 35.0 to 49.0% ] , tlc 6 10/l l [ normal range 5 to 12 10/l l ] , dlc - n 62 [ normal range 6070% ] , l 27% [ normal range 2040% ] , plt 274 10/l l [ normal range 100 to 300 10/l ] , urea 14 mg / dl [ normal range 825 mg / dl ] , cr 0.6 mg / dl [ normal range 0.51.7 mg / dl ] , na / k 139/4 [ normal range 135147/ 3.55 meq / dl ] , urinalysis revealed no bacteriuria and pyuria , tsb / dsb 0.77/0 [ normal range 0.11.0/ < 0.2 mg / dl ] , sgot / pt 44/23 [ normal range 1147/ 753 iu / l ] , stoolr / e , 2d mri scan of brain did not reveal acute stroke . the patient 's neurological status improved and three days later he was again alert and oriented . the proposed mechanisms include a decrease in -amino butyric acid ( gaba)-mediated inhibition and cephalosporin - mediated release of cytokines . in fact , cephalosporins may decrease gaba release from nerve terminals , increase excitatory amino acid release , and exert a competitive antagonism with gaba . alternatively , cephalosporin treatment has been proposed to induce endotoxin release , which generates cytokines liberation , such as tumor necrosis factor- , a proinflammatory cytokine implicated in septic encephalopathy . pre - existing cns abnormalities have been indicated as a risk factor for -lactams encephalopathy . in this was not the case in our patient , who presented with enteric fever and dehydration corrected with intravenous fluids . in fact , the temporal association of the encephalopathy induction and resolution with ceftriaxone administration and withdrawal makes this antibiotic highly likely to be responsible for the encephalopathy . moreover , the temporal pattern is in accordance with previous publications reporting cephalosporin neurotoxicity , with a latency of one to ten days after drug initiation and regression of all neurological symptoms within two to seven days following ceftriaxone treatment suspension . we could establish a probable causal relationship between ceftriaxone and the encephalopathy ( naranjo score 6 ) . the severity assessment revealed the adr to be moderate , suggesting that required therapeutic intervention and hospitalization prolonged by 1 day but resolved in 24 h or change in drug therapy or specific treatment to prevent a further outcome . since this patient did not have a history of any such reaction due to ceftriaxone , this adverse drug reaction was unpreventable . we describe a case of ceftriaxone - induced acute reversible encephalopathy in a patient treated for enteric fever infection . early recognition of this complication is particularly relevant as discontinuation of ceftriaxone reverts the neurological syndrome .

ceftriaxone is a commonly used , third - generation cephalosporin . encephalopathy is a rare side effect of third- and fourth - generation cephalosporins . renal failure and previous disease of the central nervous system predispose to this neurotoxicity . we describe a case of acute transient encephalopathy in a patient treated with ceftriaxone for enteric fever infection . early detection of this complication is relevant given that stopping the drug usually reverts the neurological syndrome .

Introduction Case Report Discussion Conclusion

in the field of medicine , stress is one of the most common complaints among patients1 . stress may negatively influence the affective state of an individual , which in turn may exert direct adverse effects on biological processes or behavioral patterns , thereby increasing the individual s disease risk and pathogenesis of a disease2 . psychosocial stress is believed to contribute to musculoskeletal disorders of the neck , shoulders , and other areas . previous studies have shown that mental stress induces a significant increase in muscle tension3 . recently , it has been demonstrated that the same motor units are activated by mental stress as by physical stress , which means that mental stress may also result in low - threshold motor units remaining active during breaks at work and outside of work3 . thus , numerous therapeutic approaches have been used to treat musculoskeletal diseases caused by psychological stress4,5,6 . the utilization of alternative medicine has been increasing in recent years , with massage documented as being one of the most frequently treatments for musculoskeletal disease7 . manual lymph drainage ( mld ) is a procedure that consists of several techniques derived from traditional massage8 . mld has become increasingly popular in recent years , not least because of the enormous amount of publicity it has recieved8,9,10,11,12 . moreover , the spread of information about edema among doctors , physical therapists , and patients , and the efforts for managing its cosmetic aspects have also resulted in its popularity10,11,12 . the frontal region of the brain has been associated with affective states , and different affective states have been associated with different eeg patterns in this region13 . normal subjects experience positively valenced emotions , such as happiness and joy , together with greater relative left frontal eeg activation . in contrast , the experience of negative emotions , including depression , is associated with greater relative right frontal eeg activation14 , 15 . there are some studies that have investigated the effects of different manual techniques on frontal eeg activation4 , 5 , 16 , 17 ; however , mld is not one of the techniques that have been investigated . therefore , the present preliminary study investigated the effects of mld in subjects with psychological stress and greater relative right frontal eeg activation , with mld expected to shift eeg patterns toward symmetry . the subjects included 52 university students , chosen according to the following criteria : ( 1 ) no history of mental illness , ( 2 ) not currently taking any medication known to affect eeg signals , and ( 3 ) no known heart- or muscle - related disease . they were all free of pulmonary , cardiac , and metabolic disease , as well as other disease states , which may cause brain dysfunction . all subjects responded to the stress response questionnaire ( sri)18 and returned it to the authors . according to the results of the questionnaire , 13 female subjects aged 19 to 23 years with an sri score > 80 and informed , written consent was obtained from each subject after the experimental procedures had been explained . all test protocols were approved by the ethics committee of the physical therapy faculty of kangwon national university . all interventions were completed in a supine position on a massage table with a pillow placed under the knees to relax the lower back muscles . data acquisition and mld were performed in a quiet , temperature - controlled environment ( 2224 c ) . stimuli such as conversation , phone calls , and noise that could increase the activity of sentinel nodes were minimized , and the subject s body was covered with a soft and thin sheet to avoid discomfort from body exposure . mld was conducted by a well - trained certified mld therapist , and applied twice to the neck area . the protocol was standardized , in that , the massage stroke category ( type ) and time was the same for all participants . subjects were allowed to rest comfortably for at least 5 min prior to the baseline recording procedure . eeg data were acquired for 5 min before , and immediately following , mld . a total of 6 channels of eeg were recorded : inclusive of fp1fp2 , f3f4 , and f7f8 . there were two other electrodes , a ground electrode and reference electrode , which were placed on both zygomatic bones . participants were then asked to close their eyes and refrain from talking , falling asleep , or making exaggerated body movements , in order to observe the cortical electrical activity without any external stimuli , minimizing possible visual artifacts during eeg measurement . the eeg signal was acquired over a 5-min period , followed by computerized fourier analysis of the eeg waves using the telescan software package ( laxtha , daejeon , south korea ) . the signal was sampled at a rate of 256 hz and was digitally filtered using a 150-hz band - pass filter . after data acquisition and storage , all statistics were computed to extract asymmetry values for the alpha frequency band in the frontal area of the cerebral cortex19,20,21 . the asymmetry index was calculated by subtracting the log - transformed absolute alpha power of the left hemisphere from the analogous log - transformed right hemisphere alpha power ( log right log left ) . as alpha power is inversely associated with cortical activation , a negative asymmetry score , which denotes greater alpha activity on the left and less alpha power on the right , would suggest greater right - sided activation . on the other hand , the asymmetry index was compared prior to , and after , mld application using a paired t - test , and the collected data were analyzed using a statistical package program ( spss v. 19.0 ) . mld generally caused an activation shift from the right hemisphere to the left hemisphere in all frontal electrode pairs analyzed in this study . statistical analysis comparing pre- and post - mld asymmetry values in channels f7f8 showed a statistically significant increase in hemispheric asymmetry : fp1fp2 , p = 0.082 ; f3f4 , p = 0.065 ; and f7f8 , p = 0.032 . alpha asymmetry scores for each regionareabefore- mldafter- mldfp1fp20.130.060.030.18f3f40.150.050.020.16f7f80.120.040.090.25 * all variables are means sd , mld : manual lymph drainage , * p<0.05 . in this preliminary study , frontal lobe eeg activity patterns in response to mld of the neck were examined to better understand alpha wave processing in subjects with psychological stress . many studies have shown that eeg asymmetry can be attenuated by massage , relaxation techniques , and musical therapies22,23,24 . however , to date , there are no studies investigating the effects of mld on frontal asymmetry . mld application acts as a very light stimulator against psychological stress ; we predicted that mld would be associated with frontal eeg asymmetry and state , and specifically result in greater left frontal hemisphere activation in the alpha frequency band . in this study , the alpha band ( typically in the range of 813 hz ) was chosen as an electrophysiological marker because , as previously discussed , it is relatively stable21 , 25 and inversely related to activation26,27,28 . the measurement of alpha asymmetry has been shown to be effective and reliable in discriminating positive and negative emotions23 , 29 . since previous investigations have demonstrated a relationship between brain electrophysiology in the frontal area and measures of affective states19 , 20 , 30 , 31 , we have also focused our analysis on the three anterior electrode pairs , fp1fp2 , f3f4 , and f7f8 . recent studies have suggested that greater left - sided anterior activation , indexed by decreased alpha activity in the left hemisphere , is associated with a higher degree of positive affectivity , a feeling of well - being , and a reduction in anxiety28 , 32 . our results indicate that mld is associated with a significant increase in left - sided anterior activation , thus supporting our hypothesis that mld can attenuate frontal eeg asymmetry . these results are also in line with findings from previous studies that have demonstrated the therapeutic effects of massage4 , 5 , 20 . although this study showed demonstrable effects of mld , there are several limitations and suggestions for future research . firstly , this study only examined the acute electrophysiological changes produced after mld in subjects with psychological stress . in addition to replicating the present findings with a bigger sample size , further studies are needed to investigate the effects of mld on other clinical populations across different conditions and diseases under a more controlled experimental design . in conclusion , our preliminary study showed that mld can significantly increase left - sided anterior activation . these results provide evidence to support the hypothesis that mld gives rise to positive affectivity .

[ purpose ] the purpose of this preliminary study was to investigate the effect of manual lymph drainage ( mld ) of the neck on frontal electroencephalogram ( eeg ) asymmetry in subjects with psychological stress . [ subjects ] thirteen subjects with psychological stress participated in the study . [ methods ] subjects received mld of the neck for 15 min . [ results ] analysis of the frontal asymmetry index showed that the energy shift in the alpha frequency band from the left hemisphere to the right hemisphere after mld resulted in greater left - side activation ( positive asymmetry values ) , which could be related to the positive emotional state observed particularly in the f7f8 area . [ conclusion ] these preliminary findings suggest that frontal eeg asymmetry was significantly attenuated after mld .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

it is well known that the commutator \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[a , b]=a^{-1}b^{-1}ab$$\end{document}[a , b]=a-1b-1ab of two elements \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a$$\end{document}a and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b$$\end{document}b of a group \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf g $ $ \end{document}g can be seen as a measure how far are \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a$$\end{document}a and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$b$$\end{document}b from commuting according to the group operation of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf g $ $ \end{document}g . thus , the normal subgroup \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[g , g]$$\end{document}[g , g ] generated by all such commutators measures how far is the group \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf g $ $ \end{document}g from an abelian group . namely , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[g , g]=0$$\end{document}[g , g]=0 if and only if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf g $ $ \end{document}g is abelian . the concept of commutators of normal subgroups has been generalized to a binary operation of the congruence lattice of arbitrary algebras in congruence modular varieties by smith , freese , mckenzie , hagemann , herrmann, ... one can find more details in . a malcev term of an algebra \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a is a ternary term operation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d$$\end{document}d of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a that satisfies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d(x , y , y)=d(y , y , x)=x$$\end{document}d(x , y , y)=d(y , y , x)=x for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x , y\in a$$\end{document}x , ya . an algebra with a malcev term we call a malcev algebra . what \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$=0$$\end{document}=0 in malcev algebras means has been answered by gumm , hagemann and herrmann , see [ 4 , theorem 13.4 ] . they proved that a malcev algebra is abelian\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(=0)$$\end{document}(=0 ) if and only if it is polynomially equivalent to a module over a ring . here , we call two algebras \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf b $ $ \end{document}b on the same domain polynomially equivalent if they have the same set of polynomial operations . in 2001 , a. bulatov has generalized the binary commutator operations to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n$$\end{document}n - ary commutator operations \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\bullet , \bullet , \ldots , \bullet ] $ $ \end{document}[,, , ] on congruence lattices of malcev algebras , for each \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n\in \mathbb n $ $ \end{document}nn , see . the aim of this note is to answer what \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$=0$$\end{document}=0 means in malcev algebras . we want to characterize all malcev algebras with such a property , because it could be expected that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$=0$$\end{document}=0 implies that the algebra is polynomially equivalent to a well studied structure . according to the property ( hc3 ) in we know that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\le $ $ \end{document}. therefore , we have that each abelian malcev algebra satisfies the condition \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$=0$$\end{document}=0 . in theorem 3.3 we prove that every malcev algebra with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$=0$$\end{document}=0 is an expanded group . in accordance with [ 2 , definition 7.1 ] , malcev algebras that satisfy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$=0$$\end{document}=0 are called 2-supernilpotent . as it has been defined in algebras that satisfy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[1,]=0$$\end{document}[1,]=0 are called 2-nilpotent . in expanded groups we deal with ideals rather then with congruences , because the corresponding commutator operations act in the same way according to [ 2 , corollary 6.12 ] . an expanded group \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v is 2-nilpotent if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[v,[v , v]]=0$$\end{document}[v,[v , v]]=0 and 2-supernilpotent if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[v , v , v]=0$$\end{document}[v , v , v]=0 . a polynomial of an algebra \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a is an operation obtained by composition of projections , fundamental and constant operations of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a , see [ 6 , definition 4.4 ] . the set of all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n$$\end{document}n - ary polynomials of an algebra \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a we denote by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathsf{pol}_n\mathbf a $ $ \end{document}polna for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n\in \mathbb n $ $ \end{document}nn . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v = ( v,+,-,0,f)$$\end{document}v=(v,+,-,0,f ) be an expanded group and let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n\in \mathbb n $ $ \end{document}nn . we call an \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n$$\end{document}n - ary polynomial \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } 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\begin{document}$$p(x_{1},\ldots , x_{n})=a$$\end{document}p(x1, ,xn)=a whenever there exists an \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i\in \{1,\ldots , n\}$$\end{document}i{1, ,n } such that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x_i = a_i$$\end{document}xi = ai . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v be an expanded group with a group reduct 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\usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\pi _ i^n : v^n\rightarrow v$$\end{document}in : vnv be the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i$$\end{document}i - th projection . then the group \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(\mathsf{pol}_n\mathbf{v } , + , -,0)$$\end{document}(polnv,+,-,0 ) is generated by constants and\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned}&\bigcup _ { k=1}^n\{f(\pi _ { i_1}^n,\ldots , \pi _ { i_k}^n):f \text { is a nonzero absorbing polynomial of } \mathbf v \text { of degree } k,\\&\qquad i_1,\ldots , i_k\in \{1,\ldots , n\ } \}. \end{aligned}$$\end{document}k=1n{f(i1n, ,ikn):fis a nonzero absorbing polynomial ofvof degreek , i1, ,ik{1, ,n}}. see [ 1 , lemma 3.1 ] . in [ 1 , p. 260 ] each \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n$$\end{document}n - ary commutator polynomial is exactly a polynomial \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f(\pi _ { i_1}^n,\ldots , \pi _ { i_k}^n)$$\end{document}f(i1n, ,ikn ) where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k\le n$$\end{document}kn and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f$$\end{document}f is a nonzero \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k$$\end{document}k - ary absorbing polynomial defined in definition 2.1 or constant . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\square $ $ \end{document} let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v = ( v,+,-,0,f)$$\end{document}v=(v,+,-,0,f ) be an expanded group such that\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f$$\end{document}f is the set of at most binary absorbing operations on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v$$\end{document}v , every absorbing operation in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathsf{pol}_2(\mathbf v ) $ $ \end{document}pol2(v ) is distributive with respect to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$+$$\end{document}+ , and\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2$$\end{document}2-nilpotent.then \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2$$\end{document}2-supernilpotent . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f$$\end{document}f is the set of at most binary absorbing operations on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v$$\end{document}v , every absorbing operation in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathsf{pol}_2(\mathbf v ) $ $ \end{document}pol2(v ) is distributive with respect to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$+$$\end{document}+ , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2$$\end{document}2-nilpotent . first , we show that for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$k\in \mathbb n $ $ \end{document}kn and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f\in \mathsf{pol}_k(\mathbf v ) $ $ \end{document}fpolk(v ) there exist \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c\in v$$\end{document}cv , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_i\in p_0(\mathbf v ) $ $ \end{document}pip0(v ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q_{ij}\in cp(\mathbf v ) $ $ \end{document}qijcp(v ) for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$1\le i < j\le k$$\end{document}1i < jk such that2.1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } f(x_{1},\ldots , x_{k})=c+p_1(x_1)+\cdots + p_k(x_k)+\sum _ { 1\le i ,xk)=c+p1(x1)++pk(xk)+1i < jkqij(xi , xj).let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_k$$\end{document}pk be the set of operations as in ( 2.1 ) . obviously \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_k\subseteq \mathsf{pol}_k(\mathbf \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathsf{pol}_k(\mathbf v ) \subseteq p_k$$\end{document}polk(v)pk , we need to show that the set \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_k$$\end{document}pk contains projections and constants and that is closed under the basic operations of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v . projections are in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_0(\mathbf v ) $ $ \end{document}p0(v ) and therefore in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_k$$\end{document}pk . constants are in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_k$$\end{document}pk because \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_0(\mathbf v ) $ $ \end{document}p0(v ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$cp(\mathbf v ) $ $ \end{document}cp(v ) contain zero polynomials . clearly , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_k$$\end{document}pk is closed under \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$+$$\end{document}+ and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$-$$\end{document}- since \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[c , p(x_i)]=r(x_i)$$\end{document}[c , p(xi)]=r(xi ) , for some \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r\in p_0(\mathbf v ) $ $ \end{document}rp0(v ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[c , q_{ij}(x_i , x_j)]=s(x_i , x_j)$$\end{document}[c , qij(xi , xj)]=s(xi , xj ) for some \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s\in cp(\mathbf{v } ) $ $ \end{document}scp(v ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[p(x_i),p(x_j)]=t(x_i , x_j)$$\end{document}[p(xi),p(xj)]=t(xi , xj ) for some \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t\in cp(\mathbf{v } ) $ $ \end{document}tcp(v ) . furthermore , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[q_{ij}(x_i , x_j),p_l(x_l)]$$\end{document}[qij(xi , xj),pl(xl ) ] and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[q_{ij}(x_i , x_j),q_{lt}(x_l , x_t)]$$\end{document}[qij(xi , xj),qlt(xl , xt ) ] are zero polynomials because \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2$$\end{document}2-nilpotent . therefore , in the sum of two operations in the form of ( 2.1 ) one can permute all summands to obtain again the form as in ( 2.1 ) , because \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_0(\mathbf v ) $ $ \end{document}p0(v ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$cp(\mathbf v ) $ $ \end{document}cp(v ) are closed under \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$+$$\end{document}+ and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$-$$\end{document}- . to show that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_k$$\end{document}pk is closed under all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q\in cp(\mathbf{v } ) $ $ \end{document}qcp(v ) we note that for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q\in cp(\mathbf{v } ) $ $ \end{document}qcp(v ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f , f'\in p_k$$\end{document}f , fpk we have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(f , f')\in p_k$$\end{document}q(f , f)pk by the distributivity of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q$$\end{document}q and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2$$\end{document}2-nilpotence of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v . it remains to show that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_k$$\end{document}pk is closed under any unary operation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g\in f$$\end{document}gf . note that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g'(x , y):=g(x+y)-g(x)-g(y)$$\end{document}g(x , y):=g(x+y)-g(x)-g(y ) is in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$cp(\mathbf v ) $ $ \end{document}cp(v ) , hence distributive . by induction it follows that for every \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n$$\end{document}n there exist \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c_{ij}\in cp(\mathbf v ) $ $ \end{document}cijcp(v ) such that2.2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } g(x_1+\cdots + x_n)=g(x_1)+\cdots + g(x_n)+\sum _ { 1\le i < j\le n}c_{ij}(x_i , x_j ) . \end{aligned}$$\end{document}g(x1++xn)=g(x1)++g(xn)+1i < jncij(xi , xj).for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f$$\end{document}f as in ( 2.1 ) this yields2.3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } gf(x_1,\ldots , x_k)&= g(c)+gp_1(x_1)+\cdots + gp_k(x_k)+\sum _ { 1\le i < j\le k}gq_{ij}(x_i , x_j)\nonumber \\&+\sum _ { 1\le i\le k}c_i(c , p_i(x_i))+\sum _ { 1\le i < j\le k}c_{ij}(p(x_i),p(x_j ) ) \end{aligned}$$\end{document}gf(x1, ,xk)=g(c)+gp1(x1)++gpk(xk)+1i < jkgqij(xi , xj)+1ikci(c , pi(xi))+1i < jkcij(p(xi),p(xj))for some \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c_i , c_{ij}\in cp(\mathbf v ) $ $ \end{document}ci , cijcp(v ) . note that the other terms vanish by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[v,[v , v]]=0$$\end{document}[v,[v , v]]=0 . thus \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$gf\in p_k$$\end{document}gfpk . we have proved \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p_k=\mathsf{pol}_k(\mathbf v ) $ $ \end{document}pk = polk(v ) . now we know that for every ternary polynomial operation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p$$\end{document}p on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v there exist a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c\in v$$\end{document}cv , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f , g , h\in p_0(\mathbf v ) $ $ \end{document}f , g , hp0(v ) and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r , s , t\in cp(\mathbf{v } ) $ $ \end{document}r , s , tcp(v ) such that2.4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } p(x , y , z)=c+f(x)+g(y)+h(z)+r(x , y)+s(x , z)+t(y , z ) \end{aligned}$$\end{document}p(x , y , z)=c+f(x)+g(y)+h(z)+r(x , y)+s(x , z)+t(y , z)for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x , y , z\in v$$\end{document}x , y , zv . using the absorbing property of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p , f , g , h , r , s$$\end{document}p , f , g , h , r , s and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t$$\end{document}t we obtain the following . first , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c=0$$\end{document}c=0 because \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(0,0,0)=0$$\end{document}p(0,0,0)=0 from ( 2.4 ) . then , we substitute \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$y = z=0$$\end{document}y = z=0 in ( 2.4 ) and obtain \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f(x)=0$$\end{document}f(x)=0 . analogously , we have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$g(y)=h(z)=0$$\end{document}g(y)=h(z)=0 . it remains \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(x , y , z)=r(x , y)+s(x , z)+t(y , z)$$\end{document}p(x , y , z)=r(x , y)+s(x , z)+t(y , z ) . we have \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$t(y , z)=0$$\end{document}t(y , z)=0 , because \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0=p(0,y , z)$$\end{document}0=p(0,y , z ) . analogously , we obtain \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$r(x , y)=s(x , z)=0$$\end{document}r(x , y)=s(x , z)=0 . whence , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2$$\end{document}2-supernilpotent , by [ 2 , corollary 6.12 ] . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\square $ $ \end{document} ( cf . [ 5 , corollary 7.4 ] ) let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a be a nilpotent malcev algebra with a malcev term \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d$$\end{document}d and let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$o\in a$$\end{document}oa . then for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_1,a_2,b_1,b_2\in a$$\end{document}a1,a2,b1,b2a there exist \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x , y\in a$$\end{document}x , ya such that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d(x , o , a_1)=b_1$$\end{document}d(x , o , a1)=b1 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d(a_2,o , y)=b_2$$\end{document}d(a2,o , y)=b2 . the function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x\mapsto d(x , o , a_1)$$\end{document}xd(x , o , a1 ) is bijective for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_1\in a$$\end{document}a1a , by [ 5 , corollary 7.4 ] . hence , the equation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d(x , o , a_1)=b_1$$\end{document}d(x , o , a1)=b1 has a unique solution for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_1,b_1\in a$$\end{document}a1,b1a . we know that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d(x , y , z):=d(z , y , x)$$\end{document}d(x , y , z):=d(z , y , x ) for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x , y , z\in a$$\end{document}x , y , za is also a malcev term of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a . therefore , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$y\mapsto d(y , o , a_2)$$\end{document}yd(y , o , a2 ) is bijective for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_2\in a$$\end{document}a2a by [ 5 , corollary 7.4 ] . hence , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$y\mapsto d(a_2,o , y)$$\end{document}yd(a2,o , y ) is bijective for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_2\in a$$\end{document}a2a . whence , the equation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d(a_2,o , y)=b_2$$\end{document}d(a2,o , y)=b2 has a unique solution for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_2,b_2\in a$$\end{document}a2,b2a . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\square $ $ \end{document} ( cf . [ 7 , theorem 1.2 ] ) every semigroup \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(g,+)$$\end{document}(g,+ ) such that the equations \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_1+x = b_1$$\end{document}a1+x = b1 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$y+a_2=b_2$$\end{document}y+a2=b2 are solvable for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_1,a_2,b_1,b_2\in a$$\end{document}a1,a2,b1,b2a , is a group . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\square $ $ \end{document} for a malcev algebra \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a the following are equivalent:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2$$\end{document}2-supernilpotent \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(=0)$$\end{document}(=0)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a is polynomially equivalent to an expanded group \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v = ( a,+,-,0,f)$$\end{document}v=(a,+,-,0,f ) such that\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f$$\end{document}f is a set of at most binary absorbing operations on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v , every absorbing operation in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathsf{pol}_2(\mathbf v ) $ $ \end{document}pol2(v ) is distributive with respect to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$+$$\end{document}+ on both arguments , and\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2$$\end{document}2-nilpotent . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2$$\end{document}2-supernilpotent \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(=0)$$\end{document}(=0 ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a is polynomially equivalent to an expanded group \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v = ( a,+,-,0,f)$$\end{document}v=(a,+,-,0,f ) such that\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f$$\end{document}f is a set of at most binary absorbing operations on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v , every absorbing operation in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathsf{pol}_2(\mathbf v ) $ $ \end{document}pol2(v ) is distributive with respect to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$+$$\end{document}+ on both arguments , and\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2$$\end{document}2-nilpotent . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f$$\end{document}f is a set of at most binary absorbing operations on \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v , every absorbing operation in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathsf{pol}_2(\mathbf v ) $ $ \end{document}pol2(v ) is distributive with respect to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$+$$\end{document}+ on both arguments , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2$$\end{document}2-nilpotent . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(1)\rightarrow ( 2)$$\end{document}(1)(2 ) using [ 2 , ( hc8 ) ] we obtain \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[1,]=0$$\end{document}[1,]=0 . therefore , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a is a 2-nilpotent malcev algebra . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$o\in a$$\end{document}oa and let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d$$\end{document}d be a malcev term . we define \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$+:a^2\rightarrow a$$\end{document}+:a2a by\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } x+y:=d(x , o , y ) \end{aligned}$$\end{document}x+y:=d(x , o , y)for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x , y\in a$$\end{document}x , ya . from proposition 3.1 we know that the equations \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_1+x = b_1$$\end{document}a1+x = b1 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$y+a_2=b_2$$\end{document}y+a2=b2 are solvable for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a_1,a_2,b_1,b_2\in a$$\end{document}a1,a2,b1,b2a . let us show that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } we observe that the polynomial\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } p(x , y , z):=d(d(d(x , o , y),o , z),d(x , o , d(y , o , z)),o ) \end{aligned}$$\end{document}p(x , y , z):=d(d(d(x , o , y),o , z),d(x , o , d(y , o , z)),o)for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x , y , z\in a$$\end{document}x , y , za is an absorbing polynomial at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(o , o , o)$$\end{document}(o , o , o ) with value \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$o$$\end{document}o . therefore , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(p(a , b , c),o)\in $ $ \end{document}(p(a , b , c),o) for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a , b , c\in a$$\end{document}a , b , ca by [ 2 , lemma 6.9 ] . using the assumption \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$=0$$\end{document}=0 , we obtain \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(a , b , c)=o$$\end{document}p(a , b , c)=o . equivalently,\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } d(d(d(a , o , b),o , c),d(a , o , d(b , o , c)),o)=o . \end{aligned}$$\end{document}d(d(d(a , o , b),o , c),d(a , o , d(b , o , c)),o)=o.by [ 5 , corollary 7.4 ] we have that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x\mapsto d(x , d(a , o , d(b , o , c)),o)$$\end{document}xd(x , d(a , o , d(b , o , c)),o ) is a bijective mapping of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a$$\end{document}a . hence , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d(d(a , o , b),o , c)=d(a , o , d(b , o , c))$$\end{document}d(d(a , o , b),o , c)=d(a , o , d(b , o , c ) ) , because \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$d(d(d(a , o , b),o , c),d(a , o , d(b , o , c)),o)=d(d(a , o , d(b , o , c)),d(a , o , d(b , o , c)),o)$$\end{document}d(d(d(a , o , b),o , c),d(a , o , d(b , o , c)),o)=d(d(a , o , d(b , o , c)),d(a , o , d(b , o , c)),o ) . using the recently introduced notation we have proved \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(a+b)+c = a+(b+c)$$\end{document}(a+b)+c = a+(b+c ) . now using lemma 3.2 we obtain that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$+$$\end{document}+ is a group operation with the neutral element \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$o$$\end{document}o . we denote the inverse of an element \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a\in a$$\end{document}aa by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$-a$$\end{document}-a . this is a polynomial operation given by\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } -x:=d(o , d(d(o , x , o),o , x),d(o , x , o ) ) \end{aligned}$$\end{document}-x:=d(o , d(d(o , x , o),o , x),d(o , x , o))for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x\in a$$\end{document}xa , by [ 5 , lemma 7.3 ] . we have proved that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a is polynomially equivalent to an expanded group \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v with the group reduct \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(a,+,-,o)$$\end{document}(a,+,-,o ) . now , we shall prove that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a is polynomially equivalent to the expanded group \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf{v } : = ( a,+,-,o , f)$$\end{document}v:=(a,+,-,o , f ) , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f:=p_o(\mathbf{a } ) \cup cp(\mathbf{a } ) $ $ \end{document}f:=po(a)cp(a ) . we have already that all fundamental operations of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf v $ $ \end{document}v are polynomials of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a . by [ 2 , ( hc3 ) ] , we know that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$[\,\underbrace{1,\ldots , 1}_n\,]=0$$\end{document}[1, ,1n]=0 for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n\ge 3$$\end{document}n3 . hence , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f=0$$\end{document}f=0 for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n$$\end{document}n - ary absorbing polynomial operations \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f$$\end{document}f of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$n\ge 3$$\end{document}n3 , by [ 2 , corollary 6.12 ] . hence , the set of all non - constant absorbing polynomial operations of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$f:=p_o(\mathbf{a } ) \cup cp(\mathbf{a } ) $ $ \end{document}f:=po(a)cp(a ) . using lemma 2.2 we obtain that each polynomial of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a is also a polynomial of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } 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, y ) \end{aligned}$$\end{document}q(x , y , z):=f(x , y+z)-f(x , z)-f(x , y)for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x , y , z\in a$$\end{document}x , y , za is absorbing at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(o , o , o)$$\end{document}(o , o , o ) with value \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$o$$\end{document}o . therefore , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(q(a , b , c),o)\in $ $ \end{document}(q(a , b , c),o) by [ 2 , corollary 6.9 ] for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a , b , c\in a$$\end{document}a , b , ca . hence , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$q(a , b , c)=o$$\end{document}q(a , b , c)=o or equivalently,\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{aligned } f(a , b+c)=f(a , b)+f(a , c ) \end{aligned}$$\end{document}f(a , b+c)=f(a , b)+f(a , c)for all \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a , b , c\in a$$\end{document}a , b , ca , by the assumption \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$=0$$\end{document}=0 . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(2)\rightarrow ( 1)$$\end{document}(2)(1 ) polynomially equivalent algebras have the same congruence lattice and the same commutator operations acting on the congruence lattice , by [ 2 , corollary 6.11 ] . therefore , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\mathbf a $ $ \end{document}a is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$2$$\end{document}2-supernilpotent by lemma 2.3 . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\square $ $ \end{document}

in this note we prove that a malcev algebra is 2-supernilpotent ( [ 1 , 1 , 1 ] = 0 ) if and only if it is polynomially equivalent to a special expanded group . this generalizes gumm s result that a malcev algebra is abelian if and only if it is polynomially equivalent to a module over a ring .

Introduction Special expanded groups Malcev algebras

tumors of the nail apparatus often present a challenge to diagnosis as well as treatment due to the specialized nail anatomy . the nail plate may conceal the tumor as well as alter the tumor growth pattern . however , alterations in the color , thickness , curvature of the nail plate may often provide a clue . a 40-year - old male presented with yellowish discoloration , thickening and altered curvature of the right great toe nail for the past 2 years . there was history of mild pain on walking and application of pressure for the past 2 weeks . , there was a markedly thickened nail plate , increased proximal transverse curvature and yellowish band - like discoloration of medial two thirds of the nail ( xanthonychia ) [ figure 1 ] . however , there was no friability of the nail plate . there was swelling and brownish discoloration of medial two thirds of the proximal nail fold . there was minimal pain on applying pressure on medial part of nail and nail fold . markedly thickened nail plate , increased proximal transverse curvature and yellowish band like discoloration ( xanthonychia ) of medial two thirds of the right great toe nail . also , note swelling of medial two thirds of proximal nail fold the patient underwent a total nail avulsion which revealed a well - defined tumor extruding from below the proximal nail fold toward the distal end of the nail bed [ figure 2a ] . the proximal nail fold was everted and the tumor was excised in entirety . the excised tumor ( 3 cm 2 cm 0.5 cm ) was firm in consistency with parallel ridges corresponding to the ridges on the nail plate [ figures 2b d ] . a differential diagnosis of onychomatricoma , fibrokeratoma of the nail matrix , superficial acral fibromyxoma , fibroma and glomus tumor was considered . histopathology of proximal transverse section [ figure 3 ] revealed a polypoid fibroepithelial tumor with a foliated pattern , acanthosis , papillomatosis and deep epithelial invaginations of multilayered basal and suprabasal cells with elongated nuclei oriented perpendicular to the basement membrane , absent granular layer with clear v - shaped clefts . at places , the stroma had a superficial cellular , fibrillary and vascular layer and a deep , relatively , acellular layer with denser collagen . a distal longitudinal section [ figure 4a and b ] showed a different pattern of the tumor : glove finger - like / monodigitate pattern with marked papillomatosis and multiple thick and deep epithelial ridges . on immunohistochemical analysis , the fibrous stroma showed diffuse staining for cd34 and negative staining for cd99 [ figures 5a and b ] . ( a ) post nail avulsion ; a well - defined tumor extruding from below the proximal nail fold toward the distal end of the nail bed . ( b ) the excised tumor showing parallel ridges ( r ) corresponding to the ridges on the nail plate . ( c ) dorsal view of the avulsed nail plate showing thin proximal portion and thickened yellowish distal portion ( d ) ventral view showing ridges on the nail corresponding to the tumor histopathology : proximal transverse section h and e ( 10 and 40 ) ( a ) a polypoid fibroepithelial tumor with a foliated pattern ; acanthotic , papillo matous epithelium with v - shaped clefts ( v ) corresponding to the avulsed nail . the stroma had a superficial cellular , fibrillary and vascular layer and a deep relatively acellular layer with denser collagen . ( b ) the tumor was lined by mature malpighian epithelium resembling normal matriceal epithelium ; absent stratum granulosum with multilayered basal and suprabasal cells with elongated nuclei oriented perpendicular to the basement membrane . at places , ( c ) numerous mast cells ( m ) in the stroma immunohistochemistry : ( 10 ) fibrous stroma showing diffuse staining for cd34 ( a ) and negative staining for cd99 ( b ) histopathology ( h and e ) a ( 10 ) , b ( 40 ) . a distal longitudinal section showing a glove finger - like / monodigitate pattern with marked papillomatosis and multiple thick and deep epithelial ridges onychomatricoma ( om ) is a rare onychogenic tumor of the nail matrix , described mostly in caucasians . the usual clinical presentation is a thickened nail with xanthonychia , increased transverse curvature and proximal splinter hemorrhages ( due to vascular stroma ) . perrin et al . have defined two types of om based on tumor and nail characteristics : ( 1 ) onychomatricoma of ventral matrix ( om type i ) : characterized by a single large fibroepithelial tumor . the nail plate is thinned out proximally and thick distally giving the appearance of a porch roof . ( 2 ) onychomatricoma of the proximal nail fold ( om type ii ) : characterized by a tumor with multiple fibroepithelial projections . om type ii often presents with unusual clinical features such as pterygium , fibrokeratoma like om , total onychodystrophy and verrucous band - like pattern ( suggesting wart or bowen 's disease ) . histopathologically , om type i shows two different architectural patterns in transverse and longitudinal sections . on proximal transverse sections , a lobulated / foliated pattern is observed while a glove finger monodigitate pattern is observed on longitudinal distal sections . awareness of these different patterns is important when only fragmented specimens are available for histological analysis . a three - dimensional model using proximal and distal transverse and longitudinal sections helps in better understanding and visualization of the morphology of this rare tumor . om is characterized by deep epithelial invaginations with a thick v - shaped keratogenous zone overlying the prekeratogenous zone . on avulsion of the nail plate , this is seen as an optically empty v - shaped zone . the epithelium corresponds to the matriceal epithelium with absent granular layer , basal cells with thin elongated nuclei and multi - layered suprabasal cells with oval nuclei . keratin markers , k5 and 17 , are positive through the epithelium ( indicative of matriceal differentiation ) , while k 85 stains the prekeratogenous zone . the stroma contains several mast cells and is arranged in two layers : a superficial cellular , vascular layer with fibrillary collagen and a deeper relatively less cellular layer with dense collagen bundles . the two - layered stroma is more frequently observed in the proximal portion than in the distal zone of the tumor . based on the proportion of epithelial and mesenchymal components and presence / absence of atypia in the stroma , ko et al . have proposed a different nomenclature with three categories : ( 1 ) unguioblastoma ( predominant epithelial component ) ( 2 ) unguioblastic fibroma ( predominant stromal component ) , and ( 3 ) atypical unguioblastic fibroma ( nuclear pleomorphism , atypia in the stroma ) . differential diagnosis of om includes fibrokeratoma , fibroma , superficial acral fibromyxoma ( saf ) , onycholemmal horn , malignant proliferating onycholemmal cyst , subungual warts , subungual keratoacanthoma , subungual squamous cell carcinoma and bowen 's disease . all of these are easily distinguishable by histopathology except fibrokeratoma , fibroma and saf , which need more careful differentiation . in distal longitudinal sections , the structure of om tends to resemble a fibrokeratoma . however , absence of a horny corn , multiple fibroepithelial digitations and two layered stroma ( on transverse section ) rules out fibrokeratoma . the stroma of om may resemble a fibroma , which can however be excluded by the hyperplastic and onychogenic epithelium of om . saf has a close resemblance to om due to highly vascular collagenous stroma , presence of mast cells and epidermal hyperplasia with papillomatosis . also , a single case of om with myxocollagenous stroma ( superficial acral fibromyxoma like om ) has been reported . saf shows diffuse cd34 , cd99 expression , while om shows diffuse expression of cd34 , but is cd99 negative . treatment of om is complete surgical excision . despite the atypical nature of stroma reported in some cases , no frank malignant variant has been described to date . in conclusion , onychomatricoma is a rare fibroepithelial tumor mostly arising from the nail matrix . it needs to be distinguished from other subungual / periungual tumors ; immunohistochemical markers cd34 and cd99 are useful adjuncts in the diagnosis of om . a three - dimensional spatial reconstitution of om helps in better understanding of its variable morphological characteristics . histopathological patterns of onychomatricoma vary in proximal and distal sections ; foliated pattern on proximal transverse sections and a glove finger - like pattern on the distal longitudinal section.study of onychomatricoma in multiple sections and planes is important for a three - dimensional spatial reconstitution of the tumor and differentiation from other subungual / periungual tumors.patterns of stromal immunohistochemical markers , cd34 and cd99 are especially useful in differentiating onychomatricoma from superficial acral fibromyxoma , a subungual tumor with several similar histological features as onychomatricoma . histopathological patterns of onychomatricoma vary in proximal and distal sections ; foliated pattern on proximal transverse sections and a glove finger - like pattern on the distal longitudinal section . study of onychomatricoma in multiple sections and planes is important for a three - dimensional spatial reconstitution of the tumor and differentiation from other subungual / periungual tumors . patterns of stromal immunohistochemical markers , cd34 and cd99 are especially useful in differentiating onychomatricoma from superficial acral fibromyxoma , a subungual tumor with several similar histological features as onychomatricoma .

onychomatricoma is a tumor of the nail matrix which often presents with alterations in the nail plate while the tumor itself is concealed beneath the nail plate . it is a benign , biphasic fibroepithelial tumor which has to be differentiated from other subungual and periungual tumors . we report a rare case of onychomatricoma and describe a three - dimensional histopathological analysis and immunohistochemical patterns of onychomatricoma .

Introduction Case Report Discussion

between 1903 and 1906 , oswaldo cruz , director - general of public health , had devoted his energies to improving sanitary conditions in rio de janeiro , a government priority under the administration of francisco de paula rodrigues alves ( 19026 ) . if yellow fever put a damper on the country s business prospects and image abroad , malaria made it virtually impossible to build the infrastructure needed to connect the country s inland regions to its ports . to control it , researchers new to the ranks of the instituto soroterapico federal ( renamed the instituto oswaldo cruz in 1908 and also known as the instituto de manguinhos ) pulled together the knowledge that had been amassed on the disease , creatively adapting it to the realities they encountered in the field . carlos chagas , a young doctor recently admitted to the institute , had been dispatched in 1905 to fight malaria in itatinga , in so paulo state , where a dam was under construction to provide power for the port in santos , then the largest exporter of coffee . chagas identified which species of anopheles were present in the region , describing some that were new to science . he chose to administer quinine preventively , drain and landfill wetlands , and provide collective protective measures for the workers in the form of screens in their lodgings , while keeping the chronically ill in isolation and providing treatment for acute cases . in early 1907 , chagas and another researcher recently admitted to the institute , arthur neiva , were sent to the vast swampy plains near rio de janeiro , known as baixada fluminense . chagas stayed there just three months before being sent to to fight malaria in minas gerais , where the central do brasil railway was being extended . there , he came across a new human trypanosomiasis , which was later named after him : chagas disease . when they reached the baixada fluminense plains , they found almost all the workers suffering from malaria and a few work sites already shut down . spanish and portuguese immigrants were taken straight from the port to the work site by rail to prevent alarming news of the state of affairs from reaching them and scaring them off . much of the land where pipes were being laid was swampy , and the workers were often up to their waists in water . by 1908 , there were close on four thousand workers there . many brought their families to the region , which also attracted travelling salesmen , merchants and other migrants . a wooden hospital protected by mosquito screens was built at the end of the railway . the general inspectorate of public works was responsible for all water treatments ; insect larvae were killed by the spraying of oil , water tanks in houses were covered , and larva - eating fish were introduced . as in railway construction projects and scientific and military expeditions across malaria zones , quinine was the mainstay of the campaign , being used for both treatment and prevention . the workers soon protested against having to take quinine for prophylactic purposes , prompting neiva to impose a strict , compulsory system : either the workers took the alkaloid or they would lose their jobs . quininisers , who had to make sure they had actually swallowed the capsules , rather than palming them or hiding them under their tongue . the daily dose of 30cg recommended in the manuals by patrick manson , botto scheube and carl mense had already proved insufficient in itatinga . from february to august 1907 , the workers at xerm started to be given 50cg of quinine hydrochloride , produced by merck , every three days , which yielded promising results . however , from late august and into september , neiva started to see a relatively large number of first - time infections amongst the workers who had taken the higher dose of quinine preventively . he first put it down to higher individual susceptibility , since the cases then being recorded were of workers who had failed to follow protocol regarding the taking of quinine . the morbidity rate was just 0.59% , which seemed to suggest they were on the right track , even though the protocol could not be applied to workers families and other migrants , amongst whom the number of cases was very high . the natives of the region , carriers of chronic infections , were a constant source of contamination from anopheles . much as robert koch had seen in africa , neiva encountered infected children whose only pathological manifestation of the disease was a dilated spleen . there was something else that drew his attention : workers who had taken quinine regularly and had no symptoms in xerm were struck by malaria when they visited rio for a few days without taking the alkaloid . from september 1907 onwards , such occurrences became increasingly commonplace . so it was that in october neiva upped the dose to 50cg every other day . the cases of first - time infection vanished wherever quinine administration was strict , but the change in dosage did not halt the rise in the number of workers who fell ill when they were away from the worksite temporarily . in november 1907 , first - time infections started to appear amongst people who had taken quinine preventively every two days . in response , neiva started administering it every day at the same dose of 50cg . this seemed to work , but now the disease struck those who were taking lower doses . and even amongst those who were taking the full daily dose , there were still cases of workers who would fall ill or have a relapse when they went to the capital . by january 1908 , the four thousand workers were receiving medication either every day or every other day , depending on where they were : in some locations , wrote neiva , we have never , for various reasons , managed to arrange things so that we could provide safe quinine administration. the only safe form of treatment was , then , 50cg taken without the exception of even a single day. even so , if treatment was interrupted for one or two days , malaria would strike . neiva himself , upon moving away from the region and stopping taking quinine preventively , was struck with a benign tertian fever at his home in rio de janeiro . in may 1908 , he was dispatched to head an anti - malaria campaign in the region to be crossed by the brazilian north - western railway , which stretched from the main coffee production area of so paulo across the mato grosso until the border with bolivia and paraguay . the article he published two years later shows , from its somewhat muddled structure and imprecise data , that he had not had enough time to test experimentally the deductions he had made in the months he was working on the problem . neiva wrote that he had the opportunity to observe a great number of people and thus a large quantity of human material in conditions that virtually corresponded to those of laboratory research . perfect contrast : the workers , who took quinine , and the women , children and other migrants almost as numerous as the workers who took no quinine and were totally attacked by malaria. it is likely that part of this second contingent took poor quality medications , including ones based on quinine . meanwhile , there were differences amongst the workers because of the different dosages and frequencies with which they took the drug , depending on where they were based , and also because some workers had relapses , showing the group to be less homogeneous than it was first believed because there were infected people in its midst . neiva s interpretation of the situation was that the malaria haematozoon had developed resistance to quinine . it says that when exposed to the constant action of quinine over a more or less lengthy period of time , the parasite became adaptation opportunity for the haematozoa as they fed on blood with different degrees of chemical richness. resistance was the result of the haematozoa s adaptation to organic media containing different quinine levels , which ultimately made them resistant , so that , over the generations , they acquired the capacity to be differentiated clearly in quinine - resistant races. in a third explanatory hypothesis , neiva highlights the relationship between non - infected individuals and victims of relapses in a situation of homogeneous chemical richness , supposing that the haematozoa got accustomed to the alkaloid when they encountered it in high concentrations throughout the whole man the resistant plasmodium races were formed by their cultivation in a quinine - rich environment , both in man and in vectors : as such , growing from gametes accustomed to living in media where quinine was always present , the haematozoa in another cycle , from ookinete to the final feeding that released the sporozoite in the bloodstream , already continued to develop even in the presence of quinine . as such , growing from gametes accustomed to living in media where quinine was always present , the haematozoa in another cycle , from ookinete to the final feeding that released the sporozoite in the bloodstream , already continued to develop even in the presence of quinine . while this may not have been the first time such a phenomenon had been observed by tropical medicine , it seems to have been the first theory in the scientific literature on malaria to explain it . from his experience in xerm , neiva concluded that constant quinine administration amongst malarial population groups , when it is not administered equally to all the inhabitants , will give the haematozoa the means to acquire specific resistance to the point of splitting into different races. in other words , he believed resistance to be a phenomenon which , from a clinical perspective , was manifested in the failure of quinine to cure patients , but whose origin lay in the development of parasite strains resistant to the drug . the publication of neiva s theory is related to oswaldo cruz s trip to the amazon on the service of the madeira - mamor railway , or the devil s railway , as it became known for the many thousands of people who lost their lives to the disease during its construction . there , the director of the instituto de manguinhos noted that huge doses of quinine , up to 3 g a day , still failed to prevent malaria from breaking out amongst people working on the railway . oswaldo cruz reached the region where the tracks were being laid in july 1910 , after the first section of the railway had been opened . in september , he delivered a report to the company containing an analysis of the sanitary conditions and his recommendations for their improvement . notwithstanding the range of diseases that plagued the workers , the main culprit for crippling the madeira - mamor works was malaria . it was so prevalent that it inverted the ratio between normal and pathological : the people , wrote oswaldo cruz , have no idea what a healthy state is. morbidity was high , but mortality was far lower . the most widespread form of malaria was the most serious : aestival - autumnal , or tropical ( 70% ) , caused by plasmodium falciparum . as in the campaigns run by chagas and neiva , emphasis was put on using quinine and providing protection against mosquito bites . oswaldo cruz considered the doses used in xerm and minas gerais to be ineffectual in such circumstances , and proposed the daily administration of 23 g , even though he noted that a daily dose of over 0.75 or 1 g produced signs of toxicity . in his report to the company building the railway , oswaldo cruz endorsed neiva s theory about the formation of quinine - resistant races of the plasmodium . some patients continued to contain parasites in their blood 24h after an intravenous dose of 6 g of the alkaloid ! despite noting this , he did not make any progress in his theoretical formulations about this biological phenomenon , which would go on to intrigue german researchers with broad experience in malaria prevention and treatment . german researchers followed closely the scientific theories and innovations produced on the other side of the atlantic . the intense commercial and diplomatic relations between germany and brazil , and the increasing flow of german emigrants to a country long ruled by a monarchy linked to the habsburgs created favourable conditions for scientific interchange . the archiv fr schiffs- und tropen - hygiene [ naval and tropical hygiene archive ] frequently commented on or summarised articles published by brazilians in local or foreign journals . one of the progeny of german science was adolpho lutz , an important actor in the institute of microbiology and medical zoology in brazil . and oswaldo cruz himself , when studying medicine in the 1880s , belonged to a group of germanists who would meet up to learn the language in order to keep up with the latest medical breakthroughs published in german journals . the successful campaign against yellow fever led by oswaldo cruz in rio de janeiro attracted two researchers from hamburg s institute for maritime and tropical diseases in 1904 . their mission was to observe the methods used to fight the disease and to study as yet obscure aspects of its aetiology and epidemiology . in the brazilian capital , hans moritz otto and rudolf otto neumann kept in close contact with the manguinhos institute s staff . the expedition was sponsored by shipping companies that connected hamburg and south america and coffee exporters like theodor wille : the hanseatic port was one of the main destinations for brazil s most important export . later , otto and neumann would apply the methods used in the brazilian capital to control yellow fever in togoland , then a german protectorate in west africa . henrique da rocha lima , a founding staff member at the instituto de manguinhos and oswaldo cruz s right - hand man , had specialised in microbiology and pathology in berlin from 1901 to 1903 , and in munich from 1906 to 1908 . in 1909 he was hired by the hamburg institute and from then until his death in 1956 , rocha lima worked actively as a mediator of german , he helped organise brazil s exhibits at the exhibition appended to the 14th international congress of hygiene and demography held in berlin in 1907 . the brazilian delegation presented materials illustrating the campaign against yellow fever , histopathology collections related to tropical diseases and other innovations , such as the technology used to produce bubonic plague serum and a description of the exoerythrocytic cycle of the protozoan haemoproteus columbae , an important breakthrough in malaria studies made by henrique arago . these contributions earned brazil a gold medal , awarded by the german empress , and awakened the curiosity of more german scientists about the research being done there . henrique arago and alcides godoy , another researcher at the instituto oswaldo cruz , both did postgraduate studies in germany in 1907 . the following year , the brazilian institution received two researchers from the hamburg institute , the protozoologist stanislas von prowazek and the chemist gustav giemsa . in their six - month stay , they did research and trained young scientists at the instituto oswaldo cruz . in 1910 , max hartmann , then on the staff of berlin s institute for infectious diseases , also spent some months there . two years later , hermann duerck helped to consolidate the anatomical pathology department at the instituto oswaldo cruz . the period in which prowazek and giemsa were working at the instituto oswaldo cruz coincided with neiva s malaria studies and his campaigns in inland brazil . neiva and prowazek even undertook a joint expedition to the tiet river in the state of so paulo to study its flora and fauna and also the epidemiology of malaria in the region . during prowazek and giemsa s sojourn at the instituto oswaldo cruz , the institution was also visited by ernst rodenwaldt , an army physician also connected to hamburg s institute for maritime and tropical diseases . he was journeying along the coast of south america as a ship s doctor on a vessel owned by the hamburg it is therefore more than likely that giemsa , prowazek and rodenwaldt learnt of neiva s observations of quinine resistance in rio de janeiro s swampy lowlands before he systematised and published his findings in the first volume of memrias do instituto oswaldo cruz . the fact that this and other articles were published in both german and portuguese in the institute s journal shows not only germany s leading position at the time in biomedical research , but also the close dialogue maintained by german and brazilian specialists . it was interrupted during the first world war but was soon re - established , along with an exchange of merchandise and capital and an influx of german migrants to brazil . the point we want to stress is that brazil s involvement in the production of knowledge on tropical diseases was more complex than is suggested in traditional representations of its role in international capitalism as a mere exporter of primary goods and destination of capital and manufactured products from europe and north america . scientific relations between brazil and germany in the field of tropical medicine were very dynamic , with knowledge and innovations being produced on both sides . not beholden to an imperial power , brazil had developed a strong tradition in medical research that focused on local diseases that threatened both the imperial and the republican elites nation - building and modernisation projects . this tradition created the institutional and cultural basis for its reception of cultural and scientific initiatives from the empire of wilhelm ii , including those from its chemical and pharmaceutical industry . in hamburg , the director of the institute for maritime and tropical diseases , bernhard nocht , and the head of the institute s clinical department , heinrich werner , would have the chance to observe resistance amongst the german workers who had been repatriated to that hanseatic city , as mentioned before . in an article also published in 1910 , they stated that they had been prepared for some quinine resistance in the malaria contracted in inland brazil , but they had been taken aback by its strength . the reason nocht and werner were so surprised at the failure of quinine in patients from the madeira - mamor region was simply that the treatment they had used until then had been successful . since the founding of the hamburg institute and the sailors hospital , the disease that had been most studied and treated there had been malaria . nocht had developed a new method for administering quinine that differed from the method suggested by koch , which consisted of administering 1 g of the drug on the ninth and tenth day of the disease . inspired by the italian method of fractionated dosage , nocht was in favour of administering a total of 1 g every day in five separate doses for eight days running , after which time he would increase the gap between the doses ( for instance , the same five doses a day for just two days ) until the patient was cured . this method had proved successful until the influx of workers from the madeira region of brazil . according to the tropical medicine specialist ernst rodenwaldt , amongst the specialists with experience of working in the tropics or on board ship , the way to treat malaria was taken for granted by all. the success of their methods had been proven by the constant improvement in morbidity and mortality rates amongst europeans in the colonies . several colleagues , wrote rodenwaldt in 1919 , will still remember the time when a malaria diagnosis in the tropics or at home would somehow lift our spirits about the patient , for we were certain we could help and promise a definite cure. amongst the workers being repatriated from the amazon , outbreaks of fever were exacerbated by symptoms of diarrhoea , dysentery and repeated relapses . in 90 cases observed by nocht and werner ( a number that included the patients and their relapses ) , 56 had tertian fever , 15 had tropical fever and 19 had mixed infections . unlike previous experience , the tropical form was more likely to relapse than the tertian forms , which normally was caused by a species of plasmodium that tended to produce more obstinate relapses . as they observed these anomalies , nocht and werner progressively raised the dose of quinine they administered twofold and even more . the initial dose rose to 2 g and , in the complementary treatment phase , between pauses of increasing length , they would administer quinine for two or three days so that the first pause would last two days , the second three , the next four and so on until eight days break was reached . however , the higher dose did nothing to improve the results : neither the frequency nor the seriousness of the relapses diminished . this quite unusual circumstance led them to experiment with other products . compounds containing arsenic had been tested by patrick manson in 1902 and by peter mhlens a year later . koch observed the effect atoxyl had on the malaria parasite , but as it also affected eyesight , its use in treating the disease was not pursued . the usefulness of another compound , methylene blue , had first been reported in 1891 by paul ehrlich and paul guttmann , and had been recommended afterwards for children . miguel couto also recommended it for brazilian malaria , which encouraged neiva to test it in xerm . he did find it to have some effect against the plasmodium , but its action was very slow . incontestable the effect of ehrlich hata preparation 606 ( salvarsan ) in five cases of quinine - resistant malaria . werner tested the preparation again on brazilian malaria , but it failed to prevent relapses in any case . werner also sought to demonstrate how the amazonian variant of the disease differed from other regions in the world . with nocht , he had observed several clinical peculiarities , especially the more frequent disturbance of the intestine . in no patients they also found it notable that the nervous system was affected in two serious cases , both with symptoms of beriberi . and the spleen , while only slightly dilated in several very serious cases of malaria , was found to be alterations of the lungs were also a characteristic of patients from brazil : fourteen had more or less serious cases of bronchitis , which disappeared as the malaria treatment progressed . the doctors from hamburg also reported temperature variations that had not previously been seen . in six cases , shortly after the fever had been abated by quinine , the patients temperature had risen to for several days without any parasites being detected in the peripheral blood . pseudo - relapse. faced with these singularities , nocht and werner started to wonder whether the species of parasites involved in brazilian malaria were different , not just in their virulence and resistance , but also morphologically . the parasites in the tertian form were observed to be slightly different to the ones found in other parts of the world . in an article published in 1910 , the tropeninstitut researchers did not put forward any explanation for the development of quinine resistance . only the following year did werner discuss it theoretically , differentiating two forms of resistance : that acquired during the passage of the plasmodium through warm - blooded animals that is human hosts and that acquired in the passage through insects . the first form , resulting from the repeated effect of quinine on the plasmodium , was correlated to the prolonged use of the alkaloid in malaria treatment . this would partially explain the resistance seen in the cases from the amazon , including the peruvian amazon , where quinine had been used to fight malaria for centuries . meanwhile , werner thought it unlikely that the second form during the passage through the insect existed . for neiva , the high levels of quinine absorbed by the insects as they fed on human blood were enough to assure the continued or even increased resistance of the parasites in their passage through the vector . according to werner , research by gustav giemsa and heinrich schaumann had demonstrated that the level of alkaloid in human blood was too low for a new generation of resistant parasites to develop from the fertilisation of the insect . the hamburg researcher believed it was impossible to reach any firm conclusions about the phenomenon of resistance based on deductions that were primarily theoretical. the tests made by nocht and werner with methylene blue and salvarsan show that they were attuned with the chemotherapy developed by paul ehrlich and his collaborators . the resistance of trypanosomes to atoxyl studied by ehrlich from 1907 onwards influenced in large measure the analysis of the phenomenon now observed with malaria parasites . according to christoph gradmann , ehrlich was interested in the trypanosomes resistance to atoxyl not so much for its clinical importance as for the fact that it could furnish evidence about the existence of cell receptors and to support his theory about the drug s action on the cell , which he compared with the relationship between toxins and antitoxins . both bernhard nocht and ernst rodenwaldt wrote that the results obtained in experiments with protozoan parasites of the blood like trypanosomes could not be extrapolated to intracellular parasites . according to nocht ( 1919 ) , the trypanosomes resistance to drugs was stable , and was maintained after many passages , while quinine resistance disappeared in plasmodia after prolonged interruption of medication in the same host . rodenwaldt was not so sure of this , but added that the disappearance of alterations in the malaria plasmodia was in part due to the fact that their asexual reproduction was more complex than that of trypanosomes . new theories emerged during the first world war , where the havoc wreaked by malaria in the balkans , turkey and syria made resistance to quinine a top priority . the action of quinine often left much to be desired , both therapeutically and prophylactically, wrote the army physician wilhelm hoffmann as he weighed up the impact of malaria on german troops . the initial optimism after the discovery of its aetiology and transmission and the success of experiments to control the disease in specific geographical contexts was badly shaken during the first world war , forcing the specialists in the field to review the epidemiological and scientific studies on the disease . in macedonia in particular , british , french and german troops were struck by particularly devastating epidemics and caught off - guard by the inefficacy of the classic preventive and therapeutic methods . the infection rate amongst the local people was 6095% and the very high density of mosquitoes made it impossible to control the vector . in late 1916 , french army doctors were alarmed to find that sixty thousand men had lost their lives to malaria in the region , half of the french military contingent . malaria starts to appear as a serious sanitation issue in german medical records in early 1916 . let us turn to the testimony of ernst rodenwaldt : first of all , prevention seemed to fail , then the cure . everything indicated that quinine was not the specific medication one had hoped for , or was being administered incorrectly , or that there must be interferences that reduced or prevented its expected efficacy. some doctors even defended the idea that in regions like macedonia , albania or the taurus mountains there was a different form of malaria that could not be treated with quinine . its prophylactic use , considered by the doctors working in the tropics to be essential , started to be seen with increasing scepticism by the doctors working on the front . they argued that it fostered a certain resistance and worsened the chances of treatment with the alkaloid when infection hit . the drug s side effects queasiness , weakness , bleeding under the skin , impaired sight and hearing , chest pain and blackwater fever made it hard to get the soldiers to stick to the treatment . for this reason , peter mhlens , head of the clinical section at the tropeninstitut , recommended , as neiva had in the baixada fluminense lowlands in brazil , strict supervision by the sanitation officials to make sure they actually ingested the quinine . the servicemen would often hide the drug and later exchange it for cigarettes or alcohol . certain authors suggested that many supposed cases of ( biological ) quinine resistance were not just down to human resistance and the inadequate use of the drug , but also to the general state of the patients , who suffered from malnutrition , stress and fatigue because of the physical and emotional impact of the war . at the time of the war , few german doctors were familiar with the clinical aspects of malaria and how to treat and prevent it , because , according to rodenwaldt , having been eliminated from germany half a century ago , except for small enclaves , malaria was no more than a curiosity in medical teaching before the war. this explains why so many of them were so little prepared for dealing with it during the conflict . to make matters worse , it seems that the military sanitation organisation failed to give clear , standard guidelines on how to fight the disease . nevertheless , the new circumstances provided an unprecedented opportunity for verifying findings from previous , more sporadic observations of malaria on a far larger scale , albeit with little control over the conditions . ultimately , the war served as a great experimental field for malaria research and especially for evaluating the prophylactic and therapeutic efficacy of quinine. whenever quinine was taken preventively , the doses used varied greatly . in some regions around 1 g quinine was administered a day , while in others the troops would receive 1.8 g in ten days . for rodenwaldt , treatment had to be continued for two to three weeks after the malaria - infested region was left , warned mhlens , but this rarely took place . there were also disagreements over the best way of using quinine : whether in tablets , in a solution , or in subcutaneous or intramuscular injections . apart from the hypothesis that there was a specific form of the disease in the balkans and turkey , other theories were also put forward to account for the failure of treatment or prevention using quinine . in a bid to make some sense of the arguments and opinions that abounded , martin mayer distinguished two forms of resistance : chininabstmpfung dulling or insensitivity to quinine due to factors within the patient s body that made the individual unresponsive to the alkaloid and chiningewhnung adaptation to quinine referring to what took place inside the parasite . in the former case , physiological processes were responsible for the drug s diminished effectiveness , witnessed by the reduced excretion of quinine in the urine or its reduced concentration in the blood . gustav giemsa and joseph halberkann , from the hamburg institute , questioned these results : the excretion of quinine did not prove that it had failed to exert any action against the parasite in the organism . however , the products of the alkaloid s decomposition could be involved in the process , and this was something giemsa turned his attention to . nocht felt that the inefficacy of ever larger doses of quinine was not related to the emergence of strains of quinine - resistant parasites , as neiva had suggested . rather , he attributed the diminished or non - existent effect of quinine to the weakening of the organism s defences and the prolonged and inappropriate use of the drug . the fact is that the parasite s or human organism s resistance to quinine involved complex issues that were beyond the grasp of medical science at the time . indeed , several aspects of the epidemiology and physiopathology of malaria would remain obscure . resistance to quinine became a polysemic concept , employed in different circumstances depending on the author s principles or procedures . if before the war neiva and werner spoke of a property developed in the parasites , during the war the phenomenon started to be related to the inefficacy of the alkaloid caused by incorrect prophylaxis , organic factors like immunity , the social resistance of troops ascribable to the drug s side effects , and so on . in an attempt to avoid the ambiguity suggested by the word resistance , martin mayer , from the hamburg institute , proposed a distinction between insensitivity to quinine , referring to the patient s organism , and quinine adaptation , a characteristic developed by the parasite . the hypothesis of direct action was refuted by laboratory evidence that the concentration of the alkaloid in the blood was far lower than that needed to kill the microbe . repulsion theory , proposed by julius morgenroth in 1917 , was that quinine would build up inside erythrocytes and would there repel the parasites by chemostatic action , preventing them from penetrating the cell . free in the blood stream , their life cycle was interrupted and they were destroyed . however , if they were only repelled by erythrocytes , argued rodenwaldt , it was hard to imagine how these plasmodia would adapt to a drug that had no direct effect on them . morgenroth viewed the action of quinine in the light of the principle of selective action proposed by ehrlich , with whom he had worked . when it came to the life cycle of the plasmodium , the process by which it resisted immune reactions and the effects of quinine , remaining quiescent in the organism until the disease relapsed , was still a mystery . it was not known where in the body it remained , nor whether it was capable of being maintained inside other cells than erythrocytes . the asymptomatic carriers of parasites and the chronically ill were held accountable for the introduction of the disease to areas prone to epidemics . incite the manifestation of hidden infections , using techniques that included submitting individuals suspected of being infected to temperature shocks or physical exertion followed by hot and cold baths . in a chapter on malaria in the respected handbuch der tropenkrankheiten [ handbook on tropical diseases ] by carl mense , published between 1917 and 1918 races in certain regions like brazil , which could have existed previously or have developed in response to the overuse of the drug . finally , the third form was indirect resistance via the failure of the patients defences . ziemann had summarised the different explanations , but had made no progress in developing a theory , thereby corroborating nocht and mhlens statements that the abundant literature on malaria published after the war discussed specific problems concerning the epidemiology , physiopathology , treatment and prevention of the disease , but failed to put forward any satisfactory solution for most of the enigmas the doctors on the front line had been faced with . neiva and werner s theoretical speculations continued to be the most comprehensive attempts to explain quinine resistance , even if they were riddled with inaccuracies and gaps . however , the idea that the parasite acquired resistance to the alkaloid during its life cycle in humans and/or in mosquitoes , and that it was able to transmit resistance to its offspring , was refuted by ernst rodenwaldt , who in 1919 wrote one of the most robust works on the subject , presented as a thesis to the university of heidelberg . why have our skills failed in the balkans and turkey if before the war tropical medicine had so many successful experiences? he wondered . let us , then , examine the ideas he galvanised to explain the complex biological process by which the malaria parasites became resistant to quinine . writing in his memoirs about the german retreat from asia minor , where he had worked as a hygiene adviser , rodenwaldt relates that he took advantage of the six - week wait for a passage to europe to study wilhelm johannsen and erwin baur s theories of heredity . upon reaching germany , he decided to enrol at the medicine department of heidelberg university , where he wrote a thesis about the question that had intrigued him since he had been in africa : the resistance of the malaria plasmodium to quinine . in his memoirs he adds that in his thesis he took pains to apply the notions of genetics to the issue of infection , being at the time a staunch believer in august weissmann s theories of heredity . a defender of darwin s theories and critic of the inheritance of acquired characteristics , an idea associated with lamarck but admitted by darwin himself , weissmann , a german biologist , proposed the almost absolute constancy and autonomy of what was known as the germinative plasma , an element responsible for carrying the hereditary factors in a way that was completely immune to the influence of the sum , the rest of the body , which merely transmitted the germinative plasma from one generation to the next . germinative plasma would only change under exceptional circumstances , so that any alteration the organism went through during its life would not be transmitted by hereditary means . the blending of weissmann and mendel s conceptions in the early years of the twentieth century meant that heredity was seen as a fixed condition that was inherent to the biological characteristics of individuals. but rodenwaldt s formulations about quinine resistance were more strongly influenced by wilhelm johannsen and erwin baur , whose insights contributed towards a reworking of gregor mendel s ideas from the early 1900s onwards , especially concerning the emergence of species . different theories were expounded to explain the mechanisms by which different variants and therefore new species emerged , as well as the processes involved in transmitting these variations through successive generations . mutationists , who rejected the role of natural selection as a mechanism of evolution , holding that only mutations responsible for major modifications could have some role in the emergence of new species ; and the biometricians , like karl pearson , who not only recognised natural selection as the principal driving force for evolution , but also the idea that small , continuous variations led to the development of new species . in the midst of this debate came the experiments by a dane , wilhelm johannsen , on pure lines of beans , showing that natural selection was only induced by genetic differences , not environmental ones . johannsen distinguished the genotype , or the set of factors responsible for heredity , from the phenotype , which were the observable characteristics of individuals . individuals with pure lineages that is , with the same genetic material , resulting from self - fertilisation could have different phenotypes in response to different environmental conditions , but their genotype would remain the same . he concluded that an organism s appearance was the result of the influence of mendelian traits and also a response to its environment . mendelian factors only determined the breadth of an organism s reactions to its environment . like johannsen , baur distinguished environmentally - induced changes from heredity . he conceded that mutations could modify genetic traits , but not on the scale proposed by the mutationists. they took place on a small scale , resulting in such subtle changes that they often went unnoticed . these small mutations were important in the formation of new races , but not of new species . in einfhrung in die experimentellen vererbungslehre [ introduction to the experimental doctrines of inheritance ] , a book published in 1914 and re - edited in 1919 , baur mentions the resistance of the plasmodium to quinine . he did not see this property as being an inheritance of acquired modifications : in this case , as in others , it is naturally far more reasonable to concede that isolated races of malaria parasites had differing resistances to quinine , and that by treating an infected person with the drug , a more resistant race that already existed was simply cultivated in the pure form . in this case , as in others , it is naturally far more reasonable to concede that isolated races of malaria parasites had differing resistances to quinine , and that by treating an infected person with the drug , a more resistant race that already existed was simply cultivated in the pure form . in a similar vein and also drawing on weissmann , johannsen and baur s ideas , rodenwaldt sought to refute neiva and werner s lamarck - inspired interpretation . they both held that the parasite developed resistance to quinine over several generations , maintaining or increasing its resistant features in its passage through mosquitoes . for neiva , the quinine the mosquito absorbed when it fed on blood was in some way implicated in the process , although this is an idea that werner ruled out as he thought that the quantity of alkaloid in the blood was too small for this . for rodenwaldt , none of these hypotheses explained the failure of quinine in brazil , the balkans or turkey , nor the contrast with successful cases of tropical medicine in the colonies . neither did they explain the failure of the prophylaxis , nor the way the preparations were used , nor the circumstances of war , nor factors relating to the human organism , nor even the existence of a specific form of malaria in the combat zones . rodenwaldt believed that plasmodium strains that had different levels of susceptibility to quinine , but the same physical features , emerged spontaneously in nature . most of the parasites quickly succumbed to the action of the alkaloid , while a few races were selected . it is not a matter of the creation or cultivation of quinine - resistant strains , as most authors would have us believe , but pre - existing quinine - resistant strains that are selected. selection took place when quinine was used in too small quantities in regions where malaria was widespread and where the arrival of non - immune people provoked more virulent forms of the disease . this process could take place in regions where quinine had been used for centuries , such as the south american mountain ranges , or over a short period of time where a large population group was submitted to the same pattern of administration of the drug . the incomplete prevention or cure of patients contributed to the development of chronic malaria , as in the regions where growing resistance to quinine had been observed . this dependence of the selection of quinine - resistant races on the greater or lesser efficacy of therapeutic treatments explained why the problem did not occur in regions like germany , where malaria had been endemic for a long time , or italy , which had had a systematic process for fighting the disease for some time : the main consequence of treatment in a country with doctors and where the people are used to the therapy until the disease is eradicated was that the selection of quinine - resistant races was prevented , because the use of high doses of quinine until the parasites were killed made it impossible for epidemic levels to be reached . the main consequence of treatment in a country with doctors and where the people are used to the therapy until the disease is eradicated was that the selection of quinine - resistant races was prevented , because the use of high doses of quinine until the parasites were killed made it impossible for epidemic levels to be reached . in the tropics , selection did not take place because the native population had a degree of immunity to the disease and almost never took quinine , while europeans were generally treated correctly . there , the rare relapses and quinine resistance occurred only in people who had the bad luck of being infected with an already existing strain that had not passed through selection . but although there were signs that the plasmodia from regions where there was quinine resistance presented morphological particularities , this was not essential for proving that there were different races of the parasite , rodenwaldt argued . his acceptance of johannsen s views is evident in the distinction he makes between genotype and phenotype to interpret the phenomenon of resistance . alterations to the phenotype did not in any way alter the genotype , whose constitution remained constant . therefore , the alterations could not be transmitted by hereditary means : in a morphologically identical population of plasmodia , but with a different genotypical behaviour towards quinine , it is possible that quinine - resistant races can be isolated by selection ; that is , plasmodia that have the same phenotypical appearance , but which behave differently towards quinine , could lead to the selection of a quinine - resistant race with common phenotypical and genotypical features . in a morphologically identical population of plasmodia , but with a different genotypical behaviour towards quinine , it is possible that quinine - resistant races can be isolated by selection ; that is , plasmodia that have the same phenotypical appearance , but which behave differently towards quinine , could lead to the selection of a quinine - resistant race with common phenotypical and genotypical features . if rodenwaldt owed johannsen the distinction between variations in visible characteristics and variations in hereditary material , the core of his theory was based on baur s deductions . the environment that selected quinine - resistant strains was understood in a broad sense as being an ecological and social environment . in the balkans and turkey , the environment was war - ravaged , crossed by people who were physically and psychically weakened , where troops came into contact with villages rife with chronic malaria and other diseases , and where army doctors had little familiarity with treating malaria , a key ingredient in view of the importance rodenwaldt gave to the correct administration of preventive and therapeutic quinine in his theory . similar experiments to johannsen s would have to be done on pure lines of plasmodia , that is ones with homogeneous genotypes . in 1919 , in the archiv fr schiffs- und tropen - hygiene , rodenwaldt published an article about the plasmodium s resistance to quinine . the following year , werner rosenthal ( 1920 ) , from gttingen , author of tierische immunitt [ animal immunity ] , praised the heidelberg professor for his contribution , but criticised how he had presented the concepts of immunity and resistance in malaria . rodenwaldt s response leads us to a new ramification of the debates and investigations about this disease that experience in the war had made such a moot scientific and epidemiological issue , here approaching it not from the perspective of genetics , but immunology , a field of knowledge that went on to gain ground in experimental and practical medicine . the material researched for this paper is not yet enough to analyse these discussions about the problem of quinine - resistant malaria in any great depth . basically , rosenthal accused rodenwaldt of mistaking a non - specific form of immunity observed in people from a region with chronic malaria with the specific elements of the defence mechanisms activated by the organism in its interaction with the pathogen . europeans were prone to malaria in the tropics because they did not have this non - specific resistance and because they were weakened by the unfamiliar climatic conditions . unlike immunity in bacterial infections , he went on , malaria immunity was developed very slowly through continued exposure to the parasite , that is in the chronic form of the disease . he further stated that it did not produce long - lasting effects , as seen in bacterial diseases , and could be dissipated by internal or external factors ( other illnesses , physical debilitation , etc . ) . unstable , were exposed to a new infection , they tended to exhibit far milder symptoms which would subside more readily under drug - based therapy . rodenwaldt was involved in another far less gentlemanly exchange of views with another former student of the hamburg school , viktor schilling , who visited brazil in 1912 . as a physician and hygiene adviser in turkey and syria , schilling had observed several cases of malaria that showed resistance to mounting doses of quinine . it appears that as early as 1917 he had written a communiqu about this subject which was not published because of strict censorship at the time . his comments only became public two years later in the archiv fr schiffs- und tropen - hygiene and deutsche medizinische wochenschrift , at the same time that rodenwaldt was publishing his thesis . according to schilling : the conditions that neiva , nocht , werner , mhlens and others have presented as being propitious for the development of quinine - resistant races are valid . the parasites have always found a way to partially escape quinine , increase their virulence in new ground and adapt in some way to the alkaloid . . . generally speaking , i am less inclined to accept that the parasites develop hereditary resistance to quinine , and deem it more likely that that there is selection and increased virulence in particularly virulent , resistant strains . the conditions that neiva , nocht , werner , mhlens and others have presented as being propitious for the development of quinine - resistant races are valid . the parasites have always found a way to partially escape quinine , increase their virulence in new ground and adapt in some way to the alkaloid . . . generally speaking , i am less inclined to accept that the parasites develop hereditary resistance to quinine , and deem it more likely that that there is selection and increased virulence in particularly virulent , resistant strains . later , in 1921 , schilling modified his view slightly , bringing it more into line with rodenwaldt s : i have reached the idea of parasite races that are resistant to quinine by practical means , in a severely affected population which received an unreliable supply of the alkaloid , [ with the process observed not being due ] to developed and inherited resistance , but rather to selection and increased virulence , especially in more lethal and resistant parasite strains . rodenwaldt later tried to make selection the theoretical basis for this process , exactly in the same sense i gave to him . i have reached the idea of parasite races that are resistant to quinine by practical means , in a severely affected population which received an unreliable supply of the alkaloid , [ with the process observed not being due ] to developed and inherited resistance , but rather to selection and increased virulence , especially in more lethal and resistant parasite strains . rodenwaldt later tried to make selection the theoretical basis for this process , exactly in the same sense i gave to him . rodenwaldt reacted to this claim , which he believed diminished his contribution to the debate , thus : it is far easier , with so many and such broad possibilities , for the person to defend the idea as their own later , when one of these lines becomes more likely. he went on to reiterate his belief in the selection of pure lineages of already existing resistant strains in which , furthermore , nothing is altered , there is no increase or diminishment of virulence , and they do not adapt to anything at all. in his rebuttal , schilling accused rodenwaldt of having appropriated his ideas , which he had learnt about in smyrna , a province in south - western turkey , where they had met in 1918 . even so , schilling acknowledged that the way rodenwaldt had developed baur s ideas was an interesting , independent theoretical accomplishment which far outstrips my scant experiences derived from practice. rodenwaldt s reply ( 1923b , 404 ) was curt and was accompanied by a note by the archiv editors : the tone of the text had been moderated , as it had involved accusations of a personal nature , and the objective discussion of the matter was now closed . today , the resistance of the plasmodium to antimalarial drugs is one of the big problems facing international health care , prompting the continued development of different therapeutic compounds and combinations of drugs . in this study , we show that quinine resistance was already recorded in the early decades of the twentieth century in some regions of brazil , giving rise to different theories to explain it . at the time , the alkaloid was practically the only option available for preventing and treating outbreaks of malaria . the theory of the development of quinine - resistant races of the plasmodium put forward by arthur neiva would appear to be the first scientific work in the literature about malaria that attempted to give an explanation for the persistence of the disease even when the alkaloid was taken in ever higher doses . these preliminary hypotheses were never verified theoretically or experimentally by neiva , the researcher from the oswaldo cruz institute , whose career took a different turn . meanwhile in brazil , the supremacy of preventive approaches which focused primarily on the vector of malaria ( adopted in the campaigns started by the rockefeller foundation after the first world war ) put strategies designed to address the parasite , and the preventive and therapeutic use of quinine on the back burner . neiva s hypothesis does not seem to have inspired any particular interest amongst brazilian specialists . neiva s observations about the malaria parasite s resistance to quinine reveal the interdependency between laboratory and field work in overcoming public health problems and producing new knowledge about tropical diseases . vast field of observations and experiments. this interdependency is characteristic of the institutionalisation of experimental medicine during the brazilian belle poque . the anti - malaria campaigns were a key part of the collaborative efforts of doctors keen to make their names as scientists in conjunction with the public and private clients that sought their assistance , through both the old and the new biological and medical institutions in which they worked . they did research , ran projects and ultimately made important advances in the most pressing problems of the day , affecting not just public health , but also engineering , farming and resource exploitation . field research was essential for understanding what linked human population groups to parasites , with their complex life cycles and multiple vectors , in intricate chains of interdependency in the natural and social ecosystems . in a manner that was not unlike the scientists working for the administrations of european colonial possessions , brazilian researchers like neiva saw the unexplored brazilian hinterland as a potential repository of new scientific data . as helen tilley reports about britain s african colonies , while they [ the colonial scientists ] evoked the authority of laboratory knowledge , they simultaneously challenged the physical boundaries and natural validity on which that authority was based. the wetlands in the fluminense lowlands were not exactly a laboratory because they presented the biological phenomena in all their complexity in a space that was partially immune to the manipulations and controls inherent in experimental research . however , the field was a fine observation context for malaria because at the time the different plasmodium species related to it could not be grown artificially . according to leo slater , by different routes from those of neiva and other tropical medicine specialists , researchers working on new drugs for malaria did their best to reconstitute the complex connections between parasite , host and vector in the laboratory . in brazil s vast unexplored inland regions , researchers like arthur neiva and carlos chagas obtained new knowledge for tropical medicine . as had been the case when the theory of quinine resistance had been formulated , it was also in a campaign to fight malaria ( in itatinga , in 1905 ) that chagas formulated his hypothesis about domestic infection by anophelines , which would be called on years later to support the campaign to kill malaria vectors by using ddt inside homes . while he was engaged in a campaign against malaria in the worksites for the central do brasil railway , chagas identified the clinical , etiological and epidemiological phenomena of a new human trypanosomiasis which was later named after him . just as it had been propitious for the study of quinine resistance , the exchange between brazil and germany contributed to the communication of the discovery of chagas disease . it was in the archiv fr schiffs- und tropenhygiene that the brazilian scientist first published his findings , identifying trypanosoma cruzi , its vector , and the clinical aspects of the disease , which was so different from the well - known sleeping sickness . the recognition of the brazilian discovery came in june 1912 , when chagas was awarded the schaudinn prize by the hamburg institute of maritime and tropical diseases as the best work in the field of protozoology . interestingly , the presence of german researchers at the instituto oswaldo cruz contributed towards the theoretical research of the new trypanosomiasis , as reported by magali s . malaria , chagas disease and ancylostomiasis formed the triad of diseases which , during and after the first world war , galvanised debate in brazil about the need to extend public health services into the inland areas of the country . sanitation movement was spearheaded by intellectuals , writers and scientists , including chagas and neiva , who drew attention to the prevalence of disease amongst the people who were left at the mercy of god when it came to medical care. according to neiva , one of the factors that exacerbated malaria amongst these population groups was the incorrect use of quinine , which was sold mainly in the form of a sulphate that was effective neither for prevention nor for treatment . as head of public health in so paulo from 1916 to 1920 , neiva faced some serious malaria epidemics in inland and coastal areas of brazil s richest state . alongside measures to attack the mosquitoes , he recommended treating the sick with quinine . the difficulty of importing it during the first world war prompted him to start quinine production at the butant institute , run by the so paulo public health service . from the 1920s onwards , neiva started working mainly in the organisation and administration of scientific institutions and , from 1930 , as a politician . even though he no longer researched malaria , it continued to be a topic of interest to him , albeit now in the arena of debates and political action . and despite having witnessed quinine resistance first - hand , neiva held by the opinion that it was the most reliable method for preventing and treating the disease . in a letter he wrote in 1935 to a fellow public health expert , he criticised the aggressiveness with which the german pharmaceuticals industry was marketing its new antimalarial agents : i am sure that quinine will valiantly withstand all these tests , but responsible work must be done under strict scientific determinism , and these new products manufactured by german laboratories after experiments using bird haematozoa and launched on the market under treacherous commercial conditions must be kept at bay . i am sure that quinine will valiantly withstand all these tests , but responsible work must be done under strict scientific determinism , and these new products manufactured by german laboratories after experiments using bird haematozoa and launched on the market under treacherous commercial conditions must be kept at bay . one of the people involved in marketing the new malaria drugs was peter mhlens , director of the clinical section of the institute for tropical diseases in hamburg . in august 1925 this drug had been developed that year by werner schulemann , fritz schonhfer and august wingler in the laboratories of ig farben by modifying quinoline , one of the main components of quinine . in 1930 , ig farben also brought out atebrine , developed by hans mauss and fritz mietzsch . these synthetic malarial agents were presented as being more effective and incurring fewer side effects than quinine . mhlens deployed his network of scientific and political contacts to get testing approved in balkan nations like romania and bulgaria , and also in latin america , especially in 1931 , when he was in venezuela , guatemala and mexico . he was also in mexico in late 1926 , when he faced some resistance to the promotion of the new malaria drugs being supported by the german foreign ministry as part of its cultural diplomacy efforts . the mexican press warned mhlens that the public references to plasmoquine compromised his medical standing . a mexican military doctor also censured him for behaving not as a scientist , but as a sly travelling marketeer. in a series of articles published not just in german , but also in english , spanish , french and italian , mhlens set forth the advantages of plasmoquine and atebrine over quinine . the resistance of the plasmodium to the alkaloid was cited as one of its main disadvantages . mhlens also enumerated the main side effects of quinine as being blurred vision , nausea and ringing of the ears , and associated it with blackwater fever and quinine idiosyncrasy. in a text written with bernhard nocht , mhlens did admit that the malaria parasite may come to develop resistance to plasmoquine analogous to the so - called quinine - resistance , but went on to say that the veracity of this hypothesis remains for the present a debatable point. a publication from the following year shows that he saw resistance differently from neiva and rodenwaldt , which suggests that the latters hypotheses had not left the theoretical realm : my personal views on the matter have always been that the apparent resistance to medicine was less a characteristic of the parasite itself or even of certain strains of parasites than of the infected individual , in whom it represented a lowering of the protective powers of the organism . my personal views on the matter have always been that the apparent resistance to medicine was less a characteristic of the parasite itself or even of certain strains of parasites than of the infected individual , in whom it represented a lowering of the protective powers of the organism . in a chapter on malaria in the renowned clinical medicine handbook edited by klemperer he claimed that many cases of tropical malaria ( caused by p. falciparum ) were found to be resistant to treatment using drugs because the treatment was inadequate : this is why during the war and in the following years so much was said about the malaria parasites resistance to quinine. but this resistance was down to weakened immunity caused by factors such as malnutrition or other diseases . without doubt , the strength of the organism s defences exerts a fundamental role in any therapy . the medication alone is not capable of leading to a cure. this was equally true for quinine and plasmoquine , whose action on the plasmodium was indirect . when the national socialist party came to power in 1933 , mhlens competed for the top position at the institute of tropical medicine in hamburg . however , he was up against another key figure in this narrative , ernst rodenwaldt , the nazi party s favourite for his staunch anti - semitism and his involvement in the study and communication of theories of racial hygiene . from 1921 to 1934 , when he was working in dutch colonies in south east asia , primarily combating malaria , rodenwaldt published dozens of papers about racial hygiene or eugenics . his involvement with the problem of quinine resistance certainly influenced the campaign strategies adopted in asia . this attempt to apply modern heredity theories to the study of an infectious disease could be seen as a milestone in the development of ideas that ultimately led to his being well known as a racial hygienist. according to heiner fangerau , the combination of mendel and weissmann s ideas that formed the groundwork for rodenwaldt s analyses of resistance represented a watershed for the theories of racial hygiene , it becoming clear how predictable was the inheritance of isolated features. rodenwaldt s trajectory took a similar turn to one of his mentor s , erwin baur , the ideologue behind eugenics and author , with eugen fischer and fritz lenz , of the manual that inspired hitler s racial conceptions set forth in mein kampf ( fangerau , 2000 ) . die mestizen auf kisar ( 1927 ) , about the hereditary transmission of the biological features of a mixed - race population , earned rodenwaldt an eminent position in the international league of racial hygiene . during the years of national socialism , he was the main link between tropical medicine and the much - touted racial conceptions of the day . in the words of wolfgang eckart , notable protagonist of anthropology and racial hygiene in germany in the 30s and 40s , fervently defending racial segregation . as shown by christoph gradmann with regard to trypanosomes and arsenic compounds , the analysis of quinine resistance on the part of the plasmodium shows that this became an object of investigation far before the phenomenon gained the full attention of the medical community because of bacterial resistance to antibiotics starting in the 1960s . but unlike gradmann s findings concerning trypanosomes , the plasmodium s resistance to quinine was closely related to the frequency with which it occurred as a clinical phenomenon . years after the debates about the resistance of the malaria plasmodium to quinine , the resistance of bacteria to antibiotics prompted equally heated discussions in the field of genetics . angela creager suggests that the researchers involved in bacterial genetics in the 1940s and 50s rejected the conceptions of lamarck , by which microbes adapted to drugs and transmitted this property to their progeny . however , the bacteriologists had the chance to replicate resistance in the laboratory as they cultivated bacteria in the presence of antibiotics , unlike the researchers working on malaria resistance at the beginning of the century . the role of mutations in the emergence of variations that granted bacteria their resistance was discussed . from the perspective of mendelian genetics and darwinism , resistance was seen as the selection of mutated lineages that had developed randomly from a population of bacteria that were sensitive to the antibiotic . experimental studies and theoretical speculations , creager continues , led to the identification of the means by which the genetic inheritance was transmitted without involving the chromosome core , ultimately revealing that in the end , adaptation versus mutation turned out to be a false dichotomy. the interpretations of bacterial resistance to antibiotics were applied to the analysis of the similar phenomenon seen amongst malaria parasites : spontaneous mutations conferring reduced sensitivity to a given drug or class of drugs . nevertheless , from the 1950s onwards , the drug that drew specialists attention when it came to resistance was not quinine , but chloroquine , the most widely used drug in malaria treatment since its development . its proven efficacy considerably restricted the use of quinine , and chloroquine was itself succeeded by other drugs , including primaquine , mefloquine and proguanil . this fact helps explain why neiva , nocht , werner and rodenwaldt s observations had such a limited impact and became reduced to mere footnotes in narratives about malaria treatment . and yet , echoes of these authors deductions can be heard in the way researchers currently address the plasmodium s resistance to chloroquine and other drugs : population movements induce the introduction of resistant parasite strains , while the widespread , large - scale use of the drugs operates as a selective pressure in that it removes populations of susceptible protozoa and maintains the resistant ones . the ways the leading actors and conceptions in malariology interacted in different contexts in the twentieth century are not clear - cut and beg further exploratory study . the scientific and geopolitical cartography that emerged after the second world war was a far cry from the context when neiva , nocht and rodenwaldt were active in their fields . this war fostered appropriations of scientific ideas and actions that are not always clear or obvious . the alliance between german researchers and the german chemicals and pharmaceuticals industry , which was strengthened during the weimar republic , became tarnished during the nazi regime and the second world war . not only was rodenwaldt an active promoter of racial hygiene theories , for which he was put on trial at nuremberg , but , when the germans retreated in the face of the advancing allied troops , he was also instrumental in the destruction of italy s sanitation engineering system , which had ensured the control of malaria in the country , resulting in a significant upsurge in the proliferation of the disease . it is also important to remember that the potentialities and limitations of the work of neiva and other brazilian scientists were also related to the fact that it was produced in a country which by many was considered subaltern to europe s centres of civilisation. however , as we follow the research and disputes on both sides of the atlantic , we can see not just how internationalised tropical medicine was in those early decades of the twentieth century , but also how the interaction between the researchers on both sides took such complex and convoluted paths and dynamics . brazilian doctors like arthur neiva , miguel couto and oswaldo cruz did not restrict themselves to adopting and adapting theories and practices that circulated through publications , correspondence , conferences , exchanges of patients and biological materials , but actually formulated new concepts and theories that had an impact on the central social formations . the study of the historical records about the resistance of the malaria parasite to quinine demonstrates how important the research into the topic was to individual careers and to the formation of collective ideas , which , despite being involved in international research programmes , followed national styles in the way they studied and fought the disease , a prototypical tropical disease ,

this article addresses the discussion about quinine - resistant malaria plasmodium in the early decades of the twentieth century . observed by arthur neiva in rio de janeiro in 1907 , the biological and social resistance of malaria sufferers to preventive and curative treatment with quinine was corroborated three years later by oswaldo cruz during the construction of the madeira - mamor railway in the brazilian amazon . likewise in 1910 , ailing german workers were transferred from brazil to hamburg s institute for maritime and tropical diseases , where quinine resistance was confirmed by bernard nocht and heinrich werner . when the first world war saw failures in treating and preventing malaria with quinine along with violent outbreaks of the disease on the turkish and balkan fronts , resistance to this alkaloid became the topic of the day within the field of experimental medicine in germany . new attempts were made to account for the resistance , especially by the physician ernst rodenwaldt , who explored the topic by applying modern theories on heredity . the present article offers a preliminary survey and analysis of pronouncements about quinine resistance , shedding new light on the circulation of knowledge in the field of tropical medicine .

Malaria Campaigns in Brazil and Resistance to Quinine German Medical Doctors Prove Malaria Resistance Malaria During the First World War: Quinine Resistance on the German Medical and Scientific Agenda Ernst Rodenwalt and Quinine Resistance in the Light of Modern Genetics Concluding Remarks

ovarian cancer represents the sixth most commonly diagnosed cancer among women in the world and causes more deaths per year than any other cancer of the female reproductive system . in advanced disease which constitutes about 75% of women at presentation , this has been the approach for some decades , though the 5-year survival remains poor at about 40% . epithelial ovarian cancer constitutes the majority of disease types , and this review will focus on reports relating to advanced epithelial ovarian carcinoma . a medline database search ( january 1966 to april 2009 ) was undertaken using key words : epithelial ovarian cancer , debulking surgery , and interval debulking surgery resulting in 80 articles with 14 relevant papers . the articles in full were obtained for each of the papers and reviewed by the authors . results in terms of overall survival ( os ) and progression free survival ( pfs ) were evaluated in each study . the 80 resulting articles were screened and 14 relevant papers were retained : 3 meta - analysis [ 24 ] , 3 randomized control trials ( rtc ) [ 57 ] ( table 1 ) 2 cochrane reviews ( crs ) [ 8 , 9 ] , and 6 case / control ( cc ) reports enrolling more than 50 patients [ 1015 ] ( table 2 ) . the initial studies supporting the concept of debulking surgery were published in the 1970s by griffiths et al . . the premise for considering the potential impact of reducing intra - abdominal tumour burden was based on the findings of work by magrath et al . , which reported enhanced survival outcome by reducing intra - abdominal disease , in patients with hodgkin 's disease . griffiths undertook a retrospective analysis of just over 100 women and noted that those with residual disease masses < 1.6 cms in largest diameter had an improved survival outcome compared with patients left with a greater disease volume . a subsequent small prospective study on a heterogeneous population of patients , who underwent aggressive radical surgery , also revealed the better survival pattern associated with less tumour burden . thus , the concept of debulking surgery in ovarian cancer became the normal approach to this disease . the use of adjuvant chemotherapy , which is platinum based , is also the accepted norm in care . the question as to whether the surgical ability of the operator or the inherent tumour biology of the disease is the main factor impacting on survival remains a debate . indeed , the benefit of radical debulking has already come under criticism while some have advocated that tumour biology rather than the surgical effort might determine prognosis . in a study of 213 patients with stage iiic epithelial ovarian cancer who underwent complete cytoreduction before initiation of systemic platinum - based combination chemotherapy , eisenkop and spirtos came to the conclusion that the need to remove a large number of peritoneal implants correlates with biological aggressiveness and diminished survival , but not significantly enough to preclude long - term survival or justify abbreviation of the operative effort . regarding primary surgery , there is a plethora of published papers , all of which support the findings of griffiths , though none are randomized controlled trials , and hence , all with similar inherent biases . it is also important to note that various definitions of optimal cytoreduction have been proposed [ 2224 ] . the gynaecologic oncology group ( gog ) currently defines optimal cytoreduction as leaving residual disease less than 1 cm in maximum tumour diameter . there are 3 systematic reviews on residual disease and outcome , which have conflicting conclusions . in an analysis of 81 cohorts of patients ( over 6000 women ) with advanced - stage ovarian carcinoma treated with platinum - based chemotherapy bristow et al . found a 5.5-percent increase in median survival for every 10-percent increase in the proportion of patients achieving maximal cytoreduction . , ( again over 6000 women ) whereby the administration of platinum was deemed more important in influencing survival rather than the achievement of optimum debulking surgery . the main difference between these papers is that in bristow 's study , all patients were exposed to adjuvant platinum therapy , which was not the case in hunters study . the third and smaller study also concluded that optimum debulking was associated with improved survival patterns , though further prospective trials were necessary . at the beginning of the eighties , berek et al . noticed that secondary cytoreduction could also improve survival subsequently , the role of interval debulking surgery ( ids ) has been investigated in three prospective randomized controlled trials ( rcts ) [ 57 ] where conclusions are different . interval debulking surgery is defined as a second operation performed after 3 or 4 cycles of platinum chemotherapy in woman who had suboptimal debulking primary surgery . the trials by redman et al . and the gog by rose et al . failed to show any advantage of ids . the study by redman was closed prematurely , as no survival benefit was noted at interim analysis , and of note , optimum debulking was defined as < 2 cms residium compared with < 1 cms in the other studies . in the gog study , 550 women with suboptimally debulked stage iii / iv ovarian cancer received three cycles of paclitaxel / cisplatin and then were randomly assigned to interval cytoreduction or no surgery . a secondary attempt at cytoreduction was not associated with an improvement in progression free survival ( pfs ) ( 12.5 versus 12.7 months ) or overall survival ( os ) ( 36.2 versus 35.7 months ) . this was not the case with the eortc trial carried out by van de burg et al . , which showed that the ids group had a significantly increased median survival of 6 months compared to those who had not undergone this procedure . indeed this is still the only prospective rct showing a survival benefit with debulking surgery . nevertheless , it is important to point out some differences between these trials . at the time of the eortc trial , chemotherapy consisted of cisplatin / cyclophosphamide as paclitaxel was not available , unlike the gog trial . another major difference was that in the eortc trial , primary surgery was not necessarily performed by a trained gynaecological oncologist , resulting in different extents of debulking . the number of patients with less than 5 cm of residual tumour following primary cytoreduction in the eortc trial was less than a third , compared to 55 percent in the gog trial . surgery performed by a trained gynaecological oncologist has been shown to increase survival , and the gog study therefore concludes that with appropriate persons undertaking primary surgery , ids is not required . the term ids should be confined to patients who have had primary surgical debulking , but it has been used in situations whereby a primary surgical attempt is delayed until during chemotherapy . six large case - control studies [ 1015 ] relating to delayed primary surgery were identified , and are summarized in table 2 . one of the studies [ 10 , colombo et al . ] divided patients into 2 groups to evaluate the place of surgery in the therapeutic sequence of care : group 1 receiving upfront surgery and group 2 where first debulking was undertaken after chemotherapy . in group 1 the os was 38 months and 3 factors significantly predicted suboptimal upfront surgery : poor performance status , extensive mesenteric involvemen , and stage iv disease . the second group showed os of 26 months , and despite a response to nact in 90% of cases , there was no long - term survivors in the patients whose interval cytoreduction was suboptimal . generally , os was stated to be influenced by three main factors : the extent of the disease at the time of diagnosis , the biology of the tumour , and its chemosensitivity , and the authors concluded that optimal surgery with limited morbidity ( 14% in their case ) can be achieved in many cases at primary surgery setting . hegazy et al . found , in a population of patients with advanced ovarian carcinoma where resectability was not possible , that neoadjuvant chemotherapy helped to select patients for feasible and relatively less aggressive ids , thus preventing initial surgical failure , in terms of optimal debulking . however , morris et al . in 1989 demonstrated that patients resistant to chemotherapy during primary treatment had little benefit from ids . this was also concluded by rafii et al . as well as the selection effect of nact for the second intention surgery . in another recent study , the complete response rates after three cycles of platinum / taxane chemotherapy was 36.1% . after ids , 80% of all patients were left with optimal residuals ( < 2 cms ) . the response rate to chemotherapy given in a neoadjuvant setting was comparable to those published in literature in patients who were treated with conventional upfront tumour reduction surgery followed by adjuvant chemotherapy . they also found that residual decease after ids is the only significant predictive factor associated with prolonged pfs ( p = .003 ) . to date , there is very little good quality evidence to either support or refute the use of neoadjuvant chemotherapy in the treatment of ovarian cancer . a retrospective study between 1980 and 1997 from vergote et al . included 285 patients with stages iii and iv ovarian cancer . in the period from 1980 to 1988 , optimal primary cytoreduction ( 0.5 cm residual disease ) was achieved in 82% of cases , but patients with stage iv disease or a metastatic tumour load of > 1 kg prior to this procedure had poorer survival with high postoperative mortality ( 6% ) . between 1989 and 1997 patients received either upfront surgery or chemotherapy depending on the extent of the disease and the performance status . this subsequent management improved overall survival , despite a reduction of 25% in the rate of primary debulking . a large norwegian retrospective study ( n = 789 ) carried out at the radium hospital looked at treatment model for 1st relapse of ovarian cancer of any stage . they found that treatment free interval ( tfi ) following primary therapy is a significant prognostic factor for os in multivariate analysis . they also report age as prognostic factor for os at the time of secondary cytoreductive surgery . survival benefit was clear for patients with optimum secondary cytoreductive surgery followed by chemotherapy compared with chemotherapy alone at the time of recurrence . complete secondary cytoreductive surgery was found possible in a significant percentage of patients properly selected for this secondary surgery . guidelines at relapse for local and disseminated disease have been set up , where secondary cytoreductive surgery is recommended as independent of tfi for localized tumours and should be considered for tfi > 24 months in case of disseminated disease ( table 3 ) . selecting the right patients for the right treatment sequence has been studied [ 28 , 29 ] with one model having an 85% specificity or ability to identify patients undergoing optimal surgery . in certain situations laparoscopy is recommended as the most valuable tool for evaluating the operability in upfront or second line debulking surgery . this paper has reviewed only rcts and large series , which do reflect the findings of many other reports on the specific debates surrounding the role and timing of surgery in ovarian carcinoma . there is agreement that one of the most important prognostic factors for survival in the treatment of ovarian cancer is the amount of residual tumour after cytoreduction [ 4 , 16 ] . it is welcome to note that in more recent times surgical approaches have undergone scrutiny in rcts . indeed there is evidence of a shift from debulking for all to debulking for a select group , or put another way increased individualisation of therapy . unlike in previous decades the use of neoadjuvant chemotherapy seems to have gained some popularity , though the real impact requires the formal publication of the randomized trials eortc 55971 and chorus . the eortc study has been presented at the igcs in bangkok and generated a lot of debate , as to the role of neoadjuvant chemotherapy . another factor which can not be ignored in the debate is the inherent tumour biology where the question , raised by some and still requiring an answer , is to know if it is the surgeon 's skills or tumour biology which determines survival outcome . in this respect , opinions vary regarding its impact on the ability to surgically debulk . on the other hand , others have put forward the strong expression of the p53 tumour suppressor gene correlating with reduced likelihood of achieving complete cytoreduction . the progress and accessibility to novel technologies applied to biology will make possible in the future the assessment of new prognostic profilesbased on genetic and/or proteomic tumour characteristics .

initial surgical management is commonly accepted to date as paramount in the treatment of women presenting with epithelial ovarian cancer and permits the assessment of the disease ( staging ) , the histological confirmation of disease type and grade , and the practice of maximal debulking preceding platinum - based chemotherapy . many studies have shown that the volume of residual disease after initial surgical cytoreduction inversely correlates with survival . thus , women with optimal debulking performed by a trained specialist have improved median survival . in this review , we will focus on the answers gleaned from clinical trials on primary and interval surgery , which prompts the question on the timing of surgery in respect to chemotherapy . interval debulking surgery ( ids ) is secondary cytoreduction following primary debulking and is carried out in between the courses of chemotherapy . the major clinical trials and the latest systematic reviews seem unable to give any definitive guidance or recommendation for clinical practice . the choice of aggressive primary cytoreduction or upfront chemotherapy followed by second line surgical cytoreduction seems among others to have to be individualized according to tumour load , prediction of its resectability , and response to chemotherapy . the role of tumour biology must also be kept in mind . finally , concrete answers are awaited on the timing of surgery from the ongoing prospective randomized control trials ( chorus and eortc 55971 ) though preliminary data from the latter have already been presented at major meetings ( igcs 2008 ; sgo 2009 ) and ignited strong debate .

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

to report a rare presentation of unifocal langerhans cell histiocytosis ( lch ) simulating a limbal papilloma . a 24-year - old man presented with a limbal mass in his left eye which had initially been suspected to be a papilloma based on clinical findings . the mass was excised and a histopathological diagnosis of acute bullous inflammation with granulation tissue was made . the lesion relapsed 10 months later which necessitated repeat resection along with corneoscleral patch grafting . to the best of our knowledge , this is the second report of a rare presentation of lch in the limbus which recurred after excision of the primary mass . langerhans cell histiocytosis ( lch ) is a relatively rare disorder characterized by monoclonal proliferation of histiocytes . the condition can be unifocal with involvement of a single organ , or multifocal presenting as disseminated disease . unifocal involvement is the most common presentation of lch and the bone is the most frequently affected tissue.1 ocular involvement can be observed in 1% to 20% of lch cases.2 the condition may primarily involve the orbit , eyelid , epibulbar conjunctiva , cornea , iris , vitreous , choroid and optic nerve , or secondarily infiltrate ocular tissues from surrounding structures.3,4 intraocular involvement by lch has rarely been reported in letterer - siwe disease.5 the orbit is usually involved with unifocal lch , which has a distinct tendency for the superotemporal bone at the rim of the orbit.6 the limbus can rarely be involved in unifocal lch presenting clinically as an infiltrative solitary limbal nodule which may be misdiagnosed as amyloidosis , fibrous histiocytoma , lymphoma , and juvenile xanthogranuloma.2,7 the diagnosis of lch is based on characteristic histopathological features . electron microscopy may also assist the diagnosis by demonstrating intracytoplasmic inclusions named birbeck granules.1 the natural course of untreated lch varies from progressive and fatal systemic disease to localized self - limited lesions.8 local resection for unifocal involvement and systemic chemotherapy for extensive multifocal lesions have been proposed as treatment modalities in lch,9 however spontaneous regression has been reported in some unifocal lch cases.1 herein , we report a case of unifocal lch presenting as an unusual limbal lesion which recurred after primary excision . a 24-year - old man was referred to our center for a recurrent and painless limbocorneal lesion in his left eye . the primary lesion had been removed 10 months earlier with a clinical impression of limbal papilloma at another eye care center . the histopathological diagnosis had been acute bullous inflammation with granulation tissue composed of scattered single- and multi - nucleated histiocytes . uncorrected visual acuity was 20/20 in the right eye and 20/80 in the involved left eye . on slit lamp biomicroscopy , there was an elevated vascular mass extending from the temporal limbus to the central cornea in the left eye . intraocular pressure was within normal limits and fundus examination was unremarkable . with a clinical diagnosis of a recurrent limbocorneal papillomatous / dermoid - like lesion , the patient underwent mass resection together with corneoscleral patch grafting and lateral tarsorrhaphy . slit lamp photography was overlooked preoperatively since the lesion was thought to be a simple recurrent limbal papilloma . after surgery the patient received topical betamethasone 0.1% and chloramphenicol 0.5% eye drops along with non - preserved lubrication four times a day for four weeks . on gross histopathological examination , light microscopy disclosed non - keratinized stratified squamous epithelium overlying a diffuse inflammatory background composed of nests and clusters of large histiocytes with indented and grooved nuclei intermixed with lymphocytes , plasma cells and eosinophils ( figures 1a and 1b ) . the histopathological features were characteristic of lch with incomplete surgical excision . during follow - up and after four months , no signs indicative of recurrence lch results from proliferation of langerhans cells normally present in the epidermis.10 the condition is characterized by a wide clinical spectrum varying from spontaneous regression to rapid progression , recurrence and long - lasting sequelae.11 lch encompasses three main clinical subtypes ; unifocal lch ( eosinophilic granuloma ) , multifocal lch ( hand - schuller - christian disease ) and systemic lch ( abt - letterer - siwe disease).1 younger patients with lch , have a predisposition for multifocal involvement.12 the etiology of lch remains unclear but immune dysregulation with different cytokines has been postulated.1 the orbit is the most common site of involvement in the eye which often includes the orbital diploe . mononuclear histiocytes and multinucleated giant cells are intermixed with eosinophils , lymphocytes , plasma cells and neutrophil polymorphs.10 langerhans cells are immune reactive for s-100 protein and cd1a.1 electron microscopy demonstrates tennis racket shaped cytoplasmic inclusions named birbeck granules in histiocytes which are the gold standard for a diagnosis of lch.1,12 treatment options for unifocal lesions include observation , partial or complete resection , combined resection and low dose radiation , and intralesional corticosteroids . systemic chemotherapy may be indicated for extensive multifocal and systemic lesions.1,9 to the best of our knowledge , after the report by saxena et al2 , our patient is the second case of unifocal limbal lch presenting as a recurrent solitary vascularized nodule . despite the presence of mono- and multi - nucleated histiocytes amongst the intrastromal inflammatory cell infiltrates on histopathological examination of the primary lesion , lch was not diagnosed initially which might be due to the very rare presentation of this condition in the limbocorneal area . differential diagnoses for limbocorneal lch include dermoid , amyloidosis , fibrous histiocytoma and juvenile xanthogranuloma.2 in our case , a limbal papilloma was the initial clinical diagnosis which should be added to this list . there is only a single case report of limbal lch in which the patient remained asymptomatic for a while after complete excision of the primary lesion , but the tumor recurred 15 months afterwards.2,7 based on clinical and histopathological evaluations , the recurrent tumor partially responded to combination chemotherapy and the patient was clinically stable for 5.5 years.7 in our case , recurrence developed 10 months after resection of the primary lesion for which repeat resection together with corneoscleral patch grafting were performed and the patient has been tumor - free up to 4 months . although bakhshi et al7 stated that surgery or radiotherapy for unifocal ocular involvement improves function or cosmesis , the functional and cosmetic outcomes of surgical intervention alone were also favorable in our patient . the other treatment option for limbal lch is chemotherapy . theoretically due to poor penetration of chemotherapeutic agents into the corneal side of the tumor , such treatment may be associated with a partial response.7 whether surgical therapy , in the form of resection of the recurrent lesion along with a limbocorneal patch grafting , is associated with a low risk of recurrence or not requires longer follow - up . in summary , herein we report the second case of recurrent limbocorneal lch which had missed correct diagnosis on initial histopathological studies . the recurrent lesion was managed surgically which included mass resection and a limbocorneal patch grafting , with satisfying cosmetic and functional results .

purposeto report a rare presentation of unifocal langerhans cell histiocytosis ( lch ) simulating a limbal papilloma.case reporta 24-year - old man presented with a limbal mass in his left eye which had initially been suspected to be a papilloma based on clinical findings . the mass was excised and a histopathological diagnosis of acute bullous inflammation with granulation tissue was made . the lesion relapsed 10 months later which necessitated repeat resection along with corneoscleral patch grafting . histopathological studies of the excised lesion led to a final diagnosis of lch.conclusionto the best of our knowledge , this is the second report of a rare presentation of lch in the limbus which recurred after excision of the primary mass . the recurrent lesion was diagnosed based on histopathology and managed accordingly .

Purpose Case report Conclusion INTRODUCTION Case report DISCUSSION

a 2-year - old asian indian female presented to us with mild fever and swelling of the right upper lid of 10 days duration . there was no history of preceding viral illness or significant medical history necessitating treatment with antibiotics . cutaneous anthrax was unlikely as there was no history of unexplained cattle death in her environment . on examination , the child had low - grade fever and there were no other skin lesions . ophthalmological examination revealed right upper lid edema with a large black necrotic area of the lid which was adherent to the underlying tissues . the child was examined by a pediatrician to rule out any other focus of infection . microscopic examination of the skin biopsy revealed staphylococci and hence cutaneous anthrax was ruled out . the child was started on intravenous cefotaxime for a week with resolution of fever and the necrotic area turned to a well - defined eschar with no edema and induration . after 2 weeks , the child underwent escharotomy with wound debridement and full thickness skin graft from the groin [ fig . 2 ] . under general anesthesia , the groin area was cleaned and draped . the eschar on the lid was found to be partial thickness , was excised in toto , and the wound margins were debrided . the harvested skin was placed over the lid defect and sutured with 6 - 0 prolene . clinical photograph of the child showing large black necrotic area of the right upper lid adherent to the underlying tissues with surrounding erythema and edema and no discharge immediate postoperative clinical photograph showing full thickness skin graft from the groin postoperative photograph at 1 week showing healthy well - taken graft bacterial invasion of the arteries in the dermis and subcutaneous tissues produces a necrotizing vasculitis . the characteristic clinical appearance of eg is a red macule that progresses to a nodular or ulcerative lesion with central area of necrosis surrounded by erythema . bullae develop subsequently and become filled with mucopurulent or serosanguinous fluid . in the end stage , the lesions become hemorrhagic and slough off , leading to a necrotic eschar . progression through these stages is rapid , typically occurring within 1224 h. there are few reports of this condition developing in healthy individuals without any predisposing factors . usually , eg is associated with bacteremia , but can also occur in the absence of it . classic eg rarely involves the periocular tissues and to our knowledge , only a few such cases have been described in the literature . maccheron et al . presented a case of eg that led to orbital cellulitis and panophthalmitis . inamadar et al . described a diabetic individual who developed severe periorbital eg after suffering a laceration to the forehead . ghosheh and kathuria reported a case of bilateral periorbital eg in a diabetic male with renal failure . the mortality rate in nonsepticemic cases varies between 0% and 15% compared with 2096% for those associated with septicemia . the closest differential diagnosis in our case was necrotizing fasciitis , but on the basis of clinical features and negative blood cultures , a diagnosis of eg was entertained in this case . the diagnosis of necrotizing fasciitis depends on clinical features , blood cultures , and gram stain to identify causative organisms and these patients usually have septicemia with positive blood cultures . the eschar formed following antibiotic administration was a full thickness eschar adherent to surrounding tissues and the lesion caused ectropion and mechanical ptosis , which blocked the pupil . considering the possible complications of scarring including entropion or ectropion , trichiasis , corneal exposure , and amblyopia in the child , surgical intervention was indicated . to the best of our knowledge , there are no reports of skin grafting being done as a treatment modality for eg . our patient was atypical in that eg was due to methicillin - resistant staphylococcal infection in contrast to all the four reports where there was pseudomonas infection . the case also highlights the need of early surgical intervention in such circumstances so as the probable sequelae of scarring of upper eye lid , resulting in mechanical ptosis which can result in stimulus deprivation amblyopia can be prevented . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .

ecthyma gangrenosum ( eg ) is a cutaneous infection which usually occurs in immunocompromised patients . we report a case of eg of the eyelid treated with escharotomy and skin grafting , highlighting the importance of surgical management . a 2-year - old asian indian female presented to us with right upper lid edema with a large necrotic area . the child received intravenous cefotaxime for a week and the necrotic area turned to a well - defined eschar . escharotomy with wound debridement and skin grafting was done . the present case highlights the importance of surgical intervention to prevent the sequelae of scarring of upper lid .

Case Report Discussion Declaration of patient consent Financial support and sponsorship Conflicts of interest

in recent years , the results after surgery for rectal cancer in norway , with a 5-year overall survival ( os ) rate of 60.1% , has surpassed that of colon cancer at 57.5% . this has been achieved because the surgical technique has been standardized according to total mesorectal excision ( tme ) with subsequent dramatic reductions of local recurrences . beginning in 2007 , all colon cancers were to be reported separately to the norwegian national cancer registry in an effort to systematically survey and hopefully improve results . nevertheless , a national strategy to standardize surgical treatment along the lines of radical surgery has neither been implemented in detail nor been generally accepted [ 2 , 3 ] . in this respect , the number of lymph nodes retrieved may act as a surrogate measure of radical surgery . the survival benefit of a large lymph node harvest has been shown in several reports [ 24 ] . it has been accepted nationally to offer patients with tumor node metastasis ( tnm ) stage iii below a certain age , usually 75 years , adjuvant chemotherapy . it has been decided that a rather arbitrary level of 12 retrieved nodes is enough to obtain adequate surgery and staging . pathologists may be a key factor for optimal lymph node harvest , and a conjoined effort between surgeon and pathologist would be ideal to improve results [ 26 ] . the aim of the study was to examine , after modest radical colon surgery removing mesocolic nodes and focus on lymph node yield , what would influence survival and where surgical improvement might be possible using data from a cohort of patients from three large norwegian teaching hospitals . patients from a national cohort were operated in 2000 , and follow - up was until december 2007 , a mean of 7.5 years later . three teaching community hospitals , haraldsplass deaconal hospital , stavanger university hospital , and akershus university hospital contributed patients . all three hospitals are teaching community hospitals , and the patients were operated with an open access by a large number of surgeons . at that time , extra radical surgery was unusual , and it is fair to assume that radical surgery usually constituted a moderate mesocolic resection . if metastases were diagnosed , patients and tumor conditions were assessed regarding feasibility for resection . patients usually went to the outpatient clinic every third month for the first 2 years and then every sixth month until 5 years had passed . blood tests with carcino - embryonic antigen measurement and ultrasonography of the liver and chest x - ray were carried out . elderly patients are stead - bound and even if a few of them were not followed up frequently , they could be tracked and life status ascertained through their identity number in the official national population registry . the specimen was examined and rinsed by the surgeons on the back table before being mounted on a board and placed in a box filled with enough formaldehyde for secure fixation . the specimen was examined by a junior pathologist ; after 4872 h , assisted by the consultant . tissue was paraffin - embedded , and hematoxylin eosin staining was used routinely before sections were examined microscopically . metastatic deposits were defined as lymph nodes if these structures resembled nodes but without containing visible lymphatic tissue . patients younger than 75 years of age that were classified as tnm stage iii were offered 12 courses of adjuvant treatment with 5-fluorouracil plus calsiumfolinate ( flv ) . the regional committee for medical and health research ethics of western norway and the data inspectorate for national registries approved the study . the chi - square test was used to compare groups with respect to categorical variables and analysis of variance for continuous variables . the following variables were analysed with respect to survival : hospital , age , gender , location , no of lymph nodes and lymph node ratio ( lnr ) , t stage , and tnm stage . survival curves were estimated by the kaplan meier method and compared using the log - rank test . multiple prognostic factors were analysed with the cox proportional hazards model using the spss 17 package . all three hospitals are teaching community hospitals , and the patients were operated with an open access by a large number of surgeons . at that time , extra radical surgery was unusual , and it is fair to assume that radical surgery usually constituted a moderate mesocolic resection . if metastases were diagnosed , patients and tumor conditions were assessed regarding feasibility for resection . patients usually went to the outpatient clinic every third month for the first 2 years and then every sixth month until 5 years had passed . blood tests with carcino - embryonic antigen measurement and ultrasonography of the liver and chest x - ray were carried out . elderly patients are stead - bound and even if a few of them were not followed up frequently , they could be tracked and life status ascertained through their identity number in the official national population registry . the specimen was examined and rinsed by the surgeons on the back table before being mounted on a board and placed in a box filled with enough formaldehyde for secure fixation . the specimen was examined by a junior pathologist ; after 4872 h , assisted by the consultant . tissue was paraffin - embedded , and hematoxylin eosin staining was used routinely before sections were examined microscopically . metastatic deposits were defined as lymph nodes if these structures resembled nodes but without containing visible lymphatic tissue . patients younger than 75 years of age that were classified as tnm stage iii were offered 12 courses of adjuvant treatment with 5-fluorouracil plus calsiumfolinate ( flv ) . the regional committee for medical and health research ethics of western norway and the data inspectorate for national registries approved the study . the chi - square test was used to compare groups with respect to categorical variables and analysis of variance for continuous variables . the following variables were analysed with respect to survival : hospital , age , gender , location , no of lymph nodes and lymph node ratio ( lnr ) , t stage , and tnm stage . survival curves were estimated by the kaplan meier method and compared using the log - rank test . multiple prognostic factors were analysed with the cox proportional hazards model using the spss 17 package . two hundred sixty - nine patients , 152 ( 56.5% ) women and 117 men , with a mean age of 71 years ( range , 2093 years ) were studied . one of the hospitals operated male patients that were younger , with a mean of 67 years . tumor locations were not different between the hospitals ( p = 0.059 ) . locoregional r0 resections for a single tumor location were done in 264 patients , while double resections ( n = 4 ) and a suspected locoregional r1 resection ( n = 1 ) were done in five patients . table 1tumor locations and pathology variables of patients resected for colon cancer at three teaching community hospitals in 2000variablesall patientschi - square testn = 269p valuelocation0.059 right hemicolon115 ( 42.8 ) transverse colon including flexures44 ( 16.4 ) descending colon9 ( 3.3 ) sigmoid colon and rectosigmoid96 ( 35.7 ) multiple or r125 ( 1.9)t category0.021 t111 ( 4.1 ) t230 ( 11.2 ) t3180 ( 66.9 ) t448 ( 17.8)tnm stage0.098 i34 ( 12.6 ) ii116 ( 43.1 ) iii93 ( 34.6 ) iv26 ( 9.7)resection of two tumors in separate locationsno radical loco - regional tumor resection tumor locations and pathology variables of patients resected for colon cancer at three teaching community hospitals in 2000 resection of two tumors in separate locations no radical loco - regional tumor resection there were no significant differences between hospitals relating to tnm stage distribution : 34 patients ( 12.6% ) were stage i , 116 patients ( 43.1% ) were stage ii , 93 patients ( 34.6% ) were stage iii , and 26 patients ( 9.6% ) were stage iv . the number of lymph nodes harvested for various stages were 8.7 ( stage i ) , 10.3 ( stage ii ) , 10.9 ( stage iii ) , and 10.3 ( stage iv ) . in 11 patients , the pathologist had classified the t category and tnm stage but omitted to specify the number of lymph nodes present . twelve or more lymph nodes were examined in 41.1% ( 106/258 ) of the resected specimens . significantly fewer lymph nodes ( p < 0.001 ) were harvested at one of the hospitals . otherwise , the three patient populations had similar characteristics . urgent surgery had a mortality of 12.5% ( 3/24 ) , whereas the elective group had a mortality of 4.5% ( 11/245 ) . did not differ between the hospitals ( log - rank p = 0.372 ; fig . 1kaplan meier survival curve for 269 patients resected for colon cancer in three norwegian hospitals in 2000 kaplan meier survival curve for 269 patients resected for colon cancer in three norwegian hospitals in 2000 whether categorizing number of lymph nodes harvested in three groups ( < 10 , 1019 , 20 or more ) or in two ( < 12 , 12 or more ) , no differences was found in os ( log - rank p = 0.423 and 0.270 , respectively ) . this was still the case after adjusting for hospital or tnm stage ( log - rank p = 0.449 ) . the uncategorized number of lymph nodes was not significant in a simple cox regression ( likelihood ratio p = 0.129 ) . however , in one hospital , better survival was found when the lymph node harvest was 12 compared to < 12 ( log - rank p = 0.037 ) as shown in fig . 2 . stage ii patients had os of 72.7% with 12 nodes harvested and 58.3% with < 12 nodes ( p = 0.124 ) , and stage iii patients had a 5-year os of 61.5% and 55.6% , respectively ( p = 0.508 ) . 2kaplan meier survival curve for 103 patients resected for colon cancer in one of three norwegian hospitals in 2000 . patients with a lymph node harvest of 12 or more showed significantly better overall survival ( log - rank test p = 0.037 ) kaplan meier survival curve for 103 patients resected for colon cancer in one of three norwegian hospitals in 2000 . patients with a lymph node harvest of 12 or more showed significantly better overall survival ( log - rank test p = 0.037 ) in stage iii patients , the lymph node ratio ( lnr ) was highly significant for patient survival , and os for lnr 1 ( < 0.25 ) was 83.3.5% , lnr 2 ( 0.250.50 ) 63.3% , lnr 3 ( 0.510.75 ) 18.8% , and lnr 4 ( 0.761 ) 18.2% ( log - rank test p < 0.001 ) . two of the hospitals did more resections of t4 tumors ( chi - square test p = 0.021 ) . adjusted for hospital , t4 tumors compared to t1t3 , was a significant adverse factor for survival in the log - rank test ( p = 0.049 ) . survival according to the different tnm stages is shown in table 2 . the 5-year os was 58.1% for stage iii and 63.8% for stage ii without differences between the hospitals in uni- and multivariate analyses . os for stage iv was significantly worse ( p < 0.001 ) . table 2five - year overall survival ( os ) according to the different tnm stages for 269 patients treated with resection for colon cancer in three norwegian teaching community hospitals in 2000tnm stagen ( % ) % osi34 ( 12.6)76.5ii116 ( 43.1)63.8iii93 ( 34.6)58.1iv26 ( 9.7)7.7all stages269 ( 100)58.0 five - year overall survival ( os ) according to the different tnm stages for 269 patients treated with resection for colon cancer in three norwegian teaching community hospitals in 2000 the results of univariate and multivariate cox - regression analyses are shown in table 3 . in the univariate cox regression old age , t category , high lnr , and tnm stage age as a variable was highly significant both as a continuous variable and with a cutoff value of 69 years . locations of the tumors with regard to the various resected segments were not significant ( table 3 ) . table 3univariate ( n = 269 patients ) and multivariate ( n = 258 ) cox regression models for analysis of overall survival for patients resected for colon cancer in three norwegian teaching community hospitals in 2000 univariatemultivariatelr testhr 95% cip valuehr 95% cip valueno . of sampled lymph nodes per increments of 100.79 ( 0.58 , 1.08)0.1290.82 ( 0.57 , 1.17)0.266 t category<0.0010.129 t1t21 ( reference)1 ( reference ) t31.69 ( 0.94 , 3.03)1.84 ( 0.55 , 6.15 ) t43.38 ( 1.79 , 6.40)2.64 ( 0.78 , 8.99)tumor location0.5680.716 right colon1 ( reference)1 ( reference ) transverse colon0.89 ( 0.55 , 1.46)0.79 ( 0.47 , 1.32 ) descending colon0.97 ( 0.39 , 2.42)1.44 ( 0.57 , 3.66 ) sigmoid colon0.70 ( 0.45 , 1.10)0.81 ( 0.50 , 1.31 ) rectosigmoid0.82 ( 0.44 , 1.53)0.62 ( 0.31 , 1.26 ) other / double0.96 ( 0.23 , 3.93)0.67 ( 0.15 , 2.91 ) r25.83 ( 0.79 , 42.81)0.61 ( 0.08 , 4.94)age per 10 years1.46 ( 1.23 , 1.74)0.0011.68 ( 1.38 , 2.03)<0.001gender0.1920.031 females1 ( reference)1 ( reference ) males1.26 ( 0.89 , 1.77)1.52 ( 1.04 , 2.23)tnm stage<0.001<0.001 i1 ( reference)1 ( reference ) ii1.61 ( 0.82 , 3.18)0.91 ( 0.23 , 3.69 ) iii2.07 ( 1.05 , 4.12)1.49 ( 0.39 , 5.66 ) iv10.50 ( 4.94 , 22.32)9.26 ( 2.15 , 39.85)hospital0.3900.169 ahus1 ( reference)1 ( reference ) sus1.06 ( 0.71 , 1.60)0.72 ( 0.46 , 1.14 ) hds1.34 ( 0.87 , 2.07)0.62 ( 0.37 , 1.03)hr hazard ratio ; ci confidence interval ; lr likelihood ratio ; sus stavanger university hospital ; hds haraldsplass deaconal hospital ; ahus akershus university hospitalanalyses based on 258 patients because of lack of specified number of lymph nodesfig . 3kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to hospital ( log - rank test p = 0.372 ) univariate ( n = 269 patients ) and multivariate ( n = 258 ) cox regression models for analysis of overall survival for patients resected for colon cancer in three norwegian teaching community hospitals in 2000 hr hazard ratio ; ci confidence interval ; lr likelihood ratio ; sus stavanger university hospital ; hds haraldsplass deaconal hospital ; ahus akershus university hospital analyses based on 258 patients because of lack of specified number of lymph nodes kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to hospital ( log - rank test p = 0.372 ) in the multivariate model age , male gender , high lnr , and tnm stage were adverse factors ( figs . 4 , 5 , and 6 ) . in the fully adjusted multivariate analysis , t category was not significant , but when removing tnm stage from the multivariate model , the t category again became significant ( p = 0.047 ) . these variables were not significant : hospital , tumor location , or number of harvested lymph nodes in these categories ( < 10 , 1019 , 20 ) . lnr was also highly significant for stage iii patients ( n = 93 ) when adjusted for all the variables in table 3 in a multivariate cox regression with hrs ( 95% cis ) of 1.72 ( 0.80 , 3.67 ) , 5.16 ( 2.48 , 10.74 ) , and 4.80 ( 1.92 , 11.99 ) for lnr 24 vs. lnr 1 , respectively ( p < 0.001 ) . 4kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to age ( log - rank test p < 0.001)fig . 5kaplan meier survival curves for 93 patients resected for colon cancer in three norwegian hospitals in 2000 according to lymph node ratio groups : < 0.25 ( group 1 ) , 0.250.5 ( group 2 ) , 0.510.75 ( group 3 ) , and > 0.75 ( group 4 ) ( log - rank test p < 0.001)fig . 6kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to tnm stage ( log - rank test p < 0.001 ) kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to age ( log - rank test p < 0.001 ) kaplan meier survival curves for 93 patients resected for colon cancer in three norwegian hospitals in 2000 according to lymph node ratio groups : < 0.25 ( group 1 ) , 0.250.5 ( group 2 ) , 0.510.75 ( group 3 ) , and > 0.75 ( group 4 ) ( log - rank test p < 0.001 ) kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to tnm stage ( log - rank test p < 0.001 ) the study may have been underpowered for detection of an assumed difference between patients with a lymph node harvest of < 12 lymph nodes and those with > 12 . with the number of patients included in our study , a power of at least 0.84 would be necessary to detect a difference in 5-year survival of 50% vs. 68% . an increase in sample size to detect a difference of 50% vs. 60% with a power of 0.80 in a one - tailed chi - square test tumor locations were not different between the hospitals ( p = 0.059 ) . locoregional r0 resections for a single tumor location were done in 264 patients , while double resections ( n = 4 ) and a suspected locoregional r1 resection ( n = 1 ) were done in five patients . table 1tumor locations and pathology variables of patients resected for colon cancer at three teaching community hospitals in 2000variablesall patientschi - square testn = 269p valuelocation0.059 right hemicolon115 ( 42.8 ) transverse colon including flexures44 ( 16.4 ) descending colon9 ( 3.3 ) sigmoid colon and rectosigmoid96 ( 35.7 ) multiple or r125 ( 1.9)t category0.021 t111 ( 4.1 ) t230 ( 11.2 ) t3180 ( 66.9 ) t448 ( 17.8)tnm stage0.098 i34 ( 12.6 ) ii116 ( 43.1 ) iii93 ( 34.6 ) iv26 ( 9.7)resection of two tumors in separate locationsno radical loco - regional tumor resection tumor locations and pathology variables of patients resected for colon cancer at three teaching community hospitals in 2000 resection of two tumors in separate locations no radical loco - regional tumor resection there were no significant differences between hospitals relating to tnm stage distribution : 34 patients ( 12.6% ) were stage i , 116 patients ( 43.1% ) were stage ii , 93 patients ( 34.6% ) were stage iii , and 26 patients ( 9.6% ) were stage iv . the number of lymph nodes harvested for various stages were 8.7 ( stage i ) , 10.3 ( stage ii ) , 10.9 ( stage iii ) , and 10.3 ( stage iv ) . in 11 patients , the pathologist had classified the t category and tnm stage but omitted to specify the number of lymph nodes present . twelve or more lymph nodes were examined in 41.1% ( 106/258 ) of the resected specimens . significantly fewer lymph nodes ( p < 0.001 ) were harvested at one of the hospitals . urgent surgery had a mortality of 12.5% ( 3/24 ) , whereas the elective group had a mortality of 4.5% ( 11/245 ) . the 5-year os was 58.0% ( fig . 1 ) and did not differ between the hospitals ( log - rank p = 0.372 ; fig . 1kaplan meier survival curve for 269 patients resected for colon cancer in three norwegian hospitals in 2000 kaplan meier survival curve for 269 patients resected for colon cancer in three norwegian hospitals in 2000 whether categorizing number of lymph nodes harvested in three groups ( < 10 , 1019 , 20 or more ) or in two ( < 12 , 12 or more ) , no differences was found in os ( log - rank p = 0.423 and 0.270 , respectively ) . this was still the case after adjusting for hospital or tnm stage ( log - rank p = 0.449 ) . the uncategorized number of lymph nodes was not significant in a simple cox regression ( likelihood ratio p = 0.129 ) . however , in one hospital , better survival was found when the lymph node harvest was 12 compared to < 12 ( log - rank p = 0.037 ) as shown in fig . 2 . stage ii patients had os of 72.7% with 12 nodes harvested and 58.3% with < 12 nodes ( p = 0.124 ) , and stage iii patients had a 5-year os of 61.5% and 55.6% , respectively ( p = 0.508 ) . 2kaplan meier survival curve for 103 patients resected for colon cancer in one of three norwegian hospitals in 2000 . patients with a lymph node harvest of 12 or more showed significantly better overall survival ( log - rank test p = 0.037 ) kaplan meier survival curve for 103 patients resected for colon cancer in one of three norwegian hospitals in 2000 . patients with a lymph node harvest of 12 or more showed significantly better overall survival ( log - rank test p = 0.037 ) in stage iii patients , the lymph node ratio ( lnr ) was highly significant for patient survival , and os for lnr 1 ( < 0.25 ) was 83.3.5% , lnr 2 ( 0.250.50 ) 63.3% , lnr 3 ( 0.510.75 ) 18.8% , and lnr 4 ( 0.761 ) 18.2% ( log - rank test p < 0.001 ) . two of the hospitals did more resections of t4 tumors ( chi - square test p = 0.021 ) . adjusted for hospital , t4 tumors compared to t1t3 , was a significant adverse factor for survival in the log - rank test ( p = 0.049 ) . the 5-year os was 58.1% for stage iii and 63.8% for stage ii without differences between the hospitals in uni- and multivariate analyses . os for stage iv was significantly worse ( p < 0.001 ) . table 2five - year overall survival ( os ) according to the different tnm stages for 269 patients treated with resection for colon cancer in three norwegian teaching community hospitals in 2000tnm stagen ( % ) % osi34 ( 12.6)76.5ii116 ( 43.1)63.8iii93 ( 34.6)58.1iv26 ( 9.7)7.7all stages269 ( 100)58.0 five - year overall survival ( os ) according to the different tnm stages for 269 patients treated with resection for colon cancer in three norwegian teaching community hospitals in 2000 the results of univariate and multivariate cox - regression analyses are shown in table 3 . in the univariate cox regression old age , t category , high lnr , and tnm stage were adverse factors for survival . age as a variable was highly significant both as a continuous variable and with a cutoff value of 69 years . locations of the tumors with regard to the various resected segments were not significant ( table 3 ) . table 3univariate ( n = 269 patients ) and multivariate ( n = 258 ) cox regression models for analysis of overall survival for patients resected for colon cancer in three norwegian teaching community hospitals in 2000 univariatemultivariatelr testhr 95% cip valuehr 95% cip valueno . of sampled lymph nodes per increments of 100.79 ( 0.58 , 1.08)0.1290.82 ( 0.57 , 1.17)0.266 t category<0.0010.129 t1t21 ( reference)1 ( reference ) t31.69 ( 0.94 , 3.03)1.84 ( 0.55 , 6.15 ) t43.38 ( 1.79 , 6.40)2.64 ( 0.78 , 8.99)tumor location0.5680.716 right colon1 ( reference)1 ( reference ) transverse colon0.89 ( 0.55 , 1.46)0.79 ( 0.47 , 1.32 ) descending colon0.97 ( 0.39 , 2.42)1.44 ( 0.57 , 3.66 ) sigmoid colon0.70 ( 0.45 , 1.10)0.81 ( 0.50 , 1.31 ) rectosigmoid0.82 ( 0.44 , 1.53)0.62 ( 0.31 , 1.26 ) other / double0.96 ( 0.23 , 3.93)0.67 ( 0.15 , 2.91 ) r25.83 ( 0.79 , 42.81)0.61 ( 0.08 , 4.94)age per 10 years1.46 ( 1.23 , 1.74)0.0011.68 ( 1.38 , 2.03)<0.001gender0.1920.031 females1 ( reference)1 ( reference ) males1.26 ( 0.89 , 1.77)1.52 ( 1.04 , 2.23)tnm stage<0.001<0.001 i1 ( reference)1 ( reference ) ii1.61 ( 0.82 , 3.18)0.91 ( 0.23 , 3.69 ) iii2.07 ( 1.05 , 4.12)1.49 ( 0.39 , 5.66 ) iv10.50 ( 4.94 , 22.32)9.26 ( 2.15 , 39.85)hospital0.3900.169 ahus1 ( reference)1 ( reference ) sus1.06 ( 0.71 , 1.60)0.72 ( 0.46 , 1.14 ) hds1.34 ( 0.87 , 2.07)0.62 ( 0.37 , 1.03)hr hazard ratio ; ci confidence interval ; lr likelihood ratio ; sus stavanger university hospital ; hds haraldsplass deaconal hospital ; ahus akershus university hospitalanalyses based on 258 patients because of lack of specified number of lymph nodesfig . 3kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to hospital ( log - rank test p = 0.372 ) univariate ( n = 269 patients ) and multivariate ( n = 258 ) cox regression models for analysis of overall survival for patients resected for colon cancer in three norwegian teaching community hospitals in 2000 hr hazard ratio ; ci confidence interval ; lr likelihood ratio ; sus stavanger university hospital ; hds haraldsplass deaconal hospital ; ahus akershus university hospital analyses based on 258 patients because of lack of specified number of lymph nodes kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to hospital ( log - rank test p = 0.372 ) in the multivariate model age , male gender , high lnr , and tnm stage were adverse factors ( figs . 4 , 5 , and 6 ) . in the fully adjusted multivariate analysis , t category was not significant , but when removing tnm stage from the multivariate model , the t category again became significant ( p = 0.047 ) . these variables were not significant : hospital , tumor location , or number of harvested lymph nodes in these categories ( < 10 , 1019 , 20 ) . lnr was also highly significant for stage iii patients ( n = 93 ) when adjusted for all the variables in table 3 in a multivariate cox regression with hrs ( 95% cis ) of 1.72 ( 0.80 , 3.67 ) , 5.16 ( 2.48 , 10.74 ) , and 4.80 ( 1.92 , 11.99 ) for lnr 24 vs. lnr 1 , respectively ( p < 0.001 ) . 4kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to age ( log - rank test p < 0.001)fig . 5kaplan meier survival curves for 93 patients resected for colon cancer in three norwegian hospitals in 2000 according to lymph node ratio groups : < 0.25 ( group 1 ) , 0.250.5 ( group 2 ) , 0.510.75 ( group 3 ) , and > 0.75 ( group 4 ) ( log - rank test p < 0.001)fig . 6kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to tnm stage ( log - rank test p < 0.001 ) kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to age ( log - rank test p < 0.001 ) kaplan meier survival curves for 93 patients resected for colon cancer in three norwegian hospitals in 2000 according to lymph node ratio groups : < 0.25 ( group 1 ) , 0.250.5 ( group 2 ) , 0.510.75 ( group 3 ) , and > 0.75 ( group 4 ) ( log - rank test p < 0.001 ) kaplan meier survival curves for 269 patients resected for colon cancer in three norwegian hospitals in 2000 according to tnm stage ( log - rank test p < 0.001 ) the study may have been underpowered for detection of an assumed difference between patients with a lymph node harvest of < 12 lymph nodes and those with > 12 . with the number of patients included in our study , a power of at least 0.84 would be necessary to detect a difference in 5-year survival of 50% vs. 68% . an increase in sample size to detect a difference of 50% vs. 60% with a power of 0.80 in a one - tailed chi - square test even though several groups have pioneered radical surgery for colon cancer ( complete mesocolic excision and a central tie ) with its potential benefits similar to ( tme ) of rectal cancer surgery , such an approach has not been widely adopted in our country . therefore , we wanted to examine our background results based on a less standardized approach and potential areas for improvement before embarking on more radical procedures as championed by others . advanced tnm stage had an adverse influence on outcome , as the results for stage iv versus i iii and stage iii versus i ii were significant in the multivariate analysis . for stage iii patients , the 5-year os was 58.1% . in one hospital , the 5-year os for stage iii was worse with 47.8% . in the multivariate analysis , however , when adjusted for hospital , there was no significant survival difference . a recently published international multicenter study of a colorectal population reported an identical os of 58.8% for patients treated in 20002002 . others have found a 5-year os of 90.796.3% for stage ii and 64.671.7% for stage iii using radical surgical procedures . thus , we consider our figures to show room for improvement , although where improvement may be best realised is debatable . an advanced stage may theoretically be better treated with more radical surgery even though the presence of skip lesions in rare instances may hardly explain this . radical or complete mesocolic excision has been shown to increase both the absolute lymph node harvest and prognosis for the patients [ 2 , 3 ] . however , it has been contested that patients will benefit from more radical surgery in advanced cases . in a swedish population - based retrospective study , a median number of six lymph nodes were detected in the specimens from 1,856 patients operated between 1996 and 2000 . there were a very low number of nodes in the specimens from the left colon , whereas in the right flexure tumors , the lymph node count was not inferior . in both instances , the feeding vessel areas were not divided completely , i.e. , the inferior mesenteric artery proximally and the root of the medial colic artery . in a population - based study from the netherlands by kelder and co - workers , the median lymph node harvest was six and in only 21% of the specimens were 12 or more nodes examined . thus , we consider that the principle of radical colon surgery may have been violated in these studies when tumors were in those locations . more radical surgery for colon cancer has been recommended in reports from the usa , europe , and japan [ 2 , 15 , 16 ] . a retrospective national report based on a small number with supposedly radical colon surgery found that this approach significantly increased survival compared with a much larger and older group subject to a standard the number of lymph nodes may be seen only as a surrogate marker for the extent of surgery without a proper oncologic explanation for the importance of a large harvest [ 2 , 3 ] . the node count has been correlated to survival both for stage ii and iii disease [ 2 , 18 ] . the ontario cancer registry study showed better survival with a lymph node harvest of more than ten lymph nodes . increasing in contrast , there is good evidence reported by others that increasing the total number of lymph nodes increases survival significantly [ 2 , 4 ] . in one study , a mean harvest of more than 28 lymph nodes data from the seer database demonstrated that a cutoff value of 15 was useful , and perhaps , there is an inherent limit to the number of nodes necessary to achieve this effect on survival . nevertheless , the hypothesis that a larger ( negative ) lymph node yield is beneficial is contradicted even from highly rated institutions . in the univariate analysis , one of the hospitals achieved a significantly better survival with a harvest of more than 12 lymph nodes . however , no significant survival benefit was found with harvest of more than 12 lymph nodes in the multivariate analysis . many pathologists were involved in this trial even though the specimen handling was supposed to be equal . in a recent study , the pathologist was found to be the dependent factor in lymph node harvest in multivariate analysis , not the operating surgeon . the overall poor lymph node harvest may also indicate that more extensive surgery is one way to improve outcome as found in one of the hospitals . it may be speculated that the poorer outcome in the other two hospitals may have levelled out any benefit by an improved lymph node harvest , i.e. , more radical surgery . patients below 75 years were given flv chemotherapy , and staging was considered important for that reason . for proper staging of colon cancer , the minimum number of lymph nodes needed has been somewhat arbitrarily suggested as 12 by a national cancer institute expert panel . although some authors [ 14 , 18 ] have concluded with stage migration as an explanation for an increased survival benefit , other studies have shown that detection of positive nodes beyond six or seven lymph nodes examined had no effect on staging [ 19 , 23 ] . doubtless , a standardized surgical approach in cooperation with a dedicated pathology service is necessary if a minimum number of nodes shall be found and consequently help in outcome assessment . studies have shown that the lnr is an independent and better marker than pn+ for survival . in our study , lnr was highly significant for os when adjusted for hospitals in the multivariate analysis . five - year os varied from 83.5% in lnr group 1 , 63.3% in lnr 2 , to 18.8% in lnr group 3 . according to wang et al . [ 26 , 27 ] , the prognostic effect of lnr did not depend on the total number of lymph nodes nor on the number of positive nodes . in contrast , berger et al . found that lnr was not a significant prognostic indicator with less than ten examined nodes but became highly significant for os and disease - free survival with more than ten lymph nodes harvested . a mayo clinic study found a positive correlation between the number of positive lymph nodes and survival but did not analyze the effect of lnr . fifty - eight percent of our stage iii patients had less than 12 lymph nodes examined . still , we found a significant survival difference of 44.5% between lnr group 2 and 3 and 65.3% between group 1 and 4 . a study from new zealand found that both the absolute number of lymph nodes retrieved up to 16 , as well as the lnr , were important for prognosis . the share of stage iii compared to stage ii cancers in their patients amounted to 54.9% , higher than in most other reports . it seems obvious that a large negative lymph node yield will down - regulate lnr , but the importance of this with regard to outcome may need to be examined in more detail . another national report of colon cancer from a single institution found that an emergency operation , some colon locations , blood transfusion of more than two units , old age , and tnm stage were negative predictive factors . we did not study the effect of blood transfusion , and emergency operations were not separated from the rest partly because they were few , i.e. , 15% reported by sjo et al . , and the definition may be debatable . the different colon locations found to be statistically significant ( transverse , left flexure , and descending colon ) were not so in our multivariate analysis . we suspect that this is partly because they were too few , 20% , to make an impact . even though the number of urgently operated patients was small in this series , as expected , this small group had a higher mortality of 12.5% compared to 4.5% in the elective patients . severe co - morbidities in old age may have contributed to these figures as has been reported by others . however , it should be possible to set an optimal target of < 3% in elective cases . the average age of our study population was 71 years and that may be older than in series of selected patients . the average age of men and women in our country for those that have reached the age of 62 ( earliest oap retirement age ) is 81 and 85 years . to circumvent the analytic problem of age , sjo et al thus , their survival figures improved from 62% actuarial survival to 74% for women and 79% for men . the method is cumbersome , as it necessitates life tables to calculate this for every patient year . three - year disease - free survival has been shown to parallel overall 5-year survival . however , it requires close follow - up with ct instead of the conventional ultrasonography and chest x - ray examinations . close follow - up may even result in better treatment of metastases according to japanese results . only 41% had a lymph node harvest of 12 or more lymph nodes , and although this influenced os in one hospital , it did not overall . more radical surgery may increase the lymph node yield , but if this has the potential to increase overall survival in our patients remains to be seen in the prospective registry . mortality should be kept low with adequate assessment and treatment of co - morbidities as well as meticulous surgery to avoid complications .

backgrounda national surveillance program of colon cancer treatment was introduced in 2007 . we examined prognostic factors for colon cancer operated in 2000 with an aim of improving survival in the new program and a special focus on the merit of lymph node yield.methodsa cohort of 269 patients , 152 women ( 56.5% ) , with a mean age of 71 years , was operated for colon cancer in 2000 at three teaching hospitals and followed up for 7 years.resultsoverall 5-year survival was 58.0% , and overall hospital mortality was 5.2% , with 4.5% in elective cases and 12.5% after urgent surgery . in only 41.1% of the specimens were 12 or more lymph nodes retrieved , but this did not affect survival in the combined cohort , although one of the hospitals achieved a significantly better result with a harvest of 12 or more lymph nodes . in a multivariate analysis , old age , gender , a high lymph node ratio ( lnr ) at stage iii , and tumor node metastasis stage were adverse factors for survival.conclusionsthe operative mortality was high and should be reassessed . the lymph node count did not have a significant impact on outcome overall , whereas the lnr proved significant for stage iii . a prospective protocol using overall lymph node yield as a surrogate measure for more radical surgery , nevertheless , seems warranted to improve the lymph node harvest according to international recommendations .

Introduction Material and methods Surgery Follow-up Pathology Oncology Ethics Statistical analysis Results Surgery Pathology Survival analysis Statistical analysis Discussion Conclusion

according to the surveillance epidemiology and end results ( seer ) database , the incidence of thyroid cancer , the most prevalent endocrine malignancy , increased in the us between 1975 and 2012 , with estimated 62,450 new cases and 1950 deaths in 2015.1 a rising incidence has also been documented in many other countries.2 the most common types of thyroid cancer arise from follicular cells , which include papillary thyroid carcinoma ( ptc ) and follicular thyroid carcinoma ( ftc ) . these subtypes account for 90%95% of all cases and are collectively referred to as differentiated thyroid cancers ( dtcs).3 in general , these are indolent tumors associated with a favorable prognosis as reflected by long - term survival rates of ~90% with standard treatments of surgery and radioactive iodine therapy.4 nevertheless , patients who develop recurrent or metastatic radioactive iodine refractory disease have 10-year survival rates of only 15%20%.5 before the advent of targeted therapies , chemotherapy was the only option for the treatment of patients with advanced thyroid cancer of follicular origin refractory to radioactive iodine.6 doxorubicin ( with its known cardiac and hematologic toxicities ) alone or in combination with other compounds formed the backbone of palliative therapy for such patients.7,8 the oncogene pathway - driven approach to the understanding of the pathophysiology of thyroid cancer led to the development of clinical trials that assessed the antitumor activity of tyrosine kinase inhibitors ( tkis ) . these studies provided evidence of clinically meaningful antitumor activity that is not seen with chemotherapy and ultimately supported the us food and drug administration s ( fda ) approval of vandetanib and cabozantinib for the treatment of medullary thyroid cancer ( mtc ) and , more recently , sorafenib and lenvatinib for progressive radioiodine - refractory ptc and ftc . notwithstanding their progression - free survival ( pfs ) benefit , tkis are associated with significant and unique toxicity profiles . furthermore , the decision of when to start treatment with a tki can be challenging when encountering asymptomatic patients with slowly progressive radioiodine - refractory thyroid carcinomas , a relatively common finding in this disease . this article aims to critically review the data on antitumor activity , toxicity , and potential patient selection tools for the newly approved multikinase inhibitor lenvatinib.9 a better understanding of the molecular biology of malignancies and the advent of targeted therapies represented an unprecedented development in the therapy of several solid tumors in recent years , including non - small - cell lung cancer , breast cancer , melanoma , and gastrointestinal stromal tumors.1013 a comprehensive genetic analysis of 496 samples of ptc as part of the cancer genome atlas ( tcga ) project showed that driver genomic alterations were found i97% of cases.14 the vascular endothelial growth factor receptor ( vegfr ) was one of the first signaling pathways to be associated with the aggressiveness of thyroid cancer.1517 despite its key role in the pathophysiology of thyroid malignancies , other signaling pathways drive the thyroid cancer cell behavior . fibroblast growth factor receptor ( fgfr ) , platelet - derived growth factor receptor ( pdgfr ) , v - ras oncogene homologue ( ras ) , b - raf proto - oncogene , serine / threonine kinase ( braf ) , and ret / ptc rearrangement receptor , among others , have been recognized as important signaling pathways that are implicated in the pathophysiology of thyroid tumors.1824 until recently , sorafenib was the only kinase inhibitor approved by the us fda for the treatment of metastatic iodine - refractory dtc . sorafenib is an oral tki that abrogates signaling from numerous molecules including braf , ret / ptc , vegfr13 , pdgfr , and c - kit.25,26 the antineoplastic activity of sorafenib for the treatment of thyroid cancer was demonstrated in many trials including the decision trial , which was a phase iii placebo - controlled randomized study of 417 patients with progressive radioactive iodine - refractory , locally advanced , or metastatic thyroid cancer assigned to sorafenib 400 mg twice daily or placebo.27,28 the histological subtypes , confirmed by a central review , primarily consisted of ptc and ftc . the primary end point of the study was met with a hazard ratio ( hr ) for pfs of 0.59 ( 95% confidence interval [ ci ] 0.450.76 ; p<0.0001 ) . the overall survival ( os ) difference did not meet statistical significance , likely due to a crossover effect ( hr 0.80 ; 95% ci 0.541.19 ; p=0.14 ) . these benefits were counterbalanced by frequent treatment - related adverse events ( aes ) including hand foot syndrome ( hfs ) , diarrhea , alopecia , and rash , which were documented in ~70% of the participants . serious aes occurred in 37% of patients treated with sorafenib compared to 26% of those treated with placebo . the most common treatment - related grade 3 aes in the sorafenib arm included hfs ( 20% ) , diarrhea ( 5% ) , skin rash or desquamation ( 4.8% ) , fatigue ( 5% ) , and weight loss ( 5.8% ) . dose modifications because of adverse effects were required in ~60% of the patients , and the treatment was suspended in 18% of the patients . most recently , a meta - analysis of 24 phase ii and iii trials estimated the risk of all - grade hfs at 39% for patients treated with sorafenib.29 prophylactic treatment with urea - based cream can decrease the incidence of or delay the development of hfs in patients receiving sorafenib.30 another meta - analysis showed that all - grade diarrhea occurred in 37% of patients treated with sorafenib for different types of tumors and can negatively impact quality of life.31 in addition to sorafenib , several other kinase inhibitors have been studied for the treatment of advanced follicular origin radioiodine - refractory thyroid cancer ( table 1 ) . lenvatinib is a tki that targets vegfr13 , fgfr14 , pdgfr- , ret , and c - kit.3235 preclinical evidence indicates that lenvatinib inhibits tumor growth , tumor - induced angiogenesis , cell migration , and invasion in various human thyroid cancer xenograft and in vitro models.36,37 compared to the other multikinase inhibitors tested in patients with differentiated thyroid tumors , lenvatinib is particularly active against the fgfr family of tyrosine kinase receptors ( table 2 ) . fgfrs contain three immunoglobulin - like extracellular domains , a transmembrane region , and an intracellular domain , which is composed of a split tyrosine kinase and its carboxyterminus.38 fgfrs are overexpressed in a variety of solid tumors , including thyroid cancer.39,40 preclinical models and immunohistochemistry analyses of tumor tissues showed that fgfr1 is overexpressed in 50%75% of cells in both follicular and papillary tumors . fgfr2 seems to be expressed in normal thyroid tissues but not in tumors.19 fgfr3 is overexpressed in 25%50% of well - differentiated follicular origin thyroid cancers . cells and xenograft models were treated with the tki pd173074 , which is known to selectively inhibit fgfr phosphorylation and tyrosine kinase activity in solid tumors.41 the abrogation of fgfr1 and 3 signaling with pd173074 was associated with in vitro and in vivo growth inhibition of differentiated thyroid tumor cell lines . tpc-1 cells sustained up to 90% inhibition of cell proliferation , and xenograft models treated with pd173074 achieved a tumor size reduction of ~55%.19 these observations indicate the importance of the fgfr pathway in thyroid cancer pathogenesis and progression . next - generation sequencing analysis of the genomes of 492 samples of ptc identified this disease as having a low mutational burden compared with other carcinomas.14 fgfr3 amplification was noted in only one case , and fgfr2 gene fusions were observed in only two cases . in a dose - escalation 3 + 3 phase i trial , the maximum tolerated dose ( mtd ) of lenvatinib was investigated among a wide range of doses ( 0.520 mg twice daily ) in solid tumors , including colon , sarcoma , and non - small - cell lung cancers.43 dose - limiting toxicities of grade 3 elevations of aspartate and alanine aminotransferases and thrombocytopenia were observed at the 20 mg twice - daily dose level . mtd was 13 mg twice daily on a 2-week - on/1-week - off schedule for this trial . of note , five patients experienced six serious aes related or possibly related to the study drug.43 a larger phase i trial investigated the safety and efficacy of lenvatinib among 82 patients with advanced refractory solid tumors , which included renal , colorectal , and pancreatic tumors . grade 3 hypertension and proteinuria occurred in nine patients ( 11% ) and six patients ( 7% ) , respectively . a positive correlation between the dose of lenvatinib and hypertension and proteinuria was observed . in the expanded mtd cohort of 25 mg ( n=24 ) , grade 3 hypertension occurred in three ( 13% ) patients and grade 3 proteinuria in two ( 8% ) patients.44 based on these phase i trial results , a subsequent phase ii trial was conducted among 58 patients with differentiated radioiodine - refractory thyroid cancer who were treated with lenvatinib at 24 mg daily . prior anti - vegfr therapy and chemotherapy were reported in 29% and 14% of the patients , respectively . aes led to dose interruptions , reductions , or study drug withdrawal in 74% , 66% , and 26% of the patients , respectively . the most common grade 3 side effects were weight loss ( 12% ) , hypertension ( 10% ) , proteinuria ( 10% ) , diarrhea ( 10% ) , fatigue ( 9% ) , dehydration ( 9% ) , and arthralgia ( 5% ) . of note , two patients died after serious aes ( one arterial hemorrhage and one cardiac arrest).45 in another cohort of patients , lenvatinib was tested at the set dose of 24 mg daily in 59 patients with advanced mtc , 44% of whom received prior anti - vegfr therapy and 15% received prior chemotherapy.46 overall response rate ( orr ) was 36% , and median pfs was 9 months . dose reduction or interruption was necessary in up to 59% of the patients , and 24% of the patients discontinued treatment due to aes . the most common grade 3 side effects included diarrhea ( 14% ) , hypertension ( 7% ) , decreased appetite ( 7% ) , fatigue ( 5% ) , dysphagia ( 5% ) , and increased levels of alanine aminotransferase ( 5% ) . there were three deaths due to aes including respiratory arrest ( not otherwise specified ) , respiratory failure , and paraneoplastic syndrome ( not specified ) ; only the respiratory failure - related death was deemed to be treatment related by the treating physicians.46 in the seminal phase iii study select , a total of 392 patients with radioactive iodine - resistant progressive thyroid carcinomas of follicular origin ( defined as progression within the previous 13 months according to response evaluation criteria in solid tumors , the presence of one measurable lesion without radioiodine uptake on any iodine scan , or disease progression within 12 months of radioiodine therapy ) were randomized to either lenvatinib ( n=261 ) or placebo ( n=131).47 a significant improvement in the median pfs , the primary end point , was documented among patients treated with lenvatinib compared to placebo ( 18.3 vs 3.6 months ; hr 0.21 ; 99% ci 0.140.31 ; p<0.001 ) , with an unprecedented response rate of 64.8% . ninety - three patients in the trial received prior anti - vegf tkis ( sorafenib 77% , sunitinib 9% , pazopanib 5% , and other 9% ) . despite the previous use of targeted therapies , lenvatinib also significantly improved the pfs in this subgroup of patients ( 15.1 vs 3.6 months ; hr 0.22 ; 95% ci 0.120.41 ) and increased response rates ( 62.1 vs. 3.7 % ) .48 complete radiologic responses were seen in four patients , and prolonged stable disease ( longer than 23 weeks ) was noted in 39 patients treated with lenvatinib . while consistent with the results of phase i and ii studies , the toxicity profile in this study was significant . as noted with the use of other multikinase inhibitors , lenvatinib toxicity resulted in a significant rate of dose adjustments and clinically relevant consequences . dose reductions , dose interruptions , and treatment discontinuation were needed in 67% , 82% , and 14% of the patients , respectively . the most frequent grade 3 or higher treatment - related aes were hypertension ( 42% ) , fatigue ( 9% ) , diarrhea ( 8% ) , proteinuria ( 10% ) , arterial and venous thromboembolic effects ( 2.7% and 3.8% , respectively ) , acute renal failure ( 1.9% ) , and hepatic failure ( 0.4% ) . dose modifications due to severe diarrhea and decreased appetite were required in 22% and 18% of the patients , respectively . in this larger trial , qtc prolongation was seen in 8% of the patients , of whom 1.5% experienced grade 3 aes , defined as a qtc 501 ms . however , among a study of healthy individuals who took a single 32 mg dose of lenvatinib , there was a lack of qtc prolongation within the 24-hour continuous electrocardiogram ( ekg ) monitoring period.49 thus far , there seems to be no evidence of significant direct cardiac toxicity associated with lenvatinib . of note , six deaths in the lenvatinib group were considered treatment related : three cases resulted from unspecified causes and three were associated with pulmonary embolism , hemorrhagic stroke , and health deterioration . however , four of these deaths were nonspecific in etiology , and it is thus difficult to accurately assess the contribution of lenvatinib toxicity to these fatal events . as of yet , data on median os are not available , and no significant os benefit has been demonstrated with lenvatinib thus far ( hr 0.73 , 95% ci 0.51.0 ; p=0.10 ) . notably , 109 patients per protocol were allowed to cross over to the lenvatinib arm upon disease progression , which can confound differences in os . as of november 2013 , nonetheless , an updated survival data analysis of the select trial at a later cut - off date ( june 15 , 2014 ) was presented at the 2015 european cancer congress.50 to adjust for the crossover and estimate the true os treatment effect ( the effect that would have been observed in the absence of switching ) , a rank - preserving structural failure time model was used . after 34 months of follow - up of the trial for the lenvatinib arm , the median os was not reached for this group . for the placebo crossover arm , the reported medial os was 19.1 months ( 95% ci 14.3 , not estimable ) . the rank - preserving structural failure time - adjusted hr showed a significant difference in os between the treatment groups ( hr 0.53 ; 95% ci 0.340.82 ; nominal p=0.0051 ) as determined using the resampling method ( bootstrapping ) . of note , hypertension was the most common grade 3 or higher toxicity , affecting 42% of the patients in the select trial , while observed in only 7%10% of the patients in the phase ii trials.45,46 all - grade hypertension occurred in up to 67% of the patients treated with lenvatinib ( select ) , and it led to dose reductions or interruptions in up to 20% of the patients . in the phase ii trials , hypertension was observed in 51%76% of the patients treated with lenvatinib . because of the risk of hypertension , careful cardiovascular evaluation of patients , including performing an echocardiogram and ekg profile , should be considered prior to initiation of therapy . an ekg should also be performed prior to initiation of therapy , considering the risk of qtc prolongation associated with multikinase inhibitors in general.51 frequent blood pressure monitoring should be performed throughout treatment . according to the select study protocol , dose interruptions of up to 28 days for patients with grade 2 hypertension should be performed prior to dose reduction ( 20 , 14 , 10 mg / d ) . patients with confirmed systolic blood pressure ( bp ) 140 mmhg or diastolic bp 90 mmhg should be prescribed antihypertensive agents and monitored every 2 weeks . patients with systolic bp 160 mmhg or diastolic bp 100 mmhg , despite optimal management , should have a dose reduction . in the phase iii select study , gastrointestinal perforation or fistula was observed in 2% of patients receiving lenvatinib compared to 0.8% in the placebo group.52 in addition , aerodigestive fistula formation had also been documented in a case report in which a patient receiving lenvatinib developed significant general decline and required percutaneous gastrostomy for nutritional support.53 radiation therapy and large thoracic tumor burden are considered risk factors for aerodigestive fistula formation associated with lenvatinib and other anti - angiogenesis therapies.54,55 hemorrhagic complications were also documented in the 35% of patients treated with lenvatinib in the phase iii trial compared to 18% in the placebo group . epistaxis was the most common hemorrhagic event ( 11% grade 1 and 1% grade 2).52 the incidence of severe hemorrhage episodes ( grade 35 ) was comparable between both the groups ( 2% lenvatinib and 3% placebo ) . these results are in agreement with the risk of fatal bleeding documented with other tyrosine kinase inhibitors and highlight the importance of careful monitoring.56 the clinically meaningful benefit in pfs and tumor response rate in this disease setting led to the us fda s approval of lenvatinib in february 2015 for the treatment of progressive thyroid cancer . despite the overall response rate of > 60% across distinct histologic subtypes ( ie , papillary , poorly differentiated , follicular , and hrthle cell ) , up to 35% of the patients did not meet criteria for response under the therapy with lenvatinib.47 this is particularly important in patients who are symptomatic from their tumor burden and would benefit from tumor reduction . also , as described earlier , lenvatinib has been associated with significant toxicity and treatment - related deaths . hence , it is paramount to find predictive markers of response , which may facilitate selecting patients for therapy . the first and best - characterized point mutation in thyroid cancer is a valine - to - glutamate substitution at residue 600 ( v600e ) of the braf ( v - raf murine sarcoma viral oncogenes homolog b1 ) gene.14,57 this results in constitutive activation of the braf kinase that confers continuous activation of the mitogen - activated protein kinase ( mapk ) signaling pathway with consequent uncontrolled cell growth.58 braf mutations may have prognostic value in differentiated thyroid tumors . a recent retrospective analysis of 500 patients with ptc showed tumor recurrence rates of 25% among those with a braf v600e mutation as compared to 9.6% in mutation - negative patients.59 following braf mutations in thyroid cancers ( 40% ) , the most frequent driver mutations occur in the ras genes . the most common ras mutations in thyroid cancer occur in the nras and hras genes and lead to constitutive activation of both the mapk and phosphoinositide 3-kinase ( pi3k)/akt pathways.60,61 ras can be mutated in up to 20% of ftc and in 6%13% of patients with ptc.14,60,61 archival formalin - fixed , paraffin - embedded tissues were obtained and analyzed for braf and ras mutation hotspots in the select trial . interestingly , the benefit of lenvatinib was independent of the braf and ras mutational status of the tumor on a preplanned subgroup analysis in select.47 this could be due to the wide range of tumor cell targets affected by lenvatinib or its ability to target unique molecules such as the fgfr.19 the angiogenesis of mtc , a highly vascular tumor , is mediated principally by vegf , fgf , pdgf , and their respective receptors.62 overexpression of vegf and vegfr2 has been observed in 50%95% of mtc tumors and is associated with metastases.6365 in the aforementioned phase ii trial of lenvatinib for the treatment of 59 patients with advanced mtc , in which circulating cytokine and angiogenic factors levels were collected from 51 patients after 8 days of therapy , high baseline levels of vegf , soluble vegfr3 , and pdgf- , and low baseline levels of soluble tie-2 were associated with tumor reduction . low baseline levels of angiopoietin-2 ( ang-2 ) , hepatocyte growth factor , and interleukin-8 were associated with tumor reduction and prolonged pfs.46 in the select trial , which accrued patients with ftc and ptc , blood cytokine and angiogenic factor levels of 99% of patients were collected on cycle 1 , day 15 and day 1 of subsequent cycles . ang-2 and stie-2 levels were consistently decreased throughout the therapy with lenvatinib when compared with the placebo group . decreased ang-2 and stie-2 levels along with increased vegf levels correlated with tumor shrinkage ; at the end of the treatment , the levels of these two molecules on c2d1 increased in 78.9% and 81% of patients , respectively.66 the association between lenvatinib benefit or lack thereof and baseline angiogenic factors merits further investigation before it can affect patient treatment selection . finally , in light of lenvatinib - related toxicity , the possible interaction between age and therapy with lenvatinib was formally explored for os end point in the select trial . improved os was identified in patients older than 65 years ( median 71 years ) when treated with lenvatinib.67 at this juncture , the antitumor activity of lenvatinib , including its improvement in pfs , among patients with advanced progressive thyroid carcinomas of follicular origin supports its clinical use . recently published guidelines by the american thyroid association and the national comprehensive cancer network recommend its possible use in patients with radioiodine - refractory progressive thyroid carcinomas and as first - line kinase inhibitor therapy.68,69 thus far , no other multikinase inhibitors have displayed a comparable degree of efficacy in the treatment of advanced radioiodine - refractory thyroid cancer . taking into account all of the caveats inherent to cross - trial comparisons , the us fda approved the tki sorafenib based on the results of the decision trial , which showed a pfs of 10.8 months in the sorafenib arm vs 5.8 months in the placebo arm ( hr 0.59 ; 95% ci 0.450.76 ; p<0.0001 ) and an orr of 12.2%.27 by contrast , lenvatinib - treated patients derived a greater benefit from this tki compared to placebo in the select trial , as reflected by a pfs of 18.3 months vs 3.6 months ( hr 0.21 ; 99% ci 0.140.31 ; p<0.001 ) and an orr of 64.8%.47 both these trials included similar patient populations , but the decision trial excluded patients with previous tki exposure . the select trial included 93 patients who had received prior tki therapy ( sorafenib 77% , sunitinib 9% , pazopanib 5% , other 9% ) . the benefit of lenvatinib was not mitigated by prior multikinase treatment.48 one could hypothesize that the remarkable antitumor activity of lenvatinib is due to its tyrosine kinase inhibition of fgfr.19 fgfr family activation leads to upregulation of the ras / mapk and pi3k / akt signaling pathways.70 fgfr overexpression and phosphorylation observed in preclinical studies suggest that fgfr is an important signaling pathway in the thyroid tumors of follicular origin , as reviewed earlier.19 despite its efficacy , the toxicity of lenvatinib merits cautious consideration . up to 65%71% of the patients treated with lenvatinib experienced grade 3 or higher toxicities , having warranted dose reductions and/or delays in the majority of the patients in previous phase ii and iii clinical trials ( table 3).4547 the pattern of disease progression in radioiodine - refractory thyroid tumor should also be considered at the time of therapy initiation ( median pfs in placebo - treated groups range from 3.65.8 months ) to the extent that asymptomatic patients may take months to meet criteria for disease progression . hence , the potential for anti - tumor efficacy needs be carefully counterbalanced against its potential toxicities in asymptomatic patients prior to drug initiation . lenvatinib should be initiated at the time of rapid radiological / clinical disease progression and in the setting of symptomatic tumor burden . furthermore , patients need to be carefully selected for lenvatinib therapy , and physicians should be aware of the frequency of moderate to severe aes , including hypertension , weight loss , decreased appetite , and proteinuria associated with this medication ( table 3 ) . finally , careful consideration of locally directed therapies ( eg , surgery , radiation therapy , thermal ablation ) should be given prior to initiation and during the course of treatment with lenvatinib.68,69 this is of particular importance in the setting of mixed responses in distinct metastatic sites with imminent risk of complications related to local progression and invasion of metastasis ( figure 1 ) . formal quality of life assessments were not reported for the select trial , but sorafenib - treated patients in the decision trial had lower on - treatment scores for the functional assessment of cancer therapy : general , euroqol-5d and euroqol-5d visual analog scales , suggesting a detectable negative impact of sorafenib therapy on health - related quality of life.71 nonetheless , in light of the toxicity profile , the corollary is that physicians need to be fully aware and aggressively treat eventual aes to maintain quality of life . the role of lenvatinib as salvage therapy for patients with previous exposure to tkis , such as sorafenib , is a question of clinical relevance . a retrospective analysis of 17 patients with sorafenib - refractory thyroid cancer treated with salvage therapy , including sunitinib ( n=4 ) , pazopanib ( n=3 ) , cabozantinib ( n=4 ) , lenvatinib ( n=3 ) , and vemurafenib ( n=3 ) , revealed an orr of 41% and a median pfs of 11.4 months.72 this suggests that tkis with different targets may still be effective for refractory disease . these results are consistent with a subgroup analysis of the select trial in which patients who had been previously exposed to tkis still derived benefit from lenvatinib.48 patients with a history of previous exposure to tkis had an orr of 65.6% , whereas tki - nave patients had an orr of 62.1% . the question of whether lenvatinib or sorafenib should be the first option for first - line treatment of patients with iodine - refractory dtc still remains . the lack of head - to - head comparison of these molecules generates the need for constant individualized decision making , taking into account the estimated efficacy and toxicity for each multikinase inhibitor . there is no biomarker that facilitates the selection of or the prediction of response to lenvatinib . stratified analysis of pre - therapy braf and ras mutational status failed to show a predictive benefit of these tests in phase iii trials of sorafenib and lenvatinib.27,47 simultaneous targeting of multiple molecular pathways is a potential strategy to improve the antitumor activity of lenvatinib and to potentially increase the efficacy of and delay resistance to lenvatinib therapy in radioiodine - refractory thyroid cancer . the combined analysis of genomic variants , gene expression , and methylation patterns of 496 samples of ptc was able to cluster this tumor into two main molecular signature groups : the braf - mutant and the ras - mutant tumors with differential downstream pathway activation of mapk and pi3k / akt , respectively.14 one could hypothesize that lenvatinib s efficacy could be further increased by treatment combinations driven by pathway - enriched patient selection according to differential tumor signatures . the development of the combination treatment with the mek inhibitor trametinib and the braf inhibitor dabrafenib in recurrent thyroid cancer is ongoing ( nct01723202).73 however , to our knowledge , the multiple pathway blockade approach with lenvatinib is nonexistent at this time . also , hepatocyte growth factor ( hgf ) and met are overexpressed in the majority of ptcs ( > 90% ) , but rarely in ftcs.74 hgf overexpression correlates with invasiveness and a more clinically aggressive behavior of ptcs.7577 the met receptor is a key component of hgf pathobiology in ptc.78 among ptc cell lines , the met inhibitor pha665752 inhibited tumor cell growth and induced apoptosis.79 interestingly , preclinical data indicate that one of the mechanisms of intrinsic lenvatinib therapy resistance is mediated by hgf pathway activation in various solid tumors such as melanoma and pancreatic carcinoma.80 this can be circumvented by the combination of lenvatinib with the met inhibitor golvatinib , which showed significant reduction of lenvatinib - resistant cell proliferation in vitro.80 a phase i dose - escalation trial of the combination of lenvatinib and golvatinib in patients with melanoma and glioblastoma is currently under way ( nct01433991).81 further studies are necessary to establish the role of met inhibition in thyroid cancer . interestingly , the degree of toxicity from anti - endothelial growth factor receptor and anti - vegf drugs has been positively correlated with their efficacy among patients with solid tumors including colorectal , lung , renal cell carcinoma , and head and neck.82,83 the degree of toxicity , namely hypertension and proteinuria , could serve as a surrogate biomarker of lenvatinib activity . it has been suggested that aes such as worst grade proteinuria correlated with lenvatinib antitumor activity in a small phase i dose - escalation clinical trial.84 to our knowledge , no stratified analysis of the efficacy of lenvatinib by toxicity has been reported . in addition , dose reduction of tkis positively correlated with clinical benefit in non - small - cell lung cancer and chronic myeloid leukemia.8587 in clinical practice , it is tempting to adopt a dose reduction strategy to avoid lenvatinib - associated toxicities while offering patients effective treatment . data are not yet available on the efficacy of dose - reduced treatment of advanced thyroid cancer with lenvatinib . the results of the expanded cohort program with lenvatinib for the treatment of radioiodine - refractory dtc with three different doses , 24 , 20 , and 14 mg daily will shed light into this important question ( nct02211222).88 in conclusion , lenvatinib is a novel multikinase inhibitor that demonstrates significant responses and pfs benefit in the treatment of radioiodine - refractory dtc . consistent with the current 2015 american thyroid association and the national comprehensive cancer network guidelines , lenvatinib should be considered as the first - line therapy in the setting of clinically relevant disease progression and/or symptomatic disease burden in the setting of radioiodine - refractory disease , with the warning that physicians should be fully cognizant of its side effect profile , including potentially fatal toxicities ( figure 1).68,69 aggressive monitoring and guideline - based management of toxicities similar to those utilized with other tkis should be enforced to prevent decline in quality of life.8991 future studies should endeavor to establish biomarkers predictive of toxicity and efficacy of lenvatinib treatment in radioiodine - refractory thyroid tumors .

thyroid cancer is the most common endocrine malignancy , with over 60,000 cases reported per year in the us alone . the incidence of thyroid cancer has increased in the last several years . patients with metastatic differentiated thyroid cancer ( dtc ) generally have a good prognosis . metastatic dtc can often be treated in a targeted manner with radioactive iodine , but the ability to accumulate iodine is lost with decreasing differentiation . until recently , chemotherapy was the only treatment in patients with advanced thyroid cancer , which is no longer amenable to therapy with radioactive iodine . the modest efficacy and significant toxicity of chemotherapy necessitated the need for urgent advances in the medical field . new insights in thyroid cancer biology propelled the development of targeted therapies for this disease , including the tyrosine kinase inhibitor sorafenib as salvage treatment for dtc . in 2015 , the us food and drug administration approved a second tyrosine kinase inhibitor , lenvatinib , for the treatment of radioiodine - refractory thyroid cancer . although associated with a significant progression - free survival improvement as compared to placebo in a large phase iii study ( median progression - free survival 18.2 vs 3.6 months ; hazard ratio 0.21 ; 99% confidence interval 0.140.31 ; p<0.001 ) , the benefit of lenvatinib needs to be proved in the context of associated moderate to severe toxicities that require frequent dose reduction and delays . this article reviews the evidence supporting the use of lenvatinib as salvage therapy for radioactive iodine - refractory thyroid cancer , with a focus on the toxicity profile of this new therapy .

Introduction Multikinase inhibitors approved for the treatment of follicular origin thyroid carcinomas Lenvatinib: overview Clinical efficacy and toxicity of lenvatinib Potential predictive biomarkers for lenvatinib efficacy Discussion

stroke is a neurological disease caused by the cutoff of normal blood supply due to vessel rupture or thrombosis , and it causes brain tissue damage . motor , sense , recognition , language , and perception deficits are typical symptoms of the disease , depending on the affected areas , size , and the cause of damage1 . stroke patients mainly experience motor disorders and degeneration of balancing ability due to unbalanced posture ; this is caused by asymmetric arrangement and hemiplegia , and this disrupts independent activities of daily living ( adl)2 . the ability to balance is an individual s ability to maintain the center of gravity within a base region , keeping the body in a balanced state3 . stroke patients lack balancing ability and show degeneration of physical function due to a reduction in walking and activity caused by an inappropriate posture , proprioception malfunction , and abnormal muscle tension4 . to resolve balance problems caused by stroke , the brunnstrom approach , bobath approach , or proprioceptive neuromuscular facilitation ( pnf ) is used ; such methods focus on enhancing physical functions and balance by improving active motor control5 . pnf utilizes a typical helical or diagonal pattern to stimulate proprioceptive sensation promote a nerve root response , enhancing functional movement5 , 6 . it improves muscle strength , flexibility , and balance ; by applying it on the non - paretic side , force is transferred to the paretic side , effectively promoting muscle activity6 . davis7 suggested aquatic pnf lower extremity patterns for reinforcing muscular strength and enhancing muscle reeducation . song and kim8 applied aquatic pnf lower extremity patterns to patients who had a stroke , which stimulated proprioceptors , thus improving control and function of the nerve roots while enhancing balance . in an aquatic environment , the buoyancy allows limbs to be moved more easily with little strength , enhancing coordination of motion9 . water resistance also enables the body to move , helping increase muscle strength , joint movement , and balance10 . multiple studies had reported pnf lower extremity patterns during ground - based exercise for stroke patients , but few had reported aquatic pnf . this study aimed to investigate the effects of aquatic pnf lower extremity patterns on balance and adl of stroke patients . six months after receiving a stroke diagnosis via either computed tomography or magnetic resonance imaging , 20 patients were randomly assigned to a control group ( n = 10 ; 5 males and 5 females ) or an experimental group ( n = 10 ; 5 males and 5 females ) ; the control group was assigned to conduct pnf lower extremity patterns on the ground , and the experimental group was assigned to conduct pnf lower extremity patterns in water . all participants scored > 24 points on the mini - mental status examination ( mmse ) , could perform assignments , were capable of walking 10 m independently , and were classified as > 4 on the brunnstrom scale . the protocol was approved by the institutional review board of nambu university and was conducted in accordance with the ethical standards of the declaration of helsinki . the general features of the participants are listed in table 1table 1.general characteristics of subjectseg ( n=10)cg ( n=10)gender ( male / female)5 / 55 / 5age ( years)69.1 3.268.0 3.1height ( cm)167.6 8.2165.9 6.9weight ( kg)67.9 5.966.7 6.7paretic side ( right / left)5 / 55 / 5onset ( months)9.8 1.310.3 1.4meansd , eg : experimental group , cg : control group . the age of the experimental group was 69.1 3.2 years , height was 167.6 8.2 cm , weight was 67.9 5.9 kg , and time since stroke onset was 9.8 1.3 months . the age of the control group was 68.0 3.1 years , height was 165.9 6.9 cm , weight was 66.7 6.7 kg , and time since stroke onset was 10.3 1.4 months . meansd , eg : experimental group , cg : control group the experimental group conducted pnf lower extremity patterns using the rhythmic initiation ( ri ) method 110 cm below the water surface ; the water temperature was 3133 c . exercises were performed in a supine posture after simple stretching ; subjects wore a body ring between l5 and s1 and a neck collar . the control group conducted pnf lower extremity patterns on the ground in a supine posture after simple stretching . the ri method starts from passive exercise , proceeds to active resistance exercise , and helps increase coordination , motor sensation , and balance . the pnf lower extremity patterns consisted of patterns d1 and d2 . the d1 pattern ends at either flexion - adduction - external rotation knee flexion or extension - adduction external rotation knee extension . the d2 pattern ends at either flexion - adduction external rotation knee flexion or extension - adduction - external rotation knee extension . pnf lower extremity patterns were conducted 30 minutes / day , 5 days / week for 6 weeks . balance was measured with the berg balance scale ( bbs ) , timed up and go test ( tugt ) , functional reach test ( frt ) , and one leg stand test ( olst ) . the bbs consists of 14 items and can be categorized into sitting , standing , and postural changes . scores in each category range from 04 , with 56 possible total points ; higher scores indicate better balance . the tugt measures the time required to stand up from a chair and shuttle back and forth between the chair and a spot in front of the subject three times . the frt measures the distance one can reach with an arm from a standing posture . the olst measures how long one can stand on one foot with the eyes open without placing the other foot on the ground . adl were measured with the functional independence measure ( fim ) , which consists of 13 items related to mobility and 5 related to recognition . the items were scored on a scale of 17 with 126 possible total points ; higher scores indicate better independence . data were analyzed using spss 12.0 ( spss , chicago , il , usa ) for windows . descriptive statistics were used for the general features of the participants . a paired t - test was used to determine pre- and post - experiment differences , and an independent t - test was used to determine between - group differences . the changes in the result of the bbs , tug , frt , olst , and fim are listed in table 2table 2.comparison of the results of the bbs , tug , frt , olst , and fim between the experimental and control groupsgroupprepostd - valuebbseg42.8 1.644.9 1.7 * 2.1 1.7*cg39.9 2.040.6 1.7 * 0.7 0.8tugteg21.9 1.320.1 1.9*1.8 1.3*cg20.4 1.019.7 1.0*0.7 0.6frteg17.7 0.919.0 1.4 * 1.3 0.9*cg16.7 0.617.2 0.4 * 0.5 0.6olsteg4.3 0.85.7 1.1 * 1.4 0.9*cg3.4 0.54.1 0.7 * 0.6 0.7fimeg82.3 2.587.5 3.7 * 5.2 5.3*cg80.1 1.281.4 1.4 * 1.3 1.3meansd , * p<0.05 , d - value : difference value , eg : experimental group , cg : control group , bbs : berg balance scale , tugt : timed up and go test , frt : functional reach test , olst : one leg stand test , fim : functional independence measure . the experimental and control groups showed significant differences for all pre- and post - experiment variables ( p < 0.05 ) . in the between - group comparison , the experimental group was significantly different from the control group ( p < 0.05 ) . meansd , * p<0.05 , d - value : difference value , eg : experimental group , cg : control group , bbs : berg balance scale , tugt : timed up and go test , frt : functional reach test , olst : one leg stand test , fim : functional independence measure the purpose of this study was to investigate the effects of aquatic pnf lower extremity patterns on balance and adl of stroke patients . the experimental group showed a significant improvement in balance and adl after the aquatic pnf ( p < 0.05 ) and had significantly better balance and adl relative to the control group ( p < 0.05 ) . for example , performance of 10 weeks of aquatic proprioception exercise by seniors enhanced their motor abilities11 . in addition , kim and lee12 pnf reported that aquatic lower extremity patterns enhanced balance in healthy adults . moreover , song and kim8 reported that aquatic pnf effectively affected the balance of stroke patients . these results indicate that aquatic pnf lower extremity patterns help enhance the balance of stroke patients . pnf , used as a gradual resistance exercise that employs a helical pattern , maximizes motor unit recruitment through proprioceptive stimulation5 , 13 . aquatic pnf promotes the maximum usage of muscle fibers due to the intense circumstances caused by buoyancy and turbulence ; moreover , the water acts as a form of resistance , stimulating proprioception and thus helping to improve postural control and balance8 , 13 , 14 . the most common method for examining the adl of stroke patients is to use the fim . in the present study , the experimental group showed significant enhancement of adl after the aquatic pnf ( p < 0.05 ) . suomi and collier15 reported that the adl of arthritis patients were enhanced after conducting aquatic exercise , and sato et al.16 reported that the adl of abnormal adults were enhanced after conducting water exercise . similar to other studies , enhancement of adl was observed after conducting aquatic exercise in the present study . the enhancement of adl was the result of enabling movement with even a small amount of force . this was due to the assistance of the water buoyancy and decreased gravity , reducing the burden on muscles and joints10 , 15 . to promote functional recovery , the paretic side was spontaneously enabled , thus enhancing the adl and improving functional independence15 , 16 . the movements also stimulate shortened muscles , promoting a training effect and enhancing adl17 . a limitation of this study was that it utilized a small number of participants , and it was not confirmed that the positive effects persisted . future research should address these things when investigating the effect of aquatic pnf lower extremity patterns in stroke patients .

[ purpose ] this study investigated the effect of aquatic proprioceptive neuromuscular facilitation ( pnf ) patterns in the lower extremity on balance and activities of daily living ( adl ) in stroke patients . [ subjects ] twenty poststroke participants were randomly assigned to an experimental group ( n = 10 ) or a control group ( n = 10 ) . the experimental group performed lower extremity patterns in an aquatic environment , and the control group performed lower extremity patterns on the ground . both exercises were conducted for 30 minutes / day , 5 days / week for 6 weeks . balance was measured with the berg balance scale ( bbs ) , timed up and go test ( tugt ) , functional reach test ( frt ) , and one leg stand test ( olst ) . activities of daily living were measured with the functional independence measure ( fim ) . a paired t - test was used to measure pre- and post - experiment differences , and an independent t - test was used to measure between - group differences . [ results ] the experimental and control groups showed significant differences for all pre- and post - experiment variables . in the between - group comparison , the experimental group was significantly difference from the control group . [ conclusion ] these results indicate that performing aquatic proprioceptive neuromuscular facilitation patterns in the lower extremity enhances balance and adl in stroke patients .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

discovery of vitamin k antagonists ( vkas ) in anticoagulation occurred in the 1920s , when the veterinarian frank schofield studied the hemorrhagic disease affecting cattle consuming sweet clover . karl link and his team studied spoiled sweet clover and , in 1939 , extracted dicumarol and identified the hemorrhagic agent , which was the substance in the sweet clover affecting coagulation . the discovery of dicoumarol made it possible to inhibit thrombosis and to study anticoagulation therapy for humans in the early 1940s . in the decades 1930 and 1940 professor armand quick developed a routine prothrombin time ( pt ) coagulation test , which preceded the use of vkas . this has served as a basis for oral anticoagulant therapy ( oat ) monitoring from the onset . however this first drug for oat subsequently was found to have drawbacks by reason of its long half - life . karl link synthesized more than 150 anticoagulant compounds and found one particularly active molecule , which was named warfarin ( a 4-hydroxy compound ) after the patent holder , the wisconsin alumni research foundation . professor paul owren found factor v and developed a new pt method subsequent to the quick method to overcome its drawbacks . the owren reagent ( combined thromboplastin reagent , thrombotest ) is used mainly in the nordic countries , benelux , and japan . by reason of its different reagent composition , the quick technique is sensitive to the coagulation factors fibrinogen , ii , v , vii , and x , while the owren technique is affected by deficiencies in factors ii , vii , and x. the quick method measures factor v and fibrinogen , which are not dependent on warfarin therapy , and this constitutes a drawback for oat . warfarin is the most widely used drug for oat world - wide , and it has a predictable onset and duration of action and excellent bioavailability . the therapy is effective for a variety of clinical indications of anticoagulation . the dosage and the clinical response still vary markedly among patients , depending on genetic inheritance , age , and metabolism . warfarin is a racemic mixture of two optically active isomers , the r and s forms . it is rapidly absorbed from the gastrointestinal tract ( within 90 min ) and its half - life is 3642 h. in the circulation warfarin is bound to plasma proteins ( mainly albumin ) . warfarin interferes with the cyclic intercon - version of vitamin k and its 2,3 epoxide ( vitamin k epoxide ) and thus is a vka . vitamin k is a cofactor in the carboxylation of vitamin k - dependent coagulation factors . vka inhibits the synthesis of coagulation factors ii , vii , ix , and x in the liver , and they remain partially inactive unless 9 to 13 of the amino terminal glutamate ( glu ) residues are carboxylated to form the ca - binding -carboxyglutamate ( gla ) residues ( figure 1 ) . this carboxylation step renders the coagulation factors functionally active : binding to ca and the phospholipid surface . therapeutic dosages of warfarin decrease , by 3050% , the total amount of each vitamin k - dependent coagulation factor synthesized by the liver . the secreted molecules from the liver are under - carboxylated , resulting in diminished biological activity ( 1040% of normal ) . figure 1the enzyme reactions involved in the metabolic function of vitamin k. vitamin k - dependent carboxylase catalyzes the transformation of peptide- ( factors ii , vii , ix , and x ) bound glutamate residues ( glu ) to -carboxyglutamate ( gla ) residues in the presence of vitamin k hydroquinone , carbon dioxide , and molecular oxygen ( i ) . vitamin k hydroquinone is oxidized in the reaction to vitamin k 2,3 epoxide . the reduction of the latter to vitamin k quinone is catalyzed by vitamin k epoxide reductase , which can use certain dithiols as the reductants ( ii ) . vitamin k quinone can be reduced to vitamin k hydroquinone in the reactions catalyzed by either a dithiol - dependent ( iii ) or an nad(h)p - dependent ( iv ) entzyme . ( uotila l. recent findings on the functions and requirements of vitamin k in humans . klinlab 1998 ; 3 : 97101 ) the enzyme reactions involved in the metabolic function of vitamin k. vitamin k - dependent carboxylase catalyzes the transformation of peptide- ( factors ii , vii , ix , and x ) bound glutamate residues ( glu ) to -carboxyglutamate ( gla ) residues in the presence of vitamin k hydroquinone , carbon dioxide , and molecular oxygen ( i ) . vitamin k hydroquinone is oxidized in the reaction to vitamin k 2,3 epoxide . the reduction of the latter to vitamin k quinone is catalyzed by vitamin k epoxide reductase , which can use certain dithiols as the reductants ( ii ) . vitamin k quinone can be reduced to vitamin k hydroquinone in the reactions catalyzed by either a dithiol - dependent ( iii ) or an nad(h)p - dependent ( iv ) entzyme . ( uotila l. recent findings on the functions and requirements of vitamin k in humans . klinlab 1998 ; 3 : 97101 ) warfarin treatment reduces the number of gla residues ( normal , 9 to 13 ) per clotting factor molecule , with a concomitant fall in coagulant activity . when the number of residues decreases from 13 to 9 , only 70% of the activity of the clotting factor remains ; when one molecule contains six carboxylated residues , only 2% activity is present . the liver excretes both active and inactive coagulation factors to the plasma and both affect international normalized ratio ( inr ) measurement , which possibly has escaped notice in the world health organization ( who ) recommendation for the prothrombin time methodology . the calculation formula is for : inr = ( samplesec / normalsec ) , where isi is the international sensitivity index . when the isi is near 1.0 , the reagent is sensitive and isi has little meaning in the inr calculation . the quick and owren pt methods are the most common and generally accepted means of monitoring vka therapy . a comparison between the pt methods has been published in a recent article and the owren pt was superior . who recommends the use of the inr to harmonize pt results and therapeutic ranges globally both for patient care in clinical practice and in the scientific literature , as the units used formerly proved inadequate for international communication . the challenge is for global clinical laboratories to harmonize inr testing further to a level where inr results are consistent regardless of the methods used . the use of the inr system still involves difficulties with sample citrate concentration , different reagents and thromboplastins , and instruments , and with isi and local isi calibration to harmonize results . horsti and colleagues measured 150 samples from patients on oral anticoagulation using seven commercial reagents and four different calibrator kits . the reagent manufacturer informs the isi value or the laboratory can measure the isi with an isi calibration kit ( local calibration ) for a pt reagent lot . the meaning of isi as power in the inr calculation equation is displayed in the previous section . first , the aim in the original recommendation was to harmonize inr results by calibrating reagent isi values with human combined , which was the primary reference preparation ( irp code 67/40 ) . thromboplastins from different sources ( human brain , rabbit brain , rabbit lung , and ox brain ) yield quite different levels of pt . the hierarchy of reference thromboplastin preparations was presented by van den besselaar and associates . as the who calibration procedure was complex and demanding in the second stage , the recommendation for isi calibration was local calibration using certified lyophilized plasmas . this alternative means of determining the isi involves the use of freshly pooled plasmas from 20 normal individuals and 60 patients receiving coumarin ( oat ) . such numbers of samples are necessary to obtain a precise calibration line for isi calculation . freshly pooled plasma can be used to determine reagent or instrument isi with acceptable precision , or as good a result as with the who calibration model . poller and colleagues ( european concerted action on anticoagulation , ecaa ) have compared local isi calibration and direct inr in the correction for locally reported inrs . for local inr correction ( harmonization ) it is clear that plasmas from patients on warfarin therapy always involve varying amounts of inactive coagulation factors , depending on the level of anticoagulation and individual patient characteristics ( see section on warfarin ) . all these former calibration models involve the principle that the calibrator contains an average amount of inactive coagulation factors ( inhibition ) , which means an average correction for patient inr results . is the average correction of inactive coagulation factors appropriate for individual patient samples in a measuring range from 1 to 5 inr ? is it a fact that calibrators , according to biochemical principles , contain inactive coagulation factors ( inhibition ) ? do different reagents behave identically to active and inactive coagulation factors ? in our earlier studies we measured inactive coagulation factors ( inhibition ) in four different kits and noted conspicuous variation in inhibition . isi calibration should be based on normal plasma and normal plasma dilutions without inactive coagulation factors . in principle today the quick and owren pt methods measure the sum of active coagulation factors ( fii , fvii , fx ) and inhibition by their inactive coagulation factor counterparts . the new - generation pt method can measure separately both active coagulation factors ( fii , fvii , fx ) and the inhibition caused by inactive or partially inactive coagulation factors . two measurements for one sample are needed and the cost per sample is twice as much . in addition , the number of inactive coagulation factors varies markedly in different calibrator kits ( manufactured or local ) . the harmonization of inr results using different pt methods and reagents is problematic if attempted according to the who recommendation . new medications for oat have been developed over a number of years and anticipated without laboratory test control in an effort to displace warfarin medication . the new molecules are too expensive for global use or involve serious side - effects and possibly will never be as popular as warfarin , which is an old and cheap means widely used and accepted globally . the only drawback with warfarin medication is regular laboratory control . it would be important to develop warfarin therapy with greater attention to laboratory control , which helps patient care . different reagents and thromboplastins react variably with inactive coagulation factors and cause difficulties in calibration using patient plasmas that contain inhibiting coagulation factors . every new anticoagulant therapy patient should be tested for inhibition at the commencement of therapy . the accuracy and harmonization of patient inr results for different reagents is of the utmost importance for scientific publications , medication , and patient care . today 's measuring principle is taking the sum of active coagulation factors ( fii , fvii , fx ) , and inhibition of inactive coagulation factors is not a satisfactory approach from the standpoint of accurate patient care . the errors in inrs are too great , which in many ways affects the success of medication and patient well - being , also using the same calibration ( local calibration ) . the new - generation pt offers possibilities of more accurate inr results and patient care based on control of active coagulation factors . the therapeutic inr ranges guiding anticoagulant therapy using old methods are based on the principle that both calibrators and patient samples have inactive coagulation factors ( inhibition ) on average , which compensate for each other in the final inr result . the therapeutic range lies between thrombosis and bleeding , and complications are serious and general . the medication and care of oat patients must be rendered better and safe using a more sensitive pt test .

warfarin is the most widely used medicine for oral anticoagulant therapy ( oat ) . it inhibits the synthesis of coagulation factors ii , vii , ix , and x in the liver and results in the production of inactive or partially active versions of these factors . inactive coagulation factors interfere with prothrombin time measurement ( quick and owren pt ) measuring the sum of coagulation activity and inhibition . the narrow therapeutic range here involves a danger of serious complications and the risk of bleeding or thrombosis . the new - generation pt method can measure coagulation activity and inhibition separately . this new technique promotes patient care and anticoagulant medication ( warfarin , dicoumarol ) based on coagulation activity in vivo . both therapy and laboratory controls should be unquestionably accurate and based solely on in vivo coagulation activity . inactive coagulation factors ( inhibition ) render measurement , calibration , and harmonization . the use of the new - generation pt method based on measurement of coagulation activity in vivo could develop vitamin k antagonist ( vka ) therapy for the marked benefit of patients .

History Warfarin International Normalized Ratio ISI calibration The new-generation prothrombin time method Conclusions

recent times have witnessed much turmoil regarding the ' life is sacred at any cost ' maxim . current technology is capable of indiscriminately maintaining some of the vital functions of the body , but the same technology does not necessarily allow us to heal underlying disease processes . an unintended side effect of modern technological advances has been the plausibility of maintaining moribund patients in a state of suspended animation for prolonged and sometimes indefinite periods . also , advanced resuscitation techniques make it possible to convert death into life - in - death . patients may be stalled in suspended animation ; they are not alive in the sense the we enjoy life but neither are they able to die as long as nutrition , hydration , ventilation , and perfusion are assured . in many cases reanimation of such patients this conundrum is created because we must be prepared to apply life - sustaining technology to patients when the benefit appears to outweigh the risk and when there is a reasonable chance for an outcome that the patient would desire . it frequently seems reasonable to buy sufficient time to see whether the disease will respond to aggressive treatment by instituting the most invasive life support technology . however , if organ system failure is not reversible , then the reasoning behind life support technology becomes moot . we must then be prepared to remove supportive technology when it appears that inevitable death is being delayed , rather than meaningful life prolonged . the courts have repeatedly affirmed competent a patient 's authority to regulate their medical treatment , regardless of their reasoning . however , when the patient becomes incapacitated , family surrogates are granted authority to make decisions regarding treatment options because of their proximate knowledge of what the patient would have wanted before they became incompetent . this position is based on the postulate that any attempt to interject physician paternalism into the surrogate decision - making equation is ethically unacceptable . most rational surrogates are unwilling to continue life support after a reasonable trial has demonstrated that its benefit has passed the point of diminishing returns . however , there is a continuing trend of surrogates demanding that moribund patients be kept on life support after prevailing medical opinions concur that there is no meaningful chance of reanimation . some reasons why this occurs are as follows : 1 . physicians tell surrogates that they can make any decision they want as an open - ended ideal . this puts them in the position of being buyers in a consumer 's market . by asking them to make a choice an observer 's primal reaction to the vibrant external appearance of a body supported in an intensive care unit ( icu ) is radically different from that to a corpse on a morgue slab . as long as the patient ' looks viable , it is emotionally easier to accept the pie in the sky bye and bye long shot cure ' . if the patient can just be maintained comfortably for long enough , then a cure may eventually become possible . 3 . surrogates dislike being in a position of making decisions that directly result in the death of a loved one . once life - supporting care is instituted , the patient has options for ' survival ' that they did not have before , even though they are dependent on ' life support ' . there are now variables that decision makers control , and it is much easier to avoid decisions that may hasten death . instead of yielding to inevitable death , the potential now exists to manipulate it . physicians do not have an exceptional track record in explaining end - of - life issues to patients and their families . it is not uncommon for physicians to ask loaded questions in their quest for end - of - life decisions . for example , ' this is your grandmother 's 17th transfer from a skilled nursing facility in 3 months for sepsis and respiratory failure , and now she 's in kidney failure as well . what do you want to do : everything or let her die ? ' given that choice , most surrogates would opt for doing something rather than nothing , even if ' something ' perpetuated open - ended pain and discomfort . the popular media , especially the tabloids , frequently feature anecdotal articles describing patients who have awakened after years of coma . most if not all of these patients ' conditions have been embellished to generate public interest , and frequently subsequent investigators can not find these patients . accordingly , some families feel that if life support systems can maintain vital signs for a day or a week , then ' suspended animation ' should be possible indefinitely , until a cure is found . the notion of ' medical futility ' as an end - stage process in which vital signs can not be supported further is poorly understood by both physicians and surrogates . in fact , any medical treatment capable of sustaining hemodynamics , ventilation , and metabolism is not technically futile if it achieves that limited goal . therefore , if a patient in a progressive , inevitable death spiral is placed on mechanical ventilation , it is not technically futile if vital signs are sustained , however briefly . it is medically inappropriate but not technically futile . under the current rules , the only test of futility is that embodied by the question , ' will this treatment result in sustained life ? ' if the answer is ' yes ' , then virtually any treatment is fair game , even if it will do nothing to revitalize the patient . perhaps the most effective way of dealing with strong familial incentives to tread the path of least resistance in end - of - life care is twofold . first , in end - of - life issue discussions , we must strive for ' consensus without consent ' . discussions with surrogates should strive for concordance and understanding but not extend to soliciting their consent for medically inappropriate care . second , we should strive to emphasize what streat and coworkers termed , ' the large risk of unacceptable badness ' , rather than a vanishingly small potential for benefit . there are far worse things than death , and many of them occur in icus when futility maxims are circumvented . there is a population of icu patients who will die no matter what treatment is rendered them . medically inappropriate care causes pain , suffering , and discomfort . the fundamental maxim for these patients should be comfort .

when patients or their families rarely request inappropriate end of life care in the icu for capricious reasons . end of life treatment decisions that only prolong discomfort and death are usually emotional and based on unrealistic expectations . i explore some of those reasons in this paper .

None Abbreviations Competing interests

adult onset still s disease ( aosd ) is a chronic systemic inflammatory disorder in which high spiking fever , typical skin rash , and polyarthritis occur . the main biological features are neutrophilic leukocytosis , hyperferritinemia , and negative rheumatoid factor ( rf ) or antinuclear antibodies ( ana ) . others may include splenomegaly , pleuritis , pericarditis , and hepatic abnormalities . even though functional prognosis essentially depends on articular involvement , life - threatening prognosis depends on serious complications , such as hepatic failure , disseminated intravascular coagulopathy , hemophagocytosis , infections , amyloidosis , and cardiomyopathy . in this article we suggest a successful combined therapy of prednisolone ( pd ) , colchicine ( col ) , and cyclophosphamide ( ctx ) and review the literature . a 25-year - old korean woman was diagnosed with aosd four years ago after experiencing a high spiking fever , maculopapular rash , and polyarthritis in her hands , elbows and knees . in laboratory findings , the leukocyte count was 19,900/ l , the serum ferritin level was 719.3 ng / ml ( 10240 ) , and rf and ana were negative . during a follow - up , typical skin rash had disappeared after administration of pd , sulfasalazine or hydroxychloroquine and methotraxate ( mtx ) , but either high fever or polyarthitis was wax and wane , and occasionally , intra - articular injections of corticosteroid were administered . in july 1998 , she was admitted to our hospital because of slowly increasing proteinuria over a 7 month period without pitting edema or hypertension . she was single and had no family history of any rheumatic disease or drug history , such as gold or d - penicilliamine . the results of laboratory data showed that the white - cell count ( wbc ) was 12,800 / l , hemoglobin ( hb ) was 10.6 g / dl , platelet was 610,000 / l , esr was 61 mm / hr , and c - reactive protein ( crp ) was 9.50 mg / dl ( < 0.8 ) . the serum protein and albumin had decreased to 4.5 g / dl ( 6.48.5 ) and 2.1 g / dl ( 3.25.5 ) , respectively . twenty - four hour urine protein excretion was 6.9 g / day , and urine creatine clearance was 93.3 ml / min . in radiographic findings , chest pa was normal and bony erosions were detected in both wrist joints . in the sonography , the sizes of both kidneys were 11.5 cm on the right and 11 cm on the left side with increased renal parenchymal echogenicity . renal biopsy showed amorphous deposits in the mesangial areas , some glomerular capillary walls and in the vascular poles ( figure 1 ) . the amyloid deposit stained with congored displayed apple - green birefringence under the polarizing light . immunofluorescence study for igg , igm , iga , c3 , c1 , c4 , fibrinogen , albumin , and light chains showed segmental positive staining for igm and c3 in the mesangium and trace ( ) positive staining for light chain . electron microscopic examination confirmed amyloid deposits in the mesangial areas , which were characterized by non - branching fibrils arranged in a random array ( figure 2a , 2b ) . serum protein electrophoresis showed a decrease in both total protein and total albumin without a monoclonal spike . urine protein electrophoresis showed an elevated total protein level and an elevated total albumin level . we maintained a high oral daily dose of pd ( 45 mg / day , 1 mg / kg ) , ctx ( 100 mg / day ) , and col ( 1.2 mg / day ) . a follow - up 15 months later , laboratory tests showed that the wbc was 6,000 /ul , hb 13.2 g / dl , platelet 297,000 / l , esr 11 mm / hr , crp 0.11 mg / dl , and the serum protein and albumin had increased to 6.2 g / dl and 4.2 g / dl , respectively . the serum ferritin was 24.3 ng / ml . the urine protein clearance was decreased from 6.9 g / day to 92 mg / day . currently , she is taking pd 7.5 mg , ctx 50 mg , and col 1.2 gm orally . the association of amyloid deposits and aosd is rather unusual and should be considered a serious complication . the development of amyloidosis often occurs in patients with longstanding persistent inflammatory diseases . according to previous histopathologic reports on aosd we have experienced 45 patients with aosd in our hospital and so far , found one case ( 2.2% ) associated with renal amyloidosis , which had developed four years after the onset of aosd . when this complication occurs , amyloid material is preferentially deposited in the glomeruli , and proteinuria and nephrotic syndrome are the most common initial manifestations . helin et al . demonstrated in a retrospective study of nephropathy in rheumatoid arthritis ( ra ) that the most common histopathologic finding was mesangial glomerulonephritis ( gn ) , followed by amyloidosis . amyloidosis was the most common finding in nephrotic syndrome . in a patient with isolated proteinuria , amyloidosis , membranous gn , and mesangial gn were almost equally common . membranous gn was closely related to gold or d - penicillamine therapies , whereas mesangial gn probably was related to ra itself . biopsy is thus a valuable tool in differential diagnosis , assessment of prognosis , and decision - making with regard to treatment . although either nsaid or an oral steroid is usually effective in aosd , some patients with complications of the renal amyloidosis may require sustained therapy with corticosteroid and a cytotoxic agent . the col was also effective in a patient with severe ankylosing spondylitis and nephrotic syndrome due to amyloidosis . in review of 10 cases with aosd complicated by renal amyloidosis , combined drug therapy with pd , col , ctx , azathioprine , and mtx were used . in two of those cases , they were treated with steroids and dialysis due to renal failure . in our experience , it has persistently decreased proteinuria as well as recovered polyarthritis or fever , without any severe complications , during the combined therapy of pd , col , and ctx ( table 1 ) . in conclusion although the mechanism of renal amyloid deposition is unknown , early diagnosis and treatment with pd , col , ctx may produce excellent results .

we report a 25-year - old korean woman with adult onset still s disease ( aosd ) presented with renal amyloidosis , which had developed four years after disease onset . we successfully treated her with prednisolone , colchicine and cyclophosphamide . a review of the literature uncovered about 10 cases , most of which were treated by various regimens that resulted in poor outcomes . renal amyloidosis should be suspected in patients with aosd who have unexplained proteinuria . although the mechanism of renal amyloid deposition is not well known , earlier histopathologic diagnosis and choice of regimen may affect prognosis .

INTRODUCTION CASE DISCUSSION

epilepsy is associated with a two- to three - fold increase in mortality among patients compared with the general population . sudden unexpected death in epilepsy ( sudep ) is one of the most frequent causes of death among patients with epilepsy . there is strong evidence suggesting that sudden unexpected death in epilepsy ( sudep ) is a seizure - related phenomenon , , , . the first description of this phenomenon was introduced by russell in 1906 . since then , several cases have been reported in the literature presenting with a drop in heart rate or asystole during the seizure . bradycardia and asystole resulted from increased parasympathetic flow through the vagus nerve , which originates in the nucleus ambiguous and dorsal nucleus of the vagus in the medulla . on the basis of one study , the incidence of sudep ranges from 1:1000 and 1:2000 person - years to 1:200 person - years , . according to a recent revised definition , sudep consists of sudden , unexpected , witnessed or unwitnessed , nontraumatic and nondrowning death in patients with epilepsy , with or without evidence of a seizure , excluding documented status epilepticus ( seizure duration > 30 min or seizures without recovery in between ) and asphyxia ; if postmortem examination does not reveal a cause of death , the diagnosis is definite sudep , and if there is a preexisting condition before or after autopsy , which could have contributed to the death , it is classified as sudep plus . strong risk factors for sudep include young age , early onset of seizures , the presence of generalized tonic clonic seizures , male sex , and bedtime occurrence . less significant risk factors for sudep include the prone position , one or more subtherapeutic blood levels , sleep occurrence , and a structural brain lesion . the underlying pathophysiologic mechanisms for sudep are not completely understood , but autonomic dysfunction ; ictal arrhythmias , ictal bradyarrhythmia , and asystole , ; neurogenic pulmonary edema ; and ictal central or obstructive apnea , , were introduced in the literature . in this report , we describe two patients with seizure - associated asystole monitored by simultaneous video electroencephalography electrocardiography . a thirteen - year - old right - handed male with seizure disorder was admitted for a presurgical assessment . there was a history of neonatal hypoglycemia during an apparently normal vaginal delivery . he was started on antiepileptic drugs , but the second seizure appeared six months later . the seizures typically consisted of blurred vision and upward gaze followed by a loss of consciousness . his mri showed near symmetrical signal abnormality at parietooccipital regions bilaterally ( parasagittal aspect ) accompanied by mild gliosis and volume loss ( fig . , there was bilateral rhythmic activity maximum to the left associated with right - side clonic jerk and head and eye deviation to the right that secondarily generalized and was followed by 16 s of asystole at the end of the seizure ( fig . 1 , fig . 2 , fig . 3 ) . interictal abnormality consisted of bilateral spike - and - wave and bilateral slow activity maximum in the right posterior head region . at the end of monitoring , an anticonvulsant drug regimen of valproic acid and levetiracetam was started , and cardiology consultation was suggested . a 42-year - old right - handed male with seizure disorder was admitted for presurgical assessment . his epilepsy was due to penetrating head trauma in the left frontal lobe from a shell injury . since then , he had been experiencing episodes of intense fear followed by generalized tonic clonic movements . neurologic examination included mild paresis in the right upper extremity and in the distal part of the right lower extremity in the range of 12/5 . the very first clinical manifestation was after the initial eeg changes and consisted of a generalized tonic afterwards , the sa arrest took place and lasted about 1 min ( see fig . 5 , fig . 6 , the very first eeg change started with 5-hz spike slow waves over the left parasagittal area with the maximum amplitude on c3 and f3 . the interictal abnormality consisted of delta waves seen at p3 , c3 , and f3 . considering his cardiac arrest during the seizure , a cardiac consultation was done , and a pacemaker was implanted . during 24 months of antiepileptic regimen , the frequency of seizures was reduced remarkably . theoretically , these asystoles could have a role in the incidence of sudep , meaning that the presence of ictal bradycardia is a risk factor for sudep . furthermore , in cases of epileptic cardiac dysrhythmia , isolated eeg or ecg recording may prove insufficient , and prolonged simultaneous eeg / ecg monitoring may be required . only simultaneous eeg and ecg recording will reveal a possible cerebral origin of arrhythmias in these patients who are , typically , young . attaining the correct diagnosis is essential because appropriate treatment may prevent cardiogenic sudep , which is thought to be related to potentially lethal arrhythmias , such as asystole induced by epileptic seizures , and to prevent the cardiac side effects of specific antiepileptic drugs . in conclusion , cardiological investigation should be included in epilepsy management to search for abnormalities of hr or ischemic events . in addition , it can provide an appropriate guideline in pharmacotherapy since certain types of drugs including carbamazepine , phenytoin , benzodiazepine , and barbiturates should only be used with caution by patients with epilepsy who have cardiac dysfunction . increasing knowledge about sudep risk factors can have a significant preventive role . moreover , strategies such as taking a detailed cardiovascular history to get the comprehensive clinical picture including a detailed history of symptoms , risk factors , and prior cardiac findings should be undertaken .

ictal asystole is a rare , probably underestimated manifestation of epileptic seizures whose pathophysiology is still debated . this report describes two patients who had cardiac asystole at the end of their seizure . the first patient was a 13-year - old boy with complex partial seizures .. his mri showed symmetrical signal abnormality in the bilateral parietooccipital lobe accompanied by mild gliosis and volume loss . during a 3-day long - term video - eeg monitoring , he had cardiac arrest at the end of one of his seizures that was secondarily generalized . the second one was a 42-year - old veteran with penetrating head trauma in the left frontal lobe due to shell injury . during long - term video - eeg monitoring , he had one generalized tonic clonic seizure accompanied by bradycardia and cardiac asystole . asystoles could have a role in the incidence of sudden unexpected death in epilepsy ( sudep ) , meaning that the presence of ictal bradycardia is a risk factor for sudep . in cases of epileptic cardiac dysrhythmia , prolonged simultaneous eeg / ecg monitoring may be required . cardiological investigation should be included in epilepsy management .

Introduction Case1 Case2 Discussion

this condition , which is estimated to affect more than 2 million persons in the u.s . alone , has increased mortality and morbidity compared to the general population [ 2 , 3 ] . individuals with schizophrenia typically suffer from a combination of debilitating symptoms including hallucinations and delusions and treatment - resistant symptoms , such as social withdrawal . the disease affects males and females , although there is evidence to support a number of sex differences in the characteristics of schizophrenia . compared to females , males may be more likely to develop schizophrenia with ~1.4 : 1 ratio [ 5 , 6 ] , have an earlier age at onset [ 68 ] , poorer premorbid social and intellectual functioning , poorer course and medication response , greater structural brain abnormalities , more negative , symptoms and fewer affective symptoms . twin , family , and adoption studies together suggest that schizophrenia is a complex disorder involving both genes and environment [ 11 , 12 ] . further , the evidence suggesting that schizophrenia arises from a process involving prenatal environmental conditions is compelling [ 1321 ] . numerous case - control studies have demonstrated that individuals with schizophrenia are more likely to have been exposed to prenatal / obstetric complications than their unaffected siblings , normal controls , or psychiatric controls , and a meta - analysis of twelve twin studies demonstrated that a nontrivial proportion of liability to schizophrenia can be accounted for by a common or shared environmental effect ( 11% ; 95% ci 3%19% ) . because the environments of twins are most similar in utero , the role of common environment effects on liability for schizophrenia would most likely occur very early in life . suspected fetal environmental risks include exposure to maternal stress , influenza [ 2426 ] , infection , famine or prenatal nutritional deficiency [ 16 , 2831 ] , and obstetric complications [ 14 , 15 , 18 , 19 , 32 ] . several reviews [ 8 , 21 , 33 ] , including a meta analysis , support the involvement of ( i ) pregnancy complications such as rhesus d incompatibility and pre - eclampsia , ( ii ) abnormal fetal growth and development , and ( iii ) delivery complications that produce fetal hypoxia as risk factors and suggest that obstetric complications contribute approximately a 2-fold increased risk for schizophrenia . prenatal / obstetric complications are believed to disrupt normal fetal neurodevelopment and their involvement in schizophrenia susceptibility is consistent with the neurodevelopmental hypothesis of schizophrenia . this hypothesis posits that brain development is disrupted early in life and that subsequent maturational events in combination with other environmental factors leads to the emergence of psychosis during adulthood [ 3436 ] . support for the important role of prenatal / obstetric complications in schizophrenia also comes from neuroimaging studies . as an example , there is evidence that fetal hypoxia has a differential effect on the hippocampus of schizophrenics and their first degree relatives , suggesting that this temporal lobe region may be sensitive to prenatal environmental conditions [ 3739 ] . furthermore , anatomical deficits in the medial temporal lobe structures are more severe among patients with schizophrenia who have a history of hypoxia - associated obstetric complications . hence , not only do these studies suggest that factors that produce prenatal / obstetric hypoxia have an effect on the medial temporal lobe structure , but that genetic liability to schizophrenia also plays a role in predisposing an individual to schizophrenia . this evidence has produced a variety of hypotheses regarding how genetic and environmental influences aggregate to increase susceptibility to schizophrenia , with gene - environment interaction , gene - environment covariation , or direct environmental effects , that is , phenocopy model , as ( potentially overlapping ) models . to date , there is very little evidence to support a phenocopy model or a gene - environment covariation model to explain the role of prenatal / obstetric complications in schizophrenia , although additional investigation of these models is warranted . in contrast , evidence that prenatal / obstetric complications increase risk for schizophrenia through a gene - environment interaction model is accumulating . in addition to the studies cited earlier [ 3740 ] , a recent study found that risk of schizophrenia was greatest among individuals at highest familial liability who were exposed to maternal infection ( consistent with an interaction model ) . as another example , significant interaction between suspected hypoxia - regulated / vascular - expression genes and serious obstetric complications ( predominantly hypoxia ) although the causes for prenatal complications are quite heterogeneous , their diversity does not exclude a final common pathway and there is increasing discussion that the common pathway involves both the immune and vascular systems in the pathogenesis of schizophrenia [ 43 , 44 ] . in an excellent review of this theory of schizophrenia , hanson and gottesman describe a process in which ubiquitous environmental factors that normally trigger genetically - influenced inflammatory response ( infection , trauma , hypoxia ) in individuals will trigger abnormal inflammatory processes in individuals with particular genotypes at these inflammatory response loci which results in damage to the microvascular system in the brain . this vascular - inflammatory theory not only accommodates the diversity of prenatal complications associated with schizophrenia , but also specifies an interaction between genes and environment . the latter point helps to explain why most people who experience prenatal / obstetric complications do not eventually develop schizophrenia [ 19 , 21 ] , and has received empirical support through the increasing number of studies demonstrating gene - environment interactions in schizophrenia [ 27 , 3740 , 42 ] , particularly the recent study that identified an interaction between serious obstetric complications and hypoxia - regulated / vascular - expression genes . not surprisingly , there remains considerable interest in identifying fetal environmental risk factors and elucidating their role in schizophrenia . however , in addition to their heterogeneity , prenatal / obstetric complications can be difficult to document reliably through medical records or maternal recall , making it difficult to test the role of environmental insults in the pathogenesis of schizophrenia . of interest , there is some evidence that prenatal complications that increase susceptibility to schizophrenia cluster within schizophrenia families , which raises the possibility that some of these complications may have a genetic basis and that these risk genes and hence , adverse prenatal environment , can be measured directly through genetic analyses rather than through medical records or maternal recall . a benefit of direct measurement of the adverse prenatal environment through genetic analysis is that it can facilitate hypothesis testing regarding the role of prenatal / obstetric complications in the pathogenesis of schizophrenia . maternal - fetal genotype incompatibility , first described by palmer et al . to describe a mechanism that confers risk for schizophrenia through maternal - fetal genotype combinations which produce a maternal immunological reaction that creates an adverse prenatal environment , as will be described , maternal - fetal genotype incompatibility can occur when maternal and fetal genotypes differ from one another , or when maternal and fetal genotypes are too similar to each other . incompatibility genes for each of these scenarios have been implicated as risk factors for schizophrenia and are reviewed below . importantly , maternal - fetal genotype incompatibility is explicitly genetic in nature and so has the potential to be measured directly through genetic analyses even years after the adverse prenatal event has occurred . the teratogenic antibody hypothesis posits that a pregnant female can develop antibodies in response to some antibody producing stimulus ( e.g. , contact with paternal antigens ) that can interfere with normal fetal neurodevelopment . one general mechanism that is consistent with the teratogenic antibody hypothesis involves maternal - fetal genotype combinations that adversely affect the developing fetus by inducing a maternal immunological attack . this mechanism is a form of maternal - fetal genotype incompatibility , where the development of maternal antibodies can be the result of a mother 's genotype that is different from the fetus ' genotype . in some cases , a maternal immunological reaction can lead to hypoxic ischemia , a condition found to be associated with schizophrenia [ 17 , 18 ] , and hypothesized to trigger abnormal inflammatory processes in individuals with vulnerable genotypes at inflammatory response loci resulting in damage to the micro - vascular system in the brain and increasing risk for schizophrenia . conditions that can produce fetal or neonatal hypoxia include maternal - fetal genotype incompatibilities at genes that produce red blood cell antigens , such as the rhd locus . the rhd gene produces a red blood cell antigen called the rhesus d factor . an individual who is determined to be rhesus d positive has red blood cells ( rbcs ) with this antigen , while someone classified as rhesus d negative lacks this antigen . individuals who are rhesus d positive are either homozygous or heterozygous for an allele that produces the antigen ( referred to here as d / d or d / d ) . individuals who are rhesus d negative are homozygous for a null allele ( d / d ) . in caucasian populations , rhd maternal - fetal genotype incompatibility during pregnancy occurs when a pregnant woman is rhesus d negative ( d / d ) and her fetus is rhesus d positive ( d / d ) . because the rbcs of a rhesus d negative pregnant female do not possess the rhesus d antigen , maternal anti - d ( igg ) antibodies are created in response to detection of fetal rbcs in the maternal blood stream . these antibodies destroy the fetal rbcs in the maternal blood stream , cross the placenta , and destroy fetal rbcs . because rbcs carry oxygen throughout the fetus ' body , including the brain , an attack on the fetal rbcs increases risk for fetal hypoxia which could affect developing tissue , including brain tissue . a byproduct of the destruction of rbcs is bilirubin ; thus hyperbilirubinemia , or jaundice can occur , as well as kernicterus , which is deposition of bilirubin in the brain . bilirubin is a known neurotoxin [ 50 , 51 ] to which undifferentiated glial cells are sensitive [ 52 , 53 ] , and glial cell abnormalities also have been associated with schizophrenia [ 54 , 55 ] . an infant is said to have rhesus hemolytic disease of the newborn when clinical complications arise due to the rhd maternal - fetal genotype incompatibility . because maternal sensitization usually does not occur until delivery of the first rhd incompatible pregnancy , it is not until second- and later - incompatible pregnancies that risk for a maternal immune attack becomes appreciable . around 1970 prophylaxis against maternal isoimmunization became available , which has made a dramatic impact on the morbidity and mortality associated with rhd maternal - fetal genotype incompatibility . however , even in an era of prophylaxis , there continue to be cases of rhesus hemolytic disease of the newborn , either due to lack of prophylaxis use [ 56 , 57 ] , or because its use is not 100% effective at preventing maternal sensitization [ 58 , 59 ] . evidence to support involvement of rhd maternal - fetal genotype incompatibility in schizophrenia comes from both nongenetic and genetic studies performed on samples in which individuals with schizophrenia predominantly were born prior to 1970 [ 15 , 19 , 47 , 6066 ] and reviewed in . the nongenetic studies are based on serotype data ( rhesus d negative , rhesus d positive ) or evidence of hemolytic disease of the newborn in mother - child pairs [ 19 , 32 , 60 , 61 , 63 , 64 , 66 ] , while the genetic studies are based on genotype data ( d / d , d / d , d / d ) and nuclear families [ 47 , 65 , 67 ] . collectively , these studies have provided evidence that rhd maternal - fetal genotype incompatibility is a risk factor for schizophrenia with relative risk ranging from 1.4 to 2.26 , a magnitude , that is , comparable to the relative risk of schizophrenia due to obstetric complications in general or due to most genes for which an association with schizophrenia has been observed ( see [ 68 , 69 ] for reviews ) . it is remarkable that studies of rhd maternal - fetal genotype incompatibility and schizophrenia that differ in design and population cohort should arrive at similar relative risk estimates ; this consistency suggests that the relative risk of schizophrenia due to rhd maternal - fetal genotype incompatibility , although small , is substantively meaningful and worthy of more investigation . another way to look at the magnitude of the rhd maternal - fetal genotype incompatibility effect is to compute the population attributable fraction , that is , the number of cases which would not occur if the risk factor is eliminated . based on formulas found in , for rhd maternal - fetal genotype incompatibility , the population attributable fraction is ~3% , as estimated from the fraction of cases that have an rhd maternal - fetal genotype incompatibility ( 7.8% ) and the relative risk due to the incompatibility ( using the most conservative estimate of 1.5 in ) . based on a population prevalence for schizophrenia of 1% and assuming that the allele frequencies for the rhd locus are homogeneous in the u.s . , this attributable fraction suggests that more than 100,000 schizophrenia cases in the u.s . would not have occurred but for the rhd maternal - fetal genotype incompatibility this is not a trivial number , and for comparison , it has been estimated that more than 100,000 cases of schizophrenia in the u.s . would not have occurred but for the val allele of the comt gene . thus , these two loci , that is , comt and rhd , could potentially account for an effect of similar size at the population level . of course , the allele frequencies at the rhd locus differ across populations , with the d allele being less common in some populations than others ( e.g. , p(d ) = .76 in a study conducted in nairobi compared to p(d ) = .66 in the finnish population ) . thus , the frequency of rhesus negative mothers having rhesus positive children will vary across populations . this variability in allele frequencies will not affect the relative risk of disease due to rhd maternal - fetal genotype incompatibility , but will affect the fraction of schizophrenia cases that are attributed to the rhd locus across populations . since most individuals who are exposed to rhd maternal - fetal genotype incompatibility do not develop schizophrenia , it is highly unlikely that exposure to this adverse prenatal environment alone , that is , a phenocopy model , explains risk for schizophrenia . furthermore , the lack of evidence for violation of hardy - weinberg equilibrium in the founder alleles from the family - based rhd genetic studies [ 47 , 67 ] is inconsistent with a gene-environment covariation model because it suggests that mate selection in the schizophrenia families occurred independently of rhd genotype , at least among the founders . to date there have been no empirical studies to determine whether the association between rhd maternal - fetal genotype incompatibility and schizophrenia is explained through a gene-environment interaction model . there also is emerging evidence in studies based on serotype data and those based on genotype data that risk of schizophrenia due to rhd maternal - fetal genotype incompatibility may depend on offspring sex [ 61 , 66 , 67 ] , with a relative risk of 1.64 in male incompatible offspring and 1.07 in female incompatible offspring based on a recent meta - analysis . furthermore , a nonsignificant trend suggesting that male offspring are at higher risk than female offspring for schizophrenia due to maternal - fetal genotype incompatibility at another rbc antigen locus , abo , has also been identified based on serotype data . abo maternal - fetal genotype incompatibility occurs when a pregnant woman has type o blood and her fetus has type a or b . as with rhesus d incompatibility , maternal igg antibodies can be produced against the fetal antigens and result in hemolytic disease of the newborn , although in this case the risk is the same for all pregnancies [ 75 , 76 ] . these sex - dependent findings allow for hypotheses that address why the schizophrenia effect of an rbc antigen associated maternal immune response is so much greater for male offspring compared to female offspring . it is unlikely to be the case that rhd maternal - fetal genotype incompatibility is more likely to occur in pregnancies with male offspring , nor is there evidence that its related condition of hemolytic disease of the newborn is more likely to occur in pregnancies with male fetuses compared to female fetuses . however , there is evidence that the clinical manifestations of rhd maternal - fetal genotype incompatibility are more severe in pregnancies with male fetuses than with female fetuses . thus one hypothesis is that specific schizophrenia - effects of rhd maternal - fetal genotype incompatibility ( hypoxia , hyperbilirubinemia ) can affect female fetuses but that they are less likely to surpass the threshold of severity compared to male fetuses ( threshold effect ) . there also is evidence that the clinical effects of rhd maternal - fetal genotype incompatibility may occur earlier in gestation for male fetuses compared to female fetuses . coupled with research supporting sex - differences in brain maturational rates , with males exhibiting a slower pace of cerebral development compared to females , another hypothesis is that male and females are equally vulnerable to the specific effects of rhd maternal - fetal genotype incompatibility , but that these effects must occur at sex - dependent times during development . this hypothesis further suggests that female fetuses may be at increased risk for schizophrenia when subject to prenatal / obstetric complications that produce hypoxia or hyperbilirubinemia , but that these effects must occur earlier in the gestational period to increase their risk of schizophrenia ( a timing effect ) . a third hypothesis is that male fetuses , but not female fetuses , experience schizophrenia effects due to hypoxia or hyperbilirubinemia ( a specific effect ) . although there have not yet been studies addressing whether risk of schizophrenia due to rhd maternal - fetal genotype incompatibility in male and female fetuses is a function of a threshold effect or a timing effect , there have been studies addressing potential sex differences in rates of hypoxia - related in males and females , with conflicting results [ 79 , 80 ] . the involvement of the rhd gene in the form of maternal - fetal genotype incompatibility as a risk factor for schizophrenia susceptibility is further substantiated by analyses that showed no evidence to support the idea that this locus is simply linked / associated with a nearby schizophrenia susceptibility locus or that this gene acts through the maternal genotype alone . furthermore , there is empirical evidence consistent with the hypothesized biological mechanism that previous rhd maternal - fetal genotype incompatible pregnancies increase risk for maternal isoimmunization in subsequent pregnancies in the two schizophrenia rhd maternal - fetal genotype incompatibility studies that tested this hypothesis [ 61 , 65 ] . using serotype information , hollister et al . divided their birth cohort sample into firstborn rhesus d - incompatible and rhesus d - compatible males , and second- or later - born rhesus d - incompatible and rhesus d - compatible males . consistent with a birth order effect , they found the rate of schizophrenia among the second- or later - born rhesus d - incompatible males was significantly higher than the second- or later - born rhesus d - compatible males ( 2.6% versus 0.8% , p = .05 ) ; but that there was no significant difference in the rate of schizophrenia between the firstborn rhesus d - incompatible and d - compatible males ( p = .64 ) . in the second study , kraft et al . tested hypotheses about a birth order effect using nuclear families with at least one individual with schizophrenia and rhd genotype data . the model in which there is increased risk only to second- or later - born incompatible children fit the data well , with a significant point estimate of 1.7 relative risk to second- or later - born incompatible children ( p = .014 ) . the other model that fit the data well assumed an increased risk for all incompatible children regardless of birth order ; the point estimate for the relative risk of schizophrenia in this model was 1.5 ( lower than the former model ) . however , one issue with the latter model is that it forced the risk to first - born incompatible children to be identical to risk to later - born incompatible children , and essentially produced an average relative risk across birth order groups . since the relative risk was estimated at 1.7 in a model that assumed no risk to first - born , and then the relative risk was lowered to 1.5 in a model that averaged over all incompatible children , the authors concluded that the effect of including the first - born incompatible children was to artificially lower the relative risk relative risk estimates of rhd maternal - fetal genotype incompatibility for the later - born children . it is important to note that neither study had information on pregnancies that did not go to full term , for example , spontaneous abortions . the potential effect of this lack of information is to misclassify some rhd maternal - fetal genotype incompatible individuals as first - born ( and at very low risk from maternal sensitization ) when in fact they were later - born and at heightened risk due to previous maternal sensitization . such misclassification would serve to underestimate the difference between groups and bias results toward the null hypothesis of no birth order effect . in light of the challenges of truly examining a birth order effect with rhd maternal - fetal genotype incompatibility , it is striking that the two schizophrenia studies that chose to test birth order hypotheses found evidence in support of such an effect . however , further examination of this hypothesis is warranted due to the findings that suggest that the risk of schizophrenia associated with rhd maternal - fetal genotype incompatibility is limited to male offspring . in this case , one would expect to observe an increased risk among second- or later - born incompatible males , but not females . the involvement of rhesus d incompatibility in schizophrenia was initially provided by studies that inferred genotype status through serotype data . importantly , the evidence from these non - genetic studies provided the impetus for conceptualizing maternal - fetal genotype incompatibility as a more general non - mendelian mechanism involved in the etiology of complex disorders such as schizophrenia . the first candidate gene study to test the hypothesis of rhd maternal - fetal genotype incompatibility as a risk factor for schizophrenia provided the proof of principle that this non - mendelian mechanism can be tested with genotype data . further , it facilitated the development of statistical methods and study designs based on a candidate gene approach and nuclear families for addressing hypotheses about the role of maternal - fetal genotype incompatibility in disease [ 65 , 8185 ] . such innovations are important because , as illustrated in the next section , not all incompatibility genes can be inferred through serotype data . human leukocyte antigens ( hlas ) play an important role in the control of immune responses and there has long been a belief that hlas play a role in schizophrenia susceptibility , although with conflicting results from genetic studies examining the hypothesis of a high risk allele acting through the affected individual 's genotype . another way to conceptualize the role of hla in schizophrenia susceptibility is to consider its role(s ) in pregnancy . there is strong evidence for maternal recognition of paternally - derived fetal hlas during pregnancy because maternal antibodies against these fetal antigens have been detected . however , maternal recognition of paternally - derived fetal hlas that differ from maternal hlas is believed to be beneficial to implantation and maintenance of pregnancy because maternal antibodies to fetal antigens have been observed in a large number of healthy pregnancies . in contrast , lack of maternal recognition , which is the result of paternally derived hlas that are not perceived as different from the maternal hlas , may lead to adverse reproductive outcomes . the underlying biological mechanism for poor reproductive outcomes is not yet known , however , an immunological intolerance hypothesis posits that hla similarity between mother and fetus fails to stimulate an adequate maternal immune response that is necessary for proper implantation and maintenance of pregnancy . there is some empirical evidence that situations where maternal sensitization would not occur , that is , hla matching between couples or between mother and fetus , increases the risk of fetal loss [ 8992 ] , preeclampsia [ 9396 ] , low birth weight [ 97100 ] , newborn encephalopathy , and seizures . importantly , low birth weight and preeclampsia are complications that have been associated with schizophrenia [ 15 , 19 , 102104 ] . the mechanism(s ) by which low birth weight or preeclampsia increase risk for schizophrenia is not yet known . however , a current theory regarding preeclampsia hypothesizes that this condition gives rise to abnormal fetal blood flow that results in chronic fetal hypoxia or malnutrition and both of these conditions are associated with schizophrenia [ 15 , 16 , 2831 , 102 , 103 ] . furthermore , preeclampsia involves a generalized inflammatory response in the mother as a result of the oxidatively stressed or hypoxic placenta , and inflammatory processes are hypothesized to damage the microvasular system of the brain [ 43 , 44 ] and increase risk of schizophrenia . two additional lines of evidence implicate maternal - fetal hla matching in schizophrenia . first , evidence supporting the relevance of hla matching to neurodevelopmental disorders comes from a study that found that parents of children with autism were significantly more likely to share at least one hla - a , -b , or -c antigen in common compared with parents of unaffected children . second , circumstantial evidence supporting the relevance of hla matching specifically to schizophrenia comes from the literature on mate selection and the literature on olfaction in schizophrenia . specifically , in the mate selection literature there is some evidence to support disassortative mate selection with respect to hla loci [ 108111 ] and that olfaction plays a role in this process [ 109 , 110 , 112 ] . however , studies of individuals with schizophrenia and their unaffected first degree relatives reveal impairments in olfaction [ 113116 ] . hence , mate selection in this subgroup of individuals may be less likely to be guided by the ability to sniff out a mate with hla dissimilarity , and thus more likely to result in the construction of couples with hla similarity for whom maternal - fetal hla matching is more likely to occur . because risk of schizophrenia is associated with prenatal / obstetric complications , including preeclampsia and low birth weight , maternal - fetal hla matching has been associated with these and other pregnancy / obstetric complications , maternal - fetal hla matching has been observed in another neurodevelopmental disorder , and maternal - fetal hla matching may occur more frequently in families of individuals with schizophrenia for biological reasons , a candidate gene study was conducted to assess maternal - fetal genotype incompatibility , that is , matching , at the hla - a , -b , and -drb1 loci as a risk factor of schizophrenia . for this study , palmer and colleagues hypothesized that maternal - fetal genotype incompatibility increased risk of schizophrenia through a general allele - matching phenomenon rather than through specific allele combinations . for each locus , mother and offspring were considered to match if the offspring 's alleles were identical to the maternal alleles or if the offspring 's alleles were a subset of the maternal alleles . in either of these cases , maternal sensitization to fetal antigens would not occur because they would be perceived to be the same as the maternal antigens . the maternal - fetal genotype incompatibility test for multiple siblings was modified to accommodate analyses involving a general allele - matching phenomenon and missing parental genotypes . there was no evidence for violation of hardy - weinberg equilibrium in the founder alleles , consistent with random mating with respect to these three loci . there was no evidence for hla - a or -drb1 maternal - fetal genotype matching effect on schizophrenia . in contrast , there was significant evidence for an hla - b maternal - fetal genotype incompatibility effect ( p = .01 ) where inspection of the parameter estimates revealed that maternal - fetal genotype matching produced a higher risk for female offspring ( 1.74 , 95% ci : 1.222.49 ) than for male offspring ( 1.11 , 95% ci : 0.761.61 ) . of note , in the mate selection literature , hla - b appears to be particularly influential . as this is the first study to demonstrate an association between hla - b matching and schizophrenia , much more research is needed to determine the mechanism through which this form of maternal - fetal genotype incompatibility increases risk for schizophrenia . one possibility is that hla - b matching increases risk for adverse reproductive outcomes such as preeclampsia or low birth weight . this hypothesis could be tested by examining prenatal and birth records in a sample of females with schizophrenia stratified by hla - b matching status , and comparing the rates of preeclampsia , low birth weight , and other pregnancy / obstetric complications between the two groups . it also currently is unclear why female offspring would be more vulnerable to effects of hla - b matching than male offspring . one possibility is that female fetuses are more likely to survive the putative effects of hla - b matching , such as preeclampsia and hence to be observed in a study , than male offspring . although the sex - dependent finding is intriguing in light of the work demonstrating that rhd maternal - fetal genotype incompatibility as a schizophrenia risk factor is limited to males , replication and investigation of hypothesized clinical manifestations of hla - b matching ( low birth weight , preeclampsia , other complications ) are warranted because other published studies reveal conflicting results regarding sex differences in the rates of low birth weight and preeclampsia among individuals with schizophrenia [ 102 , 104 ] . because the firstborn child of a couple is at highest risk for preeclampsia , one could also seek further evidence in support of an hla - b matching preeclampsia relationship by testing for a birth order effect . future research must provide additional evidence for an association between hla - b matching and schizophrenia , determine if there are clinical outcomes of hla - b matching , for example , prenatal / obstetric complications , whether hla - b matching increases risk through a phenocopy model or a gene-environment interaction model , or is simply associated through a gene-environment covariation model , and determine the basis for a sex - dependent risk . it will be particularly important to distinguish between a gene-environment covariation model and a gene-environment interaction model given the a priori basis for expecting higher rates of hla - b matching in schizophrenia as a function of olfaction deficits . based on this review , there are a variety of hypotheses that could be tested in future research to further elucidate the role of rhd and hla - b maternal - fetal genotype incompatibility in schizophrenia . one important area of research would focus on conducting studies that add to the evidence that these maternal - fetal genotype incompatibilities are risk factors for schizophrenia . as examples , since rhd maternal - fetal genotype incompatibility is genetic in origin , one would expect more clustering of schizophrenia in families with rhd maternal - fetal genotype incompatibility than in schizophrenia families without rhd maternal - fetal genotype incompatibility . if rhd maternal - fetal genotype incompatibility is a risk factor specifically for males , one would expect to observe that schizophrenia risk is associated with a birth order effect with male offspring exposed to rhd maternal - fetal genotype incompatibility , but not for female offspring . if hla - b matching is involved in predisposition to pre - eclampsia , low birth weight , or other prenatal / obstetric complication , one would expect higher rates of these prenatal / obstetric complications in individuals with schizophrenia with hla - b maternal - fetal genotype matching compared to those without hla - b maternal - fetal genotype matching . if hla - b matching is involved specifically in predisposition to pre - eclampsia , one would expect to observe that schizophrenia risk is associated with a birth order effect with female offspring exposed to hla - b maternal - fetal genotype incompatibility , but not for male offspring . a second area of research would focus on if / how the maternal - fetal genotype incompatibility integrates with genetic liability for schizophrenia ( phenocopy , gene - environment covariation , and gene - environment interaction ) . the phenocopy and gene - environment covariation models are unlikely to explain the association between rhd maternal - fetal genotype incompatibility and schizophrenia . however , one possible explanation for the finding that most people with a history of rhesus d incompatibility do not develop schizophrenia is that the schizophrenia - producing effect of rhd maternal - fetal genotype incompatibility manifests only in individuals with genetic predisposition to schizophrenia . if this is the case , then one would expect different risks for schizophrenia based on family history and rhd maternal - fetal genotype incompatibility , with greatest risk of schizophrenia among genetically high risk individuals who are exposed to rhd maternal - fetal genotype incompatibility . following the recent work of clarke et al . , one could test for synergism between rhd maternal - fetal genotype incompatibility and family history of psychosis by comparing the rates of schizophrenia across four groups : no rhd maternal - fetal genotype incompatibility and no family history of psychosis , rhd maternal - fetal genotype only , family history of psychosis only , and rhd maternal - fetal genotype incompatibility and positive family history of psychosis . the same interaction hypothesis could be tested for hla - b maternal - fetal genotype incompatibility ; however additional research is needed also to test the phenocopy and gene - environment covariation models with respect to the association between hla - b matching and schizophrenia . a third area of research would focus on hypotheses that hypoxia is the prominent schizophrenia - producing effect of rhd / hla - b maternal - fetal genotype incompatibility . as examples , if the schizophrenia risk effect of rhd maternal - fetal genotype incompatibility is the result of hypoxia , then one would expect to observe an interaction between rhd maternal - fetal genotype incompatibility and hypoxia - regulated / vascular - expression genes . the same hypothesis can be tested with hla - b maternal - fetal genotype incompatibility . if the schizophrenia risk effect of rhd maternal - fetal genotype incompatibility is the result of hypoxia , then one would expect to observe smaller hippocampal volume in individuals with schizophrenia exposed to rhd maternal - fetal genotype incompatibility compared to those not exposed . the same hypothesis can be tested with hla - b maternal - fetal genotype incompatibility . if the schizophrenia risk effect of rhd maternal - fetal genotype incompatibility is the result of hypoxia , then one would expect rhd maternal - fetal genotype incompatibility to be associated with neurocognitive functions that may be sensitive to the effects of prenatal hypoxia in schizophrenia , for example , verbal learning and memory . the same hypothesis can be tested with hla - b maternal - fetal genotype incompatibility . a fourth area of research would focus on hypotheses to further examine offspring sex - dependent differences in the schizophrenia - producing effects of rhd maternal - fetal genotype incompatibility . for this area of research , hypotheses regarding sex - dependent differences in amount of exposure ( threshold effect ) , gestational timing of exposure ( timing effect ) , and type of exposure , that is , hypoxia and hyperbilirubinemia ( specific effect ) are likely best tested using animal models which can systematically vary conditions of hypoxia and hyperbilirubinemia . similar investigations can be performed when the prenatal effects of hla - b maternal - fetal genotype incompatibility are better elucidated . the attributable risk associated with these maternal - fetal genotype incompatibilities is limited to populations in which the incompatibility occurs with appreciable frequency . as one example , the rhesus d negative allele is less common in african and asian populations than european caucasian populations [ 72 , 120 , 121 ] , hence rhd maternal - fetal genotype incompatibility is less likely to contribute to schizophrenia susceptibility in those populations . however , rhd is not the only blood antigen locus for which a maternal - fetal genotype incompatibility could arise . other blood antigens exist , including abo , rhce , kell [ 123 , 124 ] , duffy , kidd , and mn [ 125 , 126 ] and maternal - fetal genotype incompatibilities for these antigens can give rise to a maternal immune response that is similar , although smaller in magnitude , to the rhd incompatibility response . in addition , other genes that could lead to fetal hypoxia , hyperbilirubinemia , or other prenatal conditions associated with schizophrenia , whether through maternal - fetal genotype incompatibility , maternal genetic effects alone , or fetal genetic effects alone , should be examined . prenatal environmental factors are quite heterogeneous and difficult to document reliably , making it difficult to test the role of environmental insults in the pathogenesis of schizophrenia . however , there is growing evidence that many prenatal / obstetric complications have a genetic basis , and one stream of research has focused on identifying combinations of maternal - fetal genotypes , that is , maternal - fetal genotype incompatibilities , that predispose to prenatal / obstetric complications . maternal - fetal genotype incompatibility can occur when maternal and fetal genotypes differ , for example , rhd maternal - fetal genotype incompatibility , or when they are too similar , for example , hla - b maternal - fetal genotype incompatibility . thus far , the rhd , abo , and hla - b genes have been implicated as risk factors for schizophrenia , with increasing evidence that male and female offspring may be differentially vulnerable to the effects of maternal - fetal genotype combinations involving these genes . a growing number of studies demonstrate that an interaction between prenatal / obstetric complications and putative susceptibility genes increases risk for schizophrenia . thus , these maternal - fetal genotype incompatibilities are likely to be part of a complex mixture of factors ( genetic and environmental ) , which together act on the brain in ways yet to be identified to result in schizophrenia . the empiric data demonstrating a relationship between these maternal - fetal genotype incompatibilities and schizophrenia provide hypotheses for future investigations to further our understanding of their role in increasing risk of schizophrenia . until recently , studies to understand the role of maternal - fetal genotype incompatibility in schizophrenia ( or any complex disorder ) have inferred immunologically relevant genotypes solely from birth records and for the single phenomenon of hemolytic disease . as illustrated in this review , maternal - fetal genotype incompatibility at other loci , such as hla loci , may also increase risk for schizophrenia . however , because these loci do not result in hemolytic disease of the newborn it may be challenging , a priori , to examine their role through information gleaned from birth records . hence , the development of study designs and statistical methods to study prenatal risk factors based on genotype data are essential for further delineating maternal - fetal genotype incompatibility as a non - mendelian mechanism in complex disease . in fact , genetic studies that do not model non - mendelian patterns of inheritance directly may be one contributing reason that current genome scans have not found striking and highly replicable results in complex disorders that otherwise are so highly familial . the approach described here integrates the investigation of genes and environment in an innovative manner and provides empirical data that fits within and can be further tested in a genetic - inflammatory - vascular hypothesis of schizophrenia . there are several reasons why it is important to further investigate maternal - fetal genotype incompatibly as a risk factor for schizophrenia : ( 1 ) it is a new research approach that allows precise identification of a putative high - risk prenatal environment , even years after the adverse environment has occurred ; ( 2 ) using a genetic approach , it is possible to simultaneously evaluate alternative explanations of allelic effects that act solely through the genotype of the mother or child ; ( 3 ) if certain maternal - fetal genotype incompatibilities , for example , rhd , do increase risk for schizophrenia , then efforts could be launched to increase prevention of the effects of this class of risk factor ; ( 4 ) this approach could serve as a model for studying other complex disorders for which maternal - fetal genotype incompatibilities may be involved , for example , diabetes [ 127 , 128 ] and rheumatoid arthritis [ 129131 ] .

prenatal / obstetric complications are implicated in schizophrenia susceptibility . some complications may arise from maternal - fetal genotype incompatibility , a term used to describe maternal - fetal genotype combinations that produce an adverse prenatal environment . a review of maternal - fetal genotype incompatibility studies suggests that schizophrenia susceptibility is increased by maternal - fetal genotype combinations at the rhd and hla - b loci . maternal - fetal genotype combinations at these loci are hypothesized to have an effect on the maternal immune system during pregnancy which can affect fetal neurodevelopment and increase schizophrenia susceptibility . this article reviews maternal - fetal genotype incompatibility studies and schizophrenia and discusses the hypothesized biological role of these incompatibility genes . it concludes that research is needed to further elucidate the role of rhd and hla - b maternal - fetal genotype incompatibility in schizophrenia and to identify other genes that produce an adverse prenatal environment through a maternal - fetal genotype incompatibility mechanism . efforts to develop more sophisticated study designs and data analysis techniques for modeling maternal - fetal genotype incompatibility effects are warranted .

1. Introduction 2. 3. 4. Future Research 5. Conclusion

seventeen obese subjects ( bmi 41.0 1.5 kg / m , age 35.1 1.0 years , 15 female and 2 male , 13 african american and 4 caucasian ) , who were not markedly insulin resistant based on homeostasis model assessment of insulin resistance score 2.6 ( 21 ) , participated in the study . potential subjects were interviewed with a questionnaire used in previous studies ( 22 ) that inquired about 1 ) the type of sweetener used for coffee , tea , and other drinks ; 2 ) current intake of diet beverages ( including soda , juice , ice tea , and flavor water ) , 3 ) current intake of yogurt , pudding , gelatin , or other snacks foods sweetened with nns ; and 3 ) current use of gums containing nns . for each type of product , potential participants were asked whether they had used it in the past month and , if so , on how many days per week and how many servings per day . subjects who reported consuming more than one can of diet beverage or one spoonful of nns a week ( or its equivalent from foods ) were excluded . in addition , those who smoked cigarettes in the last six months ; were pregnant or breastfeeding ; had a history of malabsorptive syndromes , bariatric surgery , or inflammatory intestinal disease ; or were taking any medication that might affect glucose metabolism were excluded . this study was approved by the institutional review board at washington university school of medicine in st . subjects were studied on two separate occasions , 7 days apart , in a crossover design . for each study , subjects were admitted in the morning to the clinical research unit at washington university school of medicine at 0700 h after subjects fasted overnight ( 12 h ) at home . a catheter was placed in a hand vein and heated in a warming box ( 55c ) to obtain arterialized venous samples ( 23 ) . blood samples were obtained to assess plasma glucose , insulin , c - peptide , glucagon , glucose - dependent insulinotropic polypeptide ( gip ) , and active glp-1 concentrations at 20 , 15 , 10 , 6 , and 2 min before and at 10 , 20 , 30 , 40 , 60 , 90 , 120 , 150 , 180 , 240 , and 300 min after ingesting 75 g glucose . in randomized order , subjects drank 60 ml of 2 mmol / l sucralose ( i.e. , 48 mg sucralose ) or an equivalent volume of distilled water 10 min before glucose ingestion . this concentration of sucralose was used because it is the effective concentration needed to stimulate glp-1 secretion in human intestinal cells in vitro ( 4 ) and it matches the sweetness of a typical diet soda ; i.e. , it approximates the amount of sucralose in a standard 12-oz can of diet soda if it is all sweetened with sucralose ( 24 ) . plasma glucose was measured immediately after collection by using an automated glucose analyzer ( ysi 2300 stat plus ; yellow springs instruments , yellow spring , oh ) . blood samples were also collected in chilled edta tubes containing a protease inhibitor cocktail ( millipore , billerica , ma ) . these samples were placed on ice and centrifuged at 4c , and the plasma was stored at 80c for subsequent analyses . plasma active glp-1 and gip were measured by using commercially available immunoassay kits from meso scale discovery ( gaithersburg , md ) and millipore , respectively . plasma c - peptide was measured by using a solid - phase two - site chemiluminescent immunometric assay ( siemens medical solutions diagnostics , los angeles , ca ) , plasma insulin concentrations were determined by using a two - site immunoenzymatic assay ( dxi 800 ; beckman instruments , chaska , mn ) , and plasma glucagon was measured by a direct , double - antibody radioimmunoassay ( millipore ) . the incremental areas under the curve ( aucs ) above baseline concentrations for glucose , insulin , c - peptide , glucagon , gip , and glp-1 were calculated by using the trapezoid method ( 25 ) . the insulin sensitivity index [ si : dl kg min/(pmol / l ) ] was determined from a minimal model of the glucose concentration as a function of the insulin concentration ( 26 ) . insulin clearance rate from plasma was calculated by dividing the mean insulin secretion rate ( isr ) by the mean plasma insulin concentration ( 27 ) . plasma c - peptide and glucose concentrations were used to determine the isr in response to the oral glucose load and the sensitivity of the -cell response ( isr ) to changes in plasma glucose by using a minimal model ( 25 ) . this model provides an estimate of the total amount of insulin secreted in response to plasma glucose as a function of time ( i.e. , total isr in pmol / min ) and partitions this total response into a dynamic component ( isrdynamic ) , which represents the rapid release of a readily releasable pool of insulin secretory granules in response to the rate of increasing plasma glucose concentration , and a static component ( isrstatic ) , which represents the slower release of a reserve pool of insulin secretory granules in response to the ambient plasma glucose concentration ( 28 ) . the -cell response sensitivity parameters ( total , dynamic , and static ) corresponding to the total , dynamic , and static isr in response to changes in plasma glucose were determined ( 29 ) . the statistical significance of the effect of sucralose on glucose , insulin , c - peptide , glucagon , gip , and active glp-1 concentrations and isr after a glucose load was determined by conducting separate repeated anovas for each outcome variable with condition ( sucralose and water ) and time point as within - subject factors . when differences in values were statistically significant , a post hoc bonferroni adjustment to fisher least significant differences analyses was conducted . active glp-1 , si , and static data were positively skewed and required logarithmic transformation to approximate a normal distribution . the significance of differences in incremental peaks and aucs , si , and insulin clearance was evaluated by using a paired t test or wilcoxon matched pairs test , as appropriate . data in the tables and figures are presented as means sem or median ( semi - interquartile range : [ 75th25th percentile]/2 ) for skewed datasets . all analyses were performed with statistica 8.0 ( statsoft , tulsa , ok ) , and criterion for statistical significance was p < 0.05 . plasma glucose was measured immediately after collection by using an automated glucose analyzer ( ysi 2300 stat plus ; yellow springs instruments , yellow spring , oh ) . blood samples were also collected in chilled edta tubes containing a protease inhibitor cocktail ( millipore , billerica , ma ) . these samples were placed on ice and centrifuged at 4c , and the plasma was stored at 80c for subsequent analyses . plasma active glp-1 and gip were measured by using commercially available immunoassay kits from meso scale discovery ( gaithersburg , md ) and millipore , respectively . plasma c - peptide was measured by using a solid - phase two - site chemiluminescent immunometric assay ( siemens medical solutions diagnostics , los angeles , ca ) , plasma insulin concentrations were determined by using a two - site immunoenzymatic assay ( dxi 800 ; beckman instruments , chaska , mn ) , and plasma glucagon was measured by a direct , double - antibody radioimmunoassay ( millipore ) . plasma glucose was measured immediately after collection by using an automated glucose analyzer ( ysi 2300 stat plus ; yellow springs instruments , yellow spring , oh ) . blood samples were also collected in chilled edta tubes containing a protease inhibitor cocktail ( millipore , billerica , ma ) . these samples were placed on ice and centrifuged at 4c , and the plasma was stored at 80c for subsequent analyses . plasma active glp-1 and gip were measured by using commercially available immunoassay kits from meso scale discovery ( gaithersburg , md ) and millipore , respectively . plasma c - peptide was measured by using a solid - phase two - site chemiluminescent immunometric assay ( siemens medical solutions diagnostics , los angeles , ca ) , plasma insulin concentrations were determined by using a two - site immunoenzymatic assay ( dxi 800 ; beckman instruments , chaska , mn ) , and plasma glucagon was measured by a direct , double - antibody radioimmunoassay ( millipore ) . the incremental areas under the curve ( aucs ) above baseline concentrations for glucose , insulin , c - peptide , glucagon , gip , and glp-1 were calculated by using the trapezoid method ( 25 ) . the insulin sensitivity index [ si : dl kg min/(pmol / l ) ] was determined from a minimal model of the glucose concentration as a function of the insulin concentration ( 26 ) . insulin clearance rate from plasma was calculated by dividing the mean insulin secretion rate ( isr ) by the mean plasma insulin concentration ( 27 ) . plasma c - peptide and glucose concentrations were used to determine the isr in response to the oral glucose load and the sensitivity of the -cell response ( isr ) to changes in plasma glucose by using a minimal model ( 25 ) . this model provides an estimate of the total amount of insulin secreted in response to plasma glucose as a function of time ( i.e. , total isr in pmol / min ) and partitions this total response into a dynamic component ( isrdynamic ) , which represents the rapid release of a readily releasable pool of insulin secretory granules in response to the rate of increasing plasma glucose concentration , and a static component ( isrstatic ) , which represents the slower release of a reserve pool of insulin secretory granules in response to the ambient plasma glucose concentration ( 28 ) . the -cell response sensitivity parameters ( total , dynamic , and static ) corresponding to the total , dynamic , and static isr in response to changes in plasma glucose were determined ( 29 ) . the incremental areas under the curve ( aucs ) above baseline concentrations for glucose , insulin , c - peptide , glucagon , gip , and glp-1 were calculated by using the trapezoid method ( 25 ) . the insulin sensitivity index [ si : dl kg min/(pmol / l ) ] was determined from a minimal model of the glucose concentration as a function of the insulin concentration ( 26 ) . insulin clearance rate from plasma was calculated by dividing the mean insulin secretion rate ( isr ) by the mean plasma insulin concentration ( 27 ) . plasma c - peptide and glucose concentrations were used to determine the isr in response to the oral glucose load and the sensitivity of the -cell response ( isr ) to changes in plasma glucose by using a minimal model ( 25 ) . this model provides an estimate of the total amount of insulin secreted in response to plasma glucose as a function of time ( i.e. , total isr in pmol / min ) and partitions this total response into a dynamic component ( isrdynamic ) , which represents the rapid release of a readily releasable pool of insulin secretory granules in response to the rate of increasing plasma glucose concentration , and a static component ( isrstatic ) , which represents the slower release of a reserve pool of insulin secretory granules in response to the ambient plasma glucose concentration ( 28 ) . the -cell response sensitivity parameters ( total , dynamic , and static ) corresponding to the total , dynamic , and static isr in response to changes in plasma glucose were determined ( 29 ) . the statistical significance of the effect of sucralose on glucose , insulin , c - peptide , glucagon , gip , and active glp-1 concentrations and isr after a glucose load was determined by conducting separate repeated anovas for each outcome variable with condition ( sucralose and water ) and time point as within - subject factors . when differences in values were statistically significant , a post hoc bonferroni adjustment to fisher least significant differences analyses was conducted . active glp-1 , si , and static data were positively skewed and required logarithmic transformation to approximate a normal distribution . the significance of differences in incremental peaks and aucs , si , and insulin clearance was evaluated by using a paired t test or wilcoxon matched pairs test , as appropriate . data in the tables and figures are presented as means sem or median ( semi - interquartile range : [ 75th25th percentile]/2 ) for skewed datasets . all analyses were performed with statistica 8.0 ( statsoft , tulsa , ok ) , and criterion for statistical significance was p < 0.05 . mean peak plasma glucose concentration was higher and the subsequent nadir was lower after sucralose than after water ingestion ( fig . 1a and table 1 ) . peak plasma insulin and c - peptide concentrations were also higher after sucralose than after water ingestion ( fig . no significant differences in the incremental auc of glucose or c - peptide were detected in response to the glucose load after sucralose and water ingestion . however , the incremental auc of insulin was 20 8% greater after sucralose than after water ingestion ( p < 0.03 ) ( table 1 ) . plasma glucagon concentration and the decremental glucagon auc after the glucose load were similar after sucralose and water ingestion ( fig . although average plasma gip concentrations tended to be higher after sucralose than after water ingestion ( 20 8 vs. 18 7 pmol / l ) , the difference was not statistically significant ( p = 0.08 ) ( fig . plasma active glp-1 concentration , incremental glp-1 auc , and incremental gip auc after the glucose load were not different after sucralose or water ingestion ( fig . mean plasma glucose ( a ) , insulin ( b ) , c - peptide ( c ) , and glucagon ( d ) concentrations in obese subjects after drinking either sucralose or water 10 min before ingestion of a 75-g glucose load ( given at time = 0 min ) . metabolic response to an oral 75-g glucose load preceded by either sucralose or water ingestion mean plasma gip ( a ) and active glp-1 ( b ) concentrations in obese subjects after drinking either sucralose or water 10 min before ingestion of a 75-g glucose load ( given at time = 0 min ) . sucralose ingestion decreased the insulin clearance rate after ingesting the glucose load by 7 4% ( p < 0.05 ) . the median si value was 23 20% lower after sucralose than after water ingestion ( p < 0.01 ) ( table 1 ) . total isr aucs in response to the oral glucose load and the sensitivity of insulin secretion to plasma glucose ( total , dynamic , and static ) were not different between sucralose and water conditions ( table 1 ) . however , differences in plasma glucose concentration between conditions caused a higher peak in the isr after sucralose than after water ( fig . 3a ) , which was exclusively due to an average increase of 22 4% in the static isr between 60 and 105 min ( p < 0.005 ) ; the dynamic isr curves were the same after sucralose and water ingestion ( fig . isr in response to a glucose load after subjects drank either sucralose 10 min before ingestion of 75 g glucose ( given at time = 0 min ) . mean peak plasma glucose concentration was higher and the subsequent nadir was lower after sucralose than after water ingestion ( fig . 1a and table 1 ) . peak plasma insulin and c - peptide concentrations were also higher after sucralose than after water ingestion ( fig . no significant differences in the incremental auc of glucose or c - peptide were detected in response to the glucose load after sucralose and water ingestion . however , the incremental auc of insulin was 20 8% greater after sucralose than after water ingestion ( p < 0.03 ) ( table 1 ) . plasma glucagon concentration and the decremental glucagon auc after the glucose load were similar after sucralose and water ingestion ( fig . although average plasma gip concentrations tended to be higher after sucralose than after water ingestion ( 20 8 vs. 18 7 pmol / l ) , the difference was not statistically significant ( p = 0.08 ) ( fig . plasma active glp-1 concentration , incremental glp-1 auc , and incremental gip auc after the glucose load were not different after sucralose or water ingestion ( fig . mean plasma glucose ( a ) , insulin ( b ) , c - peptide ( c ) , and glucagon ( d ) concentrations in obese subjects after drinking either sucralose or water 10 min before ingestion of a 75-g glucose load ( given at time = 0 min ) . metabolic response to an oral 75-g glucose load preceded by either sucralose or water ingestion mean plasma gip ( a ) and active glp-1 ( b ) concentrations in obese subjects after drinking either sucralose or water 10 min before ingestion of a 75-g glucose load ( given at time = 0 min ) . sucralose ingestion decreased the insulin clearance rate after ingesting the glucose load by 7 4% ( p < 0.05 ) . the median si value was 23 20% lower after sucralose than after water ingestion ( p < 0.01 ) ( table 1 ) . total isr aucs in response to the oral glucose load and the sensitivity of insulin secretion to plasma glucose ( total , dynamic , and static ) were not different between sucralose and water conditions ( table 1 ) . however , differences in plasma glucose concentration between conditions caused a higher peak in the isr after sucralose than after water ( fig . 3a ) , which was exclusively due to an average increase of 22 4% in the static isr between 60 and 105 min ( p < 0.005 ) ; the dynamic isr curves were the same after sucralose and water ingestion ( fig . isr in response to a glucose load after subjects drank either sucralose 10 min before ingestion of 75 g glucose ( given at time = 0 min ) . the results from the current study demonstrate that the ingestion of sucralose alters the metabolic response to an oral glucose load in obese people who are not regular consumers of nns . the peak increase plasma glucose , c - peptide , and insulin concentrations and total insulin auc after an oral glucose load were greater when subjects consumed sucralose than when they consumed water before glucose ingestion . in addition , insulin clearance from plasma was slower after sucralose than after water ingestion . these data suggest that sucralose ingestion is not physiologically inert but affects the glycemic response to an oral glucose load and potentiates glucose - stimulated insulin secretion in obese people . the finding that glucose - induced glucagon suppression was the same after both sucralose and water ingestion makes it unlikely that glucagon was responsible for the differences between conditions . the mechanisms responsible for the sucralose effect on plasma glucose after an oral glucose load are not clear but must involve an alteration in the rate of glucose absorption , disposal , or endogenous production . data from previous studies conducted in animal models showed that sucralose augments glucose absorption by increasing intestinal glucose transport ( 11,12 ) . our results support this notion because sucralose increased the early peak in plasma glucose but did not affect the indices of -cell sensitivity ( total , dynamic , and static ) to plasma glucose . we found that si decreased after sucralose ingestion , suggesting that sucralose caused insulin resistance . however , the minimal model used to calculate si from a modified ogtt is unable to determine whether the decrease in si was caused by insulin resistance in a specific organ ( e.g. , liver or skeletal muscle ) or multiple organs . additional studies that involve the use of glucose tracers to quantify the effect of sucralose on insulin - mediated suppression of endogenous glucose production and insulin - stimulated muscle glucose uptake are needed to further explore our findings . sucralose ingestion did not affect the glp-1 response to a glucose load in our subjects , which is consistent with the data reported in previous studies conducted in human subjects ( 15,18 ) . this finding suggests that the observed increase in the isr observed in our subjects was mediated by a glp-1independent mechanism . there was a trend suggesting that gip contributed to the potentiated glucose - stimulated insulin secretion , and a confirmatory study is needed . in contrast , the data from previous studies found that the oral ingestion of a nns before a glucose load augmented glp-1 ( 19,20 ) but did not affect gip secretion . however , in those studies total glp-1not the biologically active form of glp-1was measured ( 19,20 ) . although it is possible that the discrepancy between glp-1 findings of those and our study is the result of this methodological difference , we think it is unlikely because active and total glp-1 are highly correlated with each other ( 30 ) . a second methodological difference is that in those studies a diet cola sweetened with both sucralose and acesulfame potassium ( 19,20 ) was used , so it is unclear whether the enhanced glucose - stimulated glp-1 response was mediated by acesulfame potassium , a synergistic effect of both sweeteners , or other ingredients contained in the carbonated drinks ( 20 ) . additional studies that examine the metabolic effects of different nns and potential interactions with other dietary ingredients are needed . this finding suggests that intestinal sweet taste receptors ( 4 ) , which are activated by nns , are involved in regulating insulin metabolism . this observation also raises the possibility that sweet taste receptors contribute to the unexplained reduction in insulin clearance observed after an oral , but not intravenous , glucose load ( 31,32 ) . a series of studies conducted in human subjects have reported that sucralose does not affect the glycemic or hormonal responses to intraduodenal ( 15 ) or oral administration of glucose or other carbohydrates ( 16,18,33 ) ( rev . in 13 ) . the reason(s ) for the discrepancy between findings may be related to study subject selection . to increase sample homogeneity , we only included subjects who were obese , were insulin sensitive based on a homeostasis model assessment score 2.6 , and were not regular users of nns . by decreasing variability , it is likely that our study had a greater statistical power than previous studies to detect a sucralose effect . in addition , most subjects in previous studies were caucasian ( 15,16,18,20 ) , whereas most subjects in our study were african american . we believe this study is the first to evaluate the acute effects of sucralose in subjects who are not regular users of nns . data from studies conducted in animal models have shown that chronic inclusion of nns in the diet upregulates the expression of sodium - dependent glucose transporter isoform 1 , which in turn increases the initial rate of na - dependent glucose uptake ( 5,10 ) and increases glycemic responses after an oral glucose tolerance test ( 34 ) . therefore , we speculate that regular users of nns would have a higher glycemic response after an oral glucose tolerance test on the control day than irregular users and that the acute effects of sucralose intake would be blunted because differences between water and sucralose conditions would be smaller in regular than in irregular users of nns . in conclusion , the results from our study demonstrate that sucralose affects the glycemic and hormonal responses to an oral glucose load in obese people who do not normally consume nns . these findings support the notion that sucralose is not metabolically inert but has physiologic effects . additional studies of other nns , conducted in distinct study populations , including children and chronic nns users , and that evaluate the effect of nns on the metabolic response to mixed - meal ingestion are needed .

objectivenonnutritive sweeteners ( nns ) , such as sucralose , have been reported to have metabolic effects in animal models . however , the relevance of these findings to human subjects is not clear . we evaluated the acute effects of sucralose ingestion on the metabolic response to an oral glucose load in obese subjects.research design and methodsseventeen obese subjects ( bmi 42.3 1.6 kg / m2 ) who did not use nns and were insulin sensitive ( based on a homeostasis model assessment of insulin resistance score 2.6 ) underwent a 5-h modified oral glucose tolerance test on two separate occasions preceded by consuming either sucralose ( experimental condition ) or water ( control condition ) 10 min before the glucose load in a randomized crossover design . indices of -cell function , insulin sensitivity ( si ) , and insulin clearance rates were estimated by using minimal models of glucose , insulin , and c - peptide kinetics.resultscompared with the control condition , sucralose ingestion caused 1 ) a greater incremental increase in peak plasma glucose concentrations ( 4.2 0.2 vs. 4.8 0.3 mmol / l ; p = 0.03 ) , 2 ) a 20 8% greater incremental increase in insulin area under the curve ( auc ) ( p < 0.03 ) , 3 ) a 22 7% greater peak insulin secretion rate ( p < 0.02 ) , 4 ) a 7 4% decrease in insulin clearance ( p = 0.04 ) , and 5 ) a 23 20% decrease in si ( p = 0.01 ) . there were no significant differences between conditions in active glucagon - like peptide 1 , glucose - dependent insulinotropic polypeptide , glucagon incremental auc , or indices of the sensitivity of the -cell response to glucose.conclusionsthese data demonstrate that sucralose affects the glycemic and insulin responses to an oral glucose load in obese people who do not normally consume nns .

RESEARCH DESIGN AND METHODS Laboratory assessments Biochemical measurements. Calculations Metabolic response to glucose ingestion. Insulin sensitivity and clearance. -Cell function. Statistical analyses RESULTS Plasma glucose and hormonal responses to a glucose load were altered by sucralose Insulin clearance and insulin sensitivity were altered by sucralose -Cell response to glucose ingestion is altered by sucralose CONCLUSIONS

oral infections of geotrichum candidum are clinically similar to candidiasis and commonly associated with diabetes mellitus and hiv infection , . cases of dissemination and fungemia are reported in patients with chronic and acute myeloid leukemia , , , , , , . old women post - partum with isolated renal calculi and renal fungal bezoar attributed to geotrichum candidum and to illustrate the diagnostic dilemmas . old women presented with history of left flank pain and intermittent fever since 15 days . she was evaluated elsewhere with contrast enhanced computerized tomography ( ct ) scan which revealed contracted left kidney with 2 calculi in the lower and middle calyx of 89 mm each with intrapelvic mass and multiple air pockets in the renal pelvis ( fig . 2 ) . she had undergone cytoscopy and left dj stenting elsewhere but continued to have fever and flank pain when she was presented to us . after routine investigation , patient was started on 3rd generation cephalosporin and she underwent left percutaneous nephrolithotripsy ( pcnl ) which revealed brownish gray material with 2 calculi . gross specimen consists of multiple irregular gray brown tissue bits , largest measuring 0.5 cm0.5 cm and cut portion showed gray brown areas . section showed fungal ball containing aggregates of macerated , distorted fungal hyphae with some showing acute angle branching surrounded by cell debris and neutrophils . both urine and biopsy material sent to mycology laboratory for culture investigation were inoculated on sabourauds dextrose agar ( hi - media laboratories ltd . , mumbai ) and incubated at 37 c and 28 c which grew a rapidly growing fungus with flat , white to creamy having a smooth texture later becoming hairy consistent with geotrichum candidum ( fig . geotrichum candidum was morphologically identified by the presence of true hyphae , hyaline smooth , one - celled , subglobose to cylindrical , slimy arthroconidia and the lack of blastoconidia . the arthroconidia vary in size and germinate at one end giving a hockey stick appearance ( fig . 4 ) . biochemical identification was carried out in the mycology laboratory , kasturba medical college , manipal using both conventional and api 20c yeast identification system ( biomerieux inc . ) . it was further differentiated from trichosporon by the absence of urea utilization and inability to assimilate carbohydrate ; maltose , sucrose , lactose , cellobiose , inositol raffinose and trehalose . antifungal susceptibility testing for the isolate was performed according to the clinical and laboratory standards institute ( clsi ) document m38-a2 . the mic90 ( minimum inhibitory concentration ) for amphotericin , fluconazole , itraconazole and voriconazole were found to be 0.125 g / ml , 16 g / ml , 4 g / ml and 0.25 g / ml respectively . patient continued to have fever in the post - operative period and responded only after starting intravenous itraconazole 200 mg bd for 2 days followed by oral itraconazole 200 mg bd and continued for 6 weeks . during the follow up , a repeat ct done showed complete clearance of the fungal material from the left kidney . the saprophytic colonization of a preformed cavity by conglomerate of fungal mycelia without invasion of adjacent tissue is termed as fungal ball or fungal bezoar . renal colic can be caused by passage of fungal ball that obstruct the collecting system . genitourinary tract is rarely a site of primary fungal infection with exception of candida species , however it may be involved as a result or part of systemic infection . candidal infection can cause pyelonephritis , abscess , papillary necrosis and obstruction with fever and flank pain , . genitourinary fungal infections are usually encountered as a part of disseminated disease in immunocompromised host ( aids , corticosteroids , malignancy , neutropenia ) , , , , , , . fungal balls also called and fungal bezoars or accretions are known to cause ureteral and uretro - pelvic junction obstruction . diagnosis is established by identification the of fungi in urine and imaging studies using ( computerized tomography , ultra sonography , intravenous urography ) that document obstructive uropathy and soft tissue density with in renal collecting system . have successfully managed the removal of bilateral renal pelvis mycotic bezoars using a mechanical thrombectomy device followed by antifungal renal pelvis irrigation . percutaneous nephrostomy , tract dilation and fluoroscopically guided extraction of renal fungal ball under epidural anesthesia is described by doemeny et al . . the outcome of geotrichum infections depend on the degree of tissue invasion by the organism and the immune status of the host . sheehy et al . suggested that geotrichum lack virulence and ability to colonize renal tubules based on the rarity of disseminated disease , lack of tissue invasion and their rapid clearance in most of the case reported . its incidence may be under reported since it can be misdiagnosed histopathologically as candida , aspergillus or trichosporon . this possibility of misinterpretation highlights the importance of obtaining repeated fungal cultures in addition to histopathological examination . we hereby reiterate the pathogenic potential of geotrichum candidum and report its role in causing renal fungal ball .

geotrichum candidum is yeast like fungi that cause infections in immunocompromised patients . we report a case of renal fungal ball with geotrichum candidum in a 27 yr . old women post - partum . this case to our knowledge is the first case of renal fungal bezoar due to geotrichum candidum reported in india .

Introduction Case Discussion Conflict of interest

granulosa cell tumours ( gcts ) though accounting for approximately 70% of malignant sex - cord stromal tumors are rare , comprising only 25% of all ovarian neoplasms [ 13 ] . these tumours arise from granulosa cells that are hormonally active stromal elements in close association with ovarian oocytes , which are responsible for the production of estradiol . the exact etiology of this malignancy remains unknown , with no identification of specific defined risk factors . the typical clinical scenario of a gct is an older postmenopausal woman with menstrual abnormalities who is found to have a singular pelvic mass that is curable by en - bloc resection . while such typical cases do exist , characteristics of gcts in clinical practice do not always fit within the confines of these parameters . as such , seeing past this myth to recognize and identify uncommon realities is necessary for the accurate diagnosis and management of gcts . the aim of this study is to discuss the myths and realities of gcts through a series of indexed cases in the context of relevant evidence - based literature . further , a comprehensive fourteen - year ( 19972011 ) search using the laboratory information system ( lis ) identified additional patients with gcts . patient demographics including age , sex , and tumour site were collected for analysis . a literature search using the national library of medicine interface pubmed was conducted using the search terms granulosa cell limited to the english language . this study was conducted with ethics approval from the university of saskatchewan biomedical research ethics review committee . ultrasonography confirmed the mass to be a 2 cm lesion , and an orchiectomy was carried out . on histological examination , the tumour was solid with regions of cystic spaces ( figures 1(a ) and 1(b ) ) . amphophilic cytoplasm with indistinct cell borders and an extensive fibrocollagenous stroma were identified ( figure 1(c ) ) . the lesional cells were positive to inhibin ( figure 1(d ) ) , confirming the sex - cord stromal nature of this neoplasm . twenty years post - operatively he is doing well , with no evidence of recurrence . seven days after birth , a female neonate developed increased abdominal distension and constipation . a large mass was palpable on abdominal exam that prompted ultrasonography , which detected a 10 cm multiseptated cystic , mass . this was further confirmed by computed tomography ( ct ) scan , which showed the mass to be surrounded by a large volume of fluid ( figure 2(a ) ) . hematological investigations showed high levels of estradiol ( 310 ) , prolactin ( 34.9 ) , and thyroid - stimulating hormone ( tsh , 11.05 ) with normal levels of luteinizing hormone ( lh ) , follicle - stimulating hormone ( fsh ) , -fetoprotein , and -human chorionic gonadotropin ( -hcg ) . the young girl was taken to the operating room for a laparotomy , which revealed a large cystic mass of the left ovary with torsion ( figure 2(b ) ) . an oophorectomy with preservation of the left fallopian tube was carried out . on follow - up abdominal and pelvic ct , no evidence of residual disease was detected , and estradiol and tsh levels normalized . eighteen years post - operatively she is doing well , with no evidence of recurrence . on histological examination , a multicystic lesion lined by granulosa cells was identified ( figure 2(c ) ) , without the typical theca internal layer lining the cysts . solid proliferations of granulosa - like cells were recognized in the ovarian stroma , and focal areas with a solid insular pattern and nongrooved nuclei were present ( figure 2(d ) ) . histological features were between those common to adult gct ( agct ) and juvenile gct ( jgct ) . an almost exclusive macrofollicular pattern typical of agct was contrasted by small areas of the solid , insular pattern as seen in jgct . the unusual facet in this case is the clinical presentation of an unsuspected gct in a seven - day - old neonate . a 76-year - old woman presented with a growth at the umbilicus and bowel obstruction . ct scan showed a complex mass arising from the anterior bladder wall / dome of the bladder ( figure 3(a ) ) . with the clinical suspicion of a urachal carcinoma , the patient was taken to the operating room where she underwent a partial cystectomy and anterior abdominal wall mesh reconstruction . the mass was smooth and solid cystic ( figure 3(b ) ) , with extension from the bladder 's dome to the umbilicus . at microscopic examination , sheets of spindle cells in focal retiform - like areas with uniform grooved nuclei were identified ( figure 3(c ) ) . immunohistochemical analysis found the mass to be positive to vimentin , with focal positivity to inhibin a ( figure 3(d ) ) and cytokeratin . an incidental pelvic mass was detected in a 64-year woman with a past history of total abdominal hysterectomy and bilateral salpingooophrectomy for a gct 16 years prior . a ct scan confirmed the presence of a large heterogenous solid - cystic pelvic mass ( figure 4(a ) ) . the preoperative diagnosis of this mass included abscess , gastrointestinal stromal tumor , and extracolonic mass . she underwent laparotomy with pelvic washings for cytology and en - bloc resection of the left ovarian mass . on gross examination , necrosis reminiscent of colonic adenocarcinoma admixed with diffuse , microfollicular , and cords of neoplastic cells were observed ( figure 4(b ) ) . deeper sections confirmed the presence of the glandular neoplasm originating from a diverticular outpouching of the overlying colonic mucosa , confirming the presence of colonic adenocarcinoma arising in a diverticulum of the large bowel . this was further supported by immunohistochemistry that confirmed the two components of this collision tumour with inhibin positivity in the gct component ( figure 4(c ) ) and cytokeratin positivity in the adenocarcinoma component ( figure 2(d ) ) . a 45-year - old woman presented to the emergency room with a two - year history of menorrhagia and acute dyspnea . an enhanced ct scan revealed a giant mass ( estimated size , 36 cm craniocaudal ) filling the pelvis and abdomen and causing displacement of the bowel loops . the patient developed imminent renal failure and her hemoglobin levels continued to drop despite receiving 9 units of blood . it was suspected that her mass was haemorrhaging , and she was taken to the operating room . a 3540 cm , multilobulated , haemorrhagic mass was found in her right ovary , and a total abdominal hysterectomy with bilateral oophorectomy , with en - bloc mass resection and partial omentectomy were carried out . microscopic examination showed the presence of sheets of neoplastic cells interspersed with rupture and acute hemorrhage ( figure 5(a ) ) . the ovarian tumour showed granulosa cells arranged in solid sheets , trabeculae , tubules , and microfollicles that included call - exner bodies ( figures 5(b ) and 5(c ) ) . immunohistochemical analysis found the neoplastic cells to stain positive for vimentin , inhibin ( figure 5(d ) ) , cd99 , and afp . the unique presentation of this gct is an acute abdomen due to tumor rupture with hemorrhage . surgical review a 14-year ( 19972011 ) surgical pathology review yielded 37 cases of gct , with an overall prevalence of 0.0041% ( 37/902 100 ) . of these cases , 36 were females and one male ( index case 4 ) . within the female population , patient 's age ranged from 7 days to 85 years , with a mean of 52 years ( median 48 years ) . locations of gcts included 17 cases in the right ovary ( 47.2% ) , 15 in the left ovary ( 41.7% ) , and one ( 2.8% ) in each of the bowel , abdominal wall , and omentum . only four ( 10.8% ) concurrent pathologies included leiomyoma of the uterus ( 21% ) , benign cysts ( 14% ) , adenomyosis ( 7% ) , coexisting ovarian adenocarcinoma ( 3% ) , and endometrial carcinoma ( 3% ) . a 14-year ( 19972011 ) surgical pathology review yielded 37 cases of gct , with an overall prevalence of 0.0041% ( 37/902 100 ) . of these cases , 36 were females and one male ( index case 4 ) . within the female population , patient 's age ranged from 7 days to 85 years , with a mean of 52 years ( median 48 years ) . locations of gcts included 17 cases in the right ovary ( 47.2% ) , 15 in the left ovary ( 41.7% ) , and one ( 2.8% ) in each of the bowel , abdominal wall , and omentum . only four ( 10.8% ) concurrent pathologies included leiomyoma of the uterus ( 21% ) , benign cysts ( 14% ) , adenomyosis ( 7% ) , coexisting ovarian adenocarcinoma ( 3% ) , and endometrial carcinoma ( 3% ) . granulosa cell tumours ( gcts ) were first described in 1855 by rokitansky as chronicled in chew et al . gcts are rare sex - cord stromal tumors that are thought to arise from the normal proliferating granulosa cells of the late preovulatory follicle as they share many morphological and biochemical features with these cells . additionally , theca cells , interstitial cells , and stromal fibroblasts have also been delegated as cells of origin for gct . . recently , there is growing evidence to suggest that granulosa stem cells ( gscs ) do exist . identification of normal and neoplastic gscs and the factors that regulate their behavior will determine the treatment of all ovarian cancers including gcts in the future . however , currently , little is known regarding the molecular and genetic changes associated with gcts . they have been associated with a variety of genetic abnormalities such as trisomy 12/14 , monosomy 22 , and > 90% of adult gct 's have a missense somatic c134w mutation in the foxl2 gene . in men , the typical clinical scenario of a gct is usually a middle - aged female presenting with a pelvic mass who is cured by en - bloc resection of the mass . these characteristics however are not generalizable to all gcts as these lesions can present in many different facets and have a tendency to behave unpredictably , thus complicating both diagnosis and therapeutic management . accurate recognition of the true realities of multifaceted gct is therefore vital for the precise diagnosis and management of these lesions . some of the existing myths of gcts will now be contrasted with their true realities in the context of evidence - based literature . this cancer can be divided into three types based on the cell of origin ( germ , epithelial , and stromal ) with each conferring different histopathological features and clinical outcomes . stromal tumours are further classified based on the tissue types involved as sertoli , leydig , theca , and granulosa . granulosa cell tumours ( gcts ) account for 1 - 2% of all ovarian tumours and arise from the granulosa cells that normally surround the oocytes and line the developing follicle . these include a ) these neoplasms are derived from the mesenchyme of the developing genital ridge and b ) these neoplasms arise from precursors within the mesonephric and coelomic epithelium . the presence of extraovarian gct 's as seen in our case 3 supports the latter theory . to date though chromosomal anomalies and/or autocrine and endocrine signalling abnormalities are proposed aetiologies , the current etiology postulated is one of multifactorial origin . these lesions are considered a low - grade type malignancy , with 7090% of neoplasms being diagnosed at stage 1 . the high detection rate at an early stage may be due to the endocrine symptoms that often present early in the functioning tumors . low staging at diagnosis confers an excellent prognosis , with 5-year survival rates reported between 7595% ( stage 1 ) ; however , these rates drop to 5575% and 2250% for stages ii and iii / iv respectively . this may be partially due to limited treatment options for advanced and recurrent disease . although predominantly occurring in the granulosa cells of the female ovary , gcts are also reported to arise within the male testis , as seen in our index case 1 . testicular sex - cord stromal tumours are rare , comprising only 4% of all testicular tumours . juvenile gct ( jgct ) is far more common than adult gct ( agct ) within the testicle with no preferred laterality within the testis [ 9 , 10 ] . approximately half of testicular jgcts are diagnosed within the first month of life , and over 95% within the first year as seen in our case 1 . the differential diagnosis of testicular jgct includes yolk sac tumour , undifferentiated sex - cord stromal tumour , gynandroblastoma , and gonadoblastoma . typically , males present with a painless indolent testicular swelling . due to estrogen hypersecretion , patients may be impotent , and 25% have gynaecomastia [ 9 , 13 ] . an intra - abdominal mass of an undescended testis and/or a testicular torsion may additionally be present . jgcts in undescended testis are benign and do not reach an adequate size to cause pressure on other organs . though patients with lymph node metastases usually have a longer survival period , the presence of distant metastases is usually associated with a dismal prognosis . histologically , testicular gct resembles ovarian , presenting as a solid , cystic mass with microfollicular , gyriform , insular , and trabecular patterns . cells are typically immunopositive for vimentin , inhibin , smooth muscle actin , cd99 , and s-100 [ 9 , 18 ] . genetically , chromosomal abnormalities such as an atypical y chromosome and mosaicism may be present . most patients with gcts are perimenopausal or early postmenopausal , with a median age of diagnosis between 5054 years . nevertheless , as our surgical review demonstrates , gcts may arise in neonates ( index case 2 ) or in patients over the age of 80 ( series ) . gcts in neonates , as in index 2 , are a rare occurrence , with few reported cases in less than one year of age . a common misconception is that the two subclassifications of gcts , adult - type gct ( agct ) and juvenile - type gct ( jgct ) , refer to the age of development . though agct and jgct occur more often in adults and children , respectively , either form may present throughout the entire population . the majority ( 95% ) are agcts , more commonly seen in adults [ 22 , 23 ] . agct has been reported in children ; however , less than 1% of these lesions occur in prepubertal girls . upto 90% of jgcts are diagnosed in patients under the age of 30 with half of jgct cases seen in less than 10 years of age [ 22 , 27 ] and 10% occurring in infants less than one year . differences between agct and jgct are distinct ; therefore , accurate identification is critical to guide patient management . unlike agct , jgct is considered by many to be a relatively benign tumour and in infantile males is the common type of sex - cord stromal tumour of the testis . cells producing hormones such as estradiol are present in 70% of jgcts . as such , in young patients , clinical evidence of isosexual precocious pseudo - puberty including breast enlargement , pubic and axillary hair development , vaginal secretions , irregular uterine bleeding , advanced somatic / skeletal developments , and secondary sex characteristics may all be associated with jgct [ 2 , 5 ] . these patients often have elevated estradiol levels , though this is not a requirement for precocious puberty . the risk of precocious puberty is especially high in jgct patients under one year of age , but gct in this age range is rare . such symptoms are present in 8090% of patients under the age of 8 with jgct . on radiologic imaging , jgct is often indistinguishable from other ovarian neoplasms , and identification of histopathology features such as a nodular / diffuse cellular growth and/or macrofollicules with eosinophilic / basophilic fluid in their lumen is often required for correct diagnosis . as these are younger women , uterine sparing surgery with conservation of the contralateral tube and ovary is recommended at the outset for maintenance of fertility . advanced - stage disease is responsive to combination chemotherapy with platinum agents [ 5 , 6 ] . the overall prognosis for jgct is excellent , with reports of survival as high as 97% in one study with a 3.5-year followup . after - resection , gross examination of a jgct is similar to that of an agct . on average , microscopically , these tumours are solid / cellular , with follicle formation , edema , and loose stroma . hyperchromatic granulosa cells with rounded nuclei surrounded by eosinophilic or vacuolated cytoplasm and high mitotic rates are additionally seen [ 2 , 5 ] . unlike agct , few call - exner bodies are identified . estradiol is one of the first hormones to be secreted by gcts and is responsible for clinical manifestations . among females , pre - pubescent girls may experience isosexual precocious puberty as a result of increased estrogen levels caused by hyperestrogenism . though hyperestrogenism is the common form of endocrine abnormality in gcts , it is important to consider juvenile gct in females presenting with androgen excess and precocious puberty . the adult gct appears to be the most common type of gct associated with virilisation , suggesting a propensity for increased androgen secretion in agcts . by contrast , these elevated estrogen levels in adults can cause abnormal uterine bleeding , menstrual irregularities , menorrhagia , or amenorrhea [ 20 , 27 ] . patients with virilising gcts can present with hirsutism , clitoromegaly , increased abdominal size , amenorrhea , and deepening of voice . rare case reports document jgcts presenting with paraneoplastic syndrome of hypercalcemia and meig 's syndrome of pleural effusion with ascites . adult patients may present with vaginal bleeding caused by endometrial hyperplasia or uterine cancer as a result of prolonged exposure to tumor - derived estrogen . in addition , gct is a vascular tumor that may occasionally rupture and result in abdominal pain , hemoperitoneum , and hypotension , mimicking an ectopic pregnancy in younger patients . tumor rupture is often attributed to hemorrhagic cysts in upto 1015% of the cases [ 2 , 27 ] . occasionally gcts and theca cell tumours have been found in ovaries which show no enlargement and are therefore clinically occult [ 35 , 36 ] . gcts are the most common estrogen - producing neoplasms in females and are found to produce estradiol in approximately 4060% of patients . this estradiol production is directly related to the release of testosterone secreted by the theca cells . however , not all gcts are hormonally active or have theca cells that secrete testosterone , and therefore diagnostic testing for these hormones lacks sensitivity and specificity . normal granulosa cells are responsible for production not only of estradiol but also peptide hormones including inhibin , activin , follistatin , and antimullerian hormones . gct patients usually present with elevated levels of inhibin , a negative feedback regulator of fsh secretion ; however , this hormone is not specific for these tumours [ 2 , 3 , 37 ] . mom et al . evaluated the sensitivities and specificities of serum inhibin levels in 30 women with granulosa cell tumors ( inhibin a was 67 and 100% and inhibin b was 89 and 100% , resp . ) . serum inhibin levels are currently available for diagnosis and clinical followup of women with granulosa cell tumors of the ovary it can , therefore be used to monitor response to therapy or to detect recurrences . mullerian inhibitory substance ( mis ) which is produced in the developing follicles is often elevated in gcts , though it is not specific for diagnosis [ 2 , 3 ] . this hormone is produced exclusively by granulosa cells in postnatal females and both prenatally and postnatally by the sertoli cells in the male testis . this hormone functions in male fetuses to induce regression of the mullerian system . normally , mis is found in low levels in reproductive - aged females and functions as a paracrine inhibitory factor that decreases the response of the resting ovarian follicle to follicle - stimulating hormone ( fsh ) thus ensuring the emergence of a single dominant follicle . serum mis may be a marker of ovarian reserve and typically disappears from the serum after menopause or bilateral oophorectomy . however , in patients with gcts , levels have been shown to parallel the extent of disease . serum mis levels are not routinely available for clinical use in the context of gct diagnosis and followup . however , a commercial version of the ultrasensitive elisa assay has become available and may lead to wider clinical use of mis in the future . serum mis levels thus correlate well with tumor presence in patients with gcts and elevated levels are considered highly specific for gct in postmenopausal or oophorectomized women . it may also be elevated in women with sertoli - leydig cell tumors of the ovary , but is not typically produced by other gonadal or extragonadal tumors . this is in sharp contrast to inhibin and estradiol levels , both of which may be elevated in a variety of other extraovarian disorders . this marker may thus eventually be used for both diagnosis and follow - up evaluations of patients with gcts . preclinical research is also ongoing to evaluate the clinical use of targeting the mis receptor for therapy in cancers expressing this receptor . follicle regulatory protein is secreted by the granulosa cells and is elevated in some patients with gcts . however , the clinical significance of this marker is still undetermined [ 20 , 27 ] . gcts have been reported in coexistence with a number of pathologies including mucinous cystadenoma [ 7 , 41 , 42 ] , cystic teratoma , ovarian fibroma , ovarian angiosarcoma , adenosarcoma , cystadenosarcoma , sclerosing peritonitis , gastric signet - ring cell carcinoma , and cervical lipoleiomyoma . in the indexed case 4 gct was found to coexist with colonic adenocarcinoma as a unique collision tumor which has been discussed previously in detail . the presence of such coexiting pathologies may contribute to increased confusion and be a deterrent to the accurate clinical and pathological recognition of this uncommon neoplasm . uterine pathologies that have been reported to occur with gct include glandular hyperplasia , atypical adenomatous hyperplasia , adenocarcinoma insitu , and invasive carcinoma . endometrial hyperplasia is a common finding alongside gct , occurring in 2550% , which may occur due to estrogen produced from the gct stimulating the endometrium . a simultaneous uterine carcinoma can be found in 510% of patients with gct and are often well differentiated and in an early stage . perhaps due to the diagnosis at an earlier stage , patients with synchronous endometrial and ovarian cancers have a better prognosis than patients with a single malignancy that is typically detected once it has become more extensive . the rarity of these lesions prevents randomized control trials to determine the specific best consensus practice guidelines for the management of gct . the mainstay treatment for gct is the same as for epithelial ovarian cancer , that is , surgical excision [ 6 , 20 , 27 ] . diagnostic laparoscopy has been described for the identification of tumour origin , extent , and resectability ; however , currently , laparoscopic resection is not advocated for gcts . as the incidence of bilateral disease is quite low , for women with reproductive function less than 40 years old and of reproductive age , fertility sparing surgery of unilateral salpingooophrectomy with endometrial biopsy is recommended , while women under 40 without reproductive function and those over 40 require a total abdominal hysterectomy ( tah ) as well as a bilateral salpingooophrectomy ( bso ) [ 3 , 20 , 27 ] . in patients with more advanced disease , tah and bso with complete tumour debulking are suggested . improved survival with palliative debulking hepatectomy for an unusual case of a grade i , stage i granulosa cell tumor that recurred 21 years following initial surgery has also been reported . peritoneal exploration , washing cytology , peritoneal biopsy , and partial omentectomy have been suggested as part of the staging procedure in all gct patients . careful examination of the contralateral ovary and tube , intra - abdominal organs , and peritoneum with sampling of the pelvic and para - aortic lymph nodes are recommended . aside from being a treatment option , surgery is also necessary for staging and accurate tissue diagnosis [ 3 , 27 ] . these parameters are important to determine , as poor prognostic features include a tumour size greater than 1015 cm , a high mitotic index , tumour rupture , and lymphatic invasion [ 3 , 6 , 31 , 50 ] . three forms of adjuvant therapy have been suggested to use in combination with surgery : hormonal therapy , radiotherapy , and chemotherapy . hormonal therapy is believed to act directly by affecting the tumour and/or indirectly by suppressing gonadotropins or endogenous steroids . aromatase inhibitors such as anastrozole and letrozole inhibit the conversion of androstenedione to estrone , and estradiol and testosterone to estradiol , reducing aromatization of androgens by upto 90% thereby enhancing the treatment of gct . gonadotropin - releasing hormone ( gnrh ) analogs like leuprolide have been used to decrease stimulation of granulosa cells through inhibition of ovarian steroidogenesis in recurrent gct . however , the fact that not all gcts respond to hormonal therapy despite nearly all gcts containing progesterone receptors indicates that multiple factors play a role in the hormonal regulation of the tumor cell . radiotherapy may be used as an adjuvant therapy or in the instance of recurrence , and is associated with an improved survival [ 27 , 29 , 52 ] . additionally , the use of palliative radiotherapy as an alternative strategy with potential disease control has been useful in symptomatic patients with localized or metastatic disease unqualified for surgery [ 2 , 20 ] . the use of chemotherapy has yielded encouraging results , associated with a longer disease - free survival . the chemotherapeutic agent cisplatin has the highest reported activity in the ovary , and when combined with doxorubicin , cyclophosphamide , bleomycin , vinblastine , or etoposide , an overall response rate of 6083% has been reported . the current standard recommended chemotherapeutic regimen for advanced , recurrent , or metastatic gct is bleomycin , etoposide , and cisplatin ( bep ) [ 3 , 20 , 27 ] . targeted identification of targets for novel therapeutic agents is also predicted in the future with increased knowledge about the molecular biology of both the normal and neoplastic gscs [ 2 , 6 , 52 ] . the cut surface is primarily solid , with haemorrhagic regions and a gray / white or yellow colour depending on the lipid content . these tumours are often filled with serous fluid or clotted blood and may be mistaken for mucinous cystadenoma or cystadenocarcinoma . gcts are sex - cord stromal neoplasms that on microscopic examination contain sex - cord - derived epithelial elements admixed with mesenchymal elements with a variety of combinations and degrees of differentiation . fibroblasts , granulosa , and theca cells make up a gct . depending on 4 variables including age at diagnosis , histology , therapy , and prognosis , cells are usually arranged around a central cavity named a call - exner body that has a microfollicular growth pattern similar to primordial follicles and contains eosinophilic materials as well as nuclear debris . . a wide variety of growth patterns have been identified , and may be divided into two categories . the well - differentiated type includes microfollicular , macrofollicular , trabecular , insular , solid tubular , and gyriform architectural patterns . the moderately differentiated type includes a diffuse , sarcomatoid growth pattern that is easily mistaken for a carcinoma or adenocarcinoma as histological mimics . nuclear characteristics are a hallmark feature of agct , including a uniform , pale , and grooved these nuclear features may be used to differentiate agct with a diffuse pattern from poorly differentiated carcinoma , as carcinomatous nuclei are hyperchromatic and not grooved , and additionally do not demonstrate nuclear atypia and multiple mitotic figures to the same extent . immunohistochemical analysis can be used to confirm the diagnosis of gcts if the lesion 's morphology is non - predictive of histogenesis . a study by nofech - mozes et al . inhibin , calretinin , cd56 , and cd99 are part of the immunoprofile for both types of gct ; however , the lack of a single specific marker necessitates a panel of antibodies for the detection of these lesions . gct cells usually stain positive for inhibin , calretinin , cd99 , cd56 , vimentin , estrogen and progesterone receptors . other markers that can be positive leading to diagnostic confusion include cam5.2 , ae1/ae3 , cd10 , s100 , wt-1 , smooth muscle actin , and desmin . however , gct 's are usually negative for cytokeratin 7 and epithelial membrane antigen ( ema ) . the absence of staining with ema has diagnostic value in distinguishing gct from its multiplicity of histological look - alikes such as metastatic or primary carcinoma . gcts are unpredictable neoplasms that have the ability to extend locally or spread by lymphatics , especially to the para - aortic lymph nodes . distant metastatic sites of gct most commonly include the lung , liver , and brain . a quarter of gct patients will have recurrences , and the mean time to their detection is 510 years [ 3 , 27 ] . 1020% of patients may develop recurrences as late as twenty to forty years after the primary diagnosis [ 57 , 58 ] . one - third ( 33% ) of gcts recur in less than 5 years , half ( 50% ) between 59 years , and 17% ten or more years after the initial diagnosis [ 49 , 59 ] . splenic rupture from metastatic gct 29 years after the original curative resection has also been reported . as such , lifelong surveillance for these neoplasms is recommended . frequent sites of recurrence include the upper abdomen ( 5570% ) and the pelvis ( 3045% ) . this suggests that recurrences in early - stage patients may be attributed to preexisting diseased peritoneum during the initial surgery . in early - stage patients , risk factors for relapse include large tumour size , high mitotic index , and tumour rupture ; therefore , these features may indicate the need for postoperative adjuvant chemotherapy . additional postsurgical risk factors include advanced stage of presentation , lymphovascular space invasion , bilaterality , and ki67/p53 overexpression . higher stage disease is also related to aggressive tumour behaviour with recurrences . the overall ten - year survival rates in patients with gct range between 60 to 90% . approximately 80% of females with advanced gct die due to the disease , which is partly related to the tendency for delayed recurrence . this unpredictability of the time interval for recurrent and/or metastatic disease indicates the requirement for a long - term clinical followup in all cases [ 3 , 27 ] . gct is best considered an unusual indolent neoplasm of low malignant potential with late recurrences that can arise in the ovaries and testicles in both the young and the old . the multifaceted clinical presentations coupled with the unpredictable biological behaviour with late relapses are diagnostic pitfalls necessitating a high degree of suspicion for accurate clinical and pathological diagnosis . surgery continues to be the primary cornerstone of initial treatment with chemotherapy and/or radiotherapy being reserved for advanced or recurrent disease states . lack of evidence - based predictive and prognostic factors continues to be a deterrent in accurately predicting the biological behaviour of individual gcts . however , long - term lifelong followup including physical / pelvic exam , abdominal / pelvic ct scan , and/or tumor markers as available is recommended in all patients with gcts as delayed tumor recurrences beyond 5 years are characteristic of this disease .

background . granulosa cell tumors ( gcts ) , representing ~2% of ovarian tumours , are poorly understood neoplasms with unpredictable and undetermined biological behaviour . design . 5 unusual presentations of gct and a retrospective 14-year ( 19972011 ) surgical pathology review based on patient sex , age , tumour type and concurrent pathology findings are presented to discuss the myths and realities of gcts in the context of relevant evidence - based literature . results . the 5 index cases included ( 1 ) a 5 month - old boy with a left testicular mass , ( 2 ) a 7-day - old neonate with a large complex cystic mass in the abdomen , ( 3 ) a 76-year - old woman with an umbilical mass , ( 4 ) a 64-year - old woman with a complex solid - cystic pelvic mass , and ( 5 ) a 45 year - old woman with an acute abdomen . pathological analysis confirmed the final diagnosis as ( 1 ) juvenile gct , ( 2 ) macrofollicular gct , ( 3 ) recurrent gct 32 years later , ( 4 ) collision tumour : colonic adenocarcinoma and gct , and ( 5 ) ruptured gct . conclusion . gct is best considered as an unusual indolent neoplasm of low malignant potential with late recurrences that can arise in the ovaries and testicles in both the young and the old . multifaceted clinical presentations coupled with the unpredictable biological behaviour with late relapses are diagnostic pitfalls necessitating a high degree of suspicion for accurate clinical and pathological diagnosis .

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

under the name of lymphoproliferative disorders various disease patterns are included which are characterized by the expansion of a lymphoid clone more or less differentiated . the application in recent times , of immunological methods for determining the phenotype of many cell components , together with the acquisitions of cytogenetic and molecular biology , as well as clinical behavior , have helped to relatively define a wide range of diseases that may present a heterogeneous clinical and morphological picture . in fact , the last classification of lymphoproliferative disorders lists 40 types of lymphoproliferative syndromes to immunophenotype b and 23 to immunophenotype t . at the level of the oral cavity , the lymphoproliferative disorder can occur in various ways , most commonly as lymphoid lesions with extranodal externalization , but sometimes , oral lesions may represent a localization of a disease spread . under the key research that sees lymphoproliferative disorders associated with injury or events at the oral cavity , the present paper proposes a comprehensive classification as listed in table 1 and deeply described below . primary extranodal involvement can be seen in 10% to 35% of cases of non - hodgkin lymphomas . these locations include the gastrointestinal tract , skin , testicles , kidneys , and bones [ 3 , 4 ] ; the nhl of the central nervous system account for 1% of cases [ 5 , 6 ] . although the oral involvement of nhl is rare , they are the second most common oral malignant disease after oral squamous cell carcinoma [ 7 , 8 ] , constituting of 2.2% of all malignancies of the head - neck , 3.5% of intraoral malignancies , 5% of tumors of the salivary glands , and 2.5% of all cases of nhl . although every other site may be affected , ring waldayer is the most commonly involved . indolent lymphoma accounts for 40% af all nhl with the most common type being follicular lymphoma ; aggressive lymphoma accounts for approximately 50% of cases and include diffuse large b - cell lymphoma and t - cell natural killer ( nk ) cell lymphoma ; highly aggressive lymphoma includes ebv - associated burkitt lymphoma and lymphoblastic lymphoma . even though each type of nhl can occur at the level of oral cavity , the most common types are large - cell lymphoma and lymphoma of small lymphocytes lymphoma ; mucosa - associated lymphoid tissue ( malt ) lymphoma , follicular lymphoma , burkitt 's lymphoma , and immunocytoma , immunoblastic lymphoma are also reported [ 8 , 11 , 12 ] . the most common signs and symptoms with which the nhl will have intraoral include tissue swelling , tooth loss , and paresthesia . oral lesions may appear as exophytic neoplasms , erythematous , asymptomatic , often with superficial ulcerations secondary to chronic trauma ( figure 1 ) . hodgkin 's disease is a form of malignancy characterized by proliferation of neoplastic cells ( hodgkin and reed - sternberg cells ) associated with a polymorphic cellular component ( lymphocytes , histiocytes , eosinophils , neutrophils , and plasma cells ) , considered to be reactive . from its initial description until the 1990s , the nature and lineage of the reed - sternberg cell and the inflammatory infiltrate that compromises hodgkin disease were debated . the application of pcr technique revealed that hodgkin and reed - sternberg cells were clonal b cells in 98% of the patients , leading to the change of the designation of hodgkin disease into hodgkin lymphoma ( lh ) in the who classification of lymphoid neoplasms . since the waldayer ring is commonly involved by nhl that sometimes shows cells of reed - sternberg cell - like , the diagnosis of lh is difficult , especially when there are only small biopsy specimens [ 15 , 16 ] . the most common site involved is the ring of waldayer followed by the lips , tongue base [ 18 , 19 ] , buccal mucosa [ 20 , 21 ] , and parotid gland [ 22 , 23 ] . the oral manifestations by plasma cell tumors can occur in three different ways : as a consequence of the local manifestation of multiple myeloma , bone plasmacytoma as solitary , or as extramedullary plasmacytoma . the primary manifestations of plasma cell neoplasms at the oral level are represented by solitary and extramedullary plasmacytoma . the plasmacytoma of bone can be considered a localized solitary myeloma ( figure 2 ) . solitary bone plasmacytoma is a malignant monoclonal gammopathy [ 25 , 26 ] ; it is a plasma cell cancer that occurs as a single osteolytic lesion without plasmacytosis of the bone marrow and that is capable of secreting monoclonal m protein [ 27 , 28 ] . this disease accounts for 10% of all plasma cell tumors and can strike [ 25 , 27 , 28 ] , although rarely , in the oral cavity , showing a predilection for the mandibular retromolar area ; its radiological appearance may have one of two patterns , as either an oval - shaped lytic image with destruction of the cortical bone , or as a hyperinsufflating lesion showing a convex bicortical bone . instead , the extramedullary plasmacytoma is a plasma cell tumor located separately from the bone marrow ; it is found in all parts of the body where this lymphoid tissue [ 3238 ] , in 90% of the extramedullary plasmacytoma , is present in the head and neck . clinically [ 3941 ] , the plasmacytoma extraosseous is present as a sessile or pedunculated exophytic neoformation , circumscribed or infiltrative , ranging in color from red - purple to gray or yellow - white [ 4250 ] . the oral manifestations of multiple myeloma ( mm ) , a disease characterized by the proliferation and accumulation in the bone marrow of a clone of plasma cells to produce a homogeneous monoclonal protein [ 13 , 30 , 51 , 52 ] , are extensively reported in the literature ; the oral manifestations are the initial sign of submission in 12% to 15% of cases of mm [ 5355 ] . the oral features include facial , oral , and dental pain , numbness and paresthesia , swelling , soft tissue neoplasms , tooth mobility , bleeding , and deposit of amyloid substance especially on the tongue [ 5760 ] . other examples of oral lesions as first manifestation of lymphoproliferative disorders may be the infiltration of the oral mucosa by a b - cell chronic lymphocytic leukemia , the lymphomatous papulosis , a condition mucocutaneous applicant , self - limited , characterized by papular eruptions , and mycosis fungoides , t - cell cutaneous lymphoma in which the involvement of the oral cavity is a rare event but well documented [ 63 , 64 ] . in addition to entering into this category , the previously treated lymphoproliferative disorders , where the diagnosis of oral lymphoid malignancy was subsequent to an indication of their early sign , mentioned in this regard a clinical - pathological entity of which we have evidence in the literature for over a century of mycosis fungoides ( mf ) , chronic lymphoproliferative disorder with predominantly cutaneous involvement characterized by the proliferation of t lymphocytes that in advanced stages of the disease can accumulate also in lymphoid organs , bone marrow , and peripheral blood ( sezary syndrome ) . involvement in oral mf is an occasional finding observed from 7.4% to 18% of patients undergoing necropsy [ 65 , 66 ] . despite these findings relatively frequent mucosal involvement in vivo is a rare event ; there is no predisposition to sex ; age of onset varies from 36 to 81 years , with an average of 61 years . oral sites most frequently involved are the tongue , palate , gingiva , buccal mucosa , lips , and oropharynx . in almost all patients , the lesions skin prior to the mucosa over a period of time ranging from 7 months to 40 years . here is a list of non - specific signs and symptoms present in association with lymphoproliferative disorders of the oral cavity : lymphadenopathy , trismus , erythema , epiphora , pain , swelling , facial asymmetry or swelling of buccal mucosa , sinusitis , increased lacrimation and abscesses of the lacrimal sac , diplopia , nasal obstructions , sepsis , fever , runny nose , prosthetic instability , headache , and paresthesia idiopathic epistaxis . suspicion of malignancy usually develops only after these inflammatory symptoms have not responded to conventional treatment protocols , upon which more accurate evaluations are required . although oral lymphomas are extremely rare [ 68 , 69 ] , they can occur earlier and be placed in the differential diagnosis with non - specific inflammatory processes . moreover , the early recognition of these subtle cancers can decrease their morbidity . paraneoplastic pemphigus ( pnp ) , or paraneoplastic autoimmune multiorgan syndrome is a rare autoimmune vesiculobullous disease first described by anhalt et al . in 1990 in patients with occult malignancies [ 71 , 72 ] . erosive eye lesions , sinuses , oral cavity , the gastrointestinal system , and respiratory and genital epithelium could affect them . clinically , lesions may occur as polymorphic , such as pemphigus , bullous pemphigoid , erythema multiforme , the graft - versus - host disease , and lichen planus [ 7375 ] ( figure 3 ) . dermatomyositis ( dm ) is a rare inflammatory microangiopathic disease that affects skeletal muscles , with clinical externalization as characteristics mucocutaneous manifestations . oral lesions in paraneoplastic dm ( leucoerythroplasia and ulcerative lesions ) have rarely been described in the literature [ 7679 ] . the dm is totally resolved if the underlying disease is treated and the resurgence of dm expresses relapse of malignancy [ 76 , 79 ] . in the literature , there are reports of a single case of multiple myeloma with first manifestation at the oral level under the clinical aspects of lichen planus , showing extensive and irregular erosions present at buccal mucosa , labial , palatal mucosa , and ventral tongue . the literature contains many works that correlate the paraneoplastic pemphigus ( pnp ) , a rare autoimmune vesiculobullous disease underlying a malignancy [ 71 , 72 ] , with typical oral lesions of lichen planus or lichenoid reactions . in the literature there are numerous works reported that associate mucous membrane pemphigoid ( mmp ) with malignancies , including lung cancer , pancreatic adenocarcinoma , gastric adenocarcinoma , and squamous cell carcinoma of the conjunctiva there are only two cases reported of mmp with oral manifestations associated to lymphoproliferative disorders : a b - cell lymphoma and chronic lymphocytic leukemia . it has been described in the literature that large amount of cases have connection between bullous pemphigoid ( bp ) and lymphoproliferative disorders such as chronic lymphocytic leukemia [ 8991 ] and lymphoblastic lymphoma . necrotizing oral processes , although rare in the general population , can rapidly evolve in devastating stages in immunocompromised patients [ 9396 ] and are often associated with periodontal disease . these are patients with lymphoproliferative disorders such as acute lymphoblastic leukemia , which developed necrotic processes at the level of the oral cavity not associated with typical ulcer - necrotizing periodontal diseases , but by bacteria in the oral cavity unusually found as pseudomonas aeruginosa . patients with impaired lymphocyte function or reduced counts of neutrophils , due to lymphoproliferative disorders , have led to the acquisition of new infections and/or exacerbation or reactivation of latent infection ( periapical periodontitis and herpes simplex ) . in many cases the clinical presentation of oral infections may be atypical when compared to those normally seen in healthy patients . the origin of odontogenous infection of the pulp and periodontal is frequently observed in patients with lymphoproliferative disorders and should be suspected when orofacial pain , extensive dental restorations , and dental caries and periapical radiopacity occur [ 97 , 98 ] . the candidiasis may present as pseudomembranous candidiasis , erythematous , hyperplastic , or angular cheilitis . deep fungal infections should be suspected in patients with solitary ulcer that does not retreat . this group includes infections such as aspergillosis , histoplasmosis , and that by zygomycetes , and the treatment requires aggressive treatment with intravenous azoles , amphotericin b , and echinocandins . primary infection or reactivation of herpes viruses is common in patients with lymphoproliferative disorders especially during the advanced stages of the disease . infection with herpes simplex is most common in these patients ; the reactivation of the varicella - zoster virus ( vzv ) is less common . epstein - barr virus infection ( ebv ) is associated with oral hairy leucoplakia , a very rare whitish lesion that occurs on the lingual lateral margins in myelosuppressive patients [ 104 , 105 ] . infection by cytomegalovirus ( cmv ) can occur on any intraoral mucosal surface in the form of an ulcer - free character of specificity , which persists for weeks or months . because of thrombocytopenia in the course of lymphoproliferative disorders , intraoral bleeding is commonly observed , which is present with petechiae , bruising , and occasionally with the formation of hematomas ( figure 4 ) . chemotherapy and radiotherapyin addition to what is included in this paper the oral infectious complications ( bacterial , fungal , viral ) , previously treated on immunosuppression induced by lymphoproliferative disorder , and here remembered as a possible consequence of drug - induced immunodeficiency , we will consider the common characteristic found in the course of oncohaematology treatment regimens . patients with lymphoproliferative disorders who are treated with high doses of radiotherapy at the head and neck level , associated with aggressive chemotherapy regimens , are at greatest risk of developing oropharyngeal mucositis . xerostomia is often present in patients with lymphoproliferative disorders treated with radiotherapy in the head and neck region due to damage of the major salivary glands , which are still included in the irradiation of tumors . as another long - term complication of radiation therapy for head and neck lymphomas in patients who underwent radiotherapy , we noticed the osteoradionecrotic maxillary bone as a result of permanent damage of the capillary bed of bone and osteocytes caused by radiation . in addition to what is included in this paper the oral infectious complications ( bacterial , fungal , viral ) , previously treated on immunosuppression induced by lymphoproliferative disorder , and here remembered as a possible consequence of drug - induced immunodeficiency , we will consider the common characteristic found in the course of oncohaematology treatment regimens . patients with lymphoproliferative disorders who are treated with high doses of radiotherapy at the head and neck level , associated with aggressive chemotherapy regimens , are at greatest risk of developing oropharyngeal mucositis . xerostomia is often present in patients with lymphoproliferative disorders treated with radiotherapy in the head and neck region due to damage of the major salivary glands , which are still included in the irradiation of tumors . as another long - term complication of radiation therapy for head and neck lymphomas in patients who underwent radiotherapy , we noticed the osteoradionecrotic maxillary bone as a result of permanent damage of the capillary bed of bone and osteocytes caused by radiation . autologous transplantationoral complications are observed in 85% of cases of patients after autologous bone marrow transplantation ( abmt ) . infections occur during the period of severe marrow aplasia and may be secondary to the conditions of the oral cavity before the transplant . patients treated for lymphoproliferative disorders are at increased risk of relapse of the primary disease so as to develop subsequent tumors both hematologic and nonhematologic in this regard we consider the oral squamous cell carcinoma as a second primary solid tumor after allogeneic transplant . a variety of circulating autoantibodies can be found from 20% to 61% of patients undergoing autologous bone marrow transplant [ 108111 ] , and a number of these patients may demonstrate autoantibody - mediated diseases such as myasthenia gravis and autoimmune cytopenias and some cases of oral diseases , such as oral vesiculobullous ( pemphigus and pemphigoid ) [ 113116].the graft - versus - host disease ( gvhd ) is a multisystem immunologic reaction resulting from the action of immunocompetent cells transplanted from a donor to an immunodeficient host . the recipients of allogeneic hematopoietic cell transplantation ( allo - htc ) are at greater risk of developing the disease from acute and chronic graft versus host . the likelihood of developing gvhd depends on the type of conditioning regimen ( ablative vs. totally reduced intensity regimens ) . gvhd in oral lesions , although infrequent , can be observed , and rarely require specific treatment . chronic gvhd typically develops after the hundredth day , with an incidence ranging from 25% to 40% after allo - hct [ 107 , 117 ] ( figure 5 ) . the mouth is one of the locations most commonly affected by events such as lichenoid mucositis ( including ulcers ) , pain , odynophagia , dysgeusia , hypofunction of the salivary glands , caries decayed teeth , and rarely sclerotic processes that lead to a hypomobility and a reduction in functionality [ 10 , 118 ] . oral complications are observed in 85% of cases of patients after autologous bone marrow transplantation ( abmt ) . infections occur during the period of severe marrow aplasia and may be secondary to the conditions of the oral cavity before the transplant . patients treated for lymphoproliferative disorders are at increased risk of relapse of the primary disease so as to develop subsequent tumors both hematologic and nonhematologic in this regard we consider the oral squamous cell carcinoma as a second primary solid tumor after allogeneic transplant . a variety of circulating autoantibodies can be found from 20% to 61% of patients undergoing autologous bone marrow transplant [ 108111 ] , and a number of these patients may demonstrate autoantibody - mediated diseases such as myasthenia gravis and autoimmune cytopenias and some cases of oral diseases , such as oral vesiculobullous ( pemphigus and pemphigoid ) [ 113116 ] . the graft - versus - host disease ( gvhd ) is a multisystem immunologic reaction resulting from the action of immunocompetent cells transplanted from a donor to an immunodeficient host . the recipients of allogeneic hematopoietic cell transplantation ( allo - htc ) are at greater risk of developing the disease from acute and chronic graft versus host . the likelihood of developing gvhd depends on the type of conditioning regimen ( ablative vs. totally reduced intensity regimens ) . gvhd in oral lesions , although infrequent , can be observed , and rarely require specific treatment . chronic gvhd typically develops after the hundredth day , with an incidence ranging from 25% to 40% after allo - hct [ 107 , 117 ] ( figure 5 ) . the mouth is one of the locations most commonly affected by events such as lichenoid mucositis ( including ulcers ) , pain , odynophagia , dysgeusia , hypofunction of the salivary glands , caries decayed teeth , and rarely sclerotic processes that lead to a hypomobility and a reduction in functionality [ 10 , 118 ] . supportive therapybisphosphonates are drugs used in combination with chemotherapy in the treatment of hypercalcemia secondary to malignancy , lytic bone metastases and multiple myeloma [ 119121 ] . starting from 2003 , an increasing number of cases that describe the correlation between osteonecrosis of the jaw ( onj ) and administration of intravenous bisphosphonates were published in the literature [ 122131 ] ( figure 6 ) . bisphosphonates are drugs used in combination with chemotherapy in the treatment of hypercalcemia secondary to malignancy , lytic bone metastases and multiple myeloma [ 119121 ] . starting from 2003 , an increasing number of cases that describe the correlation between osteonecrosis of the jaw ( onj ) and administration of intravenous bisphosphonates were published in the literature [ 122131 ] ( figure 6 ) . approximately 10% of all cases of non - hodgkin lymphomas in the united states and europe are aids related [ 132134 ] . typically , aids - related nhl has a predilection for extranodal sites and appears in the head and neck , in 50 - 60% of cases [ 135144 ] ; they have an aggressive course and a poor prognosis [ 145147 ] . in histopathology these lymphomas are b - cells derived and characterized by widespread growth , pleomorphism , high - grade morphology , frequent mitotic figures , and increased tendency to develop into necrosis [ 148153 ] . lymphomas of head and neck may involve the gingiva , palate , tongue , tonsils , nasopharynx , orbits , parotid glands , jaw breasts , soft tissues , and hypopharynx . lymphoid neoplasms are heterogeneous malignant disorders of the lymphatic system characterized by uncontrolled proliferation of lymphoid cells or their precursors . with regard to the primary localizations of lymphoproliferative disorders , a careful examination of the head and neck , oral , and oropharyngeal area is necessary in order to identify suspicious lesions and their early detection . an early diagnosis may allow treatment at early stages of the disease , resulting in a better prognosis for the patient . numerous complications have been described and frequently found at oral level , due to pathology or different therapeutic strategies . furthermore , the evaluation of patients after treatment for lymphoma is essential to prevent relapse and to recognize early any eventual secondary localizations . in all this , oral pathologists , as well as dental practitioners , have a central role in the treatment and long term monitoring of these patients , having the opportunity and the duty to examine and make a diagnosis at the level of hard and soft tissue of the oral cavity .

lymphoproliferative disorders are heterogeneous malignancy characterized by the expansion of a lymphoid clone more or less differentiated . at the level of the oral cavity , the lymphoproliferative disorder can occur in various ways , most commonly as lymphoid lesions with extranodal externalization , but sometimes , oral lesions may represent a localization of a disease spread . with regard to the primary localizations of lymphoproliferative disorders , a careful examination of the head and neck , oral , and oropharyngeal area is necessary in order to identify suspicious lesions , and their early detection results in a better prognosis for the patient . numerous complications have been described and frequently found at oral level , due to pathology or different therapeutic strategies . these complications require precise diagnosis and measures to oral health care . in all this , oral pathologists , as well as dental practitioners , have a central role in the treatment and long - term monitoring of these patients .

1. Introduction 2. Classification and Related Aspects of the Oral Pathologies Associated with Lymphoproliferative Disorders 3. Conclusions

the prevalence of diabetes in korea has increased six- to sevenfold from 1.5 to 9.9% over the past 40 years . the number of people with diabetes worldwide is expected to increase 11.4% from 366 million in 2011 to 552 million by 2030 , affecting one in 10 adults . this global increase in the prevalence of diabetes will inevitably lead to increases in the prevalence of diabetic microvascular and macrovascular diseases , and consequently , significantly increased health care expenditure . in korea , diabetic patients with microvascular disease spend up to 4.7 times as much , patients with macrovascular disease up to 10.7 times as much , and patients with both complications 8.8 times as much as those with no complications . the medical cost of diabetes mellitus covered by the national health insurance corporation is 3.2 trillion won and accounted for 19.2% of all medical costs . diabetes and its complications have become major causes of morbidity and mortality in korea . although diabetes - related mortality has decreased recently from 25.1 per 100,000 persons in 2002 to 19.6 per 100,000 persons in 2009 the rate of death among patients with diabetes is about twice as high as that among persons without diabetes . the most common cause of death is cardiovascular disease ( 30.6% ) , followed by infectious disease ( 25.3% ) , cancer ( 21.9% ) , congestive heart failure ( 7.1% ) , renal disease ( 4.7% ) , liver disease ( 2.7% ) , and diabetes itself ( 1.9% ) . despite the seriousness of diabetic complications , 30% to 70% of patients receive inadequate care , and only 40% of treated diabetic patients achieve the optimal control , defined as an hba1c level < 7% . the serious outcomes from diabetic complications and inadequate glucose control in diabetic patients prompt the need for more aggressive efforts to provide optimal metabolic control . type 2 diabetes mellitus is associated with a high rate of complications related to cardiovascular disease and diabetic nephropathy , retinopathy , and neuropathy . in 2006 , 30.3% , 38.3% , and 44.6% of patients were found to have microvascular complications such as microalbuminuria , retinopathy , and neuropathy , respectively from korean nationwide survey . the prevalence of macrovascular complications including coronary artery disease , cerebrovascular disease , and peripheral artery disease was 8.7% , 6.7% , and 3.0% , respectively . the prevalence of diabetic foot was 4.4% , and 44.8% of patients with an amputated foot had diabetes mellitus . the prevalence of macrovascular complications seems underestimated in these data . in a study of 343 patients with diabetes mellitus , the prevalence of cardiovascular complication was 23.6% , and in another study of 406 patients with diabetes , extracranial internal carotid artery stenosis 40% was detected in 5.2% of the patients . in addition , the prevalence of macrovascular complication was 10.8% from recent the korean national diabetes program ( kndp ) data and there is reference from 2005 korea national health and nutrition examination survey ( knhanes ) data in table 1 . the role of hyperglycemia in the development of microvascular complications of diabetes , such as nephropathy , retinopathy , and neuropathy , is well documented . the incidence of microvascular complications begins to increase at an hba1c level > 7.0% and increases by 30% to 40% per 1% increase in hba1c level from over 8,000 patients ( fig . microvascular complications are closely related to age , duration of diabetes , and glycemic control , and this relationship is stronger than that with macrovascular complications . diabetic retinopathy is the most common microvascular complication of diabetes mellitus , and its prevalence is strongly related to the duration of diabetes . it is the most frequent cause of new cases of blindness among adults aged 20 to 74 years . in ansung cohort study ( prospective , rural , community cohort in korea ) , the prevalence of any diabetic retinopathy was 2.9% and the proper cutoff of hba1c value for detecting any diabetic retinopathy was 6.6% ( unpublished data ) . diabetic nephropathy is characterized by albuminuria ( 300 mg / day ) and a reduced glomerular filtration rate . it is often present at the time of the diagnosis of diabetes after the kidney has been exposed to chronic hyperglycemia during the prediabetic phase . patients with microalbuminuria who progress to macroalbuminuria ( 300 mg/24 hr ) are likely to progress to end - stage renal disease ( esrd ) . diabetes is a major cause of chronic kidney disease ( ckd ) and is recognized as the most common cause of esrd in the usa and korea . about 40% of united states adults with diagnosed or undiagnosed diabetes had some degree of ckd in the 1999 to 2006 national health and nutrition examination survey . the prevalence of peripheral neuropathy has been estimated at 40.0% to 44.6% . at present , there is no specific treatment for the underlying nerve damage other than to improve glycemic control , which may slow the progression modestly . the case fatality rate after myocardial infarction is higher in patients with diabetes than in patients without diabetes . the association between diabetes and coronary heart disease is likely to become more important for two reasons . first , the incidence of type 2 diabetes is increasing among both high - risk and low - risk populations . second , although the rate of death caused by coronary heart disease in the overall population has declined markedly over the past 35 years , this has not been the case among persons with diabetes . cardiovascular disease is the major cause of morbidity and mortality for individuals with diabetes and is the largest contributor to the direct and indirect costs of diabetes . older age , high blood pressure , and smoking history are major risk factors for the development of macrovascular complications . a smoking history in males can be both a risk factor and a predictive factor for earlier development of macrovascular complications in korean patients with type 2 diabetes . microvascular complications of diabetes increase the risk of cardiovascular events in diabetic patients . although diabetic retinopathy is not associated with the presence of atherosclerotic plaque , it is associated with increased carotid intima - media thickness , and the increase in intima - media thickness is associated with the presence of plaque , which predisposes patients to cardiovascular disease . microalbuminuria and a low glomerular filtration rate ( < 60 ml / min/1.73 m ) increase the risk of major cardiovascular events and death . diabetic polyneuropathy was also independently associated with a high prevalence of cardiovascular disease in type 2 diabetic patients . these data imply that the microvascular complications of diabetes are related indirectly to the macrovascular complications of diabetes , similar in korea . intensive glycemic control has been suggested as an effective treatment for reducing the burden of cardiovascular disease and microvascular complications in people with diabetes . the united kingdom prospective diabetes study and the kumamoto study showed that early intensive glycemic control can delay the onset and progression of diabetic retinopathy , nephropathy , and neuropathy compared with conventional treatment [ 21,24 - 26 ] . intensive glucose control involving gliclazide ( modified release ) and other drugs as required lowered the hba1c value to 6.5% and reduced by 10% the relative risk for the combined outcome of major macrovascular and microvascular events , primarily because of a 21% reduction in incidence of nephropathy . in addition , the legacy effect was observed after 10 years of trial in the rate of microvascular complication and myocardial infarction . in the steno-2 study , the intensive therapy group had a 46% lower risk for all - cause mortality and a 57% lower risk of death from cardiovascular causes . one patient in the intensive therapy group progressed to esrd compared with 6 patients in the conventional therapy group , and fewer patients in the intensive therapy group required retinal laser therapy . although there are many benefits of intensive glucose - lowering treatment for preventing macrovascular and microvascular events , it remains uncertain whether these benefits outweigh the risks . intensive blood glucose control decreases the risk of developing microvascular complications but not macrovascular disease in patients with type 2 diabetes . in the action to control cardiovascular risk in diabetes ( accord ) study , an intensive glucose - lowering regimen reduced rate of 5-year nonfatal myocardial infarctions but was associated with a 22% increase in mortality . a recent meta - analysis found that intensive glucose - lowering treatment has limited effect on the rates of all - cause mortality and death from cardiovascular causes . the data are conflicting : a 9% reduction to a 19% increase in all - cause mortality and a 14% reduction to a 43% increase in cardiovascular death rates . the harm associated with severe hypoglycemia might counterbalance the potential benefit of intensive glucose - lowering treatment . the microvascular benefits of intensive therapy should be weighed against the increase in total and cardiovascular disease - related mortality , weight gain , and high risk of severe hypoglycemia . however , multifactorial approaches in high risk patients with diabetes , such as tight glucose regulation and the use of anti - hypertensive medication , aspirin , and lipid - lowering agents , have been shown to reduce the risk of nonfatal cardiovascular disease among patients with type 2 diabetes mellitus and microalbuminuria . in high risk patients with type 2 diabetes , intensive treatment approaches with multiple drug combinations and behavior modification had sustained beneficial effects with respect to vascular complications and on rates of death from any cause and from cardiovascular causes . several adipokines are implicated in the metabolic syndrome , coronary heart disease , and insulin resistance . we want to discuss some adipokines , which showed correlation with diabetes and metabolic syndrome in korea . lipocalin family proteins , including adipocyte fatty acid - binding protein ( a - fabp ) , lipocalin-2 , and retinol - binding protein 4 ( rbp4 ) , have been identified recently as adipokines associated with obesity , type 2 diabetes , and the metabolic syndrome . serum a - fabp is associated with glucose dysregulation , and its level predicts the development of type 2 diabetes and the development of the metabolic syndrome independently of adiposity and insulin resistance . serum lipocalin-2 level is significantly elevated in patients with coronary heart disease and is independently associated with coronary heart disease . these findings suggest that serum lipocalin-2 levels may be useful for assessing coronary heart disease risk . plasma rbp4 concentration is elevated in persons with impaired glucose tolerance and type 2 diabetes . high rbp4 and low plasma adiponectin concentrations are associated with the severity of glucose intolerance in women with previous gestational diabetes mellitus . a low circulating vaspin level correlates with a high fitness level , whereas physical training in untrained individuals increases vaspin serum concentration . vaspin is also correlated with metabolic syndrome in men and coronary artery stenosis in women . however , another study reported that circulating visfatin may not be a useful clinical biomarker of metabolic status . a relationship between chemerin levels , cardiometabolic parameters , and degree of coronary stenosis has been reported in korean patients with coronary artery disease . serum osteocalcin , an osteoblast - specific protein , has several hormonal features and is secreted into the general circulation by osteoblastic cells . serum osteocalcin and osteoprotegerin levels are associated with glucose metabolism and atherosclerosis parameters in people with type 2 diabetes mellitus . even though there are many studies about adipokines and metabolic diseases , we need large number , prospective studies to see the causality between the candidate adipokines and diabetic complications more clearly . the increasing prevalence of diabetes mellitus and its related complications have contributed to a substantial increase in morbidity and mortality in korea . however , the proportion of patients with type 2 diabetes achieving adequate glucose control is relatively low . multifactorial treatment approaches that target hypertension , dyslipidemia , and microalbuminuria , and that provide intensive glycemic control are urgently needed to control diabetes . these comprehensive and integrated health interventions will lead to further improvement in the management of diabetes .

the prevalence of diabetes in korea has increased six- to sevenfold over the past 40 years with its complications becoming major causes of morbidity and mortality . the rate of death among patients with diabetes is about twice as high as that among persons without diabetes and the most common cause of death is cardiovascular disease ( 30.6% ) . despite the seriousness of diabetic complications , 30 to 70% of patients receive inadequate care , and only 40% of treated diabetic patients achieve the optimal control with hba1c level < 7% in korea . in 2006 , over 30 to 40% of patients with diabetes have microvascular complications and around 10% of them have macrovascular complications from our national data . despite there are some debates about intensive glycemic control resulting in the deterioration of macrovascular complication , multifactorial treatment approaches including proper glycemic control are important to prevent diabetic complications . there have been needs for finding proper biomarkers for predicting diabetic complications properly but we still need more longitudinal studies to find this correlation with causal relationship . in this article , we wanted to review the recent status of micro- and macrovascular complications of type 2 diabetes in korea from integration of many epidemiologic studies .

INTRODUCTION COMPLICATIONS OF DIABETES MELLITUS MICROVASCULAR COMPLICATIONS MACROVASCULAR COMPLICATIONS THE EFFECT OF GLYCEMIC CONTROL ON DIABETIC COMPLICATIONS ADIPOKINE AND DIABETIC COMPLICATIONS: RISK FACTORS FOR DIABETES CONCLUSION

we make observations and based upon these observations we make empirical predictions . in the engineering sciences this is often be reduced to formulae and mathematical modelling which can be used predictively to design newer and better engineered products however orthodox medicine has been struggling to develop such methodology . despite the huge amounts of investment made in the life sciences systems biologists compile models of organs e.g. of the heart and other organs , in their efforts to understand the complexity of organ function and of associated cellular and molecular biologies . it is the equivalent of knowing the most intricate details of a computer - the hardware - yet not having the software to make it work . moreover , if there is no understanding of the mechanisms which the body uses to regulate its function and to recover from dysfunction or infection how can it be possible to assess whether a patient 's recovery is due to a medicine , medical procedure , or to the natural processes of recovery ? modern medicine evolved from the growth of the chemical industry and the massive increase of chemical research which yielded chemicals of ever greater complexity and biological significance . aspirin and paracetamol were developed which reduce the temperature of a fever and reduce the severity of headaches . such chemical discoveries were followed by drugs which reduce the symptoms of almost all conceivable diseases , and upon which huge swathes of the population are increasingly dependent , yet the burden of healthcare on society continues to increase . the cost of treating disease has grown by more than ten times since the mid-1970 's . this leads us to question basic assumptions upon which modern biomedicine is based e.g. : how accurate are biomarker techniques?it is increasingly understood that many medical conditions are polygenic and multi - systemic . the degree of coiling or uncoiling of proteins is associated with the onset of diabetes , cystic fibrosis , alzheimer 's disease , etc . accordingly , the measurement of the level of a single protein / biomarker may be a flawed concept.how accurate is a doctor 's diagnosis?the ability of the gp to provide an accurate diagnosis is questionable . many diseases are poorly defined and are often difficult to diagnose e.g. diabetes , alzheimers disease , cancers , dementia , depression , chronic fatigue syndrome , sleep disorders , etc.how effective are drugs?the ability of drugs to treat disease is typically 50% because the onset of disease involves both genotype and phenotype . any medical assessment must take into account the level and conformation of the protein , the level and conformation of the substrate , and the factors which influence the rate at which the protein reacts with the substrate e.g. ph , levels of minerals and cofactors , etc . it must also take into account that every drug alters the body 's systemic , cellular and molecular stability i.e. the body will readjust in order to maintain optimum stability . the degree of coiling or uncoiling of proteins is associated with the onset of diabetes , cystic fibrosis , alzheimer 's disease , etc . accordingly , the measurement of the level of a single protein / biomarker may be a flawed concept . many diseases are poorly defined and are often difficult to diagnose e.g. diabetes , alzheimers disease , cancers , dementia , depression , chronic fatigue syndrome , sleep disorders , etc . the ability of drugs to treat disease is typically 50% because the onset of disease involves both genotype and phenotype . any medical assessment must take into account the level and conformation of the protein , the level and conformation of the substrate , and the factors which influence the rate at which the protein reacts with the substrate e.g. ph , levels of minerals and cofactors , etc . it must also take into account that every drug alters the body 's systemic , cellular and molecular stability i.e. the body will readjust in order to maintain optimum stability . healthcare is a fashion - led industry which seeks to exploit every perceived technological advance ( penicillins , stem - cell , optogenetics , genomics , proteomics , vaccines , statins , etc ) in the commercial hope or promise that these new areas of research will lead to yet greater opportunities to diagnose and treat disease at ever greater cost to the taxpayer . a better understanding of how the body regulates its function would improve the understanding of the diagnostic or therapeutic scope of such technologies . for instance the use of stem cell implants to treat type 1 diabetes overlooks that such implants are being implanted into the same or similar biochemical conditions which were associated with the original pancreatic failure . if the patient 's regulatory system could not maintain the function of their pancreatic beta cells why do we think that a pancreatic stem cell implant will succeed ? it can also be argued that knowledge of why type 1 diabetes occurred ( e.g. viral onset ) could lead to therapies which would re - establish and reactivate the pancreatic beta cells . this illustrates that changes to dna cause physiological dysfunction of varying degrees of severity . in the case of diabetes , cited above , altered dna structure and conformation influences the expression of proteins , can be manifest as type 1 diabetes , and can also be adapted to re - establish normal function of the pancreatic beta cells . the greater the amount of changes to the genetic structures the greater will be the scope for physiological and cognitive dysfunction . at almost every step orthodox biomedical research overlooks the influence of the environment ( phenotype ) despite the clear understanding that stress is a major cause of disease e.g. depression , post - traumatic stress disorder , insomnia , cardiac arrhythmia , etc . the most glaring example is of identical twins which have different lifestyles and which have significantly different health in their advancing years . gene profiling identifies the genes which are no longer able to express a particular protein ( genotype ) and those who are genetically pre - disposed to a medical condition e.g. gauchers disease , fabry 's disease , etc ; but can not yet elucidate the complex genetic interactions ( including racial genotypes ) which are involved in polygenic diseases e.g. diabetes mellitus , alzheimers disease , cystic fibrosis , etc ; or the environmental influences ( phenotype ) which would cause this pre - disposition to be manifest as the pathology in question . how can it deal with the daily causes of stress ? how does stress influence the body 's function ? furthermore by ignoring phenotype it ignores up to 50% , perhaps more , of the disease creating process(es ) . if it can incorporate an understanding of phenotype it may be able to deal with both the cause of a disease and its symptoms . they also ignore that the body 's organs are organised into organ networks and that each of these organs has a physiological significance e.g. regulating blood pressure , blood glucose , ph , sleep , etc . perhaps the most interesting of the overlooked phenomenae are that of the eeg frequencies and the role played by light . these may be issues of the greatest medical significance i.e. they are indicative the mechanisms which the body uses to regulate its complex , multi - level function . it directly influences all of the reaction and extraction processes e.g. the levels of minerals , acidity , temperature , etc . it influences metabolic rate and cognitive dysfunction however there are few , if any , techniques which are able to measure the phenotype component of developing pathologies e.g. colour / syntonic optometry , microneurography , tilt table test , valsalva test , etc . the evidence suggests that human physiology is that of a dynamic , self - regulating system in which the brain acts to process sensory input , retain significant memories , and maintain the body 's optimum stability . it is the precise nature , and co - ordinated function of the physiological structures which determines the levels of each key systemic parameter : ph , temperature , digestion , elimination , breathing , blood pressure , blood volume , blood cell content , blood glucose , osmotic pressure , sexual function , posture and sleep . this raises blood pressure in order to maintain the appropriate level of oxygen in the brain . over time this progressively influences the stability of most , if not all , other physiological systems e.g. body temperature , blood volume , sleep , digestion , excretion , osmotic pressure , sexual function and posture . the body 's functional parameters are based upon what is required to maintain the body 's normal and/or stable function e.g. ( i ) it attempts to maintain its acidity at ph 7 because increased acidity alters the levels of minerals and , in particular , lowers the levels of key minerals ( zn , cr , mg , etc ) which are essential for normal physiology . ( ii ) it maintains heart beat and heart pressure at a level which best absorbs oxygen in the lungs and maintains the flow of oxygen to the brain i.e. high and low levels of blood pressure can lead to haemorrhage or stroke . ( iii ) it maintains temperature at circa 36.8c because this is the optimum temperature for many biochemistries i.e. at higher temperatures proteins start to denature and their reactivity is reduced whilst at lower temperatures the rate of reaction declines . ( iv ) it maintains the stability of the processes of digestion and excretion in order to ensure the flow of nutrients and the elimination of toxins which could adversely influence many biochemistries . per litre because deviations from such limits ( a ) lead to increased weight , increased release of insulin , and the onset of glycation processes in type 2 diabetes , cardiological complications , etc ; or ( b ) reduce the energy generated , lowers metabolic rate , and the development of side - effects in type 1 diabetes . ( vi ) per litre which with blood cell content , increases blood viscosity and influences the rate at which the heart can deliver oxygenated blood to the brain . increased blood viscosity leads to increased heart rate and increased risk of haemorrhage or stroke . there has for many years been interest in having a mathematical model of the physiological systems however it is only in the last 10 - 20 years that western research has recognise the significance of the physiological systems and of integrative theories linking cognition , the autonomic nervous system and visceral organs.[2023 ] this is an area in which russian researchers have excelled . a core group of researchers have researched the relationship between the physiological systems and the nervous structures for almost 50 years . this autonomic instability leads to cellular and molecular change(s ) and , ultimately , to what we know as pathologies . it uses cognitive measurements , in particular of colour perception , as the data set for this model . such methodology has been incorporated into an advanced cognitive technology which have been widely reported . this is the first technique which is able to diagnose all of the major medical conditions in a single cognitive test . it incorporates an understanding that stress / sensory input , an estimated 85% of which is in the form of light , influences cellular and molecular biology . in particular it influences the expression of proteins and the rate at which such proteins subsequently react - which can be measured . the absorption and emission of light from proteins provides the means to do so by measuring colour contrast and colour perception . virtual scanning embraces an unprecedented understanding of the mechanisms which the body employs to regulate its function e.g. to provide more information which has the potential to advances the understanding of multi - systemic etiologies e.g. migraine , diabetes , developmental dyslexia , and sleep apnoea . virtual scanning can track the emergence of typically 5 - 15 pathologies in each of over 30 organs.to illustrate the influence of both genotype and phenotype upon each pathology i.e. of ( i ) protein expression , ( ii ) the rate which expressed proteins subsequently react with their reactive substrates and ( iii ) which incorporates an understanding of the reaction conditions governing such reactions.to diagnose from presymptomatic origins . the emission of biophotons of light , which are characteristic of pathologies , occurs from the first pathological reaction . this influences colour perception from typically 50 - 100 biophotons per second ( range 10 - 10 biophotons per second ) . it contrasts with biomarker techniques which compare the analysed results with that of expected norms.to differentiate between normal and abnormal cell morphologies . altered dna and protein conformation influences their spatial orientation in the cell and their ability to conduct their cellular function e.g. facilitating the passage of glucose through the cell membranes . increased or decreased cell function ( hyperfunction or hypofunction).increased or decreased arterial or venal flow to and from each organ indicative of an inflammatory reaction or ischaemia.abnormality of a limit of cell division i.e. indications of the changes taking place to cell morphology.growth of new cells or death of old cells . by assessing the degree of system stability or instability . an advanced understanding of eegs can be used to regulate systemic stability.[83335]how is it possible to evaluate the existence or function of a system or the stability or instability of a system ? whilst it is recognised that organs function in organ networks there are no other technologies which are able to justify the stability of systems except by using the only available technology , virtual scanning.to predict the onset of further pathologies . if a system becomes destabilised this will lead to pathologies in each organ of that particular system . to provide more information which has the potential to advances the understanding of multi - systemic etiologies e.g. migraine , diabetes , developmental dyslexia , and sleep apnoea . virtual scanning can track the emergence of typically 5 - 15 pathologies in each of over 30 organs . to illustrate the influence of both genotype and phenotype upon each pathology i.e. of ( i ) protein expression , ( ii ) the rate which expressed proteins subsequently react with their reactive substrates and ( iii ) which incorporates an understanding of the reaction conditions governing such reactions . to diagnose from presymptomatic origins . the emission of biophotons of light , which are characteristic of pathologies , occurs from the first pathological reaction . this influences colour perception from typically 50 - 100 biophotons per second ( range 10 - 10 biophotons per second ) . it contrasts with biomarker techniques which compare the analysed results with that of expected norms . to differentiate between normal and abnormal cell morphologies . altered dna and protein conformation influences their spatial orientation in the cell and their ability to conduct their cellular function e.g. facilitating the passage of glucose through the cell membranes . increased or decreased cell function ( hyperfunction or hypofunction).increased or decreased arterial or venal flow to and from each organ indicative of an inflammatory reaction or ischaemia.abnormality of a limit of cell division i.e. indications of the changes taking place to cell morphology.growth of new cells or death of old cells . increased or decreased cell function ( hyperfunction or hypofunction ) . increased or decreased arterial or venal flow to and from each organ indicative of an inflammatory reaction or ischaemia . abnormality of a limit of cell division i.e. indications of the changes taking place to cell morphology . growth of new cells or death of old cells . by assessing the degree of system stability or instability . an advanced understanding of eegs can be used to regulate systemic stability.[83335 ] how is it possible to evaluate the existence or function of a system or the stability or instability of a system ? whilst it is recognised that organs function in organ networks there are no other technologies which are able to justify the stability of systems except by using the only available technology , virtual scanning . to predict the onset of further pathologies . if a system becomes destabilised this will lead to pathologies in each organ of that particular system . the purpose of this series of articles is to establish the scientific basis for virtual scanning and to prove the methodology developed by dr . they highlight that there is a more advanced technological concept , based upon an understanding of how the body responds to light , which can advance the diagnosis and treatment of disease . the problem for many is that they base their decision - making upon that which they have seen in their lifetime . this is completely natural however it illustrates the difficulties which inventors face when they develop and seek to commercialise novel and potentially disruptive technologies e.g. computers , mobile phones , etc . consequently there is resistance to anything which challenges the status quo and which could introduce new and potentially better ways . dr . grakov appears to have produced a first mathematical model of the physiological systems . this has been incorporated into a technology which uses cognitive measurements as its data sets . there is a mechanism to overcome such obstacles i.e. through clinical studies however in order to reach this stage clinicians have to be convinced of the validity of the concept . the purpose of this series of articles has been to illustrate that this technology exists , that there are precedents for a technology of this type , and that the underlying scientific methodology holds the promise of being more advanced , sophisticated and cost - effective than many of the medical techniques which are routinely employed in orthodox biomedicine .

the cost of diagnosing and treating disease continues to rise inexorably . almost every new test adds to the complexity and cost of healthcare . there is a need for better and less expensive screening , diagnostic and scanning techniques . medical scanning technologies are based upon the body 's response to an external stimulus e.g. heat , ultrasound , x - rays , magnetic resonance , etc . biomarker and histopathology tests have inherent limitations because diseases are often polygenic and/or influence the function of multiple physiological systems . the results are compared with expected norms . this makes it difficult to diagnose the onset of disease . such techniques measure only what the clinician wants or expects to see . a technique which can provide more information , regarding the influence of a medical condition upon the body 's whole function , may be invaluable to the clinician . there is not yet a clear understanding of how the body regulates its function . a greater understanding of how the body responds to sensory input , in particular to light , has been incorporated into a mathematical model of the physiological systems developed by i.g . grakov . this has been incorporated into a cognitive technology which improves the understanding of how the body regulates its function and has led to the development of a better method for the diagnosis and treatment of disease(s ) . this technique , virtual scanning , appears able to diagnose at different levels of physiological significance i.e. as systems , organs , cells ( as morphologies ) and molecular ( as pathologies ) . it may be a major scientific development , conceivably more advanced than biomarker techniques , with the potential to provide far more information about a patient 's health . it may have the potential to significantly reduce the complexity and cost of healthcare . this article reviews the available literature .

Introduction The Significance of Phenotype Neuro Regulation A Mathematical Model of the Physiological Systems Virtual Scanning Discussion

technological advances in computed tomography ( ct ) have made chest ct a fast and accurate , and therefore extensively used imaging technique in trauma patient care [ 1 , 2 ] . although the utility of ct for detection of chest injuries is primarily demonstrated in severely injured patients , this widespread use deserves reconsideration because its effectiveness might not always outweigh potential harm by radiation exposure [ 4 , 5 ] , medicalisation , time loss and the high costs . although many studies addressed the value of ct in trauma patients , few evidence - based indications for trauma ct of the chest exist . according to the american college of radiology appropriateness criteria , ct should be performed if conventional radiography ( cr ) shows signs of mediastinal bleeding suspicious for blunt aortic injury . these guidelines additionally state that thoracic spine images are now effectively obtained in all patients who undergo thoracoabdominal ct , making indications for spine imaging less important than indications for obtaining thoracoabdominal ct . the eastern association for the surgery of trauma guidelines summarise that the thoracolumbar spine can be cleared without imaging in awake trauma patients without any evidence of intoxication with ethanol or drugs , who have a normal mental status and normal physical examinations . however , these guidelines also state that aortic injuries can not be accurately ruled out by using signs of mediastinal bleeding on cr . they therefore suggest that blunt aortic injury should be considered in all patients involved in motor vehicle collisions . these recommendations reflect that little evidence exists on which patients are likely to benefit from chest ct after blunt trauma . more importantly , it remains unclear in which patients chest ct can be omitted without missing relevant injuries . the aim of this prospective study on adult blunt trauma patients is therefore to derive a set of independent clinical parameters that distinguish patients who will benefit from chest ct from patients in whom chest ct can be omitted without missing relevant injuries . we performed an observational cohort study on 1,047 consecutive blunt trauma patients who were 16 years and older . patients were prospectively included according to the inclusion and exclusion criteria in table 1 if they were primarily evaluated at the emergency department of our hospital from may 2005 until july 2008 . table 1inclusion and exclusion criteria defined before the study started inclusion criteriadefinitionslife - threatening vital problems due to trauma airway patency problemsas declared by anaesthesiologist breathing problemsbreathing frequency > 30/min circulatory problemspulse > 120/min , systolic blood pressure < 100 mmhg , capillary refill > 4 sexterior blood loss > 500 ml neurological problemsgcs 13clinical evidence of serious injuries clinically evident pelvic ring fractureas declared by attending surgeon clinical signs of unstable vertebral fractures or spinal cord compressionas declared by attending surgeonsevere mechanism of injury high - energy mechanism of injury as declared by pre - hospital emergency medical servicesfall from height > 3 mmotor vehicle accident > 50 km / hejection from vehiclecar rolloversevere impact damage to carstruck pedestrian > 10 km / hstruck bicyclist > 30 km / h high - energy crush injury to torsosqueezed under or between heavy objectsexclusion criteriact not feasible / appropriate dead soon after arrivalwithin 15 min of arrival , as declared by attending surgeon shock class iii / ivpulse rate > 120/min or systolic blood pressure < 100 mmhg and non respondent to volume therapy pregnancysuspected by history or sonographynotes : for inclusion in the study , only one criterion had to be met . gcs , glasgow coma scale inclusion and exclusion criteria defined before the study started notes : for inclusion in the study , only one criterion had to be met . gcs , glasgow coma scale all patients underwent the same diagnostic protocol according to our hospital s guidelines . this protocol consisted of physical examination ( pe ) , cr of the chest , spine and pelvis , abdominal ultrasonography and ct of the cervical spine , chest , abdomen , spine and pelvis . pe was performed and documented by residents in surgery or orthopaedics or emergency physicians who were supervised by senior trauma surgeons according to the advanced trauma life support guidelines . blood samples were collected for laboratory investigations including arterial blood gas analysis , haematological measurements and biochemistry . cr was executed on a vertix 3d system ( siemens medical solutions , forchheim , germany ) and consisted of a supine view of the chest and pelvis in an anteroposterior direction and of the thoracolumbar spine in an anteroposterior and lateral projection . abdominal ultrasonography was primarily used to detect or exclude free intraperitoneal fluid according to the principles of focused ultrasonography for trauma . cr and ultrasonography were interpreted immediately by senior residents in radiology and the supervising trauma radiologists . all patients thereafter underwent ct on a 16-detector unit ( somatom sensation 16 , siemens medical solutions , forchheim , germany ) that was located in the emergency department . ct of the cervical spine was obtained from the occipital condyles to the first thoracic vertebra . chest ct was performed as a part of a thoracoabdominal examination from the acromioclavicular joint to the lesser trochanter , with automated exposure control at a reference effective tube current time product of 200 mas , a tube voltage of 120 kv , a beam collimation of 16 1.5 mm , a median dose - length product of 1,150 mgycm and administration of 100 ml of intravenous contrast agent [ iobitridol 300 mg i / ml ( xenetix 300 , guerbet , paris , france ) ] . reconstructed section thickness was 3 mm in a bone , lung and soft tissue setting and section overlap was 1.5 , 3 and 3 mm , respectively . finally , sagittal and coronal multi - planar reformatted images of the spine were constructed . after review by radiology residents supervised by certified radiologists , the trauma team started or changed patient management as needed . finally , an effort was made to follow every patient for 6 months , either by medical consultation in the outpatient clinics or by telephone . could be waived because this was an observational study of a standard diagnostic protocol , and all patients received the same type of diagnostics and care . two unblinded investigators ( with 1 and 3 years experience in emergency medicine at the start of the study ) attended briefings and resuscitations of included patients and reviewed all charts and radiological reports . they collected data on patient characteristics , diagnoses and treatment by using standardised abstraction forms . these data had all been prospectively recorded by the trauma team members before ct was performed . if necessary the investigators re - reviewed all patients charts after 6 months to establish whether injuries were initially missed . they collected injury severity scores ( iss ) from the regional trauma registry and finally imported all data into a customised database . two outcome measures were determined before this study started : ( 1 ) presence of chest injuries on ct and ( 2 ) clinically relevant occult injuries . chest injuries on chest ct consisted of aortic injury , diaphragmatic injury , tracheobronchial tree injury , oesophageal injury , pneumothorax , haemothorax and pulmonary contusion . they also consisted of fractures of the ribs , scapula , sternum and thoracic vertebrae ( including the vertebral body and the transverse and spinous processes ) . the presence of these injuries was recorded per patient . if pneumothoraces , pulmonary contusions , haemothoraces or rib fractures were present , the investigators recorded their extent ( number ) , location and severity ( minimal , moderate and severe ) . these classifications were based on a consensus reading of 54 cases that were not included in this study . clinically relevant occult injuries were defined as injuries on ct that were not visualised by cr of the chest and thoracic spine and that had an impact on patient management . an impact on patient management was defined as the occurrence of changes in treatment as a direct result of the ct findings . these changes were determined before the study started and included additional diagnostic workup , changes in intensity of care ( care level upgrade ) and immediate interventions that were started by the trauma team . we selected dichotomous candidate predictors of injuries on ct based on a review of the literature and clinical experience . these variables could be determined during initial patient evaluation at the emergency department and were derived from pre - hospital service reports , emergency records , radiological reports of cr and abdominal ultrasonography investigations and blood sample analyses . in the literature , cervical spine fractures have been associated with thoracic spine injuries . however , because cervical spine ct reconstructions were not readily available before chest ct was obtained , we did not consider presence of cervical spine injuries a practical predictor of chest injuries in this setting . in this study , we primarily aimed to distinguish patients with injuries on chest ct from patients without injuries on chest ct by taking the following steps . first , candidate predictors of injuries on ct were combined into dichotomous composite predictors based on clinical similarity and strong biological association ( table 2 ) . if data were missing or incomplete , these were imputed as table 2definitions of composite predictors of chest injuries on ctcomposite predictordefinition : predictors were positive if any of the following conditions were fulfilledreferences55 years age 55 years or olderdangerous mechanism of injurymotor vehicle collision and any of the following:[20 , 21 , 2932]no use of constraints ejection from the vehicle death occupantpe chest breathing frequency < 10/min or > 29/min ( pre - hospital or on presentation at the ed)[2023 , 3340]pulse oximetry sao2 < 95% at presentation at the ed subcutaneous emphysema at palpation tenderness to palpation of the chest wall lacerations or haematoma of the chest wallpe circulatory problems systolic blood pressure < 90 mmhg ( pre - hospital or at presentation at the ed)[2022 , 28 , 41]heart rate > 120 beats per minute ( pre - hospital or at presentation at the ed)pe altered sensorium glasgow coma scale < 14 on initial presentation at the ed[14 , 22 , 24 , 40 , 42]orotracheal intubation before clinical evaluation at the ed clinical suspicion of drugs or alcohol intoxicationpe supraclavicular injury any fracture , laceration or haematoma above the clavicle , including the face[20 , 21]pe thoracic spine tenderness to palpation of the midline of the thoracic spine[14 , 32 , 42]thoracolumbar lacerations or haematoma neurological deficit suggesting spinal cord injurype abdomen tenderness to palpation lacerations or haematoma abdominal distension or guardingpe extremity fracture clinical suspicion of fractures of the upper or lower extremities , if cr of the extremities were obtained[2022 , 43 , 44]cr chestany of the following abnormalities identified on cr of the chest[2 , 20 , 21 , 38 , 41 , 43 , 45 , 46]pulmonary contusion haemothorax scapular fracture clavicular fracturecr thoracic spineany of the following abnormalities on cr of the thoracic spine : any fracture of the vertebral body or spinous or transverse processes spinal malalignmentcr lumbar spineany of the following abnormalities on cr of the lumbar spine:[20 , 47]any fracture of the vertebral body or spinous or transverse processes spinal malalignmentcr pelvis and abdominal ultrasonographyany of the following pelvic fractures on cr:[20 , 21 , 37]pubic bone fracture fracture acetabulum fracture illiac wing femoral head fracture symphysiolysis luxation hipabnormal abdominal ultrasound : presence of free fluidbe < 3arterial blood gas base excess less than 3 mmol / l in initial blood gas sampleshb < 6blood plasma haemoglobin concentration less than 6 mmol / lnote : ed , emergency department ; pe , physical examination ; cr , conventional radiography definitions of composite predictors of chest injuries on ct note : ed , emergency department ; pe , physical examination ; cr , conventional radiography second , univariate logistic regression analysis was used to study the ability of each composite predictor to distinguish patients with injuries from patients without injuries on chest ct . crude odds ratios ( or ) of all positive composite predictors were derived for the dependent variable presence of injuries on ct ( yes , no ) . third , multivariate regression with a forward selection procedure was used to identify independent composite predictors of presence of injuries on chest ct . a priori , we forced the composite predictor altered sensorium ( gcs < 14 , clinical suspicion of drug or alcohol intoxication , orotracheal intubation before clinical evaluation ) into the multivariate regression model , because we considered this variable to have great clinical relevance . all other composite predictors that were statistically significantly related to the risk of injuries on ct in the univariate analysis ( p < 0.05 in the likelihood ratio test ) were used as independent variables in the selection procedure . this yielded a regression model in which only statistically significant independent predictors were finally included . this model was checked for collinearity and interaction between variables by incorporating biologically plausible interaction terms in the model . discriminatory power of the final regression model was assessed with the area under the receiver - operating characteristic ( roc ) curve ( auc ) and the percentage of explained variance with the r - square . to evaluate the reliability of the regression model , an internal validation was performed with a bootstrap analysis and corrected r - square and auc measures were presented . our final aim was to construct a predictive model that defines patients in whom ct can be omitted while missing relevant injuries in as few patients as possible . we therefore chose a predicted probability cutoff point on the roc curve at which the sensitivity for injuries on ct was as high as possible with a specificity > 0 . using this cutoff point effectively meant that patients without any positive independent predictor were classified as low - risk patients , whereas patients with any positive independent predictor were all classified as high - risk patients . we evaluated the predictive model with this cutoff point by presenting the model s sensitivity and specificity for presence of chest injuries and for presence of clinically relevant occult chest injuries on ct . we performed statistical analyses with the statistical packages for microsoft windows spps , version 16.0 ( chicago , il ) , and r , version 2.6.1 ( the r project for statistical computing , www.r-project.org ) . we performed an observational cohort study on 1,047 consecutive blunt trauma patients who were 16 years and older . patients were prospectively included according to the inclusion and exclusion criteria in table 1 if they were primarily evaluated at the emergency department of our hospital from may 2005 until july 2008 . table 1inclusion and exclusion criteria defined before the study started inclusion criteriadefinitionslife - threatening vital problems due to trauma airway patency problemsas declared by anaesthesiologist breathing problemsbreathing frequency > 30/min circulatory problemspulse > 120/min , systolic blood pressure < 100 mmhg , capillary refill > 4 sexterior blood loss > 500 ml neurological problemsgcs 13clinical evidence of serious injuries clinically evident pelvic ring fractureas declared by attending surgeon clinical signs of unstable vertebral fractures or spinal cord compressionas declared by attending surgeonsevere mechanism of injury high - energy mechanism of injury as declared by pre - hospital emergency medical servicesfall from height > 3 mmotor vehicle accident > 50 km / hejection from vehiclecar rolloversevere impact damage to carstruck pedestrian > 10 km / hstruck bicyclist > 30 km / h high - energy crush injury to torsosqueezed under or between heavy objectsexclusion criteriact not feasible / appropriate dead soon after arrivalwithin 15 min of arrival , as declared by attending surgeon shock class iii / ivpulse rate > 120/min or systolic blood pressure < 100 mmhg and non respondent to volume therapy pregnancysuspected by history or sonographynotes : for inclusion in the study , only one criterion had to be met . gcs , glasgow coma scale inclusion and exclusion criteria defined before the study started notes : for inclusion in the study , only one criterion had to be met . gcs , glasgow coma scale all patients underwent the same diagnostic protocol according to our hospital s guidelines . this protocol consisted of physical examination ( pe ) , cr of the chest , spine and pelvis , abdominal ultrasonography and ct of the cervical spine , chest , abdomen , spine and pelvis . pe was performed and documented by residents in surgery or orthopaedics or emergency physicians who were supervised by senior trauma surgeons according to the advanced trauma life support guidelines . blood samples were collected for laboratory investigations including arterial blood gas analysis , haematological measurements and biochemistry . cr was executed on a vertix 3d system ( siemens medical solutions , forchheim , germany ) and consisted of a supine view of the chest and pelvis in an anteroposterior direction and of the thoracolumbar spine in an anteroposterior and lateral projection . abdominal ultrasonography was primarily used to detect or exclude free intraperitoneal fluid according to the principles of focused ultrasonography for trauma . cr and ultrasonography were interpreted immediately by senior residents in radiology and the supervising trauma radiologists . all patients thereafter underwent ct on a 16-detector unit ( somatom sensation 16 , siemens medical solutions , forchheim , germany ) that was located in the emergency department . ct of the cervical spine was obtained from the occipital condyles to the first thoracic vertebra . chest ct was performed as a part of a thoracoabdominal examination from the acromioclavicular joint to the lesser trochanter , with automated exposure control at a reference effective tube current time product of 200 mas , a tube voltage of 120 kv , a beam collimation of 16 1.5 mm , a median dose - length product of 1,150 mgycm and administration of 100 ml of intravenous contrast agent [ iobitridol 300 mg i / ml ( xenetix 300 , guerbet , paris , france ) ] . reconstructed section thickness was 3 mm in a bone , lung and soft tissue setting and section overlap was 1.5 , 3 and 3 mm , respectively . finally , sagittal and coronal multi - planar reformatted images of the spine were constructed . after review by radiology residents supervised by certified radiologists , the trauma team started or changed patient management as needed . finally , an effort was made to follow every patient for 6 months , either by medical consultation in the outpatient clinics or by telephone . could be waived because this was an observational study of a standard diagnostic protocol , and all patients received the same type of diagnostics and care . two unblinded investigators ( with 1 and 3 years experience in emergency medicine at the start of the study ) attended briefings and resuscitations of included patients and reviewed all charts and radiological reports . they collected data on patient characteristics , diagnoses and treatment by using standardised abstraction forms . these data had all been prospectively recorded by the trauma team members before ct was performed . if necessary the investigators re - reviewed all patients charts after 6 months to establish whether injuries were initially missed . they collected injury severity scores ( iss ) from the regional trauma registry and finally imported all data into a customised database . two outcome measures were determined before this study started : ( 1 ) presence of chest injuries on ct and ( 2 ) clinically relevant occult injuries . chest injuries on chest ct consisted of aortic injury , diaphragmatic injury , tracheobronchial tree injury , oesophageal injury , pneumothorax , haemothorax and pulmonary contusion . they also consisted of fractures of the ribs , scapula , sternum and thoracic vertebrae ( including the vertebral body and the transverse and spinous processes ) . the presence of these injuries was recorded per patient . if pneumothoraces , pulmonary contusions , haemothoraces or rib fractures were present , the investigators recorded their extent ( number ) , location and severity ( minimal , moderate and severe ) . these classifications were based on a consensus reading of 54 cases that were not included in this study . clinically relevant occult injuries were defined as injuries on ct that were not visualised by cr of the chest and thoracic spine and that had an impact on patient management . an impact on patient management was defined as the occurrence of changes in treatment as a direct result of the ct findings . these changes were determined before the study started and included additional diagnostic workup , changes in intensity of care ( care level upgrade ) and immediate interventions that were started by the trauma team . we selected dichotomous candidate predictors of injuries on ct based on a review of the literature and clinical experience . these variables could be determined during initial patient evaluation at the emergency department and were derived from pre - hospital service reports , emergency records , radiological reports of cr and abdominal ultrasonography investigations and blood sample analyses . in the literature , cervical spine fractures have been associated with thoracic spine injuries . however , because cervical spine ct reconstructions were not readily available before chest ct was obtained , we did not consider presence of cervical spine injuries a practical predictor of chest injuries in this setting . in this study , we primarily aimed to distinguish patients with injuries on chest ct from patients without injuries on chest ct by taking the following steps . first , candidate predictors of injuries on ct were combined into dichotomous composite predictors based on clinical similarity and strong biological association ( table 2 ) . if data were missing or incomplete , these were imputed as normal . table 2definitions of composite predictors of chest injuries on ctcomposite predictordefinition : predictors were positive if any of the following conditions were fulfilledreferences55 years age 55 years or olderdangerous mechanism of injurymotor vehicle collision and any of the following:[20 , 21 , 2932]no use of constraints ejection from the vehicle death occupantpe chest breathing frequency < 10/min or > 29/min ( pre - hospital or on presentation at the ed)[2023 , 3340]pulse oximetry sao2 < 95% at presentation at the ed decreased breathing sounds at auscultation subcutaneous emphysema at palpation tenderness to palpation of the chest wall lacerations or haematoma of the chest wallpe circulatory problems systolic blood pressure < 90 mmhg ( pre - hospital or at presentation at the ed)[2022 , 28 , 41]heart rate > 120 beats per minute ( pre - hospital or at presentation at the ed)pe altered sensorium glasgow coma scale < 14 on initial presentation at the ed[14 , 22 , 24 , 40 , 42]orotracheal intubation before clinical evaluation at the ed clinical suspicion of drugs or alcohol intoxicationpe supraclavicular injury any fracture , laceration or haematoma above the clavicle , including the face[20 , 21]pe thoracic spine tenderness to palpation of the midline of the thoracic spine[14 , 32 , 42]thoracolumbar lacerations or haematoma neurological deficit suggesting spinal cord injurype abdomen tenderness to palpation lacerations or haematoma abdominal distension or guardingpe extremity fracture clinical suspicion of fractures of the upper or lower extremities , if cr of the extremities were obtained[2022 , 43 , 44]cr chestany of the following abnormalities identified on cr of the chest[2 , 20 , 21 , 38 , 41 , 43 , 45 , 46]pulmonary contusion scapular fracture clavicular fracturecr thoracic spineany of the following abnormalities on cr of the thoracic spine : any fracture of the vertebral body or spinous or transverse processes spinal malalignmentcr lumbar spineany of the following abnormalities on cr of the lumbar spine:[20 , 47]any fracture of the vertebral body or spinous or transverse processes spinal malalignmentcr pelvis and abdominal ultrasonographyany of the following pelvic fractures on cr:[20 , 21 , 37]pubic bone fracture fracture acetabulum femoral head fracture symphysiolysis luxation hipabnormal abdominal ultrasound : presence of free fluidbe < 3arterial blood gas base excess less than 3 mmol / l in initial blood gas sampleshb < 6blood plasma haemoglobin concentration less than 6 mmol / lnote : ed , emergency department ; pe , physical examination ; cr , conventional radiography definitions of composite predictors of chest injuries on ct note : ed , emergency department ; pe , physical examination ; cr , conventional radiography second , univariate logistic regression analysis was used to study the ability of each composite predictor to distinguish patients with injuries from patients without injuries on chest ct . crude odds ratios ( or ) of all positive composite predictors were derived for the dependent variable presence of injuries on ct ( yes , no ) . third , multivariate regression with a forward selection procedure was used to identify independent composite predictors of presence of injuries on chest ct . a priori ( gcs < 14 , clinical suspicion of drug or alcohol intoxication , orotracheal intubation before clinical evaluation ) into the multivariate regression model , because we considered this variable to have great clinical relevance . all other composite predictors that were statistically significantly related to the risk of injuries on ct in the univariate analysis ( p < 0.05 in the likelihood ratio test ) were used as independent variables in the selection procedure . this yielded a regression model in which only statistically significant independent predictors were finally included . this model was checked for collinearity and interaction between variables by incorporating biologically plausible interaction terms in the model . discriminatory power of the final regression model was assessed with the area under the receiver - operating characteristic ( roc ) curve ( auc ) and the percentage of explained variance with the r - square . to evaluate the reliability of the regression model , an internal validation was performed with a bootstrap analysis and corrected r - square and auc measures were presented . our final aim was to construct a predictive model that defines patients in whom ct can be omitted while missing relevant injuries in as few patients as possible . we therefore chose a predicted probability cutoff point on the roc curve at which the sensitivity for injuries on ct was as high as possible with a specificity > 0 . using this cutoff point effectively meant that patients without any positive independent predictor were classified as low - risk patients , whereas patients with any positive independent predictor were all classified as high - risk patients . we evaluated the predictive model with this cutoff point by presenting the model s sensitivity and specificity for presence of chest injuries and for presence of clinically relevant occult chest injuries on ct . we performed statistical analyses with the statistical packages for microsoft windows spps , version 16.0 ( chicago , il ) , and r , version 2.6.1 ( the r project for statistical computing , www.r-project.org ) . eighty - one patients were excluded because of predetermined exclusion criteria and 71 patients because of protocol violation ; ct was not performed in these patients ( fig . 1 ) . 1diagram illustrating patient flow for study selection and the number of patients with chest injuries on ct , occult chest injuries on ct and occult injuries on ct with an impact on patient management . cr , conventional radiography of the chest and thoracic spine ; ct , computed tomography ; occult injuries , injuries that were only detected on ct , but not on cr diagram illustrating patient flow for study selection and the number of patients with chest injuries on ct , occult chest injuries on ct and occult injuries on ct with an impact on patient management . cr , conventional radiography of the chest and thoracic spine ; ct , computed tomography ; occult injuries , injuries that were only detected on ct , but not on cr a total of 1,047 patients were included in this study of whom 731 patients ( 70% ) were male . median iss was 14 , and mean iss was 17 ( range , 075 ) . five - hundred eight patients ( 49% ) had injuries visible on ct . in 459 ( 44% ) patients , ct detected occult injuries ( additional injuries compared with cr of the chest ) . in 183 ( 17% ) patients , these occult injuries had an impact on patient management and were therefore considered to be clinically relevant . these management changes comprised care level upgrading ( n = 60 ) , chest drain ( re)positioning ( n = 45 ) , conservative ( n = 34 ) or surgical stabilisation ( n = 19 ) of spinal fractures , epidural anaesthesia in cases of multiple occult rib fractures ( n = 15 ) , consultation with cardiologists ( n = 14 ) , angiography ( n = 8) , bronchoscopy ( n = 5 ) , interventional radiology ( aortic repair , n = 4 , embolisation , n = 1 ) , thoracotomy ( n = 2 ) and treatment for tracheal ( n = 1 ) or oesophageal rupture ( n = 1 ) . of all included patients , 43 ( 4% ) were lost to follow - up . completed follow - up revealed that in one patient with multiple chest injuries , a diaphragmatic rupture was initially missed on ct . this injury was revealed after cessation of ventilation 2 days post - trauma and was treated with a delayed laparotomy with good patient recovery . conversely , another patient with multiple chest injuries was suspected to have a diaphragmatic injury on ct . however , an emergency laparotomy , which was indicated for a splenectomy , did not confirm this injury . a third patient with multiple serious injuries on chest ct developed a pericardial tamponade 3 weeks post - trauma . although ct was therefore not 100% accurate in the detection of all specific chest injuries , completed clinical follow - up revealed that ct correctly classified patients as having or not having some injury of the chest . data were complete for all predictors except for blood analyses and cr of the thoracic spine . in 23 patients no haemoglobin and in 258 patients no be samples were obtained , mainly because patients had no respiratory or haemodynamic problems . in 46 patients , cr of the thoracic spine was not performed or was only obtained in an anteroposterior direction . table 3 shows the univariate relationships between these predictors and presence of any chest injuries on ct . this table indicates that only dangerous mechanism of injury and pe abdomen failed to demonstrate a statistically significant crude odds ratio . after multivariate logistic regression analysis on the remaining 13 composite predictors , 9 independent predictors significantly contributed to the prediction of presence of chest injuries on ct ( table 4 ) : abnormal cr of the chest , abnormal chest pe , be < 3 mmol / l , abnormal abdominal ultrasonography or pelvic cr , abnormal thoracic spine pe , age 55 years , hb < 6 , abnormal cr of the thoracic spine and altered sensorium . table 3univariate relationships between positive predictors and the presence of any chest injuries on ctpositive composite predictorsor ( 95% ci)p value55 years ( n = 208)2.37 ( 1.733.25)<0.001dangerous mechanism of injury ( n = 235)1.22 ( 0.911.63)0.209pe chest ( n = 361)4.64 ( 3.56.3)<0.001pe circulatory problems ( n = 184)2.58 ( 1.843.61)<0.001pe altered sensorium ( n = 395)2.54 ( 1.973.29)<0.001pe supraclavicular injury ( n = 615)1.79 ( 1.402.30)<0.001pe thoracic spine ( n = 134)1.51 ( 1.052.18)0.027pe abdomen ( n = 175)1.18 ( 0.851.64)0.313pe extremity fracture ( n = 514)1.40 ( 1.091.78)0.008cr chest ( n = 366)15.6 ( 11.1221.93)<0.001cr thoracic spine ( n = 129)2.55 ( 1.723.77)<0.001cr lumbar spine ( n = 86)2.64 ( 1.644.26)<0.001cr pelvis and abdominal ultrasonography ( n = 209)2.89 ( 2.093.99)<0.001be < 3 positive ( n = 351)3.81 ( 2.895.01)<0.001hb < 6 ( n = 51)7.21 ( 3.2216.16)<0.001notes : or , crude odds ratio ; 95% ci , 95% confidence intervaldefinitions of positive composite predictors are displayed in table 2table 4independent predictors of the presence of any chest injuries on ctpositive composite predictorsadjusted or ( 95% ci)55 years1.6 ( 1.12.4)pe chest3.0 ( 2.24.2)pe of the thoracic spine1.8 ( 1.12.8)pe altered sensorium1.5 ( 1.02.1)cr chest9.4 ( 6.514)cr thoracic spine1.7 ( 1.12.9)cr pelvis and abdominal ultrasonography2.3 ( 1.53.4)be < 32.0 ( 1.42.9)hb < 62.9 ( 1.17.6)note : or , odds ratio adjusted to all other predictors in the model ; ci , confidence interval . definitions of positive composite predictors are displayed in table 2 univariate relationships between positive predictors and the presence of any chest injuries on ct notes : or , crude odds ratio ; 95% ci , 95% confidence interval definitions of positive composite predictors are displayed in table 2 independent predictors of the presence of any chest injuries on ct note : or , odds ratio adjusted to all other predictors in the model ; ci , confidence interval . definitions of positive composite predictors are displayed in table 2 figure 2 shows the roc curve of the predictive model containing these nine predictors with an r - square of 0.478 and an auc of 0.85 ( 95% ci : 0.830.87 ) . after bootstrap analysis , the corrected r - square was 0.455 and the corrected auc was 0.71 . 2 includes the cutoff point at which patients were stratified into low - risk and high - risk patients . of all included patients , 855 ( 82% ) patients had one or more positive predictors and were classified as high - risk patients . one hundred ninety - two patients ( 18% ) had no positive predictor and were classified as low - risk patients . 2receiver - operating characteristic ( roc ) curve of the predictive model containing nine predictors of injuries on chest ct . the cutoff point ( dashed lines ) is located at a sensitivity of 0.95 and at a specificity of 0.31 . ( area under the curve = 0.85 ; 95% confidence interval , 0.830.87 ) receiver - operating characteristic ( roc ) curve of the predictive model containing nine predictors of injuries on chest ct . the cutoff point ( dashed lines ) is located at a sensitivity of 0.95 and at a specificity of 0.31 . ( area under the curve = 0.85 ; 95% confidence interval , 0.830.87 ) of all 508 patients with injuries on ct , our model correctly classified 484 patients as high - risk patients ( sensitivity : 0.95 ; 95% ci , 0.930.97 ) and the remaining 24 patients with injuries on ct as low - risk patients . this means that the probability of having ct injuries in the low - risk patient group was 24/192 = 13% ( 95% ci : 918% ) . these patients mainly had minimal pulmonary contusion , minimal pneumothoraces , one or two rib fractures and scapular fractures ( table 5 ) . the model correctly classified patients without injuries on ct ( n = 539 ) as low - risk in 168 patients ( specificity : 0.31 ; 95% ci , 0.270.35 ) . table 5prevalence of distinct chest injuries on ct in all patients , in patients who had 1 positive predictor in the predictive model ( high - risk patients ) and in patients who had no positive predictor of chest injury ( low - risk patients)injuries on ctno . ( % ) of low - risk patients ( n = 192)pneumothorax234(22.3)228(26.6)6(3.1)moderate pneumothorax90(8.6)89(10.4)1(0.5)severe pneumothorax35(3.3)35(4.1)0(0.0)haemothorax58(5.5)58(6.8)0(0.0)pulmonary contusion288(28)173(20.2)15(7.8)moderate or severe contusion71(6.8)71(8.3)0(0.0)oesophageal injury1(0.1)1(0.1)0(0.0)tracheobronchial injury2(0.2)2(0.2)0(0.0)aortic injury9(0.8)9(1.1)0(0.0)injury to the subclavian vein1(0.1)1(0.1)0(0.0)rib fracture317(30.3)311(36.4)6(3.1) > 2 rib fractures233(22.2)232(27.1)1(0.5)scapular fracture76(7.3)73(8.5)3(1.5)sternal fracture51(4.9)51(6.0)0(0.0)diaphragmatic injury5(0.5)5(0.6)0(0.0)any thoracic spinal fracture123(12)122(14.2)1(0.5)vertebral body fracture81(7.7)80(9.4)1(0.5)transverse process fracture59(5.6)59(6.9)0(0.0)spinous process fracture20(1.9)20(2.3)0(0.0)total ( any chest injury)508(48.5)484(56.6)24(12.5)notes : numbers in parentheses are percentages of patient groups . a patient could have multiple chest injuries prevalence of distinct chest injuries on ct in all patients , in patients who had 1 positive predictor in the predictive model ( high - risk patients ) and in patients who had no positive predictor of chest injury ( low - risk patients ) notes : numbers in parentheses are percentages of patient groups . a patient could have multiple chest injuries of all 183 patients with clinically relevant occult injuries on ct , 179 were correctly classified as high risk ( sensitivity ; 0.98 , 95% ci 0.961 ) . four out of 192 low - risk patients ( 2% ; 95% ci 15% ) had clinically relevant occult injuries : one patient had a stable fracture of the xii vertebral body that was only visualised on cr of the lumbar spine , but not on cr of the thoracic spine . a second patient had pulmonary contusion , one rib fracture and a pneumothorax of moderate size that were not visualised on chest cr . although in this patient none of the nine predictors was positive , cervical spine ct demonstrated subcutaneous emphysema . a third patient who was classified as belonging to the low - risk patient group had three rib fractures . none of the low - risk - group patients suffered from aortic injury , diaphragmatic injury , haemothoraces or large pneumothoraces ( table 5 ) . data were complete for all predictors except for blood analyses and cr of the thoracic spine . in 23 patients no haemoglobin and in 258 patients no be samples were obtained , mainly because patients had no respiratory or haemodynamic problems . in 46 patients , cr of the thoracic spine was not performed or was only obtained in an anteroposterior direction . table 3 shows the univariate relationships between these predictors and presence of any chest injuries on ct . this table indicates that only dangerous mechanism of injury and pe abdomen failed to demonstrate a statistically significant crude odds ratio . after multivariate logistic regression analysis on the remaining 13 composite predictors , 9 independent predictors significantly contributed to the prediction of presence of chest injuries on ct ( table 4 ) : abnormal cr of the chest , abnormal chest pe , be < 3 mmol / l , abnormal abdominal ultrasonography or pelvic cr , abnormal thoracic spine pe , age 55 years , hb < 6 , abnormal cr of the thoracic spine and altered sensorium . table 3univariate relationships between positive predictors and the presence of any chest injuries on ctpositive composite predictorsor ( 95% ci)p value55 years ( n = 208)2.37 ( 1.733.25)<0.001dangerous mechanism of injury ( n = 235)1.22 ( 0.911.63)0.209pe chest ( n = 361)4.64 ( 3.56.3)<0.001pe circulatory problems ( n = 184)2.58 ( 1.843.61)<0.001pe altered sensorium ( n = 395)2.54 ( 1.973.29)<0.001pe supraclavicular injury ( n = 615)1.79 ( 1.402.30)<0.001pe thoracic spine ( n = 134)1.51 ( 1.052.18)0.027pe abdomen ( n = 175)1.18 ( 0.851.64)0.313pe extremity fracture ( n = 514)1.40 ( 1.091.78)0.008cr chest ( n = 366)15.6 ( 11.1221.93)<0.001cr thoracic spine ( n = 129)2.55 ( 1.723.77)<0.001cr lumbar spine ( n = 86)2.64 ( 1.644.26)<0.001cr pelvis and abdominal ultrasonography ( n = 209)2.89 ( 2.093.99)<0.001be < 3 positive ( n = 351)3.81 ( 2.895.01)<0.001hb < 6 ( n = 51)7.21 ( 3.2216.16)<0.001notes : or , crude odds ratio ; 95% ci , 95% confidence intervaldefinitions of positive composite predictors are displayed in table 2table 4independent predictors of the presence of any chest injuries on ctpositive composite predictorsadjusted or ( 95% ci)55 years1.6 ( 1.12.4)pe chest3.0 ( 2.24.2)pe of the thoracic spine1.8 ( 1.12.8)pe altered sensorium1.5 ( 1.02.1)cr chest9.4 ( 6.514)cr thoracic spine1.7 ( 1.12.9)cr pelvis and abdominal ultrasonography2.3 ( 1.53.4)be < 32.0 ( 1.42.9)hb < 62.9 ( 1.17.6)note : or , odds ratio adjusted to all other predictors in the model ; ci , confidence interval . definitions of positive composite predictors are displayed in table 2 univariate relationships between positive predictors and the presence of any chest injuries on ct notes : or , crude odds ratio ; 95% ci , 95% confidence interval definitions of positive composite predictors are displayed in table 2 independent predictors of the presence of any chest injuries on ct note : or , odds ratio adjusted to all other predictors in the model ; ci , confidence interval . definitions of positive composite predictors are displayed in table 2 figure 2 shows the roc curve of the predictive model containing these nine predictors with an r - square of 0.478 and an auc of 0.85 ( 95% ci : 0.830.87 ) . after bootstrap analysis , 2 includes the cutoff point at which patients were stratified into low - risk and high - risk patients . of all included patients , 855 ( 82% ) patients one hundred ninety - two patients ( 18% ) had no positive predictor and were classified as low - risk patients . 2receiver - operating characteristic ( roc ) curve of the predictive model containing nine predictors of injuries on chest ct . the cutoff point ( dashed lines ) is located at a sensitivity of 0.95 and at a specificity of 0.31 . ( area under the curve = 0.85 ; 95% confidence interval , 0.830.87 ) receiver - operating characteristic ( roc ) curve of the predictive model containing nine predictors of injuries on chest ct . the cutoff point ( dashed lines ) is located at a sensitivity of 0.95 and at a specificity of 0.31 . ( area under the curve = 0.85 ; 95% confidence interval , 0.830.87 ) of all 508 patients with injuries on ct , our model correctly classified 484 patients as high - risk patients ( sensitivity : 0.95 ; 95% ci , 0.930.97 ) and the remaining 24 patients with injuries on ct as low - risk patients . this means that the probability of having ct injuries in the low - risk patient group was 24/192 = 13% ( 95% ci : 918% ) . these patients mainly had minimal pulmonary contusion , minimal pneumothoraces , one or two rib fractures and scapular fractures ( table 5 ) . the model correctly classified patients without injuries on ct ( n = 539 ) as low - risk in 168 patients ( specificity : 0.31 ; 95% ci , 0.270.35 ) . table 5prevalence of distinct chest injuries on ct in all patients , in patients who had 1 positive predictor in the predictive model ( high - risk patients ) and in patients who had no positive predictor of chest injury ( low - risk patients)injuries on ctno . ( % ) of low - risk patients ( n = 192)pneumothorax234(22.3)228(26.6)6(3.1)moderate pneumothorax90(8.6)89(10.4)1(0.5)severe pneumothorax35(3.3)35(4.1)0(0.0)haemothorax58(5.5)58(6.8)0(0.0)pulmonary contusion288(28)173(20.2)15(7.8)moderate or severe contusion71(6.8)71(8.3)0(0.0)oesophageal injury1(0.1)1(0.1)0(0.0)tracheobronchial injury2(0.2)2(0.2)0(0.0)aortic injury9(0.8)9(1.1)0(0.0)injury to the subclavian vein1(0.1)1(0.1)0(0.0)rib fracture317(30.3)311(36.4)6(3.1) > 2 rib fractures233(22.2)232(27.1)1(0.5)scapular fracture76(7.3)73(8.5)3(1.5)sternal fracture51(4.9)51(6.0)0(0.0)diaphragmatic injury5(0.5)5(0.6)0(0.0)any thoracic spinal fracture123(12)122(14.2)1(0.5)vertebral body fracture81(7.7)80(9.4)1(0.5)transverse process fracture59(5.6)59(6.9)0(0.0)spinous process fracture20(1.9)20(2.3)0(0.0)total ( any chest injury)508(48.5)484(56.6)24(12.5)notes : numbers in parentheses are percentages of patient groups . a patient could have multiple chest injuries prevalence of distinct chest injuries on ct in all patients , in patients who had 1 positive predictor in the predictive model ( high - risk patients ) and in patients who had no positive predictor of chest injury ( low - risk patients ) notes : numbers in parentheses are percentages of patient groups . a patient could have multiple chest injuries of all 183 patients with clinically relevant occult injuries on ct , 179 were correctly classified as high risk ( sensitivity ; 0.98 , 95% ci 0.961 ) . four out of 192 low - risk patients ( 2% ; 95% ci 15% ) had clinically relevant occult injuries : one patient had a stable fracture of the xii vertebral body that was only visualised on cr of the lumbar spine , but not on cr of the thoracic spine . a second patient had pulmonary contusion , one rib fracture and a pneumothorax of moderate size that were not visualised on chest cr . although in this patient none of the nine predictors was positive , cervical spine ct demonstrated subcutaneous emphysema . a third patient who was classified as belonging to the low - risk patient group had three rib fractures . none of the low - risk - group patients suffered from aortic injury , diaphragmatic injury , haemothoraces or large pneumothoraces ( table 5 ) . in this prospective study , we derived a set of variables that predict whether ct of the chest including the thoracic spine is likely to reveal relevant injuries in high - energy blunt trauma patients . these clinically intuitive predictors were derived from data that are available at initial presentation in the emergency department , including age , physical examination , laboratory analyses , cr and abdominal ultrasonography . if cts were obtained in patients with one or more positive predictors ( high - risk patients ) only , ct investigations would have been avoided in 18% of patients in this study s population , thereby decreasing ionising radiation exposure and health - care expenditure . however , our study data also suggested that if our positive predictors were implemented as scanning indications , 5% ( 24/508 ) of all patients with chest injuries on ct would not be identified . this implies that the chance of missing injuries of the chest remains 13% ( 24/192 ) in the low - risk patient group if these patients do not undergo chest ct . this risk is substantially lower compared with chest injury risk in the entire blunt trauma population , which was 49% in our study ; it is even relatively low compared with previously described low - risk populations . reported a prevalence of 39% ( 95% ci , 2751% ) of chest injuries in patients with normal cr and normal physical examination , and salim et al . reported a prevalence of 20% ( 95% ci , 1623% ) of pulmonary , mediastinal and rib injuries in patients who were clinically evaluable and had both a normal physical examination and cr one may pose the question of whether an injury probability of 13% is acceptable for a low - risk patient group . we argue that this risk can be considered acceptable , mainly because these chest injuries had no clinically relevance in most cases . the small pulmonary contusions , pneumothoraces and rib fractures rarely had an impact on patient management ( in only 2% of all low - risk patients ) and were , perhaps with the exception of the missed thoracic spine fracture , unlikely to affect patient morbidity if left unmanaged . although cost - effectiveness studies have established acceptable risks for cost - effective injury detection by using ct [ 18 , 19 ] , these , unfortunately , do not pertain to injuries of the entire chest including the thoracic spine . predicting variables that were evaluated in this study were , in part , based on previous studies on appropriate patient selection for chest ct . however , these studies only investigated distinct chest or thoracic injuries and used a case - control design [ 20 , 21 ] , or did not use ct as a standard of reference [ 14 , 2224 ] . to our knowledge , this was one of the first prospective studies to identify selection criteria to facilitate a more appropriate use of ct of the entire chest in adult blunt trauma patients . we investigated and described strong criteria that predict presence of any type of relevant chest injuries on ct . our results might not be surprising as they indicate that chest ct is warranted with abnormal pe or cr . however , this study adds to previous knowledge by defining not only in which patients chest ct is warranted , but also by defining in which patients chest ct could be safely omitted . with further validation , incorporation of these criteria into a diagnostic algorithm for patient selection for chest ct could be an important step towards optimising resource use in trauma imaging . we are aware that several centres do not use cr of the spine because it is not as sensitive as ct in injury detection or do not have laboratory analyses available in the emergency department . as long as no techniques other than cr of the spine and laboratory analyses are used to provide indications for ct imaging , these investigations seem indispensible for selective chest ct algorithms in patients who do not have other positive predictors . our study has a number of limitations . according to the oxford levels of evidence grading , good diagnostic research incorporates index tests and reference tests that are applied blindly and objectively . however , the standard of reference ( ct ) was not interpreted independently from other clinical information because in our practice , radiologists and surgeons work closely together in trauma patient care . however , we do not consider this a major source of incorporation bias because chest ct rarely misses injuries that are visualised on cr . a second limitation is that we abstracted information on potential predictors or index tests from medical records . although we used objective predictor definitions and instructed trauma team members to record all data on potential predictors prospectively ( blinded to ct outcomes ) , this introduced a retrospective component in this study . however , we minimised hindsight bias on presence of predictors by dictating which data on potential predictors had to be present in all medical records and by personally monitoring patient evaluations . researchers , therefore , only rarely had to ask trauma team members for additional information on potential predictors in retrospect . third , the findings of our study have not been externally validated in different trauma populations and settings . external validation is needed for three reasons : the performance of our model is likely to be over - optimistic : the model was created in the same sample of patients in which the performance was determined and should therefore be evaluated in a new sample of trauma patients in our centre.external validation should furthermore be carried out to evaluate our model in other centres : the definition of some clinically relevant injuries ( such as occult injuries for which the care level was upgraded or additional diagnostic work - up was needed ) is institution- and clinician-dependent.the costs , effectiveness and impact on patient quality of life of ct in chest injury diagnosis should be further investigated . the performance of our model is likely to be over - optimistic : the model was created in the same sample of patients in which the performance was determined and should therefore be evaluated in a new sample of trauma patients in our centre . external validation should furthermore be carried out to evaluate our model in other centres : the definition of some clinically relevant injuries ( such as occult injuries for which the care level was upgraded or additional diagnostic work - up was needed ) is institution- and clinician - dependent . the costs , effectiveness and impact on patient quality of life of ct in chest injury diagnosis should be further investigated . in conclusion , significant independent predictors of injuries on chest ct in high - energy blunt trauma patients are age 55 years , abnormal chest pe , altered sensorium , abnormal thoracic spine pe , abnormal chest and thoracic spine cr , abnormal abdominal us or pelvic cr , hb < 6 and be < 3 mmol / l . presence of any of these criteria can predict presence of chest injuries on ct with a sensitivity of 95% . if ct is omitted in patients without any of these criteria , the number of ct investigations can be substantially reduced , while the risk of missing relevant injuries with clinical importance seems to remain low . although our findings need external validation , a diagnostic algorithm employing these criteria has the potential to reduce unnecessary ct examinations of the chest in blunt trauma patients in the future .

purposethe purpose of this study was to derive parameters that predict which high - energy blunt trauma patients should undergo computed tomography ( ct ) for detection of chest injury.methodsthis observational study prospectively included consecutive patients ( 16 years old ) who underwent multidetector ct of the chest after a high - energy mechanism of blunt trauma in one trauma centre.resultswe included 1,047 patients ( median age , 37 ; 70% male ) , of whom 508 had chest injuries identified by ct . using logistic regression , we identified nine predictors of chest injury presence on ct ( age 55 years , abnormal chest physical examination , altered sensorium , abnormal thoracic spine physical examination , abnormal chest conventional radiography ( cr ) , abnormal thoracic spine cr , abnormal pelvic cr or abdominal ultrasound , base excess < 3 mmol / l and haemoglobin < 6 mmol / l ) . of 855 patients with 1 positive predictors , 484 had injury on ct ( 95% of all 508 patients with injury ) . of all 192 patients with no positive predictor , 24 ( 13% ) had chest injury , of whom 4 ( 2% ) had injuries that were considered clinically relevant.conclusionomission of ct in patients without any positive predictor could reduce imaging frequency by 18% , while most clinically relevant chest injuries remain adequately detected .

Introduction Patients and methods Patients Data collection Outcome measures Predictor variables Statistical analysis Results Predictive model Discussion Conclusions

many different chemotherapy regimens were therefore developed.13 however , little is known on the feasibility and efficiency of chemotherapy for these cancer types in patients with severe renal failure.46 only case reports on the pharmacology of irinotecan in patients with colon or rectal cancer are available at present.711 we present the first case of combination chemotherapy in metastatic gastroesophageal cancer in a dialysis patient . a 73-year - old patient with a longstanding history of ischemic heart disease had been on dialysis for two years for vascular renal insufficiency . in september 2004 he was admitted for gastrointestinal blood loss . ca 19.9 was very high : 24925 u / ml ( nl < 37 u / ml ) . the patient was started on chemotherapy , the regimen consisting of l - leukovorin 250 mg / m , irinotecan 50 mg / m followed by 5-fluorouracil ( 5 fu ) 2 g / m/24 h , six weeks out of eight.1213 there was neither significant nausea nor diarrhea . dialysis was continued three times a week , ( the patient was on a monday wednesday friday schedule of dialysis ) and chemotherapy was given on the monday , just after dialysis . after four weeks of chemotherapy he also underwent a right carotid endarterectomy for an intercurrent transient ischemic attack in the right carotid region . six months after starting chemotherapy the ct scan of the liver showed a complete response of the numerous metastases . ( table 1 ) nine months after initiation of treatment , however , the liver metastasis and tumor marker were progressive again . doses of chemotherapy were based on a number of case reports on paclitaxel for ovarian cancer in dialysis patients.14,15 ct scan after two months showed further progressive disease . the increase in solid tumors in a patient undergoing dialysis poses specific problems,16 especially in the choice and pharmacology of anticancer drugs , bearing in mind that all of these drugs were developed in patients with normal liver and kidney function . for gastric cancer , 5fu has always been the backbone of treatment.1,2 in chronic hemodialysis , there are some data on dose reductions with 5fu weekly.17,18 for gastric cancer , combination chemotherapy is , however , necessary to obtain prolonged disease control and even for prolonging overall survival.1,2 combinations of 5fu + cisplatin and either docetaxel or epirubicin have therefore become standard chemotherapy regimens in gastric cancer.19,20 besides the aforementioned regimens , irinotecan - based combinations were shown to be active in first21 and second line gastric cancer.22 its equivalence ( in combination with 5fu ) in first - line metastatic gastric cancer was recently established in two studies , both comparing this regimen with a combination chemotherapy with cisplatin and 5fu.23,24 irinotecan is metabolized in the liver to its active metabolite sn-38 , followed by biliary excretion.6 there is no significant renal elimination . the drug was evaluated in patients with serum creatinin between 1.6 and 5 mg / dl and no unexpected toxicities were seen.25 there are a number of case reports on the use of irinotecan during hemodialysis , all of which are on patients with metastatic colon cancer . a first report mentions the use of irinotecan at a dose of 50 mg / m without significant toxicity.7 in two other case reports on dialysis patients , both patients were started with irinotecan at 50 mg / m . both reports mention that by increasing the dose , prohibitive diarrhea was the consequence.8,11 the worst outcome in higher irinotecan doses ( above 125 mg / m ) was demonstrated in two other dialysis patients , where these dosages led to extreme gi toxicities and even death.9 it can be concluded that irinotecan in terminal renal insufficiency should not be given at a dose above 50 mg / m . korean authors have made pharmacologic evaluations on the use of irinotecan in small - cell lung cancer patients during dialysis . they noted however that these doses were only feasible in patients of korean descent.26 there is a very recent case report on the combination of irinotecan at a dose of 50 mg / m weekly combined with fu1600 mg / m/24 h / week , leading to disease stabilization at six months in a dialysis patients with diffuse bone , cerebral and liver metastases of colon cancer.10 our case report builds on this knowledge of the use of irinotecan in metastatic colorectal cancer during dialysis . this case report discusses both the weekly dose of irinotecan and the 24-hour administration of 5fu in a gastroesophageal cancer patient . this is the first report on the efficacy of irinotecan- and fluorouracil - based chemotherapy in a dialysis patient with liver metastases of a gastroesophageal carcinoma . combination chemotherapy of irinotecan and fu was extremely well tolerated , without significant delays in administration . it produced radiographically complete remission of the liver metastases , and a normalization of ca 19 - 9 tumor marker , leading to a remarkable overall survival .

we present the first case report of a complete response of metastatic gastroesophageal cancer in a chronic hemodialysis patient with irinotecan - based chemotherapy . an elderly dialysis patient presented with diffuse liver metastases by a gastroesophageal adenocarcinoma . he received combination chemotherapy with 5 fluorouracil and irinotecan . after six months of chemotherapy , liver scans show complete remission . the principles , practice , and experience of chemotherapy with irinotecan during dialysis are discussed .

Introduction and background Case report Discussion Conclusion

the fully differentiated cells of the rat mammary parenchyma , the ductal epithelial , alveolar , and myoepithelial cells , are distinguished by their ultrastructure and by their accumulation of immunocytochemically detectable marker proteins . the different cell types probably develop from primative ductal structures called terminal end buds , which are present in the developing rat mammary glands , and these structures contain relatively undifferentiated cells . clonal epithelial stem cell lines , obtained from normal rat mammary glands or benign mammary tumors , differentiate under appropriate conditions along a pathway to droplet - cell / doming cultures of primative alveolarlike cells . under different culture conditions , the epithelial stem cells differentiate along a separate pathway to myoepitheliallike cells . they accumulate some of the specific marker proteins of myoepithelial cells in vivo , including type iv collagen , laminin , and thy-1 antigen . in addition , these myoepitheliallike cells in culture contain an abundance of a potential calcium - binding protein , p9ka , which also occurs in myoepithelial cells of histological sections from mammary glands . the accumulation of type iv collagen , laminin , thy-1 , and p9ka occurs asynchronously along the pathway to the myoepitheliallike cells in vitro . furthermore , the steady - state levels of these different marker proteins arise by alterations in the controls at the transcriptional , the posttranscriptional processing , and the translational stages of their production . these results suggest a stepwise control of synthesis of myoepithelial cell marker proteins , and in the case of p9ka and thy-1 antigen , this altered control may arise through their possession of novel transcriptional promoters.imagesfigure 1 .

Images

the number of elderly people aged 65 years and older has been increasing worldwide , and the aging of the population is particularly rapid in japan . in 2007 , the japanese orthopaedic association proposed the concept of locomotive syndrome ( ls ) , which refers to the conditions under which people require or may soon require nursing - care services because of problems with the locomotor system.1 the japanese ministry of health , labour and welfare has devoted effort to increasing the percentage of individuals who know about ls in the second term of national health promotion movement in the 21st century ( health japan 21 [ second term ] ) to solve the problem of ls.2 screening tools for risk of ls in the elderly , the 25-question geriatric locomotive function scale ( glfs-25 ) and the 5-question glfs ( glfs-5 ) , which is a quick 5-item version of the glfs-25 , were developed for early detection of ls,3 and have been used to raise awareness about preventing the onset of ls . osteoarthritis ( oa ) is the most common joint disease , which is one of the causes of ls and causes pain in elderly patients . according to the research on osteoarthritis against disability , more than 25 million japanese people aged 40 years and older are estimated to have radiographic knee oa.4 similarly , the longitudinal cohort of motor system organ study reported that the prevalence of knee pain was 32.7% in the population and that it increased with age.5 it has been suggested that knee oa impairs locomotor functions because of the pain , and the biomechanical mechanism of impaired locomotor functions such as walking speed and stride length in patients with knee oa has been investigated.6,7 glucosamine - containing supplements have been used widely for improving knee pain in oa.8,9 in our previous studies , we demonstrated that glucosamine - containing supplements gcq or gcqid ( glucosamine hydrochloride , chondroitin sulfate , type ii collagen peptides , quercetin glycosides , imidazole peptides , proteoglycan and vitamin d ) were effective for relieving knee pain and/or locomotor functions in randomized , placebo - controlled trials.10,11 the effects of thermal and exercise therapies on locomotor functions and their mechanism in patients with knee oa have been elucidated with biomechanical methods,12 but the biomechanical mechanism of the effects of supplements such as gcqid on locomotor functions has not been reported . in the present study , we used a motion capture system to investigate the biomechanical mechanism for the improving effects of gcqid on locomotor functions in subjects with knee pain . an open label study was conducted to elucidate the mechanism of efficacy of gcqid supplementation for locomotor functions in japanese women and men aged 40 to 74 years . inclusion criteria were the presence of knee pain , confirmed using the walking subscale of the japanese orthopaedic association criteria ( 25 points or less for either the left or right knee joint),13 and a visual analog scale ( vas ) score for knee pain ( 20 mm or above using the first [ i ] item of the japanese knee osteoarthritis measure [ jkom]);14 kellgren lawrence grades 0ii;15 and the presence of ls confirmed with a score of 6 or higher on the glfs-5.3 all participants received an explanation about the study from the medical investigator , and written informed consent was obtained prior to enrollment in the study . exclusion criteria were the same as in our previous study,11 and typical reasons for exclusion were the following : fast walking speed ( 1.6 m / s ) ; presence of hyperuricemia , diabetes , cardiovascular disease , hepatic disease , renal disease or heart disease ; presence of rheumatoid arthritis that may cause joint pain ; surgical treatment of knee joint(s ) needed or undergone ; daily use of a cane ; daily or occasional vigorous exercise ; need for pharmacological articular treatments during the study period ; a history of osseous or articular diseases other than oa within the past 3 months ; routine use of health food or medicine that may affect the outcome of the study ; and presence of any medical condition as judged by the medical investigator . thirty participants were enrolled in the study , which was performed from february to august 2014 at a clinical services center and at tokai university in japan . all subjects were recruited around the kanto region in japan , through a volunteer bank managed by ttc co. , ltd ( tokyo , japan ) . once radiographs were obtained , kellgren lawrence grades of subjects were determined by an orthopedic surgeon . the study protocol was approved by the ethics committee of tokai university ( kanagawa , japan ) and the ethics committee of tana orthopedic surgery ( yokohama , japan ) , and was conducted in accordance with the principles of the amended declaration of helsinki and the ethical guidelines for epidemiological research ( issued by the japanese government in 2008 ) . the gcqid contained 1,200 mg of glucosamine hydrochloride , 300 mg of shark cartilage extract ( 60 mg as chondroitin sulfate , 45 mg as type ii collagen peptides ) , 90 mg of quercetin glycosides , 100 mg of fish meat extract ( 10 mg as imidazole peptides [ anserine and carnosine ] ) , 5 mg of salmon nasal cartilage extract ( 1 mg as proteoglycan ) , and 5 g ( 200 iu ) of vitamin d in six tablets . the gcqid tablets were manufactured by suntory wellness ltd ( tokyo , japan ) specifically for the purpose of the present study , and all subjects were instructed to take six tablets once a day and to record in their study diary whether they took the tablets . the indicators of efficacy of gcqid supplementation for functions of locomotor system including the knee joint were : jkom category i score ( vas score for jkom knee pain ) , total score for jkom categories ii v ( jkom total score ) , glfs-5 score , vas score for knee pain in various daily situations , and questionnaires designed for this study . jkom is a self - administered , disease - specific measure for knee oa and has demonstrated adequate validity and reliability for studies of japanese people with knee oa in terms of outcomes correlated with health - related quality of life ( qol).14 the vas score for jkom knee pain was measured on a scale from 0 to 100 , where 0 indicated no pain and 100 indicated the worst pain ever experienced . the jkom total score comprised 25 items that cover four different categories : ii : pain and stiffness in knees ; iii : conditions in daily life ; iv : general activities ; and v : health conditions . scores from the 25 items were summed , with results ranging from 0 points ( no complaint ) to 100 points ( most severe condition possible ) . the glfs-5 is also a self - administered measure , consisting of five items located in the key domain of construct structure of the glfs-25 , which covers a wide range of issues , from pain to qol.3 items are graded with 5-point scales from 0 points to 4 points , and the five item scores were summed to produce an overall score with a range of 0 ( no impairment ) to 20 points ( severe impairment ) . the vas score for knee pain in various daily situations included the pain in each knee on rest , walking or ascending / descending stairs , with scales from 0 to 100 , where 0 indicated no pain and 100 indicated the worst pain ever experienced . questionnaires addressed frequency of trips that involved walking or ascending / descending stairs , with 5-point scales from 0 ( no occurrence ) to 4 ( frequent occurrence ) . for the biomechanical mechanism of efficacy for locomotor functions , motion capture analysis in a normal walking state was conducted to obtain data on gait parameters . subjects took off their shoes and changed into dedicated full - body suits for motion capture . markers were attached to 30 locations on the body suits ( top of head , tragi , superior margin of the sternum , extremitas inferior ribs , anterior superior iliac spines , acromions , elbows , wrists , backs of the hands , great trochanters , interior and exterior sides of knees , internal and external malleoli , heels and toes ) . thereafter , subjects were instructed to walk 10 m two times at their usual speed . the time it took to walk the middle 6 m was measured and normal walking speed was calculated as the primary outcome . the position trajectories of markers attached on the suits of subjects during walking were recorded by a motion capture system ( raptor - e digital real time system ; motion analysis corporation , santa rosa , ca , usa ) at the same time . the values of step length , stride length , cadence , time in the stance phase and the swing phase , and angle of soles and toes for both feet were calculated by analyzing the data of position trajectories , and the average value of the data on both feet was evaluated . explanation of terms for some parameters in motion capture analysis is shown in figure 1 . data on functions of locomotor system including the knee joint were collected at baseline and every 4 weeks to week 16 . data on motion capture analysis were collected at baseline and at 8 and 16 weeks . exclusion criteria for efficacy assessment were as follows : taking the test supplement on fewer than 80% of the scheduled days , performing actions that affected the reliability of the efficacy assessment and noncompliance with the clinical protocol . changes in measurements during the intervention were compared with baseline using the steel test for jkom total score , glfs-5 score and questionnaires ; dunnett s test was used for the vas score for jkom knee pain , normal walking speed , and parameters measured by motion capture analysis . correlation analysis between changes in parameters analyzed by the motion capture system and locomotor functions that involve the knee joint over the 16-week intervention period was conducted , and pearson s correlation coefficient ( r value ) for parametric variables and spearman s rank correlation coefficient ( value ) for nonparametric variables were calculated . all statistical analyses were carried out using ibm spss statistics for windows , version 21.0 ( ibm corporation , armonk , ny , usa ) and ekuseru - toukei 2010 for windows ( social survey research information co. ltd , tokyo , japan ) . an open label study was conducted to elucidate the mechanism of efficacy of gcqid supplementation for locomotor functions in japanese women and men aged 40 to 74 years . inclusion criteria were the presence of knee pain , confirmed using the walking subscale of the japanese orthopaedic association criteria ( 25 points or less for either the left or right knee joint),13 and a visual analog scale ( vas ) score for knee pain ( 20 mm or above using the first [ i ] item of the japanese knee osteoarthritis measure [ jkom]);14 kellgren lawrence grades 0ii;15 and the presence of ls confirmed with a score of 6 or higher on the glfs-5.3 all participants received an explanation about the study from the medical investigator , and written informed consent was obtained prior to enrollment in the study . exclusion criteria were the same as in our previous study,11 and typical reasons for exclusion were the following : fast walking speed ( 1.6 m / s ) ; presence of hyperuricemia , diabetes , cardiovascular disease , hepatic disease , renal disease or heart disease ; presence of rheumatoid arthritis that may cause joint pain ; surgical treatment of knee joint(s ) needed or undergone ; daily use of a cane ; daily or occasional vigorous exercise ; need for pharmacological articular treatments during the study period ; a history of osseous or articular diseases other than oa within the past 3 months ; routine use of health food or medicine that may affect the outcome of the study ; and presence of any medical condition as judged by the medical investigator . thirty participants were enrolled in the study , which was performed from february to august 2014 at a clinical services center and at tokai university in japan . all subjects were recruited around the kanto region in japan , through a volunteer bank managed by ttc co. , ltd ( tokyo , japan ) . once radiographs were obtained , kellgren lawrence grades of subjects were determined by an orthopedic surgeon . the study protocol was approved by the ethics committee of tokai university ( kanagawa , japan ) and the ethics committee of tana orthopedic surgery ( yokohama , japan ) , and was conducted in accordance with the principles of the amended declaration of helsinki and the ethical guidelines for epidemiological research ( issued by the japanese government in 2008 ) . the gcqid contained 1,200 mg of glucosamine hydrochloride , 300 mg of shark cartilage extract ( 60 mg as chondroitin sulfate , 45 mg as type ii collagen peptides ) , 90 mg of quercetin glycosides , 100 mg of fish meat extract ( 10 mg as imidazole peptides [ anserine and carnosine ] ) , 5 mg of salmon nasal cartilage extract ( 1 mg as proteoglycan ) , and 5 g ( 200 iu ) of vitamin d in six tablets . the gcqid tablets were manufactured by suntory wellness ltd ( tokyo , japan ) specifically for the purpose of the present study , and all subjects were instructed to take six tablets once a day and to record in their study diary whether they took the tablets . the indicators of efficacy of gcqid supplementation for functions of locomotor system including the knee joint were : jkom category i score ( vas score for jkom knee pain ) , total score for jkom categories ii v ( jkom total score ) , glfs-5 score , vas score for knee pain in various daily situations , and questionnaires designed for this study . jkom is a self - administered , disease - specific measure for knee oa and has demonstrated adequate validity and reliability for studies of japanese people with knee oa in terms of outcomes correlated with health - related quality of life ( qol).14 the vas score for jkom knee pain was measured on a scale from 0 to 100 , where 0 indicated no pain and 100 indicated the worst pain ever experienced . the jkom total score comprised 25 items that cover four different categories : ii : pain and stiffness in knees ; iii : conditions in daily life ; iv : general activities ; and v : health conditions . scores from the 25 items were summed , with results ranging from 0 points ( no complaint ) to 100 points ( most severe condition possible ) . the glfs-5 is also a self - administered measure , consisting of five items located in the key domain of construct structure of the glfs-25 , which covers a wide range of issues , from pain to qol.3 items are graded with 5-point scales from 0 points to 4 points , and the five item scores were summed to produce an overall score with a range of 0 ( no impairment ) to 20 points ( severe impairment ) . the vas score for knee pain in various daily situations included the pain in each knee on rest , walking or ascending / descending stairs , with scales from 0 to 100 , where 0 indicated no pain and 100 indicated the worst pain ever experienced . questionnaires addressed frequency of trips that involved walking or ascending / descending stairs , with 5-point scales from 0 ( no occurrence ) to 4 ( frequent occurrence ) . for the biomechanical mechanism of efficacy for locomotor functions , motion capture analysis in a normal walking state was conducted to obtain data on gait parameters . subjects took off their shoes and changed into dedicated full - body suits for motion capture . markers were attached to 30 locations on the body suits ( top of head , tragi , superior margin of the sternum , extremitas inferior ribs , anterior superior iliac spines , acromions , elbows , wrists , backs of the hands , great trochanters , interior and exterior sides of knees , internal and external malleoli , heels and toes ) . thereafter , subjects were instructed to walk 10 m two times at their usual speed . the time it took to walk the middle 6 m was measured and normal walking speed was calculated as the primary outcome . the position trajectories of markers attached on the suits of subjects during walking were recorded by a motion capture system ( raptor - e digital real time system ; motion analysis corporation , santa rosa , ca , usa ) at the same time . the values of step length , stride length , cadence , time in the stance phase and the swing phase , and angle of soles and toes for both feet were calculated by analyzing the data of position trajectories , and the average value of the data on both feet was evaluated . explanation of terms for some parameters in motion capture analysis is shown in figure 1 . data on functions of locomotor system including the knee joint were collected at baseline and every 4 weeks to week 16 . data on motion capture analysis were collected at baseline and at 8 and 16 weeks . exclusion criteria for efficacy assessment were as follows : taking the test supplement on fewer than 80% of the scheduled days , performing actions that affected the reliability of the efficacy assessment and noncompliance with the clinical protocol . changes in measurements during the intervention were compared with baseline using the steel test for jkom total score , glfs-5 score and questionnaires ; dunnett s test was used for the vas score for jkom knee pain , normal walking speed , and parameters measured by motion capture analysis . correlation analysis between changes in parameters analyzed by the motion capture system and locomotor functions that involve the knee joint over the 16-week intervention period was conducted , and pearson s correlation coefficient ( r value ) for parametric variables and spearman s rank correlation coefficient ( value ) for nonparametric variables were calculated . all statistical analyses were carried out using ibm spss statistics for windows , version 21.0 ( ibm corporation , armonk , ny , usa ) and ekuseru - toukei 2010 for windows ( social survey research information co. ltd , tokyo , japan ) . of the 30 subjects , one female dropped out of the study because of an adverse event ( sudden deafness deemed by the medical investigator as unrelated to supplementation ) ; this subject s data were excluded from the efficacy assessment . the remaining 29 subjects were deemed eligible for efficacy assessment and their baseline characteristics are shown in table 1 . table 2 shows the changes in functions of locomotor system including the knee joint over the 16-week intervention period . the vas score for jkom knee pain , jkom total score , glfs-5 score , and all vas scores for knee pain in various daily situations significantly decreased at week 4 and thereafter ( p<0.05 or p<0.01 ) compared with the baseline . the questionnaire scores reflecting frequency of tripping while walking or ascending / descending stairs significantly decreased at week 8 and thereafter ( p<0.05 or p<0.01 ) compared with the baseline . table 3 shows the changes in parameters analyzed by the motion capture system over the 16-week intervention period . normal walking speed significantly increased at week 16 compared with the baseline ( p<0.01 ) . step length and stride length significantly increased at week 8 and at week 16 compared with the baseline ( p<0.05 , p<0.01 ) , but cadence was not significantly changed by the intervention . time in the stance phase significantly decreased at week 8 and at week 16 compared with the baseline ( p<0.01 ) , but time in the swing phase was not significantly changed by intervention . angle of soles at the end of the stance phase significantly increased at week 8 and at week 16 compared with the baseline ( p<0.05 , p<0.01 ) and angle of toe at the beginning of the stance phase significantly increased at week 16 compared with the baseline ( p<0.01 ) . table 4 shows correlations between changes in major parameters analyzed by the motion capture system and locomotor functions that involve the knee joint over the 16-week intervention period . normal walking speed significantly correlated with stride length , cadence , and angle of sole ( p<0.01 , r=0.87 ; p<0.05 , r=0.44 ; and p<0.01 , r=0.66 , respectively ) . stride length correlated with angle of sole at the end of the stance phase and with angle of toe at the beginning of the stance phase ( p<0.01 , r=0.66 ; p<0.05 , r=0.42 , respectively ) . the vas score for jkom knee pain , jkom total score , and glfs-5 significantly correlated with each other . jkom total score and glfs-5 significantly correlated with stride length ( p<0.05 , =0.42 ; p<0.05 , =0.42 , respectively ) . the present study was conducted to investigate the biomechanical mechanism for gcqid s positive effects on locomotor functions in subjects with knee pain using a motion capture system . gcqid improved functions of locomotion that involve the knee joint , as evidenced by jkom and glfs-5 scores ( table 2 ) , suggesting that gcqid supplementation exerted effects on knee pain in daily life and on qol , in accordance with our previous study.11 the motion capture analysis revealed that gcqid simultaneously increased normal walking speed and stride ( and step ) length , but that cadence was not changed ( table 3 ) . as it has been reported that walking speed is mainly controlled by stride length and cadence,16 gcqid s effect of increasing normal walking speed was likely attributable to increased stride length . this attribution is supported by the fact that the change in normal walking speed at 16 weeks was most strongly related to the change in stride length in the correlation analysis ( r=0.87 ) . in addition , gcqid increased the angle of soles at the end of the stance phase ( table 3 ) , which could indicate increased kicking force of the ground and enhanced driving power ( figure 1 ) . as the change in stride length at 16 weeks was correlated with the change in the angle of soles at the end of the stance phase ( r=0.66 ) , we considered that gcqid might increase stride length through enhanced driving power . oa patients with knee pain had shorter stride lengths compared with healthy subjects,6,7 and the driving power subjects needed to make longer strides could have been restricted because of knee pain . because the change in stride length at 16 weeks was correlated with changes in jkom total score and glfs-5 score ( =0.44 and =0.42 , respectively ) , gcqid might increase stride length through alleviated knee pain and improved functions of locomotor system in daily life . in addition , gcqid increased angle of toe at the beginning of the stance phase ( table 3 ) , which could indicate that the manner of gait control of subjects was changed to one in which toes were raised higher ( figure 1 ) . the change in angle of toe at the beginning of the stance phase at 16 weeks was correlated with the change in stride length ( r=0.42 ) , suggesting the possibility of a contribution of increased stride length to higher toe angle by gcqid . as gcqid decreased frequency of tripping while walking , according to the questionnaires ( table 2 ) , gcqid might actually change the gait in a way that makes subjects less likely to be tripped . therefore , we can not deny the possibility of a placebo effect as an explanation for the observed effect of gcqid . although improvement of scores for functions of locomotor system , such as the jkom total score , was comparatively greater than that in our previous study , the amount of improvement in normal walking speed by gcqid was very similar to that found for the gcqid group in our previous study.11 therefore , we assert that gcqid likely produced a similar effect as in our previous study , in terms of walking parameters . this point will be more apparent as future randomized , placebo - controlled trials elucidating the effect of gcqid supplement on walking parameters are conducted . second , although the biochemical mechanism of gcqid supplementation to improve locomotor functions may be explained mainly by alleviated knee pain through the anti - inflammatory and chondroprotective activities of glucosamine hydrochloride,17,18 chondroitin sulfate,19 and quercetin,20,21 in addition to direct effects on muscle such as anti - muscle atrophy effects of quercetin,22 and the effect of imidazole peptides on increasing muscle blood flow,23 as described in our previous study,11 the contribution of each component to the effects on locomotor functions in this study and our previous study have not been determined . further studies are needed in the future to elucidate the precise role of each component . third , the present study was a short - term study of 16 weeks with a small number of subjects , not enough to elucidate the influence of characteristics of subjects such as age and sex . therefore , long - term studies with larger sample size are required in the future for assessing the efficacy and safety of gcqid treatment including age- and sex - specific variations more clearly . our data based on gait analysis using a motion capture system suggest that gcqid supplements can increase walking speed through increased stride length and increased force of kicking from the ground during steps , and these improvements may be associated mainly with alleviated knee pain and direct effects on muscle .

backgroundpreviously , we demonstrated that glucosamine - containing supplementation was effective for improving locomotor functions , especially walking speed . however , the biomechanical mechanism of efficacy has not been elucidated . this study aimed to address this challenge in subjects with knee pain , using a motion capture system.methodsan open label study was conducted in 30 japanese subjects with knee pain . the subjects were administered a daily supplement containing 1,200 mg of glucosamine hydrochloride , 60 mg of chondroitin sulfate , 45 mg of type ii collagen peptides , 90 mg of quercetin glycosides , 10 mg of imidazole peptides , 1 mg of proteoglycan , and 5 g of vitamin d ( gcqid ) . the intervention continued for 16 weeks . efficacy for locomotor functions involving the knee joint was evaluated mainly using the japanese knee osteoarthritis measure ( jkom ) and the 5-question geriatric locomotive function scale ( glfs-5 ) . to examine the biomechanical mechanism of efficacy for locomotor functions , motions of subjects in a normal walking state were captured . gait analysis was conducted and efficacy for gait parameters such as normal walking speed , stride length , cadence , and angle of soles was evaluated.resultsgcqid significantly improved total scores on the jkom and glfs-5 . in gait analysis , normal walking speed , stride length , and angle of soles at the end of the stance phase were all significantly increased , but cadence did not change significantly during the intervention period . there were significant intercorrelations of changes in normal walking speed , stride length , and angle of soles at the end of the stance phase , and between changes in stride length and total jkom score.conclusiona gcqid supplement may increase walking speed through increased stride length and angle of kicking from the ground during steps , which might be mainly associated with alleviated knee pain .

Introduction Methods Study design and participants Supplementation Outcomes for efficacy assessment Statistical analysis Results Discussion Conclusion

metabolic syndrome ( mets ) is a growing concern among patients with schizophrenia1 because metabolic adverse events are widely regarded as a major risk factor for cardiovascular diseases2,3 and mortality.4,5 although the causes of metabolic adverse events are complicated , the risk factors among patients with schizophrenia are attributed to dietary habits,6 physical activity,7 antipsychotic medications,8,9 and negative symptoms of schizophrenia.10 although the opinion of psychiatrists is well known concerning this issue,1114 those of the patients most affected by metabolic adverse events are relatively unexplored ; nevertheless , they are important factors . educational programs for these patients might effectively improve their clinical outcomes.15 to date , some studies have assessed patient attitudes toward antipsychotic medications.1620 however , these studies have not primarily focused on metabolic adverse events . thus , it is necessary to accurately assess patient attitudes toward metabolic issues in a representative sample of patients . currently , japan has 28.4 psychiatric beds per 10,000 people , which is the highest ratio in the world . furthermore , the mean length of hospital stay in japan i1.5 years , which represents the longest stay among developed nations.21 because inpatients with schizophrenia receive controlled meals and occupational therapy , their lifestyles might differ from those of outpatients . a previous study conducted in japan showed that the rates of mets in outpatients and inpatients with schizophrenia were 48.1% and 15.8% , respectively.22 although the shift from inpatient care to community - based care is an ongoing challenge , the discharge of long - term psychiatric patients might elevate their risk of mets . we previously reported the psychiatrists attitudes toward metabolic adverse events in patients with schizophrenia.14 however , attitudes of schizophrenic patients themselves had not been assessed in japan . the current study investigated attitudes toward metabolic adverse events in a nationwide survey of japanese patients with schizophrenia . to our knowledge , this study uses the largest sample of these patients , and it is the first to investigate attitudes toward metabolic issues among asian patients . the joint committee of the japanese society of clinical neuropsychopharmacology and the japan psychiatric hospital association for antipsychotic treatment and physical risk prepared this survey . after reviewing the relevant literature and extant guidelines , a brief questionnaire was constructed to cover patient recognition of the following broad areas : dietary habits , lifestyle , self - monitoring , knowledge , and medical practice ( figure s1 ) . between january 2012 and june 2013 , the questionnaire was mailed to patients associated with 520 outpatient facilities and 247 inpatient facilities belonging to the japan psychiatric hospital association . participants have been selected based on the available sampling method and diagnosed with schizophrenia according to the diagnostic and statistical manual of mental disorders , fourth edition , or international classification of diseases , tenth revision . the anonymous questionnaire was the only research instrument , and a statement was included that states , the completion of the attached questionnaire will be taken as indicating your consent to participate . the ethics committee at the japan psychiatric hospital association and the hirosaki university school of medicine approved the study procedure . descriptive statistics were computed to describe the demographic and clinical variables . in order to compare the main demographic and clinical characteristics between inpatients and outpatients , the mann whitney u - test was performed to analyze continuous variables , and a chi - square test was performed to analyze categorical variables . parameter r was used to measure the effect size of mann whitney u - test , and r values of 0.3 represent medium effect sizes . strength of association on a chi - square analysis was confirmed with a medium phi and cramer s v score of 0.30 . the joint committee of the japanese society of clinical neuropsychopharmacology and the japan psychiatric hospital association for antipsychotic treatment and physical risk prepared this survey . after reviewing the relevant literature and extant guidelines , a brief questionnaire was constructed to cover patient recognition of the following broad areas : dietary habits , lifestyle , self - monitoring , knowledge , and medical practice ( figure s1 ) . between january 2012 and june 2013 , the questionnaire was mailed to patients associated with 520 outpatient facilities and 247 inpatient facilities belonging to the japan psychiatric hospital association . participants have been selected based on the available sampling method and diagnosed with schizophrenia according to the diagnostic and statistical manual of mental disorders , fourth edition , or international classification of diseases , tenth revision . the anonymous questionnaire was the only research instrument , and a statement was included that states , the completion of the attached questionnaire will be taken as indicating your consent to participate . the ethics committee at the japan psychiatric hospital association and the hirosaki university school of medicine approved the study procedure . descriptive statistics were computed to describe the demographic and clinical variables . in order to compare the main demographic and clinical characteristics between inpatients and outpatients , the mann whitney u - test was performed to analyze continuous variables , and a chi - square test was performed to analyze categorical variables . parameter r was used to measure the effect size of mann whitney u - test , and r values of 0.3 represent medium effect sizes . strength of association on a chi - square analysis was confirmed with a medium phi and cramer s v score of 0.30 . approximately 27.9% ( 4,136/14,840 ) of inpatients stated that they drank soft drinks ( eg , cola ; q1 ) every day , 34.6% ( 5,142/14,840 ) did so more than once a week , 18.3% ( 2,718/14,840 ) did so more than once a month , and 19.2% ( 2,844/14,840 ) did not drink any , whereas 27.8% ( 1,841/6,616 ) of outpatients stated that they drank soft drinks every day , 28.5% ( 1,884/6,616 ) did so more than once a week , 25.8% ( 1,706/6,616 ) did so more than once a month , and 17.9% ( 1,185/6,616 ) did not drink any ( q1 ; cramer s v=0.092 , p<0.001 ) . approximately 62.8% ( 9,324/14,841 ) of inpatients consumed 100% of three meals a day ( q2 ) , 12.2% ( 1,806/14,841 ) ate 90% , 12.1% ( 1,790/14,841 ) ate 80% , 5.8% ( 860/14,841 ) ate 70% , 4.3% ( 644/14,841 ) ate 60% , and 2.8% ( 417/14,841 ) ate 50% . approximately 82.6% ( 5,633/6,823 ) of outpatients stated that they regularly ate breakfast , lunch , and dinner . approximately 39.3% ( 5,855/14,914 ) of inpatients and 36.3% ( 2,466/6,799 ) of outpatients stated that they ate cake or other sweets more than once a day ( q3 ; phi = 0.029 , p<0.001 ) . approximately 17.0% ( 2,523/14,865 ) of inpatients were warned not to eat too many snacks ( q4 ) . approximately 36.7% ( 2,490/6,784 ) of outpatients were also warned not to eat too many meals ( q4 ) . only 6.9% ( 1,008/14,674 ) of inpatients and 6.5% ( 442/6,779 ) of outpatients stated that they generally take sweets when they go out or stay out overnight ( q5 ) . approximately 41.7% ( 6,152/14,765 ) of inpatients and 25.3% ( 1,709/6,767 ) of outpatients stated that they were always hungry ( q6 ; phi = 0.158 , p<0.001 ) . approximately 59.3% ( 3,856/6,505 ) of outpatients stated that they cook or do housework by themselves ( q9 ) . approximately 8.4% ( 1,071/12,751 ) of inpatients reported going out every day ( q8 ) , 12.2% ( 1,558/12,751 ) went out more than once a week , 16.9% ( 2,150/12,751 ) went out more than once a month , and 62.5% ( 7,972/12,751 ) did not go out at all ; these percentages were 46.6% ( 3,091/6,627 ) , 31.8% ( 2,110/6,627 ) , 13.3% ( 883/6,627 ) , and 8.2% ( 543/6,627 ) among outpatients , respectively ( q8 ; cramer s v=0.593 , p<0.001 ) . approximately 18.5% ( 2,708/14,669 ) of inpatients reported exercising every day ( q10 ) , 13.1% ( 1,923/14,669 ) did so once a week , 13.4% ( 1,969/14,669 ) did so more than once a week , and 55.0% ( 8,069/14,669 ) did not exercise at all ; these percentages were 20.2% ( 1,342/6,649 ) , 14.8% ( 986/6,649 ) , 20.2% ( 1,343/6,649 ) , and 44.8% ( 2,978/6,649 ) among outpatients , respectively ( q10 ; cramer s v=0.107 , p<0.001 ) . approximately 35.2% ( 5,231/14,864 ) of inpatients reported watching television every day ( q11 ) , 49.1% ( 7,305/14,864 ) watched television sometimes , and 15.7% ( 2,328/14,864 ) watched none at all ; these percentages were 60.4% ( 4,077/6,748 ) , 30.3% ( 2,047/6,748 ) , and 9.2% ( 624/6,748 ) among outpatients , respectively ( q11 ; cramer s v=0.236 , p<0.001 ) . in this survey , 33.1% ( 4,859/14,701 ) of inpatients and 48.4% ( 3,229/6,669 ) of outpatients reported that they gained weight over the previous year ( q7 ; phi = 0.147 , p<0.001 ) . approximately 22.9% ( 1,554/6,781 ) of outpatients stated that they weighed themselves every day ( q15 ) , 21.0% ( 1,427/6,781 ) did so more than once a week , 19.3% ( 1,306/6,781 ) did so more than once a month , and 36.8% ( 2,494/6,781 ) did not weigh themselves at all . table 2 shows the healthy habits of which patients were mindful ( q19 ) . with regard to body weight self - assessments ( q20 ) , 35.6% ( 5,261/14,794 ) of inpatients and 60.9% ( 4,116/6,760 ) of outpatients approximately 16.4% ( 2,412/14,735 ) of inpatients and 23.2% ( 1,552/6,693 ) of outpatients stated that their doctor educated them on how their medication might cause side effects such as weight gain ( q12 ; phi = 0.081 , p<0.001 ) . furthermore , 31.9% ( 4,749/14,883 ) of inpatients and 74.3% ( 5,056/6,809 ) of outpatients knew the term metabolic syndrome additionally , 9.7% ( 1,431/14,742 ) of inpatients and 22.8% ( 1,530/6,720 ) of outpatients knew the terms bmi or body mass index ( q17 ; phi = 0.176 , p<0.001 ) . approximately 10.2% ( 1,517/14,823 ) of inpatients stated that their body weight was measured at the hospital every day ( q13 ) , 13.7% ( 2,033/14,823 ) said they were weighed once a week , 74.1% ( 10,984/14,823 ) said they were weighed once a month , 0.8% ( 121/14,823 ) said they were weighed at least once every 6 months , 0.6% ( 96/14,823 ) said they were weighed at least once a year , and 0.5% ( 72/14,823 ) said that they were never weighed ; 27.9% ( 1,792/6,421 ) of outpatients stated that their body weight was measured at the hospital every visit , 19.3% ( 1,238/6,421 ) said they were weighed at least once every 3 months , 6.6% ( 423/6,421 ) said they were weighed at least once every 6 months , 10.1% ( 651/6,421 ) said they were weighed at least once a year , and 36.1% ( 2,317/6,421 ) said that they were never weighed . approximately 40.2% ( 5,814/14,473 ) of inpatients stated that they had blood tests at a hospital ( q14 ) every month , 37.5% ( 5,425/14,473 ) had blood tests at least once every 3 months , 15.5% ( 2,239/14,473 ) had blood tests at least once every 6 months , 5.5% ( 799/14,473 ) had blood tests at least once a year , and 1.4% ( 196/14,473 ) never had blood tests ; 7.2% ( 464/6,468 ) of outpatients stated that they had blood tests at the hospital every visit , 27.4% ( 1,769/6,468 ) said they had blood tests at least once every 3 months , 24.5% ( 1,587/6,468 ) said they had blood tests at least once every 6 months , 24.4% ( 1,579/6,468 ) said they had blood tests at least once a year , and 16.5% ( 1,069/6,468 ) said that they never had blood tests . table 3 lists the healthy habits that patients were told to be mindful of by their doctors ( q18 ) . to prevent weight gain and diseases such as diabetes , 51.2% of inpatients ( 7,514/14,690 ) and 60.8% of outpatients ( 4,086/6,721 ) hoped to receive blood tests regularly ( q21 ; phi = 0.090 , p<0.001 ) , and 64.5% of inpatients ( 9,514/14,744 ) and 63.3% of outpatients ( 4,260/6,729 ) hoped to weigh themselves regularly ( q22 ; phi = 0.012 , p=0.084 ) . approximately 27.9% ( 4,136/14,840 ) of inpatients stated that they drank soft drinks ( eg , cola ; q1 ) every day , 34.6% ( 5,142/14,840 ) did so more than once a week , 18.3% ( 2,718/14,840 ) did so more than once a month , and 19.2% ( 2,844/14,840 ) did not drink any , whereas 27.8% ( 1,841/6,616 ) of outpatients stated that they drank soft drinks every day , 28.5% ( 1,884/6,616 ) did so more than once a week , 25.8% ( 1,706/6,616 ) did so more than once a month , and 17.9% ( 1,185/6,616 ) did not drink any ( q1 ; cramer s v=0.092 , p<0.001 ) . approximately 62.8% ( 9,324/14,841 ) of inpatients consumed 100% of three meals a day ( q2 ) , 12.2% ( 1,806/14,841 ) ate 90% , 12.1% ( 1,790/14,841 ) ate 80% , 5.8% ( 860/14,841 ) ate 70% , 4.3% ( 644/14,841 ) ate 60% , and 2.8% ( 417/14,841 ) ate 50% . approximately 82.6% ( 5,633/6,823 ) of outpatients stated that they regularly ate breakfast , lunch , and dinner . approximately 39.3% ( 5,855/14,914 ) of inpatients and 36.3% ( 2,466/6,799 ) of outpatients stated that they ate cake or other sweets more than once a day ( q3 ; phi = 0.029 , p<0.001 ) . approximately 17.0% ( 2,523/14,865 ) of inpatients were warned not to eat too many snacks ( q4 ) . approximately 36.7% ( 2,490/6,784 ) of outpatients were also warned not to eat too many meals ( q4 ) . only 6.9% ( 1,008/14,674 ) of inpatients and 6.5% ( 442/6,779 ) of outpatients stated that they generally take sweets when they go out or stay out overnight ( q5 ) . approximately 41.7% ( 6,152/14,765 ) of inpatients and 25.3% ( 1,709/6,767 ) of outpatients stated that they were always hungry ( q6 ; phi = 0.158 , p<0.001 ) . approximately 59.3% ( 3,856/6,505 ) of outpatients stated that they cook or do housework by themselves ( q9 ) . approximately 8.4% ( 1,071/12,751 ) of inpatients reported going out every day ( q8 ) , 12.2% ( 1,558/12,751 ) went out more than once a week , 16.9% ( 2,150/12,751 ) went out more than once a month , and 62.5% ( 7,972/12,751 ) did not go out at all ; these percentages were 46.6% ( 3,091/6,627 ) , 31.8% ( 2,110/6,627 ) , 13.3% ( 883/6,627 ) , and 8.2% ( 543/6,627 ) among outpatients , respectively ( q8 ; cramer s v=0.593 , p<0.001 ) . approximately 18.5% ( 2,708/14,669 ) of inpatients reported exercising every day ( q10 ) , 13.1% ( 1,923/14,669 ) did so once a week , 13.4% ( 1,969/14,669 ) did so more than once a week , and 55.0% ( 8,069/14,669 ) did not exercise at all ; these percentages were 20.2% ( 1,342/6,649 ) , 14.8% ( 986/6,649 ) , 20.2% ( 1,343/6,649 ) , and 44.8% ( 2,978/6,649 ) among outpatients , respectively ( q10 ; cramer s v=0.107 , p<0.001 ) . approximately 35.2% ( 5,231/14,864 ) of inpatients reported watching television every day ( q11 ) , 49.1% ( 7,305/14,864 ) watched television sometimes , and 15.7% ( 2,328/14,864 ) watched none at all ; these percentages were 60.4% ( 4,077/6,748 ) , 30.3% ( 2,047/6,748 ) , and 9.2% ( 624/6,748 ) among outpatients , respectively ( q11 ; cramer s v=0.236 , p<0.001 ) . in this survey , 33.1% ( 4,859/14,701 ) of inpatients and 48.4% ( 3,229/6,669 ) of outpatients reported that they gained weight over the previous year ( q7 ; phi = 0.147 , p<0.001 ) . approximately 22.9% ( 1,554/6,781 ) of outpatients stated that they weighed themselves every day ( q15 ) , 21.0% ( 1,427/6,781 ) did so more than once a week , 19.3% ( 1,306/6,781 ) did so more than once a month , and 36.8% ( 2,494/6,781 ) did not weigh themselves at all . table 2 shows the healthy habits of which patients were mindful ( q19 ) . with regard to body weight self - assessments ( q20 ) , 35.6% ( 5,261/14,794 ) of inpatients and 60.9% ( 4,116/6,760 ) of outpatients approximately 16.4% ( 2,412/14,735 ) of inpatients and 23.2% ( 1,552/6,693 ) of outpatients stated that their doctor educated them on how their medication might cause side effects such as weight gain ( q12 ; phi = 0.081 , p<0.001 ) . furthermore , 31.9% ( 4,749/14,883 ) of inpatients and 74.3% ( 5,056/6,809 ) of outpatients knew the term metabolic syndrome additionally , 9.7% ( 1,431/14,742 ) of inpatients and 22.8% ( 1,530/6,720 ) of outpatients knew the terms bmi or body mass index approximately 10.2% ( 1,517/14,823 ) of inpatients stated that their body weight was measured at the hospital every day ( q13 ) , 13.7% ( 2,033/14,823 ) said they were weighed once a week , 74.1% ( 10,984/14,823 ) said they were weighed once a month , 0.8% ( 121/14,823 ) said they were weighed at least once every 6 months , 0.6% ( 96/14,823 ) said they were weighed at least once a year , and 0.5% ( 72/14,823 ) said that they were never weighed ; 27.9% ( 1,792/6,421 ) of outpatients stated that their body weight was measured at the hospital every visit , 19.3% ( 1,238/6,421 ) said they were weighed at least once every 3 months , 6.6% ( 423/6,421 ) said they were weighed at least once every 6 months , 10.1% ( 651/6,421 ) said they were weighed at least once a year , and 36.1% ( 2,317/6,421 ) said that they were never weighed . approximately 40.2% ( 5,814/14,473 ) of inpatients stated that they had blood tests at a hospital ( q14 ) every month , 37.5% ( 5,425/14,473 ) had blood tests at least once every 3 months , 15.5% ( 2,239/14,473 ) had blood tests at least once every 6 months , 5.5% ( 799/14,473 ) had blood tests at least once a year , and 1.4% ( 196/14,473 ) never had blood tests ; 7.2% ( 464/6,468 ) of outpatients stated that they had blood tests at the hospital every visit , 27.4% ( 1,769/6,468 ) said they had blood tests at least once every 3 months , 24.5% ( 1,587/6,468 ) said they had blood tests at least once every 6 months , 24.4% ( 1,579/6,468 ) said they had blood tests at least once a year , and 16.5% ( 1,069/6,468 ) said that they never had blood tests . table 3 lists the healthy habits that patients were told to be mindful of by their doctors ( q18 ) . to prevent weight gain and diseases such as diabetes , 51.2% of inpatients ( 7,514/14,690 ) and 60.8% of outpatients ( 4,086/6,721 ) hoped to receive blood tests regularly ( q21 ; phi = 0.090 , p<0.001 ) , and 64.5% of inpatients ( 9,514/14,744 ) and 63.3% of outpatients ( 4,260/6,729 ) hoped to weigh themselves regularly ( q22 ; phi = 0.012 , p=0.084 ) . the present study employed a national survey in japan that evaluated attitudes toward metabolic adverse events among patients with schizophrenia . more than half of outpatients felt obese , and more than half of all respondents hoped to receive regular blood tests to prevent metabolic adverse events . however , a minority of patients were mindful of eating balanced meals and having physical exercise and stated that their doctors encouraged them to have such habits . our results confirm that patients with schizophrenia are concerned about metabolic adverse events but do not have healthy lifestyle habits . the differences in dietary patterns between patients with schizophrenia and healthy controls have been reported previously.23,24 inadequate dietary patterns can cause obesity,25 mets,26 and cardiovascular events.27 in our survey , most outpatients stated that they ate breakfast , lunch , and dinner regularly . however , more than one - third of our respondents ate cake and other sweets at least once a day , and most participants stated that they were not mindful of having a healthy diet . dietary improvements in patients with schizophrenia might help prevent metabolic adverse events.28 reduced physical activity might play a role in the development of mets,29 and a previous study reported that patients with schizophrenia engaged in physical activity less frequently than healthy controls.30 in this survey , approximately half of our respondents ( 55% of inpatients and 45% of outpatients ) answered that they did not exercise at all . our results are in accordance with previous research,31 which found that 40% of individuals with schizophrenia report a lack of moderate physical activity , and 75% report a lack of vigorous physical activity . although a minority of respondents ( inpatients 29.4% , outpatients 39.5% ) were mindful of having physical exercise , several studies have indicated that regular exercise programs are possible among individuals with schizophrenia and that they can have beneficial effects on their well - being as well as their physical and mental health.32 according to our survey , more than half of outpatients reported that they felt obese . obesity is a growing public health concern , and it is becoming more prevalent among patients with schizophrenia compared with the general population.3335 previous studies have shown that being overweight is a major risk factor for mets , cardiovascular diseases , and premature death.3638 additionally , obesity among patients with schizophrenia is associated with high medication costs,39 low self - esteem , poor psychosocial adaptation,40 reduced quality of life,41,42 negative attitudes toward medication,19 and noncompliance with antipsychotic medication regimes.43 although behavioral interventions can effectively prevent and reduce antipsychotic - associated weight gain and cardiometabolic perturbations,15 most respondents in this study reported that they were not encouraged by their doctors to perform moderate physical exercise . more than half of our respondents hoped to receive blood tests regularly to prevent weight gain and diseases such as diabetes . although patients with severe mental illnesses are less aware of comorbid medical conditions such as heart disease , diabetes , and hypertension,17 patients judged disorders such as epilepsy and diabetes as worse than schizophrenia16 and described dissatisfaction or deficiency with regard to the care that they received from their health care providers.18 buckley et al showed that most psychiatrists did not routinely monitor their patients lipid profiles , blood glucose levels , or blood pressure.12 we previously reported that most psychiatrists in japan monitor the lipid profiles and blood glucose levels of their inpatients and outpatients more than twice a year.14 most psychiatrists stated that the frequency at which they monitored patients under antipsychotic treatment was based on their own clinical experience . however , only 20.6% of respondents stated that this monitoring frequency was sufficient to reduce metabolic risk . it is necessary to disseminate monitoring guidelines with regard to metabolic adverse events because they might make psychiatrists more aware of an integral approach to patients with schizophrenia , thereby increasing the physical health monitoring of these patients.44 in japan , the mental health act seeks to promote the concept of normalization , establishing mental illness as a disability and encouraging the re - assimilation of psychiatric inpatients back into the community . although inpatients were encouraged to shift to community - based care , most inpatients did not have enough knowledge of metabolic adverse events . educational programs seeking to prepare long - term hospitalized patients with schizophrenia for discharge from hospitals are warranted . firstly , our study is limited by the fact that it is cross - sectional rather than prospective in design and lacks data on the actual practices of psychiatrists . this study could not clarify a causal relationship between the psychiatrists practice and attitudes among patients with schizophrenia . secondly , several potential confounding factors , such as antipsychotic medications , duration of illness , and treatment , were not assessed by our study . age could affect the results of dietary habits , lifestyle , self - monitoring , knowledge , and medical practices . this study showed that only a minority of patients were mindful of eating balanced meals and having physical exercise ; however , more than half of patients hoped to prevent weight gain and diabetes . these findings imply that educational efforts45 and the promotion of the best pharmacotherapy and monitoring practices46 are needed for patients with schizophrenia . improving attitudes toward metabolic adverse events among these patients might contribute to physical health improvements .

backgroundmetabolic syndrome is a growing concern among patients with schizophrenia because metabolic abnormalities are widely regarded as a major risk factor for cardiovascular disease and premature death . the current study assessed attitudes toward metabolic adverse events among patients with schizophrenia.methodsa brief questionnaire was constructed to investigate patient recognition of the following broad areas : dietary habits , lifestyle , self - monitoring , knowledge , and medical practice . between january 2012 and june 2013 , questionnaires were sent to patients associated with 520 outpatient facilities and 247 inpatient facilities belonging to the japan psychiatric hospital association . all of the participants ( n=22,072 ; inpatients = 15,170 , outpatients = 6,902 ) were diagnosed with schizophrenia based on the diagnostic and statistical manual of mental disorders , fourth edition , or the international classification of diseases , tenth revision.resultsapproximately 55.0% ( 8,069/14,669 ) of inpatients and 44.8% of outpatients ( 2,978/6,649 ) reported that they did not exercise at all . although 60.9% ( 4,116/6,760 ) of outpatients reported that they felt obese , only 35.6% ( 5,261/14,794 ) of inpatients felt obese . more than half of the inpatients ( 51.2% ; 7,514/14,690 ) and outpatients ( 60.8% ; 4,086/6,721 ) hoped to receive regular blood tests to prevent weight gain and diseases such as diabetes.conclusionalthough more than half of patients hoped to prevent weight gain and diabetes , only a minority of patients were mindful of eating balanced meals and having physical exercise . educational efforts and the promotion of the best pharmacotherapy and monitoring practices are needed for patients with schizophrenia .

Introduction Methods Procedure Statistical analysis Results Patient recognition of dietary habits Patient recognition of lifestyle Patient recognition of self-monitoring Patient recognition of knowledge Patient recognition of medical practice Discussion Conclusion Supplementary material

giant cell tumor ( gct ) is a tumor found most often in the ends of long bones and is essentially located in the epiphyseal or metaphyseal or epiphyseal equivalent portions of bone . it is a locally aggressive neoplasm , generally arising in adults between the ages of 20 and 40 years , clinically possessing metastatic potential . the rib is a rare site with a reported incidence of less than one percent . even in the cases involving the rib , most were located in the posterior arc , i.e. , the head and tubercle of ribs . rare multicentric forms have been reported.[13 ] in this article , we report a case of gct originating from the anterior arc of the rib which was diagnosed on fine needle aspiration cytology ( fnac ) . a 23-year - old female presented with a six - month history of a progressively growing mass in the right anterior chest wall associated with slight pain . cytological smears were cellular and comprising aggregates of uniform appearing spindled stromal cells and innumerable osteoclast type giant cells , having variable number of nuclei . the nuclei of the stromal cells resembled those observed in the osteoclast type giant cells . the stromal cells showed high nuclear to cytoplasmic ratio , with evenly distributed chromatin and inconspicuous to small nucleoli . no significant nuclear atypia was observed either in the giant cells or the background stromal cells . mitotic figures were noted frequently [ figure 1 ] . in correlation with radiographic findings of an eccentric expanded lytic lesion with cortical erosion of anterior arc of fourth rib [ figure 2 ] , a diagnosis of aggressive giant cell tumor of rib was offered cytologically . in our case , the musculoskeletal radiologist had opined an aneurysmal bone cyst and askin tumor of anterior chest wall . aggregates of uniform appearing spindled stromal cells and innumerable osteoclast type giant cells ( h and e , 400 ) . inset showing histomorphology of the resected giant cell tumor ( h and e , 400 ) large expansile tumor mass with thinned out cortex in the anterior end of fourth rib noted on computed tomography scan in view of the cytomorphological diagnosis and radiological cortical destruction of anterior arc of fourth rib , patient was referred to a surgical oncology centre with a possibility of soft tissue extension being also considered . at the higher centre , complete resection of the tumor patient remained asymptomatic without evidence of recurrence at the end of one year following surgical intervention . at the last follow up gct of bone is an uncommon neoplasm accounting for about 45% of all primary bone tumors . in fact , they may represent stromal precursor cells that have lost some of the detectable macrophage associated antigens or mononuclear phagocyte antigens seen in the more mature cells . gct are generally considered benign but malignant cells can arise de novo or via transformation from a benign neoplastic giant cell lesion.[13 ] interestingly , gct 's are more common in females . although the role of steroid metabolism in these lesions is unclear , estrogen and progesterone receptors have been identified in the cells of this lesion . the metaphyseal or epiphyseal zones of long bones are the most common sites , with 60% arising around the knee joint . isolated cases have been reported in the scapula , sternum , patella , vertebra , skull and talus . only few cases of gct involving the ribs have been reported in the literature with most of them involving the posterior aspect . due to its rarity , gct arising from the chest wall is difficult to diagnose , especially when the tumor is located in the anterior arc of the ribs . it is also compounded by the fact that soft tissue counterpart of gct is also known.[135 ] pain and an increase in local volume are the principal forms of presentation . some patients present with pathological fracture resulting from weakening of the cortical bone . on a routine radiograph , gct presents as an initially eccentric expanded lytic lesion , without a surrounding sclerotic halo , representing the cortical bone . as the lesion grows , it can encompass the entire circumference of the bone , causing rupture of the cortical bone , but a periosteal reaction is rarely seen . roentgenographic classification schemes have been used to characterize the tumor in any given case and attempt to predict the clinical outcome . histological features : benign , aggressive and malignant , the latter having clearly pleomorphic features with abundant mitotic figures and ( ii ) . , although there were no overt anaplastic features in the tumor cells , mitotic activity was frequently detectable . with the clinical symptomatic presentation of a rapidly growing mass and radiological evidence of bone destruction and expansion , a cytomorphological diagnosis of an aggressive gct was offered in this case . a wide array of lesions may histologically mimic , depending on the quality and size of the biopsy . while examining lesions rich in these cells , if particular attention is paid to the background stromal cells and the clinico radiological data are correlated , then establishing the diagnosis becomes easier . a differential diagnosis of aneurysmal bone cyst ( abc ) , brown tumor , chondroblastoma , chondromyxoid fibroma ( cmf ) , non - ossifying fibroma ( nof ) , giant cell rich osteosarcoma and malignant fibrous histiocytoma can be considered on cytology . in gct , the giant cells are numerous in number and they are attached at the periphery of the clustered spindle cells . aspirates from an abc are generally hemorrhagic with sparse cellular yield comprised of scattered osteoclastic giant cells , spindle shaped fibroblastic cells and hemosiderin laden macrophages . in chondroblastoma , chondroid matrix and plump , spindle - shaped mononuclear cell component along with occasional osteoclastic giant cells are present . in cmf , aspirates show chondroid fragments , spindle - shaped fibroblastic cells and scattered osteoclastic giant cells in a myxoid background . nof yields groups and clusters of spindle cells , histiocytic cells with vacuolated cytoplasm and occasional osteoclastic giant cells . the cytology of brown tumor is similar to nof with characteristic spindly mononuclear cells , osteoclasts and macrophages . giant cell rich osteosarcoma and mfh are characterized by nuclear anaplasia and abnormal mitotic figures and in the gct there is no neoplastic osteoid formation which is the most pathognomonic finding of a giant cell rich osteosarcoma . except for the lack of bony involvement , primary giant cell tumor of soft tissue resembles in all its cyto - histomorphological features its osseous counterpart.[24 ] in general , all gct 's should be considered potentially aggressive and wide excision is recommended . fine needle aspiration cytology has been useful in diagnosing similar lesions at rare sites like distal fibula . a primary malignant gct of sacrum could be identified in a middle aged female as also a multifocal gct in a skeletally immature patient . serum acid phosphatase values are suggested to be a useful marker for diagnosis of gct of the bone and for evaluation of the efficacy of treatment of the tumor . the values were high in 56% of gct patients and later decreased to normal values after resection as seen in our case . to conclude , our case illustrates the fact that giant cell tumors of anterior chest wall can be mistaken for abc and other malignant tumors of bone and soft tissues . the fine needle or image - guided biopsy would be diagnostic if adequate specimen is obtained .

giant cell tumor of bone also known as osteoclastoma is a distinct clinical , roentgenographic and pathologic entity with specific characteristics . it is a benign but locally aggressive neoplasm , classically seen as a purely lytic lesion of the epiphyseal or metaphyseal - epiphyseal region of long tubular bones extending to the articular surface . it usually occurs after skeletal maturation and is one of the rare bone tumors that more frequently affects women . even though rarely it can occur in ribs , it usually occurs in the posterior aspect . we report here a case of giant cell tumor originating from the anterior arc of the rib diagnosed on fine needle aspiration cytology .

Introduction Case Report Discussion

ovarian tumors of borderline malignancy , borderline ovarian tumors ( bot ) , constitute about 10 - 15% of all epithelial ovarian malignancies ( 1 ) . however , conservative treatment might be considered in patients who want to preserve their fertility because of the excellent prognosis reported ( 2 , 3 ) . although spontaneous conceptions have been reported after conservative surgery , some of these patients suffer from infertility and require infertility treatment . some bot infertile patients undergo assisted reproductive technologies ( art ) to improve their chances of pregnancy . the influence of infertility treatment on the development of ovarian malignancies is a controversial topic . in a case - control study , it has been reported that a history of infertility increases the overall risk of ovarian cancer ( 4 ) . in addition , there are reports suggesting an association between fertility medication and bot . however , the association between fertility medication and invasive ovarian cancer is not conclusive ( 5 - 7 ) . it was suggested that high serum estradiol levels during ovarian hyperstimulation might promote tumor growth in bot , especially in estrogen receptor expression - positive cases ( 8 , 9 ) . therefore , the potential risk associated with infertility and treatment must be considered for infertile patients after conservative treatment for bot . in early - stage bot , it has been possible to consider art after conservative treatment since a multicenter study that reported 16 bot patients who had undergone in vitro fertilization ( ivf ) after conservative treatment ( 10 ) . reported that the overall success rates of ivf were satisfactory for this group of patients , suggesting no known negative impact of prior bot on pregnancy rates after ivf ( 11 ) . however , for advanced - stage bot the published reports are limited to case reports ( 12 - 14 ) . therefore , for patients with advanced bot the safety of art after conservative treatment remains anecdotal . the purpose of this study was to evaluate the outcomes of coh - ivf in infertile patients after conservative treatment for bot . a retrospective review of ivf records from january 1999 to july 2005 revealed 10 attempted ivf cycles in five patients who had been previously diagnosed with bot and had had conservative treatment to preserve fertility . bot has the histological characteristics of ovarian tumors : 1 ) epithelial proliferation with the formation of a papillary configuration , 2 ) demonstration of atypical epithelial activity , 3 ) mild or moderate atypical nuclei , and 4 ) the absence of stromal invasion , which distinguishes it from invasive carcinoma ( fig . conservative treatment is defined as preservation of the uterus and at least a portion of one ovary . in cases where the diagnosis of bot was made intraoperatively , staging was made according to the international federation of gynecology and obstetrics ( figo ) classification based on ipsilateral pelvic and paraaortic lymph node dissections , peritoneal cytology , omentectomy , and multiple peritoneal biopsies . after conservative surgery , a gynecological oncologist followed all patients every 3 months during the first year and thereafter every 6 months with a physical examination , serum ca-125 levels and transvaginal ultrasound . the main outcome measures were pregnancy outcomes such as clinical pregnancy rate ( cpr ) , implantation rate ( ir ) and live birth rate ( lbr ) after coh - ivf , and the recurrence of bot during the follow - up period . approval from the institutional review board was not obtained because this study was a retrospective case observational study . coh was performed with gonadotropin - releasing hormone agonist ( gnrh - a ) long protocol or flare - up protocol using human menopausal gonadotropin ( menogon , ferring , germany ) or recombinant follicle - stimulating hormone ( puregon , organon , netherland ) . for the long protocol , patients underwent pituitary desensitization with gnrh - a ( suprefact , hoechst , germany ) from the previous menstrual mid - luteal phase , and gonadotropins were administered after pituitary down regulation and continued up to hcg ( pregnyl , organon , netherland ) administration . for the flare - up protocol , gnrh - a was administered from the second day of the menstrual cycle and gonadotropins were administered from the third day of the menstrual cycle until hcg administration . oocyte retrieval was performed via the transvaginal approach with sonographic guidance 36 hr after 10,000 iu of hcg administration . the oocytes were incubated in human tubal fluid ( irvine scientific , irvine , ca . , u.s.a . ) medium supplemented with 10% synthetic serum supplement ( sss ; irvine scientific ) at 37 , 5% co2 in air . pregnancy was determined by serum -hcg levels above 5 miu / ml 12 days after the oocyte retrieval . a retrospective review of ivf records from january 1999 to july 2005 revealed 10 attempted ivf cycles in five patients who had been previously diagnosed with bot and had had conservative treatment to preserve fertility . bot has the histological characteristics of ovarian tumors : 1 ) epithelial proliferation with the formation of a papillary configuration , 2 ) demonstration of atypical epithelial activity , 3 ) mild or moderate atypical nuclei , and 4 ) the absence of stromal invasion , which distinguishes it from invasive carcinoma ( fig . conservative treatment is defined as preservation of the uterus and at least a portion of one ovary . in cases where the diagnosis of bot was made intraoperatively , staging was made according to the international federation of gynecology and obstetrics ( figo ) classification based on ipsilateral pelvic and paraaortic lymph node dissections , peritoneal cytology , omentectomy , and multiple peritoneal biopsies . after conservative surgery , a gynecological oncologist followed all patients every 3 months during the first year and thereafter every 6 months with a physical examination , serum ca-125 levels and transvaginal ultrasound . the main outcome measures were pregnancy outcomes such as clinical pregnancy rate ( cpr ) , implantation rate ( ir ) and live birth rate ( lbr ) after coh - ivf , and the recurrence of bot during the follow - up period . approval from the institutional review board was not obtained because this study was a retrospective case observational study . coh was performed with gonadotropin - releasing hormone agonist ( gnrh - a ) long protocol or flare - up protocol using human menopausal gonadotropin ( menogon , ferring , germany ) or recombinant follicle - stimulating hormone ( puregon , organon , netherland ) . for the long protocol , patients underwent pituitary desensitization with gnrh - a ( suprefact , hoechst , germany ) from the previous menstrual mid - luteal phase , and gonadotropins were administered after pituitary down regulation and continued up to hcg ( pregnyl , organon , netherland ) administration . for the flare - up protocol , gnrh - a was administered from the second day of the menstrual cycle and gonadotropins were administered from the third day of the menstrual cycle until hcg administration . oocyte retrieval was performed via the transvaginal approach with sonographic guidance 36 hr after 10,000 iu of hcg administration . the oocytes were incubated in human tubal fluid ( irvine scientific , irvine , ca . , u.s.a . ) medium supplemented with 10% synthetic serum supplement ( sss ; irvine scientific ) at 37 , 5% co2 in air . pregnancy was determined by serum -hcg levels above 5 miu / ml 12 days after the oocyte retrieval . two cycles out of 10 attempted ivf cycles were cancelled due to poor ovarian response during coh . table 1 shows the demographics for the five patients and the surgical findings . at the time of diagnosis with bot , the mean age of patients was 30.0 yr ( range , 24 - 40 ) , and four out of five patients were nulliparous . in three patients ( patient no . 2 , 4 , and 5 ) the diagnosis was made intraoperatively and staged as ia , ia , and iiic , respectively . in the remaining two patients ( patients no . 1 and 3 ) the diagnosis was made postoperatively without complete surgical staging . the microscopic findings in three patients showed the mucinous type , and the remaining two patients had the serous type . 2 underwent a right salpingoophorectomy ; disease recurrence occurred in the remaining ovary 9 yr after the initial diagnosis and the recurrence showed a histology and stage identical to the primary disease . 5 had right - side pelvic and paraaortic lymph node dissections , peritoneal cytology , omentectomy , and multiple peritoneal biopsies ; she was diagnosed with stage iiic disease . for the stromal microinvasion , the patient was treated with six cycles of taxol and cisplatin - based chemotherapy . at the time of the first ivf cycle the period of infertility after conservative treatment ranged from 17 to 45 months with a mean duration of 32.4 months . one cycle out of 2 attempted ivf cycles was cancelled due to poor ovarian response . because pregnancy was not achieved with her own ivf cycles , oocyte donation ( od ) was performed due to the decreased ovarian reserve . 3 had one cycle out of 4 attempted ivf cycles cancelled due to poor ovarian response . 4 had two pregnancies and delivered twice ; the first was after fresh embryo transfer and the second was after frozen - thawed embryo transfer . in 10 attempted ivf cycles , two cycles were cancelled with a 20.0% cycle cancellation rate . for eight ivf cycles , except the cancelled cycles , the mean serum estradiol level on hcg administration was 1,032.6 pg / ml ( range , 200 - 2,380 pg / ml ) . the mean number of retrieved oocytes was 5.6 ( range , 2 - 16 ) , and the mean fertilization rate was 74.4% ( range , 50.0 - 100.0% ) . the mean number of transferred embryos was 2.4 ( range , 1 - 4 ) . the cpr , ir , and lbr were 50.0% ( 4/8 cycles ) , 31.6% ( 6/19 ) , and 50.0% ( 4/8 cycles ) , respectively . there was one case of disease recurrence after conservative treatment ; this recurrence developed before ivf treatment . however , no recurrence was identified since the first ivf cycle ( table 3 ) . the follow - up period from initial diagnosis to the first ivf cycle ranged from 5 to 127 months with a mean duration of 33.0 months . since the first ivf cycle , the follow - up period ranged from 14 to 61 months with a mean duration of 29.6 months . patient no.2 had disease recurrence 108 months after the initial diagnosis and 19 months elapsed from the recurrence to the first ivf cycle . patient no.5 with stage iiic disease had no recurrence identified by exploration during cesarean section and continues to be followed up by a gynecological oncologist . since the prognosis for bot is excellent , patients of childbearing age can be treated with conservative surgery to preserve fertility ( 2 , 3 ) . unilateral adnexectomy is the optimal treatment in patients whose diagnosis of bot was made intraoperatively ; cystectomy can be considered in cases of recurrence in the remaining ovary . some patients are infertile after conservative treatment and request art in spite of the potential risk associated with infertility treatment . a history of infertility and the prior use of fertility medications have been associated with the development of ovarian tumors . recent case control studies showed that infertility per se elevates the overall risk of ovarian cancer ( 5 ) . reported an increased risk of ovarian tumors , both invasive and borderline , after the prolonged use ( > 12 cycles ) of clomiphene citrate ( 15 ) . shushan et al . reported increased incidence of epithelial ovarian tumors in patients with previous human menopausal gonadotropin ( hmg ) treatment compared to healthy controls ( 6 ) . however , the risk from ovarian hyperstimulation in patients treated for early stage bot is low . reported five pregnancies in 16 patients who subsequently underwent ivf after conservative treatment for bot and found no case of relapse during the follow - up period , 46 months on average ( 10 ) . beiner et al . suggested that art might be considered after the diagnosis of bot . recurrence occurred in 4 patients out of 7 who underwent ivf , two patients before and two patients after ivf treatment . all of the recurrences had histology identical to the initial diagnosis , borderline malignancy ( 16 ) . fasouliotis et al . reported 17 ivf cycles in five patients after conservative bot treatment . a mean of 7.9 oocytes were retrieved with a 57.1% fertilization rate , and a mean of 3.1 embryos were transferred . to date , there is no evidence in the literature to restrict the use of art in patients with early stage bot after conservative treatment . in our present study , the cpr and lbr for 7 ivf cycles in patients with early stage bot was 42.9% ( 3/7 cycles ) and 42.9% ( 3/7 cycle ) , respectively . the achieved pregnancy outcomes suggest that prior bot , diagnosis and treatment , have no perceptible negative impact on pregnancy outcomes in coh - ivf . therefore , coh - ivf can be safely offered to the patients with early stage bot . however , there are few reports on the safety of coh - ivf in cases of advanced stage bot after conservative treatment . seidman and kurman suggested that the existence of invasion in a peritoneal implant is a poor prognostic factor in patients with bot with peritoneal implants ; 16% had recurrence with a noninvasive implant , whereas 64% had recurrence with an invasive implant ( 18 ) . considering the poor prognosis of bot with invasive peritoneal implants , it seems logical to propose conservative treatment only in patients with bot who do not have invasive implants . at present however , it is not yet possible to provide guidelines for coh - ivf in patients with advanced stage bot despite successful pregnancy outcomes ( 11 - 13 , 19 ) . in our study one patient with stage iiic disease , without invasive peritoneal implant , had conservative treatment and underwent coh - ivf subsequently that resulted in a successful pregnancy and delivery . it seems that the number of coh - ivf cycles should be limited in patients with advanced stage bot because rapid progression to invasive ovarian cancer , after a successful delivery with the first ivf cycle has been reported ( 20 ) . although the pathogenic mechanisms for tumor progression remain unknown , it may be related to hormonal influences ( 9 ) . estrogen receptor expression was recently demonstrated in bot , and high serum e2 levels during coh - ivf may have a role in tumor promotion ( 10 ) . after conservative treatment of bot the recurrence rate is estimated to be 0 - 20% ( 21 ) . there is no significant difference in survival rates between conservative and radical treatment ( 22 , 23 ) . beiner et al . reported a 29% recurrence rate in an ivf treatment group that was not significantly different from the 19% recurrence in the non - ivf group ( 16 ) . all recurrences had a histology identical to the primary diagnosis ; they were conservatively treated without evidence of recurrence at the last follow - up ( 11 ) . in our study , no recurrence of bot was detected after coh - ivf ; this is a lower recurrence rate than reported in patients who underwent conservative treatment without subsequent fertility treatment . although the perod of 29.6 ( 14 - 61 ) months is not a long follow - up period , our results suggest that coh - ivf may not affect recurrence of bot , and that pregnancy per se has no effect on the course of bot . in conclusion , the current study suggests that ivf may be considered for infertile patients after conservative treatment for early stage bot . for patients with advanced stage bot , larger clinical trials with longer follow - up are necessary to evaluate the safety and efficacy of coh - ivf . all patients should be informed of the potential risks associated with ovarian hyperstimulation , and close follow - up is necessary after coh - ivf .

to evaluate the outcomes of controlled ovarian hyperstimulation ( coh)-in vitro fertilization ( ivf ) such as clinical pregnancy rate ( cpr ) , implantation rate ( ir ) and live birth rate ( lbr ) for infertile patients with borderline ovarian tumor ( bot ) after conservative treatment , 10 ivf cycles in five patients from january 1999 to july 2005 were analyzed . at the time of diagnosis with bot , the mean age of patients was 30.0 yr ( range , 22 - 40 ) . for 8 cycles out of 10 attempted ivf cycles , except for 2 cancellation cycles , the mean number of oocytes retrieved was 5.6 ( range , 2 - 16 ) with a mean fertilization rate of 74.4% . the cpr , ir , and lbr were 50.0% ( 4/8 cycles ) , 31.6% ( 6/19 ) and 50.0% ( 4/8 cycles ) respectively . the mean follow - up period after coh - ivf initiation was 29.6 ( range , 14 - 61 ) months . a gynecological oncologist followed all patients every 3 months during the first year and every 6 months thereafter . there was no recurrence during the follow - up period . our results suggest that coh - ivf may be acceptable for infertile patients with bot , especially in patients with early - stage bot after conservative treatment .

INTRODUCTION MATERIALS AND METHODS Patients COH-IVF and embryo transfer RESULTS DISCUSSION

the formation of long lasting memories appears to depend upon enduring changes in the strength of neurotransmission that alters cellular mechanisms thus reconfiguring neural circuitry and communication [ 16 ] . this review describes the relationship among extracellular matrix ( ecm ) molecules , cell adhesion molecules ( cams ) , matrix metalloproteinases ( mmps ) , and tissue inhibitors of matrix metalloproteinases ( timps ) in making possible the phenomena of long - term potentiation ( ltp ) , habituation , associative learning and memory , and perhaps drug addiction . the ecm is composed of secreted glycoproteins and proteoglycons that form scaffolding to which cells adhere . within the central nervous system this network consists of the proteins fibronectin , laminin , vitronectin , thrombospondin , tenascin , and collagen iv [ 713 ] . in addition to providing a network of scaffolding the ecm is involved in a wide range of signaling that influences cellular proliferation , growth , movement , synaptic stabilization , and apoptosis . it is now believed that these ecm molecules assist in maintaining and changing the synaptic architecture critical to neural plasticity which is believed to mediate learning and memory . these findings were anticipated by cajal more than a century ago when he hypothesized that memory storage is dependent upon alterations in synaptic connections between neurons . the interaction of cells and ecm molecules is facilitated by cell adhesion molecules ( cams ) . these molecules are cell surface macromolecules that dictate cell - to - cell and cell - to - ecm contacts by using the processes of adhesion , migration , neurite outgrowth , fasciculation , synaptogenesis , and intracellular signaling [ 8 , 15 , 16 ] . the extracellular domain of cams are targets for proteinase activity ; while their intracellular domains interact with cytoskeletal proteins . cams are functionally categorized into calcium - dependent ( integrins and cadherins ) and calcium - independent ( immunoglobulins and selectins ) proteins . integrin receptors are widely distributed dimeric transmembrane proteins with an extracellular portion that interacts with ecm molecules and cell surface proteins , and an intracellular portion that makes contact with the actin cytoskeleton via intermediate proteins such as -actinin , talin , tensin , and vinculin . thus , the binding of a ligand to the integrin receptor results in a functional link between the ecm and the actin cytoskeleton which is mediated through these intermediate proteins . these proteins trigger intracellular signaling pathways that can initiate changes in cellular shape , motility , growth , gene regulation , and apoptosis [ 17 , 18 ] . it appears that integrins are very important regarding cell - to - ecm substrate adhesion ; while cadherins , syndecans , and neural cell adhesion molecules are primarily involved with cell - to - cell adhesion . each of these cams appears to contribute to neural plasticity as related to memory formation . for additional details the reader is referred to the following excellent reviews concerning ecm molecules and cams [ 713 ] . mmps are a family of proteolytic enzymes involved with the maintenance and restructuring of the ecm [ 1921 ] . at present 25 + mmps have been identified under four major categories : collagenases , gelatinases , membrane - type , and stromelysins ( table 1 ) . many mmps require serine proteinases , such as plasmin or other mmps , for activation . a pro - peptide must be cleaved in order to reveal the catalytic domain of the mmp . mmp degradation of the ecm is tightly controlled and accomplished by three mechanisms : ( 1 ) regulation of gene transcription ; ( 2 ) regulation of pro - enzyme activation ; and ( 3 ) through the presence of timps . most mmps are nonconstituitively expressed ; however gene transcription may occur via stimulation by growth factors , oncogene products , phorbol esters , as well as cell - to - cell and cell - to - ecm interactions . these stimuli typically provoke various transcription factors including members of the c - fos and c - jun proto - oncogene families , resulting in the formation of homo- and hetero - dymeric forms of ap-1 transcription factors . such activation of mmp genes requires the combined effects of ap-1 protein and other transcription factors ( see [ 24 , 25 ] for reviews ) . at the outset mmps are maintained as inactive pro - mmp zymogenes and as such the catalytic zinc atom is bound to the cysteine residue of the pro - peptide region ( figure 1 ) . this action exposes an intermediate form of mmp capable of cleaving the pro - peptide region via autocatalysis yielding full enzymatic activity . mmp activation factors include kallikrein , plasmin , thrombin , and the tissue - type ( tpa ) and urokinase - type ( upa ) plasminogen activators , plus other mmps [ 27 , 28 ] . for example , mmp-2 , mmp-3 , and membrane - type mmps ( mt - mmps ) activate mmp-1 and mmp-9 , while mt - mmps can be activated by inhibitory pro - peptide removal , specifically accomplished by furin , also a serine protease . such characteristics of mmps make them attractive concerning their potential contribution to memory consolidation , reconsolidation , and retrieval . mmp-2 , mmp-3 , and mmp-9 reach measurable levels in the mammalian brain especially if the animal is challenged with a change in its environment ( e.g. , handling , learning tasks , lesioning , seizure ) . these mmps are also elevated in several pathologies [ 30 , 31 ] including alzheimer 's disease [ 22 , 3235 ] , and multiple sclerosis [ 22 , 3640 ] . there is accumulating evidence that mmps are essential for tumor metastasis , and cell invasion [ 9 , 19 , 24 , 41 , 42 ] . mmps are also activated during stress , brain trauma , and ischemia [ 22 , 4447 ] . for a thoughtful and informative review concerning the potential use of mmp inhibitors to treat neurodegenerative diseases see rosenberg . as mentioned above , mmps are involved in axon extension , and the control of axon guidance of receptors on the cell surface via regulated catalysis of ectodomain shedding . along these lines , the secretion of mmps by the growth cone appears to result in the laying down of a pathway through the ecm . mmps are also involved in the myelination of axons in both central and peripheral nervous systems during development and following damage from injury or disease . as with neurons , oligodendrocytes secrete mmps at the distal cell process . it appears that these mmps are also involved in clearing a path through ecm molecules permitting the growing glial tip to extend . mmp-9 and -12 null mice exhibit retarded myelination and the number of mature oligodendrocytes is reduced . increases in mmp-9 expression have been correlated with myelination of the mouse corpus callosum during postnatal development . tissue inhibitors of metalloproteinases 1 - 4 ( timp-1 - 4 ) make up a family of secreted glycoproteins ( table 1 ) . timps inhibit the proteolytic activities of mmps via the formation of tight noncovalent complexes with them [ 55 , 56 ] . timps are two - domain proteins linked by three disulfide bonds with three disulfides per domain . it appears that timps bind mmps at a 1 : 1 ratio such that when in balance the expression of timps matches that of mmps . the disruption of this timp / mmp balance impacts cns ecm - to - cell and cell - to - cell signaling . for example , timp-1 deficient mice fail to acquire an odor conditioned learning task , suggesting a dysfunction of hippocampal neuronal plasticity . subsequently , elevated timp-1 mrna and protein were measured in the hippocampus with seizure [ 61 , 62 ] . kainate - induced seizures also elevated mmp-9 mrna expression and protein within a few hours . this enhanced mmp-9 mrna expression was seen in both the dendritic layers and neuronal cell bodies primarily within the dentate gyrus . these results were interpreted to suggest that mmp-9 expression is involved in activity - dependent remodeling via influencing synaptic connections . shibayama et al . , and others [ 45 , 65 ] , have shown that following mechanical brain injury mmps , and particularly timps , are produced by microglia and astrocytes located in cortex and white matter and may play a role in neural regeneration ( or lack of ) depending upon the degree of expression and the time since injury . although our understanding of the mechanism(s ) underlying the functional remodeling of synaptic pathways remains incomplete , it is becoming clear that such reconfiguration involves alterations in the levels of mmps and timps . long - term potentiation was originally discovered in the anesthetized rabbit preparation by bliss and lomo , and then a similar electrophysiological approach was used to confirm ltp in the unanesthetized rabbit . a tetanization electrode was placed in the perforant path and a recording electrode was positioned in the dentate area . excitatory post - synaptic potentials could be progressively enhanced by short bursts of electrical stimulation applied via the perforant path electrode . ltp is now thought to represent a basic physiological mechanism of memory storage [ 6871 ] ; however others suggest that it may represent an arousal / attention mechanism . investigators subsequent to bliss and colleagues demonstrated that hippocampal ltp is , at least in part , dependent upon intact n - methyl - d - aspartate ( nmda ) receptors [ 7375 ] . the application of nmda receptor antagonists has been shown to prevent ltp and interfere with the successful performance of memory tasks mediated by the hippocampus [ 74 , 7678 ] ; however , nmda - independent ltp has been demonstrated by a number of investigators ( see [ 72 , 79 ] for reviews ) . additional studies have revealed that activation of calpain [ 8082 ] , protein kinase c [ 83 , 84 ] , calcium - calmodulin kinase type 2 [ 85 , 86 ] , and the release of ca from intracellular storage pools also contribute to hippocampal ltp . further , there is evidence that ltp may be dependent upon the release of sufficient gaba to activate gabab autoreceptors , which in turn prevents further gaba release . one form is based on the nmda receptor system which can be blocked with the nmda receptor antagonist mk-801 . the other type of ltp is dependent upon voltage - dependent calcium channels ( vdcc ) and can be blocked with the vdcc blocker verapamil . both nmda- and vdcc - ltp appear to occur during tetanus - induced ltp . further , the argument is made that a functional nmda system can mediate learning and memory for several hours ; however , the activation of the vdcc - ltp system is required for longer periods , that is , over several days . hippocampal slice cultures taken from mmp-9 knockout mice revealed impaired ltp which was restored with the addition of recombinant mmp-9 . this potential could be inhibited by blocking integrin signaling , suggesting that mmp-9 may mediate neural plasticity via integrins . using prefrontal cortex slices okulski and colleagues reported that mmp-9 is necessary for late stage ltp , and treatment with an mmp-9 inhibitor prevented the formation of late - stage ltp . . found that spine enlargement during hippocampal ltp is dependent upon mmp-9 and protein synthesis . describing unilateral lesions of the entorhinal cortex in rats followed by intracerebroventricular ( icv ) infusion of a general mmp inhibitor ( fn-439 ) . after 7 days control rats that received icv saline following lesioning revealed normal collateral sprouting , synaptogenesis , and ltp . in contrast , those rats that received icv fn-439 lost the capacity to exhibit ltp and evidenced considerable cellular debris , suggesting that mmps are a necessary component of the deafferentiation and sprouting phenomena . our laboratory has also measured impaired paired - pulse facilitation , induction and stability of ltp , and long - term depression ( ltd ) in hippocampal slices treated with fn-439 [ 97 , 98 ] . the schaffer collateral commissural projection was stimulated while field epsps were recorded from area ca1 striatum radiatum . pressure infusion of recombinant - active mmp-9 ( rmmp-9 ) into the ca1 area produced a slow , but progressive potentiation reaching maximum by 90120 minutes post - administration and remained elevated until the experiment ended at 180 minutes . it was determined that this enhancement in synaptic potentiation was not presynaptic , and once maximum potentiation to mmp-9 was achieved , the application of tetanic stimulation failed to further increase potentiation . the authors interpreted these results to indicate that tetanic stimulation , and rmmp-9 activation , share a common cellular mechanism . the intrahippocampal infusion of an mmp-2 and -9 inhibitor followed by titanic stimulation resulted in a strong potentiation comparable to control ltp . it was further determined that titanic stimulation resulted in elevated mmp-9 protein levels in the ca1 area . thus , these results indicate that mmp-9 mediated extracellular proteolysis is involved in the phenomenon of ltp in normal young adult animals . taken together , these findings support an important role for mmps in ltp and indicate that in particular mmp-9 is a necessary component in supporting the stabilization of the maintenance phase of ltp . nonassociative learning includes the phenomena of habituation , dishabituation , and sensitization and is considered to be the simplest form of learning . of these habituation is the most frequently studied and refers to a decrease in responding ( as related to frequency , magnitude , or intensity ) to a stimulus repeatedly presented , or presented for a prolonged period of time [ 99101 ] . habituation has been documented across many species and response systems ranging from the gill - withdrawal reflex in aplysia and tap withdrawal or chemotaxic response in the nematode caenorhabditis elegans , to acoustic startle response in rats and mice , schedules of reinforcement in operant conditioning [ 105 , 106 ] and feeding in humans . although the neural mechanism(s ) underlying habituation has not been identified , the hippocampus has been implicated in the control of inhibitory processes , particularly habituation [ 108110 ] . in support of this notion bilateral hippocampectomy in rats has been shown to interfere with habituation to familiar objects in an open field object recognition task [ 111 , 112 ] , severely impair the acquisition and recall of platform location in the morris water maze task , but failed to alter the habituatory pattern or rate of head - shake response ( hsr ) . the hsr consists of a rapid rotation of the head about the anterior to posterior axis in response to a mild air stimulus applied to the ear . this response follows a remarkably predictable decreasing negatively accelerated function of stimulus frequency ( figure 2 ) . our laboratory has measured hsr habituation - induced increases in mmp-3 expression in the hippocampus , prefrontal , and piriform cortices , with no change in the cerebellum . elevations in hippocampal mmp-9 activity were also measured in these habituated animals accompanied by decreases in the prefrontal cortex . to our surprise yoked control rats , introduced to the test environment but not hsr habituated , also revealed intermediate elevations in mmp-3 expression in the hippocampus and piriform cortices as compared with habituated and home cage control rats . these results suggested that elevations in mmp-3 could mediate the changes in neural plasticity that may accompany habituation ; however the introduction of the animal into a new environment also appeared to elevate mmp-3 expression in these same brain structures , but to a lesser extent . these changes in mmp-3 levels were evidenced by the yoked control animals despite efforts to minimize environmental cues ( i.e. , low ambient light and suppressed extraneous noise in a room painted black ) . given that acquisition of such spatial cues is mediated by hippocampal and prefrontal cortices ( see [ 116 , 117 ] for reviews ) it is perhaps not surprising that elevations in mmp expression were measured in these structures . however , habituation to irrelevant spatial cues is clearly an important aspect of successful performance in an associative learning task , and this too appears to be a function of the hippocampus and prefrontal cortex [ 108110 ] . as outlined above it is assumed that neural activity - dependent changes in synaptic adhesion underlie the morphological and functional plasticity of those synapses involved in learning and memory [ 118 , 119 ] . alterations in intrasynaptic ecm molecules , as influenced by cams , are presumed to be responsible for alterations in the synaptic architecture , and thus the efficiency of synaptic transmission [ 120124 ] , and to underlie neural plasticity and memory consolidation [ 125 , 126 ] . given that mmps are responsible for degrading and restructuring the ecm it is not surprising that they have been investigated with regard to seizure , associative learning , and memory . mmp-9 levels and activity increase in the hippocampus following kainic acid- and bicuculline - induced seizures [ 63 , 127129 ] and are correlated with subsequent synapse formation . in addition , mmps are known to play an important role in synaptic remodeling that results from hippocampal differentiation [ 130 , 131 ] . our laboratory noted mmp-9 elevations in the prefrontal and piriform cortices of rats tested in an object recognition task , and in the prefrontal and hippocampal cortices of rats that were successful in solving the morris water maze task . these results were confirmed and extended by meighan et al . who noted significant elevations in hippocampal mmp-3 and -9 during acquisition of the morris water maze task . the inhibition of mmp activity with mmp-3 and -9 antisense oligonucleotides , or fn-439 prevented successful performance of this task . the ability to acquire this spatial memory task was shown to result in the differential stability of cortactin , an actin - binding protein involved in regulating the dendritic cytoskeleton and synaptic efficiency . nagy et al . have reported significant elevations in hippocampal mmp-9 levels following inhibition avoidance learning in rats , peaking at 1224 hours following training and declining to baseline by three days post - training . intrahippocampal infusion of a mmp-2 and -9 inhibitor 3.5 hours following inhibitory avoidance training significantly diminished subsequent recall . similar results were obtained with the bilateral intra - hippocampal infusion of fn-439 resulting in significant interference with the acquisition of the morris water maze task . olson et al . have measured elevations in hippocampal mmp-3 beginning 1 hour following passive avoidance training in rats and returning to baselevel by 24 hours post - training . when a specific mmp-3 inhibitor was icv infused 20 minutes prior to , and 50 minutes following training , dose - dependent learning deficits were seen . finally , brown et al . found that icv infusion of fn-439 30 minutes prior to fear conditioning ( tone - foot shock paired association ) , or 30 minutes prior to a single retest session 24 hours after conditioning , disrupted successful memory retrieval of the conditioned freezing - in - place response . this reduction in freezing was not due to a decrease in overall anxiety level given that fn-439 failed to influence normal elevated plus - maze task performance . recently , we combined hsr habituation with a classical conditioning paradigm to evaluate the importance of a signaling cue that immediately preceded the onset of the air stimulus to the ear . bilateral dorsal hippocampus injections of fn-439 , or a specific mmp-3 inhibitor , interfered with acquisition of the association between a signaling tone and the hsr such that only a very weak association was present when retested 24 hours later ( figure 3 ) . these results suggest that a functioning dorsal hippocampus is critical to storage of this classically conditioned association between the signaling cue and the air stimulus to the ear that initiates the hsr . specifically , interference with activation of mmp-3 in the dorsal hippocampus appears to significantly disrupt the acquisition and memory storage of this association . there is accumulating evidence to support the notion that mmp-3 and -9 are of significant importance in the acquisition of several forms of associative learning including object recognition , spatial , passive avoidance and classical conditioning . learning and memory appear to be intimately involved in the process of drug addiction [ 137141 ] . changes in neuron morphology during and following drug addiction have been reported [ 142145 ] . to date only a few studies have focused on changes in ecm molecules accompanying drug addiction ( see [ 146 , 147 ] for reviews ) . brown and colleagues reported that icv injection of fn-439 suppressed acquisition of cocaine - induced conditioned place preference ( cpp ) in rats . this general mmp inhibitor also attenuated cocaine - primed reinstatement in extinguished animals . in agreement with these findings mash et al . have compared patterns of gene expression in human chronic cocaine abusers with drug - free control subjects . the cocaine abusers revealed 151 gene transcripts up - regulated and 91 down - regulated . one up - regulated transcript was reck , a membrane - anchored mmp inhibitor associated with angiogenesis and ecm integrity . these investigators speculated that hippocampal ecm remodeling ( or lack of ) may characterize chronic cocaine abuse and contribute to relapse . these researchers are the first to indicate an important role for mmps in the acquisition and reconsolidation of memories associated with cocaine addiction . brown et al . have also suggested that mmp inhibitors may be useful in disrupting an established cocaine - induced memory in that memory reconsolidation could be suppressed . most recently these investigators have shown that mmp-9 increased in the prefrontal cortex following cocaine reinstatement of cpp in rats . [ 150 , 151 ] used an mmp-2 and -9 inhibitor to prevent methamphetamine - induced cpp in mice . they further showed that mmp-2 and -9 deficient mice displayed attenuated sensitization and cocaine cpp when methamphetamine - primed . liu et al . have further reported that with both stimulant or toxic doses of methamphetamine brain mmp-9 gene expression was up - regulated within 5 minutes . by 24 hours mmp-9 up - regulation had returned to control levels in the stimulant treated mice but was still elevated in those mice that received the higher toxic dose . mmp-9 knockout mice were capable of evidencing methamphetamine - induced neurotoxicity suggesting that mmp-9 expression is not a contributor to the neurotoxicity . some years ago sillanaukee et al . compared serum mmp-9 levels of middle age male alcoholics ( > 1000 g / week ) and male social drinkers ( < 200 g / week ) in an attempt to identify a mechanism underlying alcohol - induced cardiovascular disease . these results are important given recent evidence that alcohol treatment not only increased mmp-1 , -2 , and -9 activity and decreased timp-1 and -2 , but also increased blood - brain barrier permeability . these researchers suggested that the elevations in mmp could be responsible for basement membrane degradation leading to a reduction in barrier tightness . our laboratory has established a relationship between ethanol - induced impairment of spatial memory ( morris water maze task ) and decreased mmp-9 levels in the hippocampus and prefrontal cortex in rats tested over a period of several days . presumably these ethanol - induced declines in active mmp-9 levels attenuated the formation of new neural pathways thus interfering with memory consolidation . these findings suggest that deviations in brain mmp activity may be prerequisite to reconfiguration of the ecm molecules that permit synaptic reconfiguration and the establishment of new memories . this appears to hold for memories associated with , and in support of , addictive drugs as well . this review brings together available information concerning the hypothesis that it is the interaction among ecm molecules , mmps , cams , and timps that permits the formation of new neural pathways in the brain . these new synaptic connections are stimulated by experiences in environments that result in learning acquisition and memory consolidation . thus , memory consolidation is presumably mediated and made possible by the process of neural plasticity . however , a number of research questions must be addressed in order for this important area of research to move forward . ( 1 ) there is accumulating evidence that ltp triggers the synthesis , release , and activation of proteases , particularly mmps . much of this work has been completed in the hippocampus , dentate gyrus , and entorhinal cortex . ( 2 ) once these synaptic connections are formed how are they maintained and protected from degradation ? ( 3 ) following memory consolidation how is this information retrieved without re - triggering synaptic reconfiguration ? ( 4 ) with the retrieval of information how is the process of short - term memory acquisition terminated such that the new memory trace can be reconsolidated and placed back into a fixed configuration ? ( 5 ) important environmental information appears to be temporarily stored in the hippocampus and then transferred to other brain structures for long - term storage . are the same molecules ( ecm , cams , mmps , timps ) involved in this transfer process ? does the ultimate storage location depend upon the type of learning and/or the sensory systems involved ? ( 6 ) what is the role of neural plasticity in drug addiction ? there are many unanswered questions regarding the influence of drugs on ltp stimulated mmp release and activation , and equally important the role of timps during ltp . beyond these issues there are additional questions regarding the influence of drug addiction on neural plasticity and memory consolidation in the hippocampus , neocortex , and amygdala as well as other brain structures . comprehensive answers to these and related questions will require significant effort but once available should provide valuable clues concerning the basic processes of memory formation and will contribute to our understanding of how failures in memory acquisition , storage , and retrieval occur . hopefully , this insight will result in clinical interventions designed to correct these deficiencies in dysfunctional memory disease states and also provide new treatment strategies for preventing drug addiction and relapse .

the premise of this paper is that increased expression of matrix metalloproteinases ( mmps ) permits the reconfiguration of synaptic connections ( i.e. , neural plasticity ) by degrading cell adhesion molecules ( cams ) designed to provide stability to those extracellular matrix ( ecm ) proteins that form scaffolding supporting neurons and glia . it is presumed that while these ecm proteins are weakened , and/or detached , synaptic connections can form resulting in new neural pathways . tissue inhibitors of metalloproteinases ( timps ) are designed to deactivate mmps permitting the reestablishment of cams , thus returning the system to a reasonably fixed state . this review considers available findings concerning the roles of mmps and timps in reorganizing ecm proteins thus facilitating the neural plasticity underlying long - term potentiation ( ltp ) , habituation , and associative learning . we conclude with a consideration of the influence of these phenomena on drug addiction , given that these same processes may be instrumental in the formation of addiction and subsequent relapse . however , our knowledge concerning the precise spatial and temporal relationships among the mechanisms of neural plasticity , habituation , associative learning , and memory consolidation is far from complete and the possibility that these phenomena mediate drug addiction is a new direction of research .

1. Introduction 2. MMPs and TIMPs 3. Categories Of Learning 4. Addictive Drugs 5. Conclusion

place the aluminum heat sink inside the 22.96 cm ( 9 " ) pie pan approximately 2 cm from the side of the pan , as shown in figure 1c . using a black marker pen , mark holes for the aluminum heat sink . note : if the aluminum block ( purchased at a local scrap metal shop or online at http://www.onlinemetals.com ) does not come with pre - drilled holes , you will need to arrange to have holes drilled and countersunk prior to step 1 to secure the aluminum block to the pie pan . we used 5 holes to secure the block to the platform and 4 extra holes to allow nitrogen gas bubbles to escape from under the aluminum . place the cryo - grid box near the location of the aluminum heat sink and mark the notch and each side . drill pilot holes for each marked position using a # 17 drill bit for the aluminum heat sink holes and a # 35 drill bit for the cryo - grid box holes . mount the aluminum heat sink using 5 - # 10 , 1.9 cm ( 3/4 " ) flat head slotted bolts and corresponding nuts as well as 15 - # 10 washers . place two washers beneath the aluminum block and the third on the backside of the pie pan for each hole . note : unsecured overhangs of the aluminum block near the viewing area can result in vibrations that limit imaging capabilities of the stage . insert 3 - # 4 , 0.95 cm ( 3/8 " ) round slotted bolts and corresponding nuts from the backside of pie pan to act as the cryo - grid box mount . wet the top and bottom surface of the cake and pie pans respectively with ddh2o . flip the pans upside down and press on the cake pan until the spacers contact the pie pan . place any weighted object atop the cake pan and let sit overnight to cure . with a serrated knife , cut away the excess dried insulating spray foam from the edge of the pans and remove the chopsticks . remove the cake pan from the pie pan . the pie pan with aluminum heat sink is now insulated and will be referred to as the " cryogenic stage " ( fig 1a ) . place a transparent plastic clipboard ( any color and greater than 3 mm thick ) over the top of the cryogenic stage . mark with a black marker the location of the cryo - grid box mount by drawing a circle approximately 1.5 times the diameter of a single round cryo - grid box . this clipboard will be referred to as the " loading screen " ( fig 1b ) . put a new transparent clipboard atop of the cryogenic stage and place on the microscope with the heat sink directly under the objective lenses . with a black marker , cut along the marked line removing a half circle from the edge of the clipboard . this can be accomplished with a jigsaw or using a 7.62 cm ( 3 " ) hole saw multiple times as shown in the video . finally cut a 1.9 cm ( 3/4 " ) hole away from the half circle but still within the area of the dish . this will be the port for refilling liquid nitrogen ( ln2 ) if levels drop while imaging . this clipboard will be referred to as the " viewing screen " ( fig 1c ) . dilute log phase yeast cells grown in synthetic complete ( sc ) media to an appropriate concentration of 1x10 cells / ml in water . 2 ml of the diluted yeast are pipetted onto a r2/1 400 mesh holey carbon coated copper cryo - em grid ( spi supplies , west chester , pa ) and allowed to adsorb for 15 seconds . blot away excess liquid from the grid surface by touching the back of the grid to a torn piece of soft cellulose tissue ( kimwipe ) for 2 seconds . if necessary , the grid can be washed with 3 l drops of water ( 1 - 3 times ) , and wicked away as in step 3 by blotting from the back . before the final wash , the sample is loaded into the pneumatic plunge freezer and the hanging grid is washed as in step 2.4 . after blotting the grid with torn tissue paper ( kimwipe ) to remove the majority of the water from the surface , the grid is plunged into ln2 cooled liquid ethane and transferred to a cryo - grid box for storage . note : it is important to leave a layer of liquid as thin as possible on the surface to keep the sample hydrated . improper blotting will either dry out the sample or leave ice that is opaque to the electron beam . fill the cryogenic stage with ln2 and quickly cover with the loading screen ( plastic clipboard with single 1.9 cm ( 3/4 " ) hole ) . once the metal reaches ln2 temperature , transfer the cryo - grid box through the 1.9 cm ( 3/4 " ) hole of the loading screen and into the transfer mount created by the 3 screws described in part a5 . unscrew the cryo - grid box from its holder and remove the holder for improved access . keep the cryo - grid box holder under ln2 in a separate dewar until step 3.10 . the cryo - grid box should also be kept under ln2 to prevent sample grid warming . refill ln2 in the cryogenic stage to just below the top of the aluminum heat sink . note : ln2 levels must be periodically checked and refilled during the imaging process to ensure ln2 is continuously in contact with the heat sink . in general , the refilling occurs every 10 minutes through the fill port in the viewing screen . pre - cool the tweezer tip in ln2 , then transfer your grid(s ) onto the flat viewing surface of the aluminum heat sink with tweezers , using the 1.9 cm ( 3/4 " ) hole of the loading screen . carefully move the cryo - stage to beneath the reflected light microscope s objective apertures . all air currents around the microscope should be minimized , including : breathing , a nearby door opening , people walking past , etc . we suggest wearing a facemask or hanging a transparent face shield below the microscope eyepieces as a precautionary measure . rotate the objective lenses into the cold nitrogen gas environment of the cryo - stage and scan for the sample on the flat heat sink viewing area . using standard bright field light microscopy techniques focus on the grid with a low magnification objective to find the center and take an overview image . specifications regarding the light microscopy lenses used for this experiment are listed in the experimental materials section . note : ln2 does not come in contact with the objective lenses and we have yet to see degradation of images or damage to the lenses from the effects of cooling . focus in high magnification on an area of interest and acquire data with bright field , dark field , polarized light or fluorescence imaging . be sure to record the location of the area of interest on the low magnification bright field image for future correlation . if all precautions listed above for refilling the ln2 are followed , then the positive pressure from the evaporating nitrogen is sufficient to maintain a contamination free environment ( independent of room humidity ) for the duration of imaging . once finished , replace the loading screen by sliding it over the top of the viewing screen . remove the cryo stage from the microscope and with pre - cooled tweezers , transfer the grid back to the cryo - grid box . screw the cryo - grid box holder into cryo - grid box to seal against the environment and transfer the holder to a ln2 dewar for storage and future imaging . following standard and established techniques , load the sample grid into a cryo - em transfer holder while keeping the specimen at liquid nitrogen temperature at all times . insert the cryo - holder with sample grid into a transmission electron microscope . in a suitable low magnification view ( 50 - 500x nominal magnification ) , find the center of the sample grid . use the previously collected low magnification cryo - lm data from step 3.7 to determine the relative orientation of the grid s central copper tabs ( as described in figure 2 ) and identify exact areas of interest for correlation . proceed with microscope alignment , low - dose cryo - em imaging and data collection . the cryogenic stage ( fig 1a ) is an effective way to gather cryo - lm data for cryo - fluorescence microscopy and correlative cryo - lm / cryo - em analysis . figure 2 shows how the combination of low and high magnification cryo - lm images allows you to build reference maps that direct you to specific areas in cryo - em . the resulting cryo - lm reference map ( fig 2b ) was utilized during cryo - em data acquisition to locate exact regions shown in figure 3 . a ) the layout of the cryogenic stage consists of a cryo - grid box transfer mount indicated with a white arrow and an aluminum heat sink . grids are transferred under ln2 from the cryo - grid box and placed directly on the heat sink s viewing area as indicated by the black arrow . the hole in the loading screen is placed directly over the cryo - grid box mount and acts as a port to transfer samples and for moving samples from the cryo - grid box to the sample viewing area on the heat sink with tweezers . c ) the cryogenic stage in position under the objective lenses with the viewing screen in place . the special cutout on the viewing screen allows the objective lenses to easily swing into position without moving the cryogenic stage . the hole in the viewing screen away from the sample area acts as a ln2 fill port to replenish ln2 levels if necessary . b ) the same image in ( a ) overlaid with a high magnification fluorescence image of an area of interest and with a filled orange polygon marking the center of the sample grid . the center consists of a group of four squares , three having an extra metal tab and the forth open . for the three squares with an extra metal tab , two pairs share the tab about the long and short axes forming an asymmetric center . this asymmetric center can be seen in the upper left corner and was used to indicate the rotation angle and handedness of the grid between cryo - lm and cryo - em . from one low magnification image many areas of interest can be marked and used as a reference map to locate identical areas in cryo - em . c ) a magnified fluorescence cryo - lm image of the area of interest in ( b ) . hta1-cfp , a c - terminal cfp histone marker , can be seen as a green punctate structure labeling the location of the nucleus . d ) a cryo - em image of the corresponding area in ( c ) . two fields of view depicting identical yeast cells correlated with cryo - bright field ( a , d ) , cryo - fluorescence ( b , e ) , and cryo - em ( c , f ) . place the aluminum heat sink inside the 22.96 cm ( 9 " ) pie pan approximately 2 cm from the side of the pan , as shown in figure 1c . using a black marker pen , mark holes for the aluminum heat sink . note : if the aluminum block ( purchased at a local scrap metal shop or online at http://www.onlinemetals.com ) does not come with pre - drilled holes , you will need to arrange to have holes drilled and countersunk prior to step 1 to secure the aluminum block to the pie pan . we used 5 holes to secure the block to the platform and 4 extra holes to allow nitrogen gas bubbles to escape from under the aluminum . place the cryo - grid box near the location of the aluminum heat sink and mark the notch and each side . drill pilot holes for each marked position using a # 17 drill bit for the aluminum heat sink holes and a # 35 drill bit for the cryo - grid box holes . mount the aluminum heat sink using 5 - # 10 , 1.9 cm ( 3/4 " ) flat head slotted bolts and corresponding nuts as well as 15 - # 10 washers . place two washers beneath the aluminum block and the third on the backside of the pie pan for each hole . note : unsecured overhangs of the aluminum block near the viewing area can result in vibrations that limit imaging capabilities of the stage . insert 3 - # 4 , 0.95 cm ( 3/8 " ) round slotted bolts and corresponding nuts from the backside of pie pan to act as the cryo - grid box mount . wet the top and bottom surface of the cake and pie pans respectively with ddh2o . flip the pans upside down and press on the cake pan until the spacers contact the pie pan . place any weighted object atop the cake pan and let sit overnight to cure . with a serrated knife , cut away the excess dried insulating spray foam from the edge of the pans and remove the chopsticks . remove the cake pan from the pie pan . the pie pan with aluminum heat sink is now insulated and will be referred to as the " cryogenic stage " ( fig 1a ) . place a transparent plastic clipboard ( any color and greater than 3 mm thick ) over the top of the cryogenic stage . mark with a black marker the location of the cryo - grid box mount by drawing a circle approximately 1.5 times the diameter of a single round cryo - grid box . this clipboard will be referred to as the " loading screen " ( fig 1b ) . put a new transparent clipboard atop of the cryogenic stage and place on the microscope with the heat sink directly under the objective lenses . with a black marker , cut along the marked line removing a half circle from the edge of the clipboard . this can be accomplished with a jigsaw or using a 7.62 cm ( 3 " ) hole saw multiple times as shown in the video . finally cut a 1.9 cm ( 3/4 " ) hole away from the half circle but still within the area of the dish . this will be the port for refilling liquid nitrogen ( ln2 ) if levels drop while imaging . this clipboard will be referred to as the " viewing screen " ( fig 1c ) . dilute log phase yeast cells grown in synthetic complete ( sc ) media to an appropriate concentration of 1x10 cells / ml in water . 2 ml of the diluted yeast are pipetted onto a r2/1 400 mesh holey carbon coated copper cryo - em grid ( spi supplies , west chester , pa ) and allowed to adsorb for 15 seconds . blot away excess liquid from the grid surface by touching the back of the grid to a torn piece of soft cellulose tissue ( kimwipe ) for 2 seconds . if necessary , the grid can be washed with 3 l drops of water ( 1 - 3 times ) , and wicked away as in step 3 by blotting from the back . before the final wash , the sample is loaded into the pneumatic plunge freezer and the hanging grid is washed as in step 2.4 . after blotting the grid with torn tissue paper ( kimwipe ) to remove the majority of the water from the surface , the grid is plunged into ln2 cooled liquid ethane and transferred to a cryo - grid box for storage . note : it is important to leave a layer of liquid as thin as possible on the surface to keep the sample hydrated . improper blotting will either dry out the sample or leave ice that is opaque to the electron beam . fill the cryogenic stage with ln2 and quickly cover with the loading screen ( plastic clipboard with single 1.9 cm ( 3/4 " ) hole ) . once the metal reaches ln2 temperature , transfer the cryo - grid box through the 1.9 cm ( 3/4 " ) hole of the loading screen and into the transfer mount created by the 3 screws described in part a5 . unscrew the cryo - grid box from its holder and remove the holder for improved access . keep the cryo - grid box holder under ln2 in a separate dewar until step 3.10 . the cryo - grid box should also be kept under ln2 to prevent sample grid warming . refill ln2 in the cryogenic stage to just below the top of the aluminum heat sink . note : ln2 levels must be periodically checked and refilled during the imaging process to ensure ln2 is continuously in contact with the heat sink . in general , the refilling occurs every 10 minutes through the fill port in the viewing screen . pre - cool the tweezer tip in ln2 , then transfer your grid(s ) onto the flat viewing surface of the aluminum heat sink with tweezers , using the 1.9 cm ( 3/4 " ) hole of the loading screen . carefully move the cryo - stage to beneath the reflected light microscope s objective apertures . all air currents around the microscope should be minimized , including : breathing , a nearby door opening , people walking past , etc . we suggest wearing a facemask or hanging a transparent face shield below the microscope eyepieces as a precautionary measure . rotate the objective lenses into the cold nitrogen gas environment of the cryo - stage and scan for the sample on the flat heat sink viewing area . using standard bright field light microscopy techniques focus on the grid with a low magnification objective to find the center and take an overview image . specifications regarding the light microscopy lenses used for this experiment are listed in the experimental materials section . note : ln2 does not come in contact with the objective lenses and we have yet to see degradation of images or damage to the lenses from the effects of cooling . focus in high magnification on an area of interest and acquire data with bright field , dark field , polarized light or fluorescence imaging . be sure to record the location of the area of interest on the low magnification bright field image for future correlation . if all precautions listed above for refilling the ln2 are followed , then the positive pressure from the evaporating nitrogen is sufficient to maintain a contamination free environment ( independent of room humidity ) for the duration of imaging . once finished , replace the loading screen by sliding it over the top of the viewing screen . remove the viewing screen by slowing sliding it beneath the loading screen . remove the cryo stage from the microscope and with pre - cooled tweezers , transfer the grid back to the cryo - grid box . screw the cryo - grid box holder into cryo - grid box to seal against the environment and transfer the holder to a ln2 dewar for storage and future imaging . following standard and established techniques , load the sample grid into a cryo - em transfer holder while keeping the specimen at liquid nitrogen temperature at all times . insert the cryo - holder with sample grid into a transmission electron microscope . in a suitable low magnification view ( 50 - 500x nominal magnification ) , find the center of the sample grid . use the previously collected low magnification cryo - lm data from step 3.7 to determine the relative orientation of the grid s central copper tabs ( as described in figure 2 ) and identify exact areas of interest for correlation . proceed with microscope alignment , low - dose cryo - em imaging and data collection . the cryogenic stage ( fig 1a ) is an effective way to gather cryo - lm data for cryo - fluorescence microscopy and correlative cryo - lm / cryo - em analysis . figure 2 shows how the combination of low and high magnification cryo - lm images allows you to build reference maps that direct you to specific areas in cryo - em . the resulting cryo - lm reference map ( fig 2b ) was utilized during cryo - em data acquisition to locate exact regions shown in figure 3 . a ) the layout of the cryogenic stage consists of a cryo - grid box transfer mount indicated with a white arrow and an aluminum heat sink . grids are transferred under ln2 from the cryo - grid box and placed directly on the heat sink s viewing area as indicated by the black arrow . the hole in the loading screen is placed directly over the cryo - grid box mount and acts as a port to transfer samples and for moving samples from the cryo - grid box to the sample viewing area on the heat sink with tweezers . c ) the cryogenic stage in position under the objective lenses with the viewing screen in place . the special cutout on the viewing screen allows the objective lenses to easily swing into position without moving the cryogenic stage . the hole in the viewing screen away from the sample area acts as a ln2 fill port to replenish ln2 levels if necessary . b ) the same image in ( a ) overlaid with a high magnification fluorescence image of an area of interest and with a filled orange polygon marking the center of the sample grid . the center consists of a group of four squares , three having an extra metal tab and the forth open . for the three squares with an extra metal tab , two pairs share the tab about the long and short axes forming an asymmetric center . this asymmetric center can be seen in the upper left corner and was used to indicate the rotation angle and handedness of the grid between cryo - lm and cryo - em . from one low magnification image many areas of interest can be marked and used as a reference map to locate identical areas in cryo - em . c ) a magnified fluorescence cryo - lm image of the area of interest in ( b ) . hta1-cfp , a c - terminal cfp histone marker , can be seen as a green punctate structure labeling the location of the nucleus . d ) a cryo - em image of the corresponding area in ( c ) . two fields of view depicting identical yeast cells correlated with cryo - bright field ( a , d ) , cryo - fluorescence ( b , e ) , and cryo - em ( c , f ) . using our cryogenic stage and sample preparation techniques , correlative studies between cryo - lm and cryo - em exhibit several benefits over traditional room temperature approaches including , but not limited to : rapid fixation , reduced photobleaching , and scanning of sample integrity prior to cryo - em or chemical fixation / embedding . one of the bottlenecks for cryo - em data collection is the time required for both transferring the sample into a tem and scanning the grid to find suitable regions to image . with cryo - lm s rapid data acquisition , time spent finding good cells can be greatly reduced . thus , we can save both time and money and mitigate this bottleneck by pre - scanning and mapping areas of interest in cryo - lm . more expensive or complex if cryo - lm imaging and sample grid transfers are performed with caution as described in the text and video , fully contamination free imaging can be accomplished . this method allows direct correlation of fluorescent and electron microscopy data of the same cell , organelle or macromolecular complex dispersed on the carbon support or contained within a thin tissue section . it should be noted that the value of this cryogenic stage extends beyond cryo - lm / cryo - em correlative studies to the field of light microscopy as a whole . this cryogenic stage is well suited for both delicate samples and fluorophore dyes / stains , quantum dots , or fluorescently tagged proteins that may be unstable during room temperature chemical fixation and embedding with glutaraldehyde and paraformaldehyde . as a result , we hope that this cryogenic stage will provide greater access to those interested in cryo - lm , who have been previously deterred by cost and insufficient access . . you may find it necessary to use other types of support grids such as continuous carbon , holey carbon , or formvar coated grids for sample support . additionally , if the sample is binding to the copper bars of the grid and not the carbon surface , it may be necessary to adjust the carbon surface chemistry by incubating the grid with a wetting agent or by glow discharging prior to sample addition . stage stability : if you notice vibration , it is likely due to the sample stage being overloaded with weight rather than the bubbling of ln2 . the added weight of the cryogenic stage , can sometimes cause low frequency vibrations to transmit along the standard 3-axis travel microscope stage , and negatively affect imaging via blurring . this can easily be corrected by supporting the microscope s travel stage from underneath with either an adjustable mini - lift or an adjustable adapter with dual - screw arms , as shown in the video . both of these adjustable support mechanisms interact with a non - moveable portion of the travel stage and therefore still permits x and y - axis translation as required for imaging .

the coupling of cryo - light microscopy ( cryo - lm ) and cryo - electron microscopy ( cryo - em ) poses a number of advantages for understanding cellular dynamics and ultrastructure . first , cells can be imaged in a near native environment for both techniques . second , due to the vitrification process , samples are preserved by rapid physical immobilization rather than slow chemical fixation . third , imaging the same sample with both cryo - lm and cryo - em provides correlation of data from a single cell , rather than a comparison of " representative samples " . while these benefits are well known from prior studies , the widespread use of correlative cryo - lm and cryo - em remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage . here we demonstrate the assembly , and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $ 40 with parts found at local hardware and grocery stores . this cryo - lm stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives . for correlative cryo - lm and cryo - em studies , we adapt the use of carbon coated standard 3-mm cryo - em grids as specimen supports . after adsorbing the sample to the grid , previously established protocols for vitrifying the sample and transferring / handling the grid are followed to permit multi - technique imaging . as a result , this setup allows any laboratory with a reflected light microscope to have access to direct correlative imaging of frozen hydrated samples .

Protocol 1. Fabrication and Assembly 2. Sample Preparation 3. Cryo-Light Microscopy 4. Cryo-Electron Microscopy 5. Representative Results Discussion Disclosures

chronic obstructive pulmonary disease ( copd ) , as a common problem in the elderly , is a major cause of chronic morbidity and mortality throughout the world.1 it is characterized by chronic obstruction of expiratory flow affecting peripheral airways , and is often associated with chronic bronchitis and emphysema , thereby accelerating the decline in lung function.2,3 previous studies have found that nutritional status is the prognostic factor that is associated with the mortality in copd.46 weight loss is the major characteristic of malnutrition in copd.7 in 2002 , nutritional risk was defined by the european society for parenteral and enteral nutrition ( espen).8 recently , nutritional risk was found to be associated with the exacerbation of copd in the patients who were hospitalized because of copd.9 meanwhile , it has been reported that nutritional risk is related to the development of copd in male smokers.10 in addition , the exercise capacity is also another factor related to mortality in copd.11 limitation of the exercise capacity seriously affects the quality of life in copd patients.12 therefore , many studies have aimed at strategies for improving the exercise capacity of copd patients.13,14 however , this effective and feasible strategy still needs further exploration . it has been reported that nutritional status could affect the exercise capacity in copd patients.15,16 moreover , nutritional supply seems to contribute to improving the exercise capacity.15,17 in view of these findings , we speculated that there might be some relationship between nutritional risk and exercise capacity . therefore , we explored this relationship in severe copd male patients in this study , which could provide evidence for the effect of nutritional supply on exercise capacity . between january 2012 and december 2013 , a total of 58 severe copd male patients ( age range : 4778 years ) who were hospitalized in the department of respiratory medicine , shanghai pulmonary hospital , affiliated to tongji university , were enrolled in the present study . all the patients were diagnosed with severe copd ( forced expiratory volume in 1 second as percentage of predicted , fev1%pred < 50 ) according to the new global initiative for chronic obstructive lung disease ( gold ) classification criteria of 2012.18 the patients were excluded if they had diseases in other organs ( such as the cardiovascular , renal , endocrine , digestive , and nervous systems ) . patients provided written informed consent before inclusion . before nutritional risk assessment , the height ( m ) and weight ( kg ) of patients were measured . body mass index ( bmi ) was calculated according to formula bmi = weight / height squared ( kg / m ) . nutritional risk assessment was conducted using the continuous sampling method according to the nutritional risk screening ( nrs , 2002 ) criteria developed by espen.19 the nrs score ( 07 ) was the sum of the scores in disease severity , nutritional status , and age . the nutritional status of each patient was scored based on the changes in body weight and bmi in the recent 13 months and food intake in the recent 1 week . besides , one score was assigned to the patients who were more than 70 years old . according to the nrs score , the patients were divided into two groups : nutritional risk group ( nrs score 3 ) and no nutritional risk group ( nrs score < 3 ) . blood samples were collected from the right forearm radial artery before conventional pulmonary function testing ( pft ) and cardiopulmonary exercise testing ( cpet ) were performed . blood gas analysis was performed to confirm whether partial pressure of oxygen in the artery ( pao2 ) and partial pressure of carbon dioxide in the artery ( paco2 ) were in the normal range ( pao2 : 80100 mmhg ; paco2 : 3545 mmhg ) using an abl 800 flex blood gas analyzer ( radiometer medical a / s , copenhagen , denmark ) . a master screen diffusion system ( jaeger corp . , hoechberg , germany ) was used for pft . prediction equations for normal lung function , which were developed in 1988 , were used in this assessment.20 the following parameters were detected in this testing : forced vital capacity ( fvc ) , forced expiratory volume in 1 second ( fev1 ) , fev1%pred , fev1/fvc ratio , residual volume ( rv ) , total lung capacity ( tlc ) , rv / tlc , and pulmonary diffusing capacity for carbon monoxide ( dlco ) . pft was performed three times for each patient and the best result was chosen for analysis . cpet was performed using a cardiopulmonary exercise testing system ( mastercreen - cpx ; jaeger corp . ) . before cpet was performed , the flow sensors and gas analyzers were strictly calibrated . in this testing , a symptom - limited incremental exercise test was performed with different loads ( 1025 w / min ) based on the conditions of the patients.21 during testing , the indicators , consisting of 12-lead electrocardiogram ( ecg ) , blood pressure , pulse oximetry , pulmonary ventilation , gas exchange of oxygen and carbon dioxide ( real - time breath - by - breath analysis ) , and anaerobic threshold ( at , v - slope analysis ) , were monitored . afterward , exercise without load was performed for 3 minutes , and then the load was gradually increased until some symptoms ( such as leg pain , exhaustion , or difficulty in breathing ) occurred . in addition , the testing would be stopped when one of the following conditions occurred : 1 ) 2 mm of st - segment depression with chest pain or 3 mm of st segment without chest pain ; 2 ) frequent ventricular premature beat ; 3 ) second- or third - degree atrioventricular block ; 4 ) systolic blood pressure 250 mmhg or / and diastolic blood pressure 120 mmhg ; 5 ) decreased blood pressure 30 mmhg compared to the baseline level ; or 6 ) pulse oxygen saturation 80% . the prediction value for cept was calculated according to the american thoracic society / american college of chest physicians statement on cpet in 2003.22,23 after testing , the following data were recorded : peak load , peak vo2 ( peak oxygen uptake ) , peak vo2%pred ( peak oxygen uptake as percentage of predicted ) , peak vo2/kg ( peak oxygen uptake per kilogram of body mass ) , peak o2 pulse ( peak oxygen pulse ) . the correlations between nrs score and peak vo2 , peak o2 pulse , or peak load were determined using pearson correlation analysis . between january 2012 and december 2013 , a total of 58 severe copd male patients ( age range : 4778 years ) who were hospitalized in the department of respiratory medicine , shanghai pulmonary hospital , affiliated to tongji university , were enrolled in the present study . all the patients were diagnosed with severe copd ( forced expiratory volume in 1 second as percentage of predicted , fev1%pred < 50 ) according to the new global initiative for chronic obstructive lung disease ( gold ) classification criteria of 2012.18 the patients were excluded if they had diseases in other organs ( such as the cardiovascular , renal , endocrine , digestive , and nervous systems ) . before nutritional risk assessment , the height ( m ) and weight ( kg ) of patients were measured . body mass index ( bmi ) was calculated according to formula bmi = weight / height squared ( kg / m ) . nutritional risk assessment was conducted using the continuous sampling method according to the nutritional risk screening ( nrs , 2002 ) criteria developed by espen.19 the nrs score ( 07 ) was the sum of the scores in disease severity , nutritional status , and age . the nutritional status of each patient was scored based on the changes in body weight and bmi in the recent 13 months and food intake in the recent 1 week . besides , one score was assigned to the patients who were more than 70 years old . according to the nrs score , the patients were divided into two groups : nutritional risk group ( nrs score 3 ) and no nutritional risk group ( nrs score < 3 ) . blood samples were collected from the right forearm radial artery before conventional pulmonary function testing ( pft ) and cardiopulmonary exercise testing ( cpet ) were performed . blood gas analysis was performed to confirm whether partial pressure of oxygen in the artery ( pao2 ) and partial pressure of carbon dioxide in the artery ( paco2 ) were in the normal range ( pao2 : 80100 mmhg ; paco2 : 3545 mmhg ) using an abl 800 flex blood gas analyzer ( radiometer medical a / s , copenhagen , denmark ) . prediction equations for normal lung function , which were developed in 1988 , were used in this assessment.20 the following parameters were detected in this testing : forced vital capacity ( fvc ) , forced expiratory volume in 1 second ( fev1 ) , fev1%pred , fev1/fvc ratio , residual volume ( rv ) , total lung capacity ( tlc ) , rv / tlc , and pulmonary diffusing capacity for carbon monoxide ( dlco ) . pft was performed three times for each patient and the best result was chosen for analysis . cpet was performed using a cardiopulmonary exercise testing system ( mastercreen - cpx ; jaeger corp . ) . before cpet was performed , the flow sensors and gas analyzers were strictly calibrated . in this testing , a symptom - limited incremental exercise test was performed with different loads ( 1025 w / min ) based on the conditions of the patients.21 during testing , the indicators , consisting of 12-lead electrocardiogram ( ecg ) , blood pressure , pulse oximetry , pulmonary ventilation , gas exchange of oxygen and carbon dioxide ( real - time breath - by - breath analysis ) , and anaerobic threshold ( at , v - slope analysis ) , were monitored . afterward , exercise without load was performed for 3 minutes , and then the load was gradually increased until some symptoms ( such as leg pain , exhaustion , or difficulty in breathing ) occurred . in addition , the testing would be stopped when one of the following conditions occurred : 1 ) 2 mm of st - segment depression with chest pain or 3 mm of st segment without chest pain ; 2 ) frequent ventricular premature beat ; 3 ) second- or third - degree atrioventricular block ; 4 ) systolic blood pressure 250 mmhg or / and diastolic blood pressure 120 mmhg ; 5 ) decreased blood pressure 30 mmhg compared to the baseline level ; or 6 ) pulse oxygen saturation 80% . the prediction value for cept was calculated according to the american thoracic society / american college of chest physicians statement on cpet in 2003.22,23 after testing , the following data were recorded : peak load , peak vo2 ( peak oxygen uptake ) , peak vo2%pred ( peak oxygen uptake as percentage of predicted ) , peak vo2/kg ( peak oxygen uptake per kilogram of body mass ) , peak o2 pulse ( peak oxygen pulse ) . the correlations between nrs score and peak vo2 , peak o2 pulse , or peak load were determined using pearson correlation analysis . among the total 58 severe copd male patients , 25 were assessed as having nutritional risk and assigned to the nutritional risk group . the other 33 patients without nutritional risk were enrolled into the no nutritional risk group . the age ( no nutritional risk group , 63.739.03 years ; nutritional risk group , 61.527.13 years ; p=0.319 ) and the height ( no nutritional risk group , 166.526.10 cm ; nutritional risk group , 167.125.83 cm ; p=0.707 ) of the patients in the nutritional risk group were similar to those in the no nutritional risk group . however , the weight ( no nutritional risk group , 64.586.31 kg ; nutritional risk group , 51.244.68 kg ; p<0.0001 ) and bmi ( no nutritional risk group , 23.302.02 m / kg ; nutritional risk group , 18.341.33 m / kg ; p<0.0001 ) of the patients in the nutritional risk group were significantly lower than those in the no nutritional risk group ( table 1 ) . before exercise , there was no statistically significant difference in pao2 ( no nutritional risk group , 73.3010.71 ; nutritional risk group , 73.6813.00 ; p=0.903 ) and paco2 ( no nutritional risk group , 42.856.82 ; no nutritional risk group , 41.957.84 ; p=0.643 ) between severe copd male patients with and without nutritional risk ( table 2 ) . there was no significant difference between severe copd male patients with and without nutritional risk in fvc ( no nutritional risk group , 2.260.53 ; nutritional risk group , 2.290.67 ; p=0.850 ) , fev1 ( no nutritional risk group , 0.860.25 ; nutritional risk group , 0.800.23 ; p=0.353 ) , fev1%pred ( no nutritional risk group , 32.408.47 ; nutritional risk group , 29.008.97 ; p=0.146 ) , fev1/fvc ( no nutritional risk group , 37.925.34 ; nutritional risk group , 36.398.47 ; p=0.433 ) , tlc ( no nutritional risk group , 7.891.39 ; nutritional risk group , 7.801.93 ; p=0.844 ) , rv ( no nutritional risk group , 5.661.31 ; nutritional risk group , 5.662.12 ; p=1.000 ) , and rv / tlc ( no nutritional risk group , 71.286.53 ; nutritional risk group , 71.4011.01 ; p=0.962 ) . nevertheless , the dlco ( no nutritional risk group , 11.984.02 ; nutritional risk group , 8.974.37 ; p=0.009 ) of the patients in the no nutritional risk group was significantly higher than that of the patients in the nutritional risk group . as shown in table 4 , the severe copd male patients in the no nutritional risk group had significantly higher peak vo2 ( no nutritional risk group , 1,068.33327.38 ; nutritional risk group , 871.24219.37 ; p=0.008 ) , peak vo2%pred ( no nutritional risk group , 60.2717.13 ; nutritional risk group , 52.0412.64 ; p=0.048 ) , and peak o2 pulse ( no nutritional risk group , 8.802.37 ; nutritional risk group , 7.321.47 ; p=0.008 ) than those in the nutritional risk group . but there was no significant difference between the groups in peak load ( no nutritional risk group , 61.2130.04 ; nutritional risk group , 49.4820.82 ; p=0.100 ) and peak vo2/kg ( no nutritional risk group , 16.574.61 ; nutritional risk group , 16.923.50 ; p=0.753 ) . figure 1 shows the correlation between the nrs score and peak vo2 , peak o2 pulse , or peak load . the results show that there are significant negative correlations between the nrs score and peak vo2 ( r=0.353 , p<0.01 ) , peak o2 pulse ( r=0.322 , p<0.05 ) , or peak load ( r=0.272 , p<0.05 ) in severe copd male patients . among the total 58 severe copd male patients , 25 were assessed as having nutritional risk and assigned to the nutritional risk group . the other 33 patients without nutritional risk were enrolled into the no nutritional risk group . the age ( no nutritional risk group , 63.739.03 years ; nutritional risk group , 61.527.13 years ; p=0.319 ) and the height ( no nutritional risk group , 166.526.10 cm ; nutritional risk group , 167.125.83 cm ; p=0.707 ) of the patients in the nutritional risk group were similar to those in the no nutritional risk group . however , the weight ( no nutritional risk group , 64.586.31 kg ; nutritional risk group , 51.244.68 kg ; p<0.0001 ) and bmi ( no nutritional risk group , 23.302.02 m / kg ; nutritional risk group , 18.341.33 m / kg ; p<0.0001 ) of the patients in the nutritional risk group were significantly lower than those in the no nutritional risk group ( table 1 ) . before exercise , there was no statistically significant difference in pao2 ( no nutritional risk group , 73.3010.71 ; nutritional risk group , 73.6813.00 ; p=0.903 ) and paco2 ( no nutritional risk group , 42.856.82 ; no nutritional risk group , 41.957.84 ; p=0.643 ) between severe copd male patients with and without nutritional risk ( table 2 ) . there was no significant difference between severe copd male patients with and without nutritional risk in fvc ( no nutritional risk group , 2.260.53 ; nutritional risk group , 2.290.67 ; p=0.850 ) , fev1 ( no nutritional risk group , 0.860.25 ; nutritional risk group , 0.800.23 ; p=0.353 ) , fev1%pred ( no nutritional risk group , 32.408.47 ; nutritional risk group , 29.008.97 ; p=0.146 ) , fev1/fvc ( no nutritional risk group , 37.925.34 ; nutritional risk group , 36.398.47 ; p=0.433 ) , tlc ( no nutritional risk group , 7.891.39 ; nutritional risk group , 7.801.93 ; p=0.844 ) , rv ( no nutritional risk group , 5.661.31 ; nutritional risk group , 5.662.12 ; p=1.000 ) , and rv / tlc ( no nutritional risk group , 71.286.53 ; nutritional risk group , 71.4011.01 ; p=0.962 ) . nevertheless , the dlco ( no nutritional risk group , 11.984.02 ; nutritional risk group , 8.974.37 ; p=0.009 ) of the patients in the no nutritional risk group was significantly higher than that of the patients in the nutritional risk group . as shown in table 4 , the severe copd male patients in the no nutritional risk group had significantly higher peak vo2 ( no nutritional risk group , 1,068.33327.38 ; nutritional risk group , 871.24219.37 ; p=0.008 ) , peak vo2%pred ( no nutritional risk group , 60.2717.13 ; nutritional risk group , 52.0412.64 ; p=0.048 ) , and peak o2 pulse ( no nutritional risk group , 8.802.37 ; nutritional risk group , 7.321.47 ; p=0.008 ) than those in the nutritional risk group . but there was no significant difference between the groups in peak load ( no nutritional risk group , 61.2130.04 ; nutritional risk group , 49.4820.82 ; p=0.100 ) and peak vo2/kg ( no nutritional risk group , 16.574.61 ; nutritional risk group , 16.923.50 ; p=0.753 ) . figure 1 shows the correlation between the nrs score and peak vo2 , peak o2 pulse , or peak load . the results show that there are significant negative correlations between the nrs score and peak vo2 ( r=0.353 , p<0.01 ) , peak o2 pulse ( r=0.322 , p<0.05 ) , or peak load ( r=0.272 , p<0.05 ) in severe copd male patients . lack of exercise capacity limits the quality of life of copd patients.12 some previous studies have found that there was an association between the nutritional status and exercise capacity in copd patients.15,16 however , it is still unclear whether a correlation exists between nutritional risk and exercise capacity in copd patients . therefore , we investigated the relationship between nutritional risk and exercise capacity in severe copd male patients . in this study , we found significantly lower weight and bmi in severe copd male patients with nutritional risk compared to those of severe copd male patients without nutritional risk , which are the characteristics of copd patients with nutritional risk.2426 besides , pft showed that dlco in severe copd male patients without nutritional risk was significantly higher than that in severe copd male patients with nutritional risk . it has been reported that reduced dlco could impair exercise capacity in patients with heart failure through increased dead space ventilation.27 meanwhile , a low dlco could impair oxygen transport by a reduction in maximum oxygen consumption.28 moreover , it has been reported that weakened exercise capacity is attributed to reduced locomotor muscle oxygen transport.29,30 normally , oxygen transport would adaptively vary with oxygen demand during exercise,31,32 and oxygen uptake would increase during exercise for biochemical adaptations.33,34 thus , the exercise capacity can decline through impaired oxygen transport , which can not adaptively regulate the oxygen uptake during exercise . as expected , the impaired oxygen transport might limit the oxygen uptake in severe copd male patients with nutritional risk , as seen in this study . cpet showed that the peak vo2 and peak o2 pulse of the nutritional risk group were significantly lower than those of the no nutritional risk group and there were significant negative correlations between the nrs score and peak vo2 or peak o2 pulse . these results indicate that the oxygen uptake of severe copd male patients with nutritional risk was significantly lower than that of severe copd male patients without nutritional risk , which might be due to the impaired oxygen transport in copd,33,35 thereby affecting the utilization of oxygen.36 it was reported that inadequate oxygen supply during exercise caused accumulation of oxygen deficit , which was responsible for the reduced exercise capacity in patients with copd.37,38 meanwhile , decreased muscle oxygen utilization could reduce the exercise capacity in patients with copd.39 thus , impaired oxygen transport may be the main reason for reduced exercise capacity in copd patients with nutritional risk . in addition , it has been reported that nutritional depletion is related to the respiratory and peripheral skeletal muscle function in outpatients with copd.40,41 nutritional supplements have been found to be useful for the prevention and treatment of exercise - induced skeletal muscle injury.42 moreover , muscle function is associated with the oxygen transport.4345 thus , patients with nutritional risk may have decreased exercise capacity due to impaired oxygen transport . given these facts , there may be a close relationship between nutritional risk and exercise capacity . furthermore , blood gas analysis indicated that the pao2 and paco2 of patients were all in normal ranges before exercise and they were similar between groups . however , the pao2 and paco2 were not measured after the exercise . the changes induced by exercise in oxygen and co2 pressure in arterial blood could not be assessed . further studies are required to investigate whether nutritional risk could affect the changes induced by exercise in oxygen and co2 pressures in arterial blood . second , we assessed only the total weight but not the fat - free mass , so further studies are required . in addition , not all of the indicators in pft and cept were found to be associated with the nutritional risk . thus , the relationship between nutritional risk and exercise capacity should be further investigated in female patients and also studies with large sample sizes for verifying the result of this study . the results of this study supported the association between exercise capacity and nutritional risk according to nrs 2002 in severe copd male patients . the impairment of oxygen transport may be the main mechanism of this relationship between nutritional risk and exercise capacity .

objectivethe nutritional status of chronic obstructive pulmonary disease ( copd ) patients is associated with their exercise capacity . in the present study , we have explored the relationship between nutritional risk and exercise capacity in severe male copd patients.methodsa total of 58 severe copd male patients were enrolled in this study . the patients were assigned to no nutritional risk group ( n=33 ) and nutritional risk group ( n=25 ) according to the nutritional risk screening ( nrs , 2002 ) criteria . blood gas analysis , conventional pulmonary function testing , and cardiopulmonary exercise testing were performed on all the patients.resultsresults showed that the weight and bmi of the patients in the nutritional risk group were significantly lower than in the no nutritional risk group ( p<0.05 ) . the pulmonary diffusing capacity for carbon monoxide of the no nutritional risk group was significantly higher than that of the nutritional risk group ( p<0.05 ) . besides , the peak vo2 ( peak oxygen uptake ) , peak o2 pulse ( peak oxygen pulse ) , and peak load of the nutritional risk group were significantly lower than those of the no nutritional risk group ( p<0.05 ) and there were significantly negative correlations between the nrs score and peak vo2 , peak o2 pulse , or peak load ( r<0 , p<0.05).conclusionthe association between exercise capacity and nutritional risk based on nrs 2002 in severe copd male patients is supported by these results of this study .

Introduction Materials and methods Patients Nutritional risk assessment Blood gas analysis PFT and CPET Statistical analyses Results Characteristics of patients Results for blood gas analysis Pulmonary function comparison between groups Results for CPET Correlation between NRS score and peak VO Discussion Conclusion

in exposure or risk assessments , both environmental and biological measurements are often used . environmental measurements are an excellent means for evaluating regulatory compliance , but the models used to estimate body burden from these measurements are complex . unless all possible routes of exposure ( i.e. , inhalation , dermal absorption , ingestion ) are evaluated , exposure to a toxicant can be underestimated . to circumvent this problem , measurements of the internal dose of a toxicant in blood , serum , urine , or tissues can be used singularly or in combination with environmental data for exposure assessment . in three separate laboratories , carbaryl or its primary metabolite , 1-naphthol , was measured in personal air , dermal samples , blood serum , and urine from farmer applicators and their families . the usefulness of both environmental and biological data has been demonstrated . for the farmer applicator , the environmental levels of carbaryl would have been sufficient to determine that an exposure had occurred . however , biological measurements were necessary to determine the absorbed dose of each member of the applicator 's family . in addition , a correlation between serum and urinary 1-naphthol measurements has been shown ; therefore , either matrix can be used to accurately evaluate occupational carbaryl exposure.imagesfigure 1.figure 2.figure 3.figure 4.figure 5 . afigure 5 . b

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fundamental concepts in psychology and philosophy of the mind are the notion of sensation and perception . when a stimulus produces an effect on different sensory receptors it induces sensation . subsequent interpretation and organization of this sensory stimulus produce a meaningful experience of the world and of one 's perception . although in most cases perception is conscious , perception without awareness does exist , that is , the interpretation of semantic stimuli . normally wakefulness and awareness are related ; one has to be awake ; that is , there has to be a certain level of consciousness to be aware of something ; that is , there is content in consciousness . in states of deep sleep , anesthesia , and coma there is little or no wakefulness and hence no awareness . in drowsiness and light sleep however , in certain states , dissociations exist between wakefulness and awareness , such as in the vegetative state , when there is wakefulness presumably without awareness ( eyes open , brain shut ) . in the dream state there is awareness ( content in consciousness ) with decreased wakefulness ( level of consciousness ) . dreams are succession of images , ideas , emotions , and perceptions without sensations that occur involuntarily in the mind predominantly during rapid eye movement ( rem ) sleep . nonpulsatile subjective tinnitus is considered a phantom perception , the conscious awareness of a percept in the absence of an external stimulus . it is characterized by the perception that the phantom sound comes from an external sound source , even though the sound might be pulled from memory [ 1 , 6 , 7 ] . this is reminiscent of a dream state , when there is awareness , with stimuli attributed to the external world but generated internally . whereas tinnitus can be considered a simple phantom percept , dreams could be considered complex phantom percepts , like hallucinations and hallucinosis [ 9 , 10 ] . however , in contrast to hallucinations and hallucinosis that occur during wakefulness , dreams occur during certain stages of sleep . stimulus - evoked auditory cortical activation does not necessarily produce conscious auditory perception , and auditory perception is possible in the absence of auditory input : more than 80% of people with normal hearing perceive phantom sounds when placed in a soundproof room . likewise , after limb amputation almost all people experience a phantom limb , whereas 70% suffer from severe phantom pain . a clear clinical analogy exists between phantom pain and disabling tinnitus [ 1 , 14 , 15 ] . there are also parallels between the pathophysiology of tinnitus and pain , as well as in the treatment [ 16 , 17 ] . however , there are also differences between tinnitus and pain . while physiological pain is mediated via nociceptive pathways , this could explain why commonly available analgesics that suppress acute physiological body pain are inefficient in ameliorating tinnitus . also , medications such as antiepileptics and antidepressants , which are effective in the treatment of neuropathic pain , tend to be ineffective for tinnitus . many to most ( 33100% ) patients who suffer from phantom limb percepts do not experience phantom limb percepts in a dream state [ 2023 ] . this has been explained as follows [ 8 , 21 , 23 ] : neural representation of the body derives from sensory and proprioceptive feedback from the body . during sleep , when the brain / mind is actively kept offline , this sensory feedback is lacking . moreover , during rem sleep and in the absence of external inputs , dreaming could activate a set of innate or early life spatial - temporal categories . so if rem sleep is a state of protoconsciousness , that is , a contextually emergent property of self - sustaining systems , the self as it appears in rem sleep dreams is no longer affected by waking experiences because it feeds from an embodied and functionally intact body scheme [ 8 , 21 ] . in view of the pathophysiological analogy between tinnitus and pain , it can be hypothesized that tinnitus is absent in the dream state as well . we therefore explored this in a group of 78 consecutive tinnitus patients attending the multidisciplinary tinnitus research initiative clinic at the university of antwerp . a recently proposed pathophysiological model of phantom sound based on a predictive brain concept with bayesian updating might explain why tinnitus is not perceived during dreaming . seventy - eight patients ( 57 males and 21 females ) with chronic , nonpulsatile tinnitus were included in this study with an average age of 48.78 years ( sd = 12.87 ) and an average tinnitus duration of 5.74 years ( sd = 6.96 ) . thirty - five patients perceive noise - like tinnitus , while 43 patients experience pure tone tinnitus . forty - three patients had bilateral tinnitus ; 12 patients perceive tinnitus holocranially , 12 on the left side and 11 on the right side . the first question asked whether the tinnitus patient recalled if they dreamed during the night ( 1 ) , followed by the question whether in their dreams they perceive tinnitus ( 2 ) . of the 78 participating patients only 2 ( 2.56% ) declared that they do not recall their dreams , while 76 ( 97.44% ) do . of those 76 patients that do recall their dreams 74 ( 97.73% ) state that they do not perceive tinnitus while dreaming or are not aware of having tinnitus during sleep . people with tinnitus do not perceive tinnitus in their dreams analogous to what is reported for many phantom limb perceptions [ 21 , 25 ] . dreams and wakefulness are both associated with awareness , but in one state of awareness there is no tinnitus ( dreams ) , whereas in the other ( wakefulness ) there is tinnitus . the reason why patients with tinnitus do not perceive tinnitus in their dream state can be theoretically explained by the bayesian brain model which has been used as an explanation for the development of tinnitus in relation to auditory deafferentation . this bayesian brain model is founded on an extension of a predictive brain model ( see figure 1(a ) ) . whereas other models ( see for an overview ) can explain the tinnitus in the presence of deafferentation the bayesian model is compatible with both the deafferentation and noise - cancelling models and provides a rationale why tinnitus develops in a wake state and not in a dream state . physiologically the brain can be conceptualized as a helmholtz machine that constantly makes one or possibly multiple predictions about the world . since the examples given to the learner are unlabeled , there is no error or reward signal to evaluate a potential solution ; in other words , there is no updating of the predictions . a bayesian brain however updates predictions based on what it actively explores in the environment by means of the senses [ 24 , 29 , 30 ] . bayesian inference can therefore be conceptualized in a way that would be familiar to john hughlings - jackson as using sensory information from the environment to update memory - based expectations ( held before acquiring sensory inputs ) to produce posterior beliefs represented as percepts . this mechanism permits decision making based on predictions updated by actively sampling the environment for confirmation or rejection of expectations ( see figure 1(b ) ) . auditory deafferentation limits the amount of information the brain can acquire to make sense of the world . the topographically specific deafferentation induces a topographically specific prediction error hypothetically based on temporal incongruity . in other words , it is inconsistent with what is stored in memory and should be updated . the model hypothesizes that deprived auditory information depends on the amount ( bandwidth ) of deafferented auditory channels . limited damage to auditory receptors causes loss of functional surround inhibition in the cortex , unmasking of latent inputs , and significantly altered neural coding . this suggests that the missing information can be obtained via access of overlapping tuning curves of the neighboring cortical cells . if the deafferentation is somewhat larger , a widening of auditory receptive fields will permit pulling the missing information from the auditory cortical neighborhood . if this is insufficient , due to a still larger deafferentation , dendritic and axonal rewiring can occur . if this is still insufficient , the missing auditory information can be pulled from ( para)hippocampal memory . when we dream , we create an image of the world that is entirely detached from sensory feedback ; that is , it can not be updated . aminergic activity is highest during waking , declines during nrem sleep , and is lowest during rem sleep . this means that the discrepancy between top - down predictions and ( the absence of ) sensory signals received will not be registered , and the auditory deafferentation will not be filled in , resulting in the absence of tinnitus in the dream state ( see figures 2(a ) and 2(b ) ) . it has been argued that the cerebellum is involved in motor , sensory , and cognitive predictions . it is therefore possible that auditory predictions are made in the paraflocculus , as removing this cerebellar structure can prevent tinnitus from arising and arrest the presence of tinnitus in animals . this conceptually suggests that removing the prediction can prevent or abolish tinnitus , which is in accordance with the concept that tinnitus could be a malprediction . however , apart from its theoretical implications , the data might also help to find the neural correlates of tinnitus . the putative on / off switch for tinnitus is to be found in these areas that differ between waking and rem state , that is , the ventrolateral prefrontal cortex / frontopolar - inferior parietal - cerebellar - parahippocampal network . these areas overlap with a recent meta - analysis of pet studies in tinnitus and provide a framework for zooming in on the pathophysiology of this enigmatic symptom . in addition to its evident benefit for tinnitus research , it could also provide clues for consciousness research , by delineating the core areas involved in the neural correlates of consciousness ; that is , minimal assembly of brain areas required for consciousness per se [ 38 , 39 ] . other potential explanations for the absence of tinnitus in the dream state have to be considered . it is possible that during the dream state there is an attention shift from the tinnitus to the dream , analogous to what is noted in patients who do not perceive their tinnitus when intensely engaged in a task . in conclusion , this report demonstrates that tinnitus perception is switched off during dream sleep even though there is awareness , like in wakefulness . this suggests that it is theoretically possible to find the neural correlates of phantom sound and thereby find a potential avenue for suppressing this enigmatic symptom .

there are pathophysiological , clinical , and treatment analogies between phantom limb pain and phantom sound ( i.e. , tinnitus ) . phantom limb pain commonly is absent in dreams , and the question arises whether this is also the case for tinnitus . a questionnaire was given to 78 consecutive tinnitus patients seen at a specialized tinnitus clinic . seventy - six patients remembered their dreams and of these 74 claim not to perceive tinnitus during their dreams ( 97% ) . this can be most easily explained by a predictive bayesian brain model . that is , during the awake state the brain constantly makes predictions about the environment . tinnitus is hypothesized to be the result of a prediction error due to deafferentation , and missing input is filled in by the brain . the heuristic explanation then is that in the dream state there is no interaction with the environment and therefore no updating of the prediction error , resulting in the absence of tinnitus .

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

atlantoaxial instability ( aai ) may be caused by trauma , arthritis , tumor , infection , or congenital malformation316 ) . atlantoaxial stabilization resulted from bony fusion is needed to correct deformities and prevent neurological impairment . the surgical method of c1 - 2 transarticular screw fixation was introduced by margerl and has been the main technique for wire fusion of c1 and c2 vertebrae358 ) . however , fusion rates may be altered by the depth of the vertebral artery ( va ) groove of the lateral mass and the width of the c2 pedicle1 ) . additionally , screw fixation might be challenging due to the high risk of injury to the va , which can lead to massive bleeding and cerebral infarction1620 ) . therefore , goel and laheri introduced c1 lateral mass and c2 pedicle screw fixation ( c1lm - c2p ) , which was popularized by harms and melcher , to reduce the risk of injury to the va56101116 ) . furthermore , in the inevitable situation of injury to the va or in the case of unfavorable bone structures , c1lm - c2p can not be performed using the bilateral method . therefore , the aim of this study is to compare the fusion rates of the unilateral and bilateral methods using the c1lm - c2p technique . between march 2009 and december 2013 , 25 consecutive patients in our institution underwent c1lm - c2p to address aai due to rheumatoid arthritis . among those patients , preoperative radiologic imaging studies were performed to obtain accurate information on the anatomy of the atlas and axis . cervical x - rays , including hyperextension and hyperflexion views , were obtained to confirm the instability of the c1 - 2 complex . the atlanto - dental interval ( adi ) was examined to determine the instability status at the preoperative ; immediate postoperative ; and 1 , 3 , 6 , and 12-month postoperative periods . moreover , computed tomography ( ct ) scans with 1-mm slices were performed to examine the potential insertion pathway of the c1 lateral mass screw and c2 pedicle screw , and ct angiography in the area of c1 - 2 was used to visualize the pathway of the va for examining potential anomalies and reducing the possible complication of va injury . magnetic resonance ( mr ) posterior c1lm - c2p screw fixation , the surgical technique of harms and melcher6 ) , was performed in all patients in this study group . after discharge , patients reported to an outpatient clinic at postoperative months 1 , 3 , 6 , and 12 to measure adis as the index of bony fusion . bony fusion was defined as a bony bridge which was seen at the c1 - 2 inter - spinous space on lateral radiograph images , no difference of adi between the immediate postoperative period and the 12 month time point , and no instrument failure during the follow - up period21 ) . after wide exposure of the c1 - 2 posterior structures , the starting point in the c1 lateral mass was formed with a high speed drill at the connection point of the posterior arch and the lateral mass . after tapping the drilled pathway , screws with a diameter between 3.5 mm and 4.0 mm were inserted into the lateral mass . before marking the entry point of the c2 screw , the medial border of the c2 pedicle was identified to prevent screw malposition in the spinal canal . after identification of the transitional corner , which is the more cephalad portion of the lamina and c2 isthmus , an entry point was made 4 mm lateral and 4 mm caudal to the transitional corner using a drill . the direction was 20 to 30 medial and cephalad , with palpation of the medial border of the c2 pars interarticularis . after drilling and tapering , a polyaxial screw with the proper length and diameter was inserted . an interlaminar spacer and wiring between c1 and c2 were tightly fastened , and the rod was applied to the screw head . preoperative and postoperative adis were measured on lateral plain radiographs using a picture archiving and communication system ( pacs ) and then compared side - by - side to evaluate the time of bony fusion and differences between unilateral and bilateral screw fixation . statistical analyses were performed with spss version 18.0 ( spss , chicago , il , usa ) . non - parametric analysis was performed in the both unilateral and bilateral screw fixation groups . the mann - whitney u test was used to compare the bony fusion rate between the unilateral and bilateral screw fixation methods . after wide exposure of the c1 - 2 posterior structures , the starting point in the c1 lateral mass was formed with a high speed drill at the connection point of the posterior arch and the lateral mass . after tapping the drilled pathway , screws with a diameter between 3.5 mm and 4.0 mm were inserted into the lateral mass . before marking the entry point of the c2 screw , the medial border of the c2 pedicle was identified to prevent screw malposition in the spinal canal . after identification of the transitional corner , which is the more cephalad portion of the lamina and c2 isthmus , an entry point was made 4 mm lateral and 4 mm caudal to the transitional corner using a drill . the direction was 20 to 30 medial and cephalad , with palpation of the medial border of the c2 pars interarticularis . after drilling and tapering , a polyaxial screw with the proper length and diameter was inserted . an interlaminar spacer and wiring between c1 and c2 were tightly fastened , and the rod was applied to the screw head . preoperative and postoperative adis were measured on lateral plain radiographs using a picture archiving and communication system ( pacs ) and then compared side - by - side to evaluate the time of bony fusion and differences between unilateral and bilateral screw fixation . statistical analyses were performed with spss version 18.0 ( spss , chicago , il , usa ) . non - parametric analysis was performed in the both unilateral and bilateral screw fixation groups . the mann - whitney u test was used to compare the bony fusion rate between the unilateral and bilateral screw fixation methods . unilateral c1 lateral mass and c2 pedicle screw fixation was performed in 11 patients ( 44% ) in this study ( fig . 1a ) . the mean age of the unilateral screw fixation group was 53.1 years ( range 30 - 74 ) with female predominance ( 90.9% ) . an anomalous course of the va was the main reason for unilateral screw fixation , which was required in eight patients . meanwhile , in the bilateral screw fixation group , the mean age was 55.9 years ( range 40 - 72 ) with a female predominance ( 85.7% ) ( table 1 , fig . one showed va stenosis compressed by the pedicle screw on digital subtraction angiography , but no other complications , such as neurologic impairment , massive bleeding , or infection , were found on further evaluation . in the 11 patients in the unilateral screw fixation group , the preoperative mean adi was 8.14 mm , and the mean immediate postoperative adi was reduced to 1.82 mm . moreover , measurements of mean adi at 1 , 3 , 6 , and 12 months postoperatively were 1.99 mm , 1.99 mm , 1.92 mm , and 1.91 mm , respectively . on the other hand , in the 14 patients in the bilateral screw fixation group , the mean preoperative adi was 8.48 mm , and that at postoperative 1 , 3 , 6 , and 12 months was 1.99 mm , 1.96 mm , 1.91 mm , and 1.90 mm , respectively ( table 1 ) . the postoperative adi values at the immediate versus 1 month postoperative time points were not statistically different ( unilateral group , p=0.241 and bilateral group , p=0.520 ) . similar results were found in the immediate versus 6 month time points unilateral group , p=0.859 and bilateral group , ( p=0.052 ) . moreover , when comparing the whole follow - up period of 12 months for both techniques , no meaningful postoperative changes in adis were found , and atlantoaxial fusion was seen in all patients included in the study ( 100% ) . according to the mann - whitney test , there was no statistically significant difference between the unilateral and bilateral screw fixation groups ( p=0.893 ) ( table 2 ) . when evaluating bony fusion at 12 months , instrument - related failure was not noted in any case . bony bridge between the interlaminar space of c1 - 2 was demonstrated on ct scans in all patients . differences in adi between the immediate postoperative and 12 month time points were not statistically significant . chronic nuchal pain with or without neurological deficits may be encountered , and improperly treated upper cervical cord injury may trigger severe neurologic impairment . the surgical method of c1 - 2 complex fixation has been introduced by many surgeons , and bilateral transarticular screw fixation using the wiring technique of gallie or brooks - jenkins has achieved the highest stability and stiffness2712 ) . however , bilateral transarticular screw fixation ( taf ) is not always possible because of va anomalies or fracture of the c1 - 2 complex1314 ) . therefore , stability achieved through bilateral taf has been compared with that of unilateral taf in a previous study . hue et al.7 ) mentioned that there were no differences in fusion rate as measured by postoperative adi change between unilateral and bilateral taf . to overcome the major complication of vascular and neural injuries , c1lm - c2p was introduced by goel and laheri in 1994 and modified by harms and melcher in 200156 ) . the superiority of c1lm - c2p fixation compared to taf is a lower risk of va injury due to the use of individual screw systems . the pathway of the screws in the taf method , which crosses c2 through c1 , is relatively complex and narrow compared with that of c1lm - c2p , which uses individual screw systems . however , some problems have been encountered in bilateral screw placement including severe anomalous va pathways and restricted pathway for the 3.5 mm screw . in other words , if the risk of injuring the va and spinal cord can be predicted preoperatively , abandoning screw placement in an ipsilateral side with anatomical variation may be a wise decision . in the 25 patients enrolled in the present study , no massive bleeding or neurologic damage was detected . vertebral canal violation without other complications was noted in one case of va stenosis caused by a c2 pedicle screw . the risk of va injury can not be completely eliminated even with c1lm - c2p . if preoperative ct angiography suggests a high risk of va injury , the screw may be inserted on the unilateral side only . therefore , our study was designed to identify differences in bony fusion between unilateral and bilateral fixation . when we compared the preoperative and postoperative adi measures , there was no statistically significant difference between unilateral and bilateral fixation ( p=0.893 ) . this suggests that unilateral and bilateral c1lm - c2p produce the same bony fusion and reduction of aai . in addition , the strength of fusion in the unilateral screw fixation groups was as strong as that in the bilateral screw fixation group . we have come into conclusion with the unilateral c1lm - c2p showed as much strength as the bilateral c1lm - c2p . likewise , the unilateral taf showed as much strength as the bilateral taf , which was mentioned by hue et al.7 ) comparison between the unilateral taf and unilateral c1lm - c2p should be made in the future study of aai . however , intraoperative reduction and smaller surgical site exposure are the advantages of c1lm - c2p which is more selective in choosing surgical technique in aai9 ) . several studies have been performed to reduce the complication rate when stabilizing the c1 - 2 complex . anatomical variations of the va and c1 lateral mass deformation due to systemic diseases like rheumatoid arthritis may increase the risk of va injury . also , as the surgical procedure is performed , the risk of injuring the va increases with every 2% when the screw is inserted3141620 ) . therefore , comprehensive understanding of the atlantoaxial and va anatomy by ct angiography can assist with successful surgical planning and reduce the complication rate related to the va . hypoglossal nerve injury may occur because of the anatomical proximity to the anterior aspect of the lateral mass315 ) . c2 dermatome anesthesia or pain may result in patients with c2 ganglion injury due to insufficient exposure of the c1 lateral mass and c1 - 2 joint34 ) . such improvements in navigation systems and monitoring devices may allow for improved accuracy when determining screw trajectory pathways1316 ) . however , these navigation systems have limitations with regard to calibration of three - dimensional ct scans with fluoroscopy - based images , inaccuracies caused by manipulating surgical instruments in the operation field , and motion of the operating field or craniocervical junction even when fixed with a head clamp16171819 ) . therefore , navigation systems require increased accuracy when operating on the c1 - 2 complex . intraoperative fluoroscopy with abundant surgical experience can reduce complications in c1 - 2 surgical techniques16 ) . if there is a risk of va damage , the choice to abandon predictable screw insertion will increase the safety and completeness of the surgical technique of c1lm - c2p screw fixation . our study illustrated that unilateral screw fixation shows similar strength , bony fusion , and reduction rate as bilateral screw fixation . even though we were able to demonstrate that unilateral screw fixation is similar to bilateral fixation in terms of strength , bony fusion , and reduction rate , several limitations of our study must be considered . the existing limitations are a lack of retrospective study , small number of enrolled cases , and deficiency of the biomechanical study . due to our small study cases , further prospective study using a multi - center design may help increasing the reliability of our unilateral surgical technique . furthermore , future biomechanical studies using animal or human cadavers might provide numeric evidence supporting the present study conclusion . c1 lateral mass and c2 pedicle screw fixation can performed in patients with aai due to rheumatoid arthritis . although bilateral fixation is the main treatment method for such cases , unilateral screw fixation can be performed if a neurovascular contraindication is predicted . in our study , by means of adi changes , unilateral fixation showed similar strength to the bilateral fixation with bone fusion . radiologic images , such as ct angiography with cervical three dimensional ct and mr images , should be performed to reduce the complications of c1lm - c2p prior to surgery .

objectivebilateral c1 lateral mass and c2 pedicle screw fixation ( c1lm - c2p ) is an ideal technique for correcting atlantoaxial instability ( aai ) . however , the inevitable situation of vertebral artery injury or unfavorable bone structure may necessitate the use of unilateral c1lm - c2p . this study compares the fusion rates of the c1 lateral mass and c2 pedicle screw in the unilateral and bilateral methods.methodsover five years , c1lm - c2p was performed in 25 patients with aai in our institute . preoperative studies including cervical x - ray , three - dimensional computed tomography ( ct ) , ct angiogram , and magnetic resonance imaging were performed . to evaluate bony fusion , measurements of the atlanto - dental interval ( adi ) and ct scans were performed in the preoperative period , immediate postoperative period , and postoperatively at 1 , 3 , 6 , and 12 months.resultsunilateral c1lm - c2p was performed in 11 patients ( 44% ) . the need to perform unilateral c1lm - c2p was due to anomalous course of the vertebral artery in eight patients ( 73% ) and severe degenerative arthritis in three patients ( 27% ) . the mean adi in the bilateral group was 2.09 mm in the immediate postoperative period and 1.75 mm in 12-months postoperatively . the mean adi in the unilateral group was 1.82 mm in the immediate postoperative period and 1.91 mm in 12-months postoperatively . comparison of adi measurements showed no significant differences in either group ( p=0.893 ) , and the fusion rate was 100% in both groups.conclusionalthough bilateral c1lm - c2p is effective for aai from a biomechanical perspective , unilateral screw fixation is a useful alternative in patients with anatomical variations .

INTRODUCTION MATERIALS AND METHODS Operative technique Statistical analysis RESULTS DISCUSSION CONCLUSION

lymphoepithelioma is a term used to designate an undifferentiated malignant epithelial tumor of the nasopharynx that is histologically distinctive because of a markedly prominent lymphoid infiltrate ( 1 ) . carcinomas with similar histological features arising outside the nasopharynx are called lymphoepithelioma - like carcinoma ( lelca ) . lelca occurs in organs such as salivary glands , the uterine cervix , the thymus , the lung , the skin , the stomach , the bladder , the prostate , and the breast ( 2 ) . it is very rare that lelca occurs in the urinary system , there is one case where it occurred in the renal pelvis , and one case where it occurred in the ureter and the kidney simultaneously , but has not been reported so far in urinary bladder in korea . the author et al . have lately experienced lelca that occurred in the urinary bladder , and thus have reported the case along with philological considerations . a 78-yr - old woman presented with gross hematuria for 2 weeks on november 23 , 2009 . the patient was a non - smoker and had not been exposed to carcinogen . physical examination and vital sign were normal at the time when the patient was admitted to this hospital , numerous rbc were observed on urinalysis . there were no notable findings on blood test and chest radiography . on cystoscopy , a frond - like mass was observed at the bladder trigone , which measured about 1 cm ( fig . 1 ) . since hematuria continued , foley catheterization was performed . on computerized tomography ( ct ) of the abdomen and pelvis , the findings of enhancement and a small - sized mass were observed on the inner surface between posterior walls in the bladder trigone . the findings of perivesical infiltration and lymph node metastasis were not observed . on the authority of above findings , the authors suspected the case to be carcinoma in situ of the urinary bladder or transitional cell carcinoma , and thus transurethral resection of bladder tumor ( turbt ) on histopathological examination , it was found that 90% of lesions were lelca and a few lesions were non - invasive transitional cell carcinoma . on microscopy , syncytial growth pattern and indistinct cytoplasmic borders were observed with the severe infiltration of lymphoid cells . tumor cells were positive for cytokeratin 7 but were negative for cytokeratin 20 ( fig . the infiltrated lymphocytes were composed of abundant cd3 positive t cells and cd20 positive b cells . for leukocyte common antigen ( lca ) , the patient has been without recurrence and metastasis for 3 months after the operation , and currently is on a follow - up . ( 3 ) in 1991 , is uncommon with a reported incidence between 0.4 and 1.3% of all bladder carcinoma ( 4 ) . in the urinary tract , they typically arise in the urinary bladder , although isolated cases have been reported in the renal pelvis , ureter , and urethra ( 5 , 6 ) . as suggested by amin et al . ( 7 ) , lelca was categorized as pure ( 100% ) , predominantly ( more than 50% ) , or focal ( less than 50% ) . if other classification is applied , lelca was classified as pure when 100% of the tumor showed lymphoepithelioma - like carcinoma pattern , and mixed when associated with usual infiltrating urothelial carcinoma , adenocarcinoma , or squamous carcinoma ( 2 ) . it has been suggested that pure / predominant lelca responds to chemotherapy and may best be treated with bladder preservation therapy ( 1 , 13 ) . according to previous reports , pure and predominant lelca is more favorable than focal lelca in prognosis ( 2 , 3 , 7 ) . this case was the predominant type that lelca accounted for over 90% of lesions and was fractionally accompanied with non - invasive transitional cell carcinoma . the differential diagnosis is usually malignant lymphoma , invasive transitional cell carcinoma , squamous cell carcinoma and small cell carcinoma . ( 5 ) note that it is imperative to distinguish between lelca and malignant lymphoma , as primary bladder lymphoma is extremely rare . therefore , immunochemical staining , such as lca and keratin , may be used for differentiation . ( 8) maintained that immunochemical staining techniques were helpful to distinguish bladder lymphoma from undifferentiated carcinoma . pooly differentiated transitional cell carcinoma ( tcc ) with a lymphoid linfiltrate should be distinguishable from lelca in that the latter is characterizied by syncytia of tumor cell , vesicular nuclei and prominent nuclei ( 7 ) . the microscopic findings of lelca are characterized by the indistinct cytoplasmic border and the syncytial growth pattern with the prominent lymphocytic infiltrate . it is important to check cytokeratin in tumor cells on immunochemical staining , in order to ascertain that cells are originated from epithelial cells ( 1 ) . the principal symptoms of lelca were mostly gross hematuria , solitary mass , and tumors measured 1 to 5 cm ( 10 , 12 ) . the epstein - barr virus is regarded as one of factors of the lelca that occurred in the thymus gland ( 9 ) . however , it has not been elucidated that lelca of the urinary system is related with ebv ( 10 ) , gulley et al . ( 11 ) reported that ebv , detected from nasophayneal carcinoma , was not detected in 9 out of 11 cases of lelca . also in 9 cases of lelca reported by holmang et al . for lelca treatment , surgical therapy and chemotherapy can be applied . in the case of small tumors that measure 5 cm and less , turbt is applied , but in the case of big or invasive tumors , radical cystectomy may be applied . as nasopharyngeal lymphoepithelioma is well reacted to chemotherapy , methotrexate , vinblastine , doxorubicin and cisplatin may be applied to chemotherapy ( 7 ) . 13 ) reported that primary chemotherapy was performed on 3 patients with muscle invasive lymphoepithelioma of the bladder and as a result their bladder functions were salvaged . it is presumed that bladder lymphoepithelioma may be well reacted to radiotherapy , like nasopharyngeal lymphoepithelioma ( 14 ) . ( 12 ) applied radiotherapy to 4 patients , but could not evaluate whether the patients got cured successfully . lelca is a rare tumor , and it is important to differentiate it from other tumors .

a 78-yr - old woman presented with gross hematuria for 2 weeks . on cystoscopy , a frond - like mass was observed at the bladder trigone . transurethral resection of bladder tumor was performed for the mass . histopathological findings showed that 90% of lesions were lymphoepithelioma - like carcinoma ( lelca ) and a few lesions were non - invasive transitional cell carcinoma . on microscopy , syncytial growth pattern and indistinct cytoplasmic borders were observed with the severe infiltration of lymphoid cells . the case was followed - up for 8 months without recurrence . this is the first report of a lelca case in korea .

INTRODUCTION CASE REPORT DISCUSSION

prostaglandins ( pg ) are a family of structurally related eicosanoids that have regulatory roles in normal physiological as well as pathological contexts . cyclooxygenase enzymes catalyze the conversion of arachidonic acid to pgh2 , which is converted to other prostanoid species including pgd2 , pge2 , pgf2 , prostacyclin ( pgi2 ) , and thromboxane ( tx ) a2 by the action of specific synthases [ 13 ] . the synthesis of pgd2 from its precursor pgh2 is catalyzed by two pgd synthases ( pgdss ) . prostaglandin d2 ( pgd2 ) is involved in a wide variety of neurophysiological functions , such as regulation of body temperature , hormone release , modulation of odor and pain responses , and regulation of the sleep - wake cycle in mammals . pgd2 acts through two receptors ( dp1 and dp2 crth2 ) , whereas 15d - pgj2 can activate peroxisome proliferator - activated receptors or inhibit a range of proinflammatory signaling pathways , including nf-b [ 1 , 2 , 5 ] . the importance of the role of pgd2 in the pathogenesis and resolution of inflammation and innate immune response is increasingly recognized [ 6 , 7 ] . however , the effect of pgd2 on inflammation is complex because pgd2 either promotes or suppresses inflammation depending on the inflammatory milieu . this is further complicated by the fact that pgd2 undergoes nonenzymatic processes to generate 15d - pgj2 , an anti - inflammatory lipid . therefore , the net effect may depend on the rate of production of distal products of pgd2 depending upon the disease process . here , we review the biology and role of pgd synthases and pgd2 in inflammation and host immune response . the arachidonate cyclooxygenase pathway can generate pgd2 by the functional linkage of a series of isoformic enzymes corresponding to phospholipase a2 , cyclooxygenase and pgds . prostanoid formation occurs when cyclooxygenase oxygenates arachidonate converting it to pgg2 , which is then reduced to pgh2 . pgh2 , in turn , is converted to five primary active metabolites , pgd2 , pge2 , pgf2 , pgi2 , or thromboxane a2 via distinct synthases such as pgd synthase and pge synthase [ 1 , 2 ] . two pgd synthases have been identified , lipocalin ( l - pgds ) and hematopoietic ( h - pgds ) [ 4 , 810 ] . l - pgds and h - pgds are quite different from each other biochemically in terms of their amino acid sequence , tertiary structure , evolutional origin , chromosomal and cellular localization , and tissue distribution and immunologically in terms of their functional relevance [ 10 , 11 ] . hematopoietic pgd synthase ( h - pgds ) was previously known as the spleen - type pgds [ 9 , 12 ] or glutathione- ( gsh- ) requiring enzyme for the production of pgd2 in the peripheral tissues [ 12 , 13 ] . h - pgds is characterized as a member of sigma class of glutathione s - transferase ( gst ) gene family that catalyze the conjugation of gsh to an electrophilic substrate . h - pgds is localized in antigen - presenting cells and mast cells of a variety of tissues and is involved in the activation and differentiation of mast cells . h - pgds isomerizes pgh2 to pgd2 selectively and effectively , whereas other gst isozymes catalyze the conversion of pgh2 nonselectively to produce pgd2 , pge2 , and pgf2. the high specificity of h - pgds for the production of pgd2 is attributed to the unique architecture of the catalytic pocket . the deep and wide catalytic cavity of h - pgds is striking in comparison with the narrow shallow cavities of other gsts . the unique 3 d architecture of the cleft leads to the putative substrate binding mode and its catalytic mechanism , responsible for the specific isomerization from pgh2 to pgd2 . h - pgds contributes to the production of the d and j series of prostanoids in the immune system and is involved in allergic inflammatory response . since h - pgds is present in mast cells , th2 cells , and other leukocytes , it is thought to be responsible for the bulk of pgd2 production during allergic responses [ 16 , 17 ] . in mouse models of asthma and allergic disease , h - pgds has a substantial proinflammatory effect , regulating many hallmark characteristics including eosinophilia , airway hyperreactivity , mucus production , and th2 cytokine levels . inhibitors of h - pgds the compound , hql-79 , is characterized as a specific inhibitor of human h - pgds and has shown to exhibit a therapeutic effect when used in animal models of allergic disease and neuroinflammation . thus , selective inhibitors of h - pgds may prove to be more useful to suppress allergic and inflammatory reactions rather than cox-1 or cox-2 inhibitors , such as aspirin , indomethacin , and coxibs because these cox inhibitors suppress the production of all prostaglandins in comparison to h - pgds inhibitors [ 2023 ] . while h - pgds is proinflammatory in allergic airway diseases , h - pgds has shown to be protective in other models of inflammation . showed that h - pgds negatively regulates the severity and duration of delayed type hypersensitivity responses . their data suggests that contrary to the role of h - pgds in driving th2-like responses in models of asthma , hpgds may act as an internal braking signal essential for bringing about the resolution of th1-driven delayed type hypersensitivity reactions . using h - pgds knockout mice showed that h - pgds synthesizes 15d - pgj2 during mammalian defense responses and together with pgd2 , acting through the dp1 receptor , plays a central role in controlling the onset of acute inflammation and its resolution by balancing pro- versus anti - inflammatory cytokines . these data highlight the anti - inflammatory and proresolution properties of cyclopentanone prostaglandins , pgd2 and dp1 receptors . lipocalin - type pgds is gsh independent and is identical to beta trace protein , which was discovered in 1961 as a major protein of human cerebrospinal fluid [ 2628 ] . because it resembles lipophilic ligand carrier proteins it was named lipocalin - type pgd synthase . l - pgds is a bifunctional protein , acting as a pgd2-producing enzyme as well as an intercellular transporter of retinoids or other lipophilic molecules . it is the only enzyme among the members of the lipocalin gene family that binds small lipophilic substances like retinoic acid , bilirubin , and ganglioside . structurally it is a monomer with a -barrel structure and a hydrophobic pocket and was initially identified as responsible for pgd2 production in the brain [ 10 , 19 , 30 ] . since then it has been shown that l - pgds is also expressed in other tissues including the heart , kidneys [ 31 , 32 ] , and lungs [ 33 , 34 ] . l - pgds is secreted into various body fluids , such as csf , plasma , seminal plasma , and urine , and functions as both a pgd2-producing enzyme and an extracellular transporter of various lipophilic substances . the l - pgds/-trace concentration in human serum fluctuates with circadian rhythmicity and exhibits a nocturnal increase and is best known because of its ability to induce sleep . deletion of l - pgds leads to accelerated glucose intolerance and induces obesity , nephropathy , and aortic thickening [ 36 , 37 ] . in animal models of ischemia lack of l - pgds confers susceptibility to injury in brain and heart [ 31 , 32 , 38 ] . l - pgds also has an inhibitory effect on progression of lung , ovarian , and colorectal cancer and some forms of leukemia . thus , it is evident that l - pgds has several key regulatory roles that extend beyond its function in the brain . similar to h - pgds in models of allergic inflammation l - pgds has shown to be proinflammatory . fujitani et al . reported that l - pgds transgenic mice exhibit strong allergic lung responses and eosinophilia with enhanced allergic airway inflammation . in a model of chronic allergic dermatitis blockade of l - pgds with an inhibitor led to significant attenuation of inflammatory response , which was also confirmed in crth2 knockout mice . the proinflammatory role of l - pgds has also been suggested in human ulcerative colitis . hokari et al . showed that the level of l - pgds mrna expression is increased in uc patients in parallel with disease activity . in a diabetic rat model ogawa et al . showed that urinary excretion of l - pgds increased preceding diabetic nephropathy and the levels of l - pgds could predict the progression of renal injury . l - pgds in the urine is being investigated as a diagnostic biomarker of acute kidney injury and inflammation associated with diabetes , hypertension , and drug - induced nephropathy . because l - pgds has a smaller molecular weight than serum albumin it may be expected to appear in the urine even before albuminuria and hence prove as a more sensitive marker for early detection of renal injury . we have studied the role of l - pgds in lps - induced inflammation and shown that l - pgds is induced in vitro in macrophages [ 33 , 34 ] and in vivo in the lung in response to lps and p. aeruginosa . our study showed that h - pgds was constitutively expressed in vitro in macrophages whereas l - pgds is induced in a time - dependent manner in response to lps or pa103 . similarly in vivo studies in mice showed that the expression of l - pgd synthase was induced in response to lps and pa103 . however , the immunomodulatory effects of l - pgds are less well studied . in a mouse model of p. aeruginosa infection we have shown that l - pgds mice have impaired host defenses whereas overexpression of l - pgds is protective in p. aeruginosa - induced pneumonia suggesting a pivotal role for l - pgds in innate immune response . pgs are a group of 20-carbon polyunsaturated fatty acids containing a unique 5-carbon ring structure . prostaglandins are all produced from arachidonic acid ( c20:4 fatty acid ) via their common intermediate , pgh2 , and are a family of structurally related eicosanoids that not only have an important role in homeostasis but also contribute to the pathology of numerous inflammatory diseases . each prostanoid is then produced from pgh2 by its specific terminal pg synthase such as pgd synthase in the case of pgd2 . pgd2 is an acidic lipid mediator derived from arachidonic acid by sequential action of cyclooxygenase and pgd2 synthases . both h- and l - pgd synthase enzymes may form pgd2 in vitro , but it is not fully understood which pgds enzyme predominates under varied conditions in vivo . pgd2 for a long time was considered as a minor and biologically inactive prostaglandin . in the 1980s , however , pgd2 was found to be the most abundant prostaglandin in the brains of rats and other mammals including humans , thus suggesting that it may have an important function in the central nervous system ( cns ) . the physiological functions of pgd2 have now been extensively defined and include regulation of sleep and body temperature , olfactory function , hormone release , and nociception in the central nervous system . it is released from mast cells as an allergic and inflammatory mediator and is responsible for the symptoms in mastocytosis patients , such as flushing , diarrhea , tachycardia , dyspnea , and deep sleep . pgd2 is further converted to 9 , 11-pgf2 , a stereoisomer of pgf2 , which exerts various pharmacological actions different from those induced by pgf2. pgd2 is also readily dehydrated in vitro and in vivo to produce prostaglandins of the j series , such as pgj2 , -pgj2 , and 15-deoxy--pgj2 . 15-deoxy--pgj2 has been identified as an endogenous ligand for a nuclear receptor ( peroxisome proliferator - activated receptor ) , and it promotes adipocyte differentiation [ 5 , 6 ] . pgd 2 has been implicated in the initiation and progression of inflammation . in mouse models of asthma and allergic disease , pgd2 has a substantial proinflammatory effect , regulating many hallmark characteristics including eosinophilia , airway hyperreactivity , mucus production , and th2 cytokine levels [ 40 , 47 ] . moreover , inhibition of pgd 2 synthesis and pgd 2 signaling blockade has a suppressive effect on neuroinflammation in mouse models of krabbe 's disease . the injection of pgd 2 into the skin has been shown to result in erythema , edema , induration , and leukocyte infiltration . pgd 2 and other vasodilator prostaglandins may also contribute to inflammation by increasing local blood flow . in contrast to these proinflammatory effects , pgd 2 and its cyclopentenone prostaglandin derivatives also have anti - inflammatory properties , with functions in resolution of inflammation . there is considerable interest in the importance of pgd2 and its distal products in the mediation and resolution of inflammation [ 3 , 57 ] . gilroy et al . showed that in a model of experimental pleuritis pgd2 significantly attenuated inflammation . similarly in a model of experimental colitis cox-2-derived pgd2 , acting via the dp receptor , was shown to attenuate neutrophilic infiltration into colonic mucosa . in a human model of an acute inflammatory response induced by administration of lps , which evokes transient flu - like symptoms with pyrexia and a hemodynamic response , song et al . have shown that tetranor pgdm increases markedly during this response and that pgj2 has antipyretic effects . although several studies have investigated the role of pgd 2 in inflammation , the role of pgd2 in host immune response has been scantly studied . an earlier study showed that pgd2 concentration , but not the pge2 or il-1 concentrations , is elevated in a time - dependent manner in the csf of patients with african sleeping sickness , caused by trypanosoma brucei . these investigators have also shown that mouse astrocytes and fibroblasts in culture induce the production of pgd2 in response to t. brucei . although the production of pgd2 was increased in vitro , the functional effects of pgd2 in this setting remain unclear . in a recent investigation zhao et al . showed that an age - related increase in pgd2 in mice led to diminished respiratory dendritic cell migration resulting in defects in virus - specific t - cell responses in vivo . they further showed that administration of pgd2 antagonist reversed this defect resulting in migration of dendritic cells with enhancement of t - cell antivirus response with increased clearance and survival . these data suggest that similar to allergic airway disease pgd2 may have immunosuppressive effects in viral infections . in a mouse model of p. aeruginosa lung infection we have shown that inhibition of cox-2 improves survival in a lethal model of p. aeruginosa infection . the bacterial clearance of p. aeruginosa was enhanced in cox-2 knockout mice whereas transgenic mice that overexpress cox-2 have an impaired bacterial clearance from the lungs . our study showed that the immunomodulatory effects of inhibition of cox-2 are related to inhibition of pge2 . we also examined the effects of administration of pgd2 in a model of p. aeruginosa lung infection . mice that were given intratracheal pgd 2 showed an enhanced clearance of p. aeruginosa from the lungs . these results were in agreement with our studies from l - pgds knockout and l - pgds overexpressing mice . we have shown that pgd2 inhibits a key proinflammatory immunoglobulin cell surface receptor trem-1 in vitro in macrophages . furthermore , we have shown that pgd2 induces the expression of nrf2 in a dp1 receptor - dependent manner . these studies provide a new paradigm and highlight a key regulatory role of pgd2 in innate immune response to bacterial infections . the role of pgds / pgd 2 in regulating inflammation in a variety of organ systems and disease process is burgeoning . the inflammatory response protects the body against infection and injury but can itself become dysregulated with deleterious consequences to the host . it is now evident that endogenous biochemical pathways such as pgds / pgd 2 get activated during defense reactions . the effect of pgds / pgd2 on inflammation is complex because they can either promote or suppresses inflammation depending on the inflammatory milieu . table 1 provides a summary of the models of different effects of pgds / pgd2 . interdiction of l - pgds , pgd2 , and dp receptors provides novel therapeutic approaches to modulate inflammation and innate immune responses .

pgd2 is formed from arachidonic acid by successive enzyme reactions : oxygenation of arachidonic acid to pgh2 , a common precursor of various prostanoids , catalyzed by cyclooxygenase , and isomerization of pgh2 to pgd2 by pgd synthases ( pgdss ) . pgd2 can be either pro- or anti - inflammatory depending on disease process and etiology . the anti - inflammatory and immunomodulatory attributes of pgds / pgd2 provide opportunities for development of novel therapeutic approaches for resistant infections and refractory inflammatory diseases . this paper highlights the role of pgd synthases and pgd2 in immune inflammatory response .

1. Introduction 2. PGD Synthases 3. Hematopoietic PGD Synthase 4. Lipocalin-Type PGD Synthase 5. Prostaglandin D 6. Conclusions

cancer dormant cell theory takes the view that activation of resting cells is the key factor causing cancer metastasis . recent studies indicate that resting cells can be activated through escaping immune surveillance when immune function is weakened and that energy used for cell revivals can be provided by the newborn vessels [ 13 ] . therefore , the study on improving body immune surveillance and restraining the energy of tumor growth will be an emergent mission as well as a breakthrough in the field of clinical treatment of cancer . chlorogenic acid ( ca ) , extracted from folium cortex eucommiae and the flower bud of lonicera confusa , is a kind of depside formed by caffeic acid and quinic acid . a large number of studies on ca have demonstrated that ca has a wide range of biological activities including inhibition of tumor cells . according to preliminary studies , we recognize that ca is able to inhibit tumor in mouse except for these with t - cells defect , which suggests that immune system can be one of the targets of tumor suppression . meanwhile , in vivo studies of our group indicate that ca also changes the advantage state of th2 drift of balb / c emt-6 mice . it significantly enhance the activities of balb / c emt-6 mice cytotoxic t lymphocyte and natural killer cells as well as strengthening macrophage phagocytosis activity and lymphocyte transcription activity , thus boosting specific and nonspecific cellular immune function to tumor cells . recent studies show that antitumor property of ca may have a connection with its ability of enhancing the activities of aryl hydrocarbon hydroxylase , suppressing formation of 8-oh - dg , carcinogen - dna adduct , and oxygen radical [ 5 , 6 ] . meanwhile , ca can guard against gastric cancer and colon cancer and even suppress related carcinogenic factors [ 7 , 8 ] . in vitro studies revealed that ca can enhance t - cell proliferation caused by influenza virus antigen and can induce the generation of ifn- and ifn- by human lymphocytes and peripheral blood leukocytes [ 9 , 10 ] . additionally , we find that ca can also activate neurocalcin to strengthen the activity of macrophagocyte . although much evidence has proved the anticancer property of ca , little is known about its exact targets on molecular level . base on the principle of complementary base pairing , microarray technology can distinguish particular genes from the mixture of genes by taking advantage of gene probes . . meanwhile , its sensitivity , accuracy , and specificity are better than those of normal pcr . to explore the expression level of genes in tumor cells , we utilized a microarray technique to detect balb / c emt-6 mice after treatment with ca , docetaxel , interferon , normal saline separately , and the differences in expression level were confirmed by rt - qpcr . time - series analysis , go analysis , and pathway analysis were used to screen out common genes and analyze the relationship between putative genes and anticancer process of ca . our data has suggested that ca is able to inhibit the growth of tumor through regulating immune system . female spf mice ( balb / c ) used in this experiment weigh 17 - 18 g on average , provided by the animal center of sichuan university . emt-6 sarcoma cells were provided by west china hospital of sichuan university department of health engineering key laboratory of transplantation and transplantation immunity . we took emt-6 cell line out of the 152c ultra low temperature refrigerator . after thawing , centrifuging , and primary culturing , we used 0.25% trypsin for digestion twice and all the collected cells were diluted with phosphate - buffered saline ( pbs ) in the end . we homogenized the tumor taken from the body of balb / c tumor - bearing mice to cell suspension and then inoculated the cell suspension to other balb / c mice . mice used for microarray analysis were injected with high - dose ca ( experimental group ) , docetaxel ( control group ) , and normal saline ( negative control group ) . gene expression of experimental group was analyzed at six time points ( 3rd , 6th , 9th , 12th , 15th , and 18th days , resp . , after administration ) , while the control group and negative control group were both analyzed at the 12th days after administration . rt - qpcr took mice injected with low , medium , and high - dose ca ( 5 mg / kg , 10 mg / kg , 20 mg / kg ) as experimental group , docetaxel and interferon as control group , and normal saline as negative control group . all extracted samples had an a260/280 ratio of between 2.0 and 2.1 and a 28s/18s ratio of 2 . agilent 2100 bioanalyzer was used for further verification and qualified rna had an rin greater than 7.0 . total rna was transcribed to double - stranded cdna using revertaid first strand cdna synthesis kit ( fermentas ) , with t7 promoter primer . crna was dyed with cy3 and purified using rneasy mini kit ( qiagen , valencia , ca , usa ) according to the protocol of the rneasy plus mini kit . cy3 crna must be fragmented in a buffer of 11 l 10 blocking agent , 2.2 l 25 fragmentation buffer and a certain amount of nuclease - free water at 60c for half an hour before hybridization . we added 55 l 2 gex hybridization buffer to the denatured and fragmented crna and then transfered them to microarrays at 65c for 17 h with constant rotation . after hybridization , the microarrays were washed twice with buffer 1 for 1 minute followed by buffer 2 at 37c for 1 minute too . in the end , the microarrays were autoscanned twice in 100% and 10% pmt , respectively , at 5 m resolution . the data was analyzed by agilent feature extraction software and the quantile normalization was finished by genespring 10.0 . after acquisition of raw data , agilent feature extraction software kit was used to eliminate the effect of background signals through automatic gridding . we took the log 2 of the normalized background adjusted values to narrow the threshold of fluorescence signal strength before quantile normalization by genespring 10.0 and then used a linear model to estimate expression values on the log scale . we chose a standard among the microarray samples thus the whole microarray data varied on it to obtain the same baseline of average density . differentially expressed genes are submitted to student 's t - test and p 0.05 . we inputted the data of probes used for gene expressing analysis into sbc analysis system ( v2010.05 ) . rna extracted from experimental group of ca , positive control group of docetaxel , and interferon , negative control group were used to measure expression of selected genes of interest by rt - qpcr . genes can , nfatc2 , nfatc3 , and nfatc2ip showed a different expression level following treatment with low , medium , and high - dose ca , respectively . primers used for rt - qpcr validation were designed according to each gene sequence and finished by sbc . the rna integrity was verified with rna formaldehyde electrophoresis and the quality was detected by spectrophotometry . the rna samples had an a260/280 ratio of greater than 2 and a 20srna/18srna ratio of greater than 1.1 . reverse transcription reactions were conducted at 42c for 30 min and at 85c for 5 min at a 20 l total reaction volume containing the following reagents : 2 l rna , 1 l oligo , 2 l ( 10 mm ) dntp mixture , 1 l ribolock rnase inhibitor , 4 l 5 reaction buffer , 9 l water , and 1 l revertaid m - mulv . pcr reaction should be taken at a temperature gradient in the range of annealing temperature of can , nfatc2 , nfatc3 , and nfatc2ip . rt - qpcr analysis was conducted to obtain crossing point ( ct ) values of each gene and standard curve was established through the linearity between ct value and log 2 of expression values . before carrying out rt - qpcr analysis , cdna and primers should be diluted in 20 l reagents containing the following : 9 l mix , 8 l h2o , 1 l sense primer ( f ) , 1 l antisense primer ( r ) , and 1 l cdna . relative content of mrna extracted from experimental group of ca , positive control group of docetaxel , and interferon negative control group was calculated by rt - qpcr detection system biorad iq5 . we carried out a student 's t - test of these probes to select differentially expressed genes ( p 0.05 ) and then drew curves reflecting different expression tendency of them at 6 time points . the smaller the p value is , the more similar each gene trend fits the curve . no.21 fitting curve had a p value close to 0 and the expression profiles of genes covered in this fitting curve were analyzed systematically here . as shown in figure 1 , each inflection point corresponds to gene expression at different time points after administration . genes covered in this curve were upregulated as seen from the whole time cycle ; in other word , ca is capable of upregulating certain genes of tumor - bearing mice . data of genes covered in no.21 was put into sbc analysis system ( v2010.05 ) and compared with kegg ( kyoto encyclopedia of genes and genomes ) database . as shown in table 2 , 96 pathways are found to be related to upregulated genes and 29 of them are found to be statistically significant including t - cell receptor signaling pathway , b - cell receptor signaling pathway , and natural killer cell mediated cytotoxicity . immune - related genes expression is speculated - changed after treatment with ca and probably have a trend of upregulation . previous studies on ca immune function verified that ca could increase the carbon clearance index and the content of serum hemolysin and enhance the phagocytic function . the release of tumor necrosis factor ( tfn ) was found reduced while the activation of cytotoxic t lymphocyte ( ctl ) which aimed at lewis lung cancer was boosted . nk cells activation and lymphocyte transformation rate of lewis lung cancer bearing mice were both significantly improved through the determination of immune function indexes . to sum up , ca has been proved capable of strengthening cellular immune functions to tumor . when 3 immune related pathways were found including upregulated genes , we inferred that the immunity of mouse had been improved after treatment of ca . gsk3b , nfatc2 , nfatc3 , can , vav2 , and nck1 were found upregulated in the 3 immune related pathways as listed in table 3 . according to the functions , genes covered in no.21 can be classified into 312 different go ( gene ontology ) and 6 of them has a p value less than 0.05 as listed in table 4 . go of statistical significance includes the following genes : cyp2a4 , cyp2c37 , cyp2c38 , cyp27b1 , cyp51 , e4f1 , fdps , gata4 , eif2ak1 , myt1l , rarg , sod1 , sp1 , taf3 , nr2c2 , hnf4 g , zdhhc3 , phf6 , mynn , zfp386 , zfp617 , zfp114 , zfp238 , zfp174 , zfp113 , zfp109 , zfp263 , pcdhb6 , pcdhb15 , pcdhb20 , pcdhb21 , cdyl , nfatc3 , pbx2 , rest , smarca5 , and nfatc2 . after doing some queries on the function of genes covered in statistically significant pathways and go , we found out immune related genes and common genes of pathway and go . nfatc2 , nfatc3 , nfatc2ip , and can were found through combing raw analysis data of gene expression profile , as shown in table 5 . relative expression level of the target genes nfatc2 , nfatc3 , nfatc2ip , and can on average is shown in table 6 . relative expression level of nfatc2 , nfatc3 , nfatc2ip , and can in high - dose ca group , nfatc2ip and can in medium - dose ca group , and can in low - dose ca group significantly improved compared with negative control group . the exact expression quantity is shown in figure 2 . as shown in figure 2 , relative expression level of nfatc2ip , nfatc3 , nfatc2 , and can in high - dose ca group improved compared with negative control group , which corresponds to the result of microarray analysis . here , a whole mouse genome oligo microarray ( 444k ) was used to analyze gene expression level of female balb / c mice and to compare the expression of corresponding target genes of each group on the time series . systematic error or accidental error can affect the accuracy of microarray analysis to some extent and the errors may come from the processes of rna extraction , rna reverse transcription , and hybridization as well as the quality of gene chips and rna . while the results of microarray analysis can reflect expressing pattern at body 's internal gene level quickly , false positive results possibly caused by systematic error or accidental error must be taken into consideration . verifying experiments like elisa test , western blot test , and pcr test should be taken for further confirmation . here , the results of pcr test help completing the study as well as making it more convincing . through the analysis of genes included in no.21 , expression of critical genes in t - cell receptor signaling pathway , b - cell receptor signaling pathway , and natural killer cell mediated cytotoxicity had changed and immune related genes nfatc2ip , nfatc3 , nfatc2 , and can were found . nfat ( nuclear factor of activated t cells ) which consists of 4 components nfatc1 , nfatc2 , nfatc3 , and nfatc4 has been proved important in lymphocyte activation and development . nfatc2 existing in cytoplasm translocates to the nucleus upon t - cell receptor stimulation and then becomes a member of the nuclear factor of activating t - cells transcription . when the body lacks nfatc2 , lymphocyte apoptosis will be significantly reduced which suggests lymphangiectasia and th2-type response . immune regulatory function will disorder when th1/th2 balance is destroyed and tumor occurrence shows the preponderance state tendency of th2 . this state will weaken antitumor immune function and will induce tumor cells free from immune surveillance and immune attack . this may be one of the immune mechanisms of tumor development and provides a new idea of tumor treating . the reverse of th1/th2 abnormal drift is in favor of recovering antitumor immunocompetence and reducing tumor recurrence and metastasis to improve long - term survival rate finally . nfatc2 and nfatc3 have been proved to synergistically regulate the reaction of t cell receptor , cell division , and th2 differentiation . th2-type reaction which suggests the secretion of il-4 , il-5 , and il-6 is increased whereas the decrease il-2 , ifn- , tnf- , and il-10 happens when the body lacks nfatc2 and nfatc3 [ 1416 ] . when b cell and t - cell lack nfatc2 and nfatc3 simultaneously , the function of t cell will be weakened but the ability of t cell receptor mediating cell proliferation still exists , while b cells over - activate and show excessive differentiation . the fact that expression of nfatc2 and nfatc3 is improved after treatment of ca indirectly indicates that ca could reverse th1/th2 drift . meanwhile , secretion of il-2 , ifn- , tnf- , and il-10 is connected to ca too . high expression of nfatc2 and nfatc3 is in favor of the secretion of these cytokines which have already been used as nonspecific immunity treatment to cancer . nfatc2ip can induce the expression of t - cell cytokines , especially enhancing il production . three splice variants existing in nfatc2ip are able to methylate nfatc2ip after its translation to produce nfatc2ip regulatory factor . expression of th1-type and th2-type cell factors will be suppressed when the methylation process is inhibited . therefore , methylation process of nfatc2ip is an important controlling point of manipulating expression of nfat - dependent cell factors but it will not have any influence on general transcription factor of th1 and th2 nor nfat activation . can ( calcineurin ) is known as the only serine / threonine protein phosphatase regulated by ca()-calmodulin so far and mainly aims at catalyzing dephosphorylation of phosphatidylserine and phosphatidyl threonine . it is a multifunctional signaling enzyme involved in function regulation of many cells and distributes in a wide range of tissues especially nerve tissue , t lymphocytes , heart , and skeletal muscle [ 18 , 19 ] . nfat regulates many genes expression as well as influencing many cells differentiation through can / nfat signaling pathway . can is able to make nfat existing in cytoplasm move into cell nucleus after dephosphorylation to finish following transcription and translation , and then nfatc can combine with ap-1 family members alone or in groups to stimulate secretion of cell factors in certain areas such as il-2 . transcription induction of il-2 is a sign of t - cells activation . as mentioned above , nfat is very important in regulating immunoreaction ; meanwhile , nfatc3 and nfatc2 play a particularly key role in correcting body 's immune function . thus , high expression of can is a benefit to dephosphorylation of nfat and indirectly has an effect upon immune system . the result of microarray analysis of differentially expressed genes of female balb / c emt-6 mice has indicated that the antitumor mechanism of ca is closely related to body 's immune system . through upregulating , the expression of can , nfatc2 , nfatc2ip , and nfatc3 , ca is able to improve the transcription of immune factors like il-2r and ifn- , stimulate proliferation and activation of t cells , nk cells , and macrophage , strengthen monitoring and killing abilities of cancer cells , and inhibit growth of tumor finally . the understanding of the anticancer mechanism of ca has provided a reliable evidence of taking advantage of ca to fight against cancer .

persistently increasing incident of cancer in human beings has served to emphasize the importance of studies on mechanism of antitumor substances . chlorogenic acid ( ca ) , extracted from folium cortex eucommiae , has been confirmed to have lots of biological activities encompassing inhibition of tumor . however , the anticancer mechanism of ca remains unclear . here , we have utilized a whole mouse genome oligo microarray ( 444k ) to analyze gene expression level of female balb / c mice ( implanted with emt-6 sarcoma cells ) after treatment with low , medium , and high - dose ca ( 5 mg / kg , 10 mg / kg , and 20 mg / kg ) , docetaxel , interferon , and normal saline separately at 6 time points ( 3rd , 6th , 9th , 12th , 15th , and 18th days after administration ) . differentially expressed genes screened out by time - series analysis , go analysis , and pathway analysis , and four immune - related genes were selected for further confirmation using rt - qpcr . the results demonstrated that ca is able to change gene expression and that the responsive genes ( can , nfatc2 , nfatc2ip , and nfatc3 ) involved in immune pathways had been significantly upregulated by ca . expression of immune factors such as il-2r and ifn- can be improved by ca to promote activation and proliferation of t cells , macrophages , and nk cells , thus enhancing their surveillance and killing abilities , further suppressing the growth rate of tumor cells .

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

the disease is endemic all over india especially in uttar pradesh , bihar , jharkhand , andhra pradesh , orissa , tamil nadu , kerala and gujarat . there are at least six million attacks of acute filarial disease per year and 45 million persons are currently having one or more chronic filarial lesions . . conventional mode of diagnosis of filariasis is by demonstration of microfilaria in peripheral blood smear . despite high incidence , it is infrequent to find microfilariae in fine needle aspiration cytology ( fnac ) smears and body fluids . the literature contains a few reports of microfilariae found in various locations including thyroid nodule , skin and soft tissue swelling , epididymis , breast,[268 ] salivary gland , cervicovaginal smear , ovarian cyst , urine , lymph node , and effusion fluids . the aim of present study was to assess the role of fnac in diagnosis of filariasis in asymptomatic patients having superficial lumps . the study was conducted at the department of pathology in collaboration with the departments of medicine and surgery at b.r.d . medical college , gorakhpur during a period of two years i.e. 2006 - 2007 . a total of 250 cases , with age ranging from 15 - 80 years , having swellings at various sites were included in the present study . aspiration was made by technique of martin and ellis . in case of cystic lesions , cyst content was aspirated and smears prepared from cyst fluid after cytocentrifugation were studied along with the aspiration performed from cyst wall . these smears were wet fixed immediately in 95% alcohol and stained by hematoxylin and eosin and papanicolaou stain . this study was conducted on 24 cases of filariasis diagnosed on routine fnac material from various sites . out of these 24 cases , maximum cases of filariasis were reported in breast swelling ( eight cases ) , followed by lymph nodes ( six cases ) , scrotal swellings ( four cases ) , thyroid swellings ( three cases ) , soft tissue swellings ( two cases ) and ascitic fluid ( one case ) . clinical presentations of these cases were variable which included swelling , pain , fever and erythema [ table 1 ] . showing clinical profile , cytological findings and associated conditions of 24 cases smears revealed sheathed microfilariae , tails of which were free from nuclei and many had graceful curves . thick and thin blood smear examination of nocturnal venous blood revealed microfilariae of wuchereria bancrofti in three out of 24 cases . microscopic examination of breast swellings showed sheathed microfilaria along with few groups of benign ductal epithelial cells , myoepithelial cells , bare nuclei , few fragments of fibrofatty tissue and inflammatory cells comprising of eosinophils and neutrophils [ figure 1 ] . two cases of breast lumps showed epithelioid non - necrotising granuloma without giant cells and plasma cells . fnac smear of breast swelling showing sheathed microfilaria along with inflammatory cells thyroid aspirates revealed few groups of follicular cells in the background of colloid . in between follicular groups , aspirate from scrotal swelling showed numerous coiled and uncoiled sheathed microfilariae along with neutrophils , eosinophils and few lymphocytes [ figure 2 ] . fnac smear of scrotal swelling showing sheathed microfilariae along with polymorphs , macrophages and eosinophils lymph node aspirates showed sheathed microfilariae in the background of mixed population of lymphoid cells comprising of mature lymphocytes , centrocytes , centroblasts , dendritic cells and few eosinophils . cytological findings of soft tissue swellings showed microfilariae along with neutrophils , eosinophils and granular debris . cell adherence of inflammatory cells and macrophages to microfilariae was seen in three out of 24 cases . filariasis is a major public health problem in tropical countries , including india . in endemic areas like eastern part of uttar pradesh , a majority of infected individuals in filarial endemic communities were asymptomatic . in the present study maximum cases ( eight out of 24 cases ) many authors have reported microfilariae in breast lumps by fnac smears.[2468 ] aspirates from lymph nodes ( five out of 24 ) , demonstrated microfilariae in a background of reactive lymphoid cells . the lymphatic vessels of spermatic cord appear to be common and perhaps the principal site of adult wuchereria bancrofti in men with asymptomatic microfilaremia . occurrence of living w bancrofti in scrotal area of men was demonstrated by noroes et al . two cases of soft tissue swellings and one case of ascitic fluid showed microfilaria along with inflammatory cell including eosinophils , lymphocytes and macrophages . out of 24 cases showing microfilariae in fnac smear examination , blood eosinophilia was present in eight cases , of which microfilaremia in nocturnal venous blood smear examination was observed in three cases only . findings are consistent with observation made by others , who reported that filariasis can exist without microfilaremia . significant adherence of inflammatory cells and macrophages to microfilariae was present in three out of 24 cases . despite high incidence of filariasis , microfilaria in fine needle aspiration cytology is not a very common finding . careful screening of fnac smears undoubtedly the demonstration of parasite , in aspirate , play a significant role in recognition of disease and institution of specific treatment , thus obviate the severe manifestations of lymphatic filariasis .

background : filariasis is a major health problem in tropical countries including india . fine needle aspiration cytology plays an important role in prompt recognition of disease.aim:to assess the role of fine needle aspiration cytology ( fnac ) in diagnosis of filariasis at all possible sites.materials and methods : total 250 cases of superficial swellings at various sites were subjected to fine needle aspiration cytology.results:out of 250 cases , 24 cases of filariasis were detected which include breast lumps ( 8 cases ) , lymph nodes ( 6 cases ) , scrotal swellings ( 4 cases ) , thyroid swellings ( 3 cases ) , soft tissue swellings ( 2 cases ) and ascitic fluid ( 1 case ) . eosinophilia was present in 8 out of 24 cases with a percentage ranging from 12 - 24% . significant adherence of inflammatory cells and macrophages to microfilariae was present in 3 out of 24 cases.conclusions:in endemic areas , it should be considered one of the differential diagnoses of a superficial swelling . careful screening of fnac smears help in detecting microfilaria even in asymptomatic patients and thus plays a significant role in recognition of the disease and institution of specific treatment .

Introduction Materials and Methods Results Discussion Conclusions

stroke accounts for approximately 11% of all deaths worldwide and is the most common cause of adult - acquired disability . among all stroke cases , ischemic stroke and intracerebral hemorrhage ( ich ) account for about 8085% and 1520% , respectively . intravenous thrombolysis by using tissue plasminogen activator ( tpa ) is the only approved treatment for acute ischemic stroke . however , tpa has very narrow time window ( within 4.5 h after onset ) of application . therefore , only a minority of patients ( 2% to 4% ) can receive timely therapy . cell therapy might be a promising strategy for stroke . bone marrow - derived mononuclear cells ( bm - mncs ) and mesenchymal stem cells ( bm - mscs ) both are bone marrow stromal cells ( bm - scs ) and are most frequently used in preclinical and clinical neurorestorative studies in stroke . bm - mncs / mscs have self - renewal capacity and pluripotency to differentiate into several mesenchymal cellular lineages , including osteoblasts , chondroblasts , adipocytes , myocytes , and fibroblasts . they can also differentiate into non - mesenchymal lineages , including neurons and glial cells . preclinical studies observed that bm - mncs / mscs transplanted either intracranially or intravascularly could migrate to damaged brain tissue and exert a neuroprotection effect by inhibiting apoptosis , decreasing peri - infarct inflammation , and promoting angiogenesis [ 79 ] . therefore , during the past decade , a series of clinical trials was performed to assess the effectiveness and safety of bm - scs transplantation after stroke . due to the small number of patients recruited in individual trials , the statistical power of the conclusions is weak . one recent single - arm meta - analysis showed this cell therapy could effectively improve national institutes of health stroke scale ( nihss ) scores , modified barthel index ( mbi ) score and modified rankin score ( mrs ) . however , without comparison with a control group , there might have observational bias . therefore , this study aimed to pool previous controlled trials to assess the effectiveness of bm - scs - based cell therapy after ischemic stroke . this study generally followed the preferred reporting items for systematic reviews and meta - analyses ( prisma ) guidelines . relevant studies published from 1 jan 2000 to 1 sept 2014 were searched among pubmed , medline , embase , and the cochrane database . we only included randomized or non - randomized controlled trials that assessed effectiveness of bm - mncs / mscs - based cell therapy in either ischemic stroke patients . the basic data extracted from original studies included : family name of the first author , year of publication , type of stoke , study design , number of patients , mean age , type of cell used , route of cell delivery , number of cells injected , time interval from stroke to therapy , follow - up , baseline nihss score , and outcome indicator measured . to assess the effectiveness of cell therapy , the outcome indicators used to assess therapeutic effectiveness include modified rankin score ( mrs ) , barthel index ( bi ) or modified barthel index ( mbi ) , and national institutes of health stroke scale ( nihss ) . original data were pooled and analyzed by using review manager 5.3 ( the cochrane collaboration ) . the risk ratio ( rr ) and corresponding 95% confidence intervals ( ci ) of mrs 2 ( cell therapy vs. control ) were estimated . for the discontinuous data , including bi or mbi and nihss score , weighted mean difference ( wmd ) and corresponding 95% ( ci ) was estimated . the chi - square based q test and i value were used to assess between - study heterogeneity , which also determines the methods used for making estimation . the random - effects model ( dersimonian and laird method ) was used when p < 0.1 in q test or i > 50% , which indicates significant heterogeneity . otherwise , the fixed - effects model based on mantel - haenszel method was applied . this study generally followed the preferred reporting items for systematic reviews and meta - analyses ( prisma ) guidelines . relevant studies published from 1 jan 2000 to 1 sept 2014 were searched among pubmed , medline , embase , and the cochrane database . we only included randomized or non - randomized controlled trials that assessed effectiveness of bm - mncs / mscs - based cell therapy in either ischemic stroke patients . the basic data extracted from original studies included : family name of the first author , year of publication , type of stoke , study design , number of patients , mean age , type of cell used , route of cell delivery , number of cells injected , time interval from stroke to therapy , follow - up , baseline nihss score , and outcome indicator measured . to assess the effectiveness of cell therapy , the outcome indicators used to assess therapeutic effectiveness include modified rankin score ( mrs ) , barthel index ( bi ) or modified barthel index ( mbi ) , and national institutes of health stroke scale ( nihss ) . original data were pooled and analyzed by using review manager 5.3 ( the cochrane collaboration ) . the risk ratio ( rr ) and corresponding 95% confidence intervals ( ci ) of mrs 2 ( cell therapy vs. control ) were estimated . for the discontinuous data , including bi or mbi and nihss score , weighted mean difference ( wmd ) and corresponding 95% ( ci ) was estimated . the chi - square based q test and i value were used to assess between - study heterogeneity , which also determines the methods used for making estimation . the random - effects model ( dersimonian and laird method ) was used when p < 0.1 in q test or i > 50% , which indicates significant heterogeneity . otherwise , the fixed - effects model based on mantel - haenszel method was applied . based on searching and screening with the preset criteria , 5 trials [ 1115 ] were finally included in this meta - analysis . the 5 trials involved a total of 228 patients , among which 104 were in the cell therapy group and 124 were in the control group . two studies used bm - mscs and 3 studies used bm - mncs [ 1315 ] . four studies transplanted cells through iv injection [ 1113,15 ] and 1 used ia injection . the time interval from stroke to cell therapy varied from several days to several months after stroke . four trials reported outcome with 6-month follow - up , while 1 study reported 5-year outcome . generally , modified rankin score ( mrs ) , barthel index ( bi ) , modified bi ( mbi ) , and national institutes of health stroke scale ( nihss ) scores are the 3 indicators most used to assess clinical outcomes of cell therapy . two studies reported bi and 2 studies reported mbi at the end of follow - up . generally , although the cell therapy group had slightly higher bi or mbi score , the mean difference was not significant between cell therapy and control group ( wmd : 2.50 , 95%ci : 4.69 to 9.68 , p=0.50 , i=46% ) ( figure 2 ) . subgroup analysis was performed by stratifying bi and mbi . subgroup using mbi as the indicator of daily activities of living reported significantly higher mbi score in the cell therapy group than in the controls ( wmd : 7.44 , 95%ci : 1.82 to 13.06 , p=0.009 , i=0% ) ( figure 2 ) , but no significant difference was observed in the bi subgroup ( wmd : 3.24 , 95%ci : 12.14 to 5.65 , p=0.47 , i=0% ) ( figure 2 ) . two studies reported nihss at the end of follow - up . generally , the mean difference of nihss score was significant lower in the cell therapy group than in the control group ( wmd : 1.85 , 95%ci : 2.77 to 0.93 , p<0.0001 , i=24% ) ( figure 3 ) . due to the non - randomized design of some studies , we only compared the proportion of patients with mrs 2 before and after cell therapy in the experimental arm . the meta - analysis did not find significant change in the proportion of patient in the mrs 2 group before and after cell therapy ( 13/86 vs. 15/86 ) ( rr : 1.81 , 95%ci : 0.37 to 8.95 , p=0.47 ) ( figure 4 ) . infection , recurrence of stroke , and death were used to assess the safety of bm - mscs transplantation . our meta - analysis did not find any difference in these 3 indicators between the cell therapy and control group ( figure 5 ) . two studies reported bi and 2 studies reported mbi at the end of follow - up . generally , although the cell therapy group had slightly higher bi or mbi score , the mean difference was not significant between cell therapy and control group ( wmd : 2.50 , 95%ci : 4.69 to 9.68 , p=0.50 , i=46% ) ( figure 2 ) . subgroup using mbi as the indicator of daily activities of living reported significantly higher mbi score in the cell therapy group than in the controls ( wmd : 7.44 , 95%ci : 1.82 to 13.06 , p=0.009 , i=0% ) ( figure 2 ) , but no significant difference was observed in the bi subgroup ( wmd : 3.24 , 95%ci : 12.14 to 5.65 , p=0.47 , i=0% ) ( figure 2 ) . two studies reported nihss at the end of follow - up . generally , the mean difference of nihss score was significant lower in the cell therapy group than in the control group ( wmd : 1.85 , 95%ci : 2.77 to 0.93 , p<0.0001 , i=24% ) ( figure 3 ) . three studies reported the change in mrs at the end of follow - up . due to the non - randomized design of some studies , we only compared the proportion of patients with mrs 2 before and after cell therapy in the experimental arm . the meta - analysis did not find significant change in the proportion of patient in the mrs 2 group before and after cell therapy ( 13/86 vs. 15/86 ) ( rr : 1.81 , 95%ci : 0.37 to 8.95 , p=0.47 ) ( figure 4 ) . infection , recurrence of stroke , and death were used to assess the safety of bm - mscs transplantation . our meta - analysis did not find any difference in these 3 indicators between the cell therapy and control group ( figure 5 ) . in animal models , transplantation of bm - mncs or mscs could reduce inflammation , decrease the infarct size in the brain , and improve neurological function in several models of stroke through multiple mechanisms [ 1618 ] . a recent meta - analysis based on 46 preclinical animal studies previous preclinical studies observed that although bm - mscs and bm - mncs could transdifferentiate into neuronal - like in vitro , they did not have basic neuronal functional properties ; this transdifferentiation seldom happens in vivo . in fact , a study based on animal models showed that only a very small proportion ( about 0.02% ) of the intravenously delivered bm - mncs migrate to the ischemic area of the brain , while most of the transplanted cells develop a macrophage / microglial phenotype . generally , the transplanted cells have a stimulating effect on release of cytokines and neurotrophic factors , including brain - derived neurotrophic factor ( bdnf ) , basic fibroblast growth factor ( bfgf ) , nerve growth factor , vascular endothelial growth factor ( vegf ) , insulin - like growth factor-1 , hepatocyte growth factor ( hgf ) , and stem cell factor . these factors can induce angiogenesis , reduce neuronal apoptosis , enhance axonal regeneration , rebuild synapses and dendrites , and promote differentiation of endogenous neural stem and progenitor cells . these effects do not necessarily require the presence of transplanted cells at the injury site in the brain . therefore , the paracrine effects of transplanted cells might be fundamental to positive clinical outcomes . however , in clinical trials , data on the exact effects of bm - mncs / mscs - based cell therapy after stroke are still conflicting . this study , based on 5 double - arms trials , demonstrated that bm - derived stromal cells might have some benefits in lowering the grade of impairment caused by ischemic stroke . in fact , a 1-point increase of nihss score decreases the likelihood of an excellent outcome by 17% . in addition , there might be some benefits in activities of daily living as measured by mbi . however , the studies involved a limited number of patients and were conducted by a same research team , so the statistical power of the finding might be weak . clinical trials usually define favorable outcome of stroke as mrs grade 2 . however , this meta - analysis failed to demonstrate significant benefits of bm - mscs / mncs - based cell therapy in increasing the proportion of mrs 2 patients . due to this study did not find any severe adverse events associated with cell therapy , suggesting it is a relatively safe intervention . firstly , the number of trials and the number of patients recruited in each trial were small . in addition , different trials reported different clinical outcomes , which makes it hard to use the same scale to summarize the results . these limitations significantly weaken the statistical power of the findings . secondly , to develop an effective cell therapy strategy , several factors , including eligibility criteria of the patients , timing , and route and dose of cell transplantation , should be considered in clinical practice . however , based on the available evidence , these factors still need to be optimized . patients with moderate , but not mild or severe , stroke might be more suitable for cell therapy , since patients with mild strokes generally have uniformly good outcome and patients with severe stroke are less likely to respond to the intervention and thus are unlikely have good outcome . the 5 trials recruited patients with basic nihss scores ranging from 4 to 20 , which means minor , moderate , and moderate - to - severe patients were all recruited . therefore although bm - mncs / mscs transplantation might generate some benefits in lowering the grade of impairment caused by ischemic stroke , large rcts are required to further confirm the effectiveness of bm - mscs / mncs - based cell therapy and to optimize the conditions required for best therapeutic effects .

backgroundautologous bone marrow stromal cells ( bm - scs ) transplantation might be a potential therapy for stroke . although a series of clinical trials were performed to assess the effectiveness and safety of bm - scs transplantation after ischemic stroke , the results are still conflicting . this study aimed to pool previous controlled trials to assess the effectiveness of bm - scs - based cell therapy after ischemic stroke.material/methodsrelevant studies were searched among online databases . barthel index ( bi ) or modified barthel index ( mbi ) , national institutes of health stroke scale ( nihss ) , and rankin score ( mrs ) were used to assess therapeutic effects . the frequencies of adverse events were extracted for assessing safety of stem cell therapy . data analysis was performed by using review manager 5.3.resultspatients who received cell therapy had significantly lower nihss score ( 1.85 ) than the controls . in addition , there might be some benefits in daily activity measured by mbi , but this meta - analysis failed to demonstrate significant benefits of bm - scs - based cell therapy in increasing the proportion of mrs 2 patients . we did not find any severe adverse events associated with bm - scs - based cell therapy.conclusionsalthough bm - mncs / mscs transplantation might generate some benefits in lowering the grade of impairment caused by ischemic stroke , large rcts are required to further confirm the effectiveness of bm - mscs / mncs - based cell therapy and to optimize the conditions require for best therapeutic effects .

Background Material and Methods Study search and selection Data extraction Statistical analysis Results The effect of BM-MSCs on BI or mBI score The effect of BM-MSCs on NIHSS score The effect of BM-MSCs on mRS Safety assessment of BM-MSCs transplantation Discussion Conclusions

percutaneous nephrolithotomy ( pnl ) is the gold standard for treating patients with kidney stones over 2 cm in diameter . it has replaced open surgeries due to equal efficacy , less morbidity , and higher patient acceptance . however , the most common complications are still urine extravasation , infection , and bleeding . the classic procedure consists of four major steps : insertion of a ureteral catheter , execution of percutaneous access to the kidney with tract dilation , lithotripsy and stone extraction , and finally protection of the tract with a nephrostomy tube . tract dilation may be conducted with the aid of metallic alken dilators , plastic amplatz dilators , or a pneumatic dilator . one - stage tract dilation is also a well - known technique ; however , it is not utilized in our department . it has been previously suggested that blood loss during renal dilation with amplatz instruments is higher . it has also been suggested that with sequential application of fascial dilators , the hemostatic pressure on the kidney parenchyma is lost , resulting in intermittent bleeding from renal vessels . on the other hand , the rate of other complications seems to be comparable . as there are various methods of tract performance ( inter / infracostal approach , various calyx punctures , utilization of access sheath , etc . ) we hypothesized that it may not be the dilator type itself but rather the way they are utilized that may influence the type of complications . the aim of our study was to compare these two methods of tract dilation in terms of efficacy and safety , and to find out what may be the source of differences in complication rates . between august 2008 and april 2016 , pnl was performed in 283 patients in our department . two methods of tract dilation were utilized : group i ( n=123 ) comprised patients where alken dilatator was used ; group ii ( n=99 ) comprised patients where the amplatz dilator was used . both procedures were conducted by two experienced endourologists ( one used the alken dilator and the other used the amplatz dilator ) . efficacy was assessed based on ultrasound and x - ray examination one month after the procedures . the presence of residual stone <3 mm in diameter was considered insignificant and patients were classified as stone free . urosepsis was diagnosed if there was positive urine and blood cultures and two out of the following four : temperature > 38.5c or < 36c , white blood cells > 12,000 cells / mm or < 4,000 cells / mm , respiratory rate > 20 breaths per minute or paco2 < 32 mm hg , and pulse over 90 beats per minute . other variables were also analyzed including : operating room time , scope time , and hospitalization time . this study was performed in accordance with the ethical standards of the 1964 declaration of helsinki . in a supine position , the patient was rotated on a table to the prone position . at an angle of 30 a hydrophilic guidewire was inserted into the needle and placed in the ureter . over the wire , after the lithotripsy , re - entry malecot catheter was inserted over the amplatz wire within the kidney and the ureteral catheter was withdrawn . our amplatz dilator technique was a standard technique , well - described in the literature where the final stages may be replaced with a tubeless option . the procedure used for the alken dilator was similar , up to the point of introduction of the amplatz wire . instead of a straight amplatz wire , a stiff wire with a coiled end was inserted into the pyelocalyceal system . subsequently , the metallic dilator was introduced over the wire to widen the tract up to 26-fr . the access sheath and safety wire were not used . at the end of the lithotripsy , a drain was left in the pyelocalyceal system and the ureteral catheter was left in place . after the procedure , each patient received 1 g paracetamol intravenously every six hours . antibiotic prophylaxis consisted of cephalosporin ( second generation ) given during induction of general anesthesia . student t - test for continuous variables with normal distribution was used . for continuous variables without normal distribution , for categorical variables , the chi - square test was used . to show dependence between categorical variables we used correspondence analysis . power analysis was conducted with the assistance of g*power statistical tool . a p value < this study was performed in accordance with the ethical standards of the 1964 declaration of helsinki . in a supine position , a 6-fr ureteral catheter was inserted into the appropriate kidney . the patient was rotated on a table to the prone position . at an angle of 30 a hydrophilic guidewire was inserted into the needle and placed in the ureter . over the wire , after the lithotripsy , re - entry malecot catheter was inserted over the amplatz wire within the kidney and the ureteral catheter was withdrawn . our amplatz dilator technique was a standard technique , well - described in the literature where the final stages may be replaced with a tubeless option . the procedure used for the alken dilator was similar , up to the point of introduction of the amplatz wire . instead of a straight amplatz wire , a stiff wire with a coiled end was inserted into the pyelocalyceal system . subsequently , the metallic dilator was introduced over the wire to widen the tract up to 26-fr . the access sheath and safety wire were not used . at the end of the lithotripsy , a drain was left in the pyelocalyceal system and the ureteral catheter was left in place . after the procedure , each patient received 1 g paracetamol intravenously every six hours . antibiotic prophylaxis consisted of cephalosporin ( second generation ) given during induction of general anesthesia . student t - test for continuous variables with normal distribution was used . for continuous variables without normal distribution , the chi - square test was used . to show dependence between categorical variables we used correspondence analysis . power analysis was conducted with the assistance of g*power statistical tool . a p value < patients in both groups did not differ in terms of age , sex , bmi , or stone size and affected side ( table 1 ) . we did not find any differences in total operative time , tract formation time , or fluoroscopy time . tract dilation failures , although more frequent in group i , were not significantly different between the groups . the hemoglobin drop and most of the procedures in group i were performed with infracostal access while in group ii more procedures were conducted with intercostal access . the number of accesses did not differ between groups . in the amplatz group ( group ii ) most accesses were through middle calyx while in group i ( alken group ) most were through the lower calyx . we found significant differences in postoperative complications as shown in table 2 and figure 1 . most grade i ( postoperative fever over 38.5c ) complications were more common in group i. similarly grade ivb complications ( urosepsis ) were more frequently observed in group i. pleural injury ( grade iva ) were more common in group ii . other complications ( ii blood transfusions , iiia extravasation , iiib arterio - venous fistula ) and non - complicated course of the disease were similarly distributed between groups . pnl is an efficacious procedure with a more than 80% stone - free rate independent of type of access . it has replaced open surgeries due to the same efficacy , less morbidity , and higher patient acceptance . still , one of the most common complications after pnl is infection , sepsis , and bleeding resulting in hemoglobin drop and occasionally the need for blood transfusion . as stated earlier , the alken dilators are allegedly safer than the amplatz dilators due to maintenance of continuous hemostatic pressure on the kidney parenchyma . in our study , both types of tract dilation were comparable in terms of blood loss . similarly , blood transfusions were not different between groups , supporting the previous reports that both types of tract dilation are equally safe . other parameters such as total operative time , fluoroscopy time , and tract formation time were not different between the two groups . similarly , the tract dilation failure rate was comparable between the two groups , which is consistent with other study outcomes . in our cohort , in the amplatz group there was insignificantly fewer failures . in our experience this may be due to the fact that in the alken group , the puncture was most often through the lower calyx and the wire was positioned in the renal pelvis . on the other hand , in the amplatz group , the puncture was usually directed at the middle calyx and the wire was positioned within the ureter . positioning the wire in the ureter makes it more stable and less prone to fall out of the kidney during tract dilation . other factors may influence tract dilation failure ( e.g. , bmi , gender ) due to kidney movement during the procedure , however , we did not observe differences in distribution of the aforementioned characteristics as showed in figure 1 , postoperative fever and sepsis in our cohort were more common in the alken group . other complications , such as contrast extravasation , arteriovenous fistula or postoperative hematoma , were comparable between groups . we may only speculate why there were differences in rates of grade i and ivb between the two groups , as there was no simple explanation . we hypothesized that the cause lies in the fact that in the alken group we did not use an access sheath . this may cause higher irrigant pressure inside the kidney during lithotripsy as most of the influent water departs through the ureter instead of the access sheath . a similar situation was described in micro - percutaneous nephrolithotomy , where there was no need for an access sheath and higher irrigant pressures were observed in comparison with classic pnl . as some of the stones were infected , this , with the combination of high irrigant pressure , may cause more septic complications after pnl . the clavien - dindo classification is usually used to evaluate major complications ( 3a ) after pnl . however , we showed that even postoperative fever ( grade i ) may vary between methods . this suggests the source of this difference may be important , and even minor complications should be reported after pnl . as more procedures in the amplatz group were conducted through the middle calyx it was not surprising that many of them were intercostal . however , this fact did not have an influence on blood loss , and supports reported evidence that inter / infracostals access is equally safe in this regard . on the other hand , intercostal access results in more frequent pleural injury ( figure 1 ) . both procedures were characterized by mild pain the day after the operation ( table 2 ) . the way in which pnl may be performed with alken and amplatz dilators differs among urological departments . indeed our study did not compare two different dilators but rather two different methods of tract dilation where both have their pros and cons . based on our study , we can not determine the superiority of one method of tract dilation over the other as these two approaches were completely different in terms of punctured calyx , inter / infracostal access , and utilization ( or not ) of a sheath . however , it is clear that irrespective of the dilator type used , the important differences result from other aforementioned factors . total operative time , tract dilation time , fluoroscopy time , and tract dilation failure were comparable between the two groups . fever over 38.5c and urosepsis were more common in the alken group . on the other hand , pleural injuries were more common after pnl with amplatz dilators . it is clear that all differences resulted from the punctured calyx , type of access to the kidney ( inter / infracostal ) , and utilization ( or not ) of an access sheath rather than the type of dilator itself .

backgroundpercutaneous nephrolithotomy ( pnl ) is the standard procedure for patients with renal stones over 2 cm in diameter . we analyzed complications after this procedure focusing on two different methods of tract dilation.material/methodsbetween august 2008 and april 2016 222 percutaneous nephrolithotomies were performed in a total of 208 patients . the group i ( n=123 ) comprised patients where alken dilatators were used , while group ii ( n=99 ) comprised patients where amplatz dilators were used . efficacy was examined based on ultrasound and x - ray examination one month after the procedure . complications were recorded using clavien dindo classification.resultsefficacy was 85.3% and 86.8% in group i and ii , respectively ( p=0.77 ) . grade i complications were present in 14.6% and 3% , grade ii were present in 9.7% and 8% , grade iiia were present in 2.4% and 2% , grade iiib were present in 1.6% and 2% , grade iva were present in 1.6% and 7% , grade ivb were present in 3.2% and 1% in group i and group ii , respectively . these differences were statistically significant ( p=0.03).conclusionsefficacy was comparable between alken dilator and amplatz dilator groups . in group i , there were more postoperative fevers > 38.5 c and a higher rate of urosepsis . on the other hand , in group ii we observed more pleural injuries . all differences resulted from the type of access to the kidney ( inter / infracostal ) , punctured calyx , and utilization ( or not ) of access sheath rather than type of dilators itself .

Background Material and Methods Compliance with ethical standard Description of procedure Statistics Results Discussion Conclusions

pop is a prevalent problem which has been reported to affect 50% of parous women . eleven percent of the women will have undergone an operation for prolapse or urinary incontinence by the age of 80 . oab is known to be a highly prevalent disorder that increases with age in both sexes and that has a profound impact on quality of life . according to the international continence society ( ics ) oab is defined as urgency with or without urge incontinence , usually with frequency and nocturia . obvious pathology. it is a matter of debate whether pop should be considered as obvious pathology. symptoms of oab are often seen in patients with pop . community based studies showed that the prevalence of oab symptoms is higher in patients with pop than without pop . the same tendency is found in hospital based studies . nevertheless , the literature about the prevalence of the combination of pop and oab is scarce . to study the relation between pop and oab , we used data from a cross - sectional study which was performed in a small dutch city about the prevalence of pelvic floor symptoms in the general population . the objective of this study is to investigate risk factors for oab and specifically to explore the relationship between oab and prolapse . this is important for clinical practice because the two diagnoses are often co - occurring which has possible consequences for diagnosis and treatment . the study was cross - sectional in a small town , brielle , in the netherlands . brielle was chosen because it has a homogenic population , where all women are registered in one of the nine general practices . all women aged 45 to 85 years registered on the patients lists of eight out of nine general practices were invited to enrol in the study , which is 95% of the women in this age group . the women were sent information about the study and informed that they could enrol by filling out an informed consent form . all women who consented non - responders received a reminder 8 weeks later that contained the same questionnaire . to check for selection bias , permanent non - responders were invited to complete a short questionnaire that comprised five questions about age , parity , presence of stress urinary incontinence ( yes / no ) , faecal incontinence ( yes / no ) and feeling of vaginal bulging ( yes / no ) . to encourage a high response to the questionnaire , we used envelopes with the name and logo of the erasmus university , coloured paper and stamped addressed return envelopes . three options were possible : women refused to participate in the study , women filled out only the questionnaire and women filled out the questionnaire and underwent vaginal examination . for the purpose of this study , data on vaginal examination are not used . the relation between symptoms and signs of vagina prolapse has been extensively described in an earlier study . 1flowchart of the study flowchart of the study the self - reported questionnaire used for this study are a composite of internationally well - known questionnaires that have been validated for the dutch language . it contains , amongst others , disease - specific questions from the validated dutch translation of the urinary distress inventory(udi ) women rated the amount of bother of various symptom on a 5-point likert scale , from 0 ( no complaints at all ) to 4 ( very serious complaints ) . in addition , questions about ethnicity , parity , pop symptoms during pregnancy , family history , menopausal status , hormone replacement therapy ( hrt ) , previous pelvic floor surgery , educational level , smoking and heavy physical work were also included . the medical ethics research committee ( metc ) of the erasmus mc in rotterdam , the netherlands , approved this study . all symptoms were dichotomized as present or absent based on responses to each symptom and degree of bother with these symptoms . women who denied the presence of a specific symptom as well as women who answered confirmative on a specific question but answered not to be bothered by it were considered as negative ( absent ) while women who indicated that they were little to severe bothered were considered as positive ( present ) . the item of pop symptoms is merged from women who reported either seeing and/or feeling vaginal bulging . for the item any oab symptoms , women who had urgency and/or frequency and/or urge incontinence symptoms were included ( see fig . 2 ) . 2any oab symptoms in relation to urgency , frequency and urge incontinence any oab symptoms in relation to urgency , frequency and urge incontinence data are presented as number of women ( percentage ) , mean ( standard deviation ) or median ( range ) as appropriate . chi - square test was used to compare the difference between the women with- versus without pop symptoms . variables with a p < 0.3 in univariate analysis were included in the multivariate analysis . we presented the odds ratio ( or ) and 95% ci for each of the oab symptoms . all data were entered and analysed in a spss 15.0 database for windows ( spss , inc . , chicago , il ) . all symptoms were dichotomized as present or absent based on responses to each symptom and degree of bother with these symptoms . women who denied the presence of a specific symptom as well as women who answered confirmative on a specific question but answered not to be bothered by it were considered as negative ( absent ) while women who indicated that they were little to severe bothered were considered as positive ( present ) . the item of pop symptoms is merged from women who reported either seeing and/or feeling vaginal bulging . for the item any oab symptoms , women who had urgency and/or frequency and/or urge incontinence symptoms were included ( see fig . 2 ) . 2any oab symptoms in relation to urgency , frequency and urge incontinence any oab symptoms in relation to urgency , frequency and urge incontinence data are presented as number of women ( percentage ) , mean ( standard deviation ) or median ( range ) as appropriate . chi - square test was used to compare the difference between the women with- versus without pop symptoms . variables with a p < 0.3 in univariate analysis were included in the multivariate analysis . we presented the odds ratio ( or ) and 95% ci for each of the oab symptoms . all data were entered and analysed in a spss 15.0 database for windows ( spss , inc . , chicago , il ) . of the 2,979 women who were eligible for this study , 1,397 ( 47% ) filled out the questionnaire . in the non - responder group , 59% filled out and returned the short questionnaire ( 620/1,051 ) , giving a total response rate of 62.7% ( fig . 1 ) . scores in the short questionnaire group were not significantly different from those in the total study group . details of the response rate are presented in a previous report . table 2 shows the prevalence of oab symptoms per age category . in table 3 , the prevalence of pop symptoms in women with and without oab symptoms is presented . table 1patients characteristics and details of previous pelvic operationsnumber of women1397age ( years ) 4555647 ( 46.9% ) 5665435 ( 31.5% ) 6675233 ( 16.9% ) 768566 ( 4.8%)parity 0120 ( 8.6% ) 1215 ( 15.4% ) 2675 ( 48.3% ) 3387 ( 27.7%)body mass index ( kg / m2 ) < 2053 ( 3.9% ) 2025599 ( 43.9% ) 2530519 ( 38.0% ) 30193 ( 14.1%)race caucasian1,351 ( 98.5% ) non - caucasian20 ( 1.5%)smoking former smoking326 ( 23.6% ) current smoking280 ( 20.3% ) postmenopausal status1,009 ( 72.2% ) hormone suppletion88 ( 6.4%)previous gynaecological surgery prolapse surgery103 ( 7.4% ) hysterectomy234 ( 16.9% ) incontinence surgery47 ( 3.4%)data are presented as number of women ( percentage)data on 16 women are missingdata on 33 women are missingdata on 26 women are missingdata on 15 women are missingdata on 30 women are missingdata on 25 women are missingdata on 18 women are missingdata on 13 women are missingdata on 14 women are missingdata on 15 women are missingtable 2prevalence of overactive bladder symptoms and any oab symptoms per age groupfrequencyurgencyurge incontinenceany of the oab symptoms4555162 ( 25.3%)193 ( 30.9%)178 ( 27.7%)298 ( 46.1%)5665121 ( 28.4%)141 ( 33.7%)116 ( 26.9%)200 ( 46.2%)667580 ( 35.9%)92 ( 40.5%)79 ( 35.0%)126 ( 54.5%)768524 ( 37.5%)33 ( 54.1%)32 ( 48.5%)42 ( 63.6%)overall393 ( 28.7%)463 ( 34.4%)411 ( 29.8%)677 ( 48.6%)data are presented as number of women ( percentage)data on age category on 16 women are missingdata on frequency on 28 women are missingdata on urgency on 52 women are missingdata on urge incontinence on 16 women are missingtable 3prevalence of prolapse symptoms in women with symptoms of oabprolapse symptomsno prolapse symptomspfrequency66 ( 41.8%)320 ( 26.9%)0.000urgency77 ( 49.7%)379 ( 32.4%)0.000urge incontinence64 ( 40.3%)340 ( 28.3%)0.003any of the oab symptoms84 ( 69.4%)588 ( 46.6%)0.000data are presented as number of women ( percentage)p value using chi - square test to compare the difference between women with versus without prolapse symptoms.data on 28 women are missingdata on 52 women are missingdata on 16 women are missingdata on four women are missingdata on prolapse symptoms on 24 women are missing patients characteristics and details of previous pelvic operations data are presented as number of women ( percentage ) data on 16 women are missing data on 33 women are missing data on 26 women are missing data on 15 women are missing data on 30 women are missing data on 25 women are missing data on 18 women are missing data on 13 women are missing data on 14 women are missing data on 15 women are missing prevalence of overactive bladder symptoms and any oab symptoms per age group data are presented as number of women ( percentage ) data on age category on 16 women are missing data on frequency on 28 women are missing data on urgency on 52 women are missing data on urge incontinence on 16 women are missing prevalence of prolapse symptoms in women with symptoms of oab data are presented as number of women ( percentage ) p value using chi - square test to compare the difference between women with versus without prolapse symptoms . data on 28 women are missing data on 52 women are missing data on 16 women are missing data on four women are missing data on prolapse symptoms on 24 women are missing in table 4 , the various possible risk factors including the presence of pop symptoms for the presence of oab symptoms are presented in a univariate logistic regression model . table 4factors of the univariate logistic regression analysis on the various oab symptomsfrequency or ( 95% ci)urgency or ( 95% ci)urge incontinence or ( 95% ci)any oab or ( 95% ci)age ( years)4555refrefrefref56651.2 ( 0.9 , 1.5)1.1 ( 0.9 , 1.5)1.0 ( 0.7,1.3)1.0 ( 0.8 , 1.3)66751.7 ( 1.2 , 2.3)1.5 ( 1.1 , 2.1)1.4 ( 1.0 , 1.9)1.4 ( 1.0 , 1.9)76851.8 ( 1.0 , 3.0)2.6 ( 1.5 , 4.5)2.5 ( 1.5 , 4.1)2.0 ( 1.2 , 3.5)parity2refrefrefref>21.3 ( 1.0 , 1.7)1.0 ( 0.8 , 1.3)1.0 ( 0.8 , 1.3)1.2 ( 0.9 , 1.5)body mass index ( kg / m2)<200.7 ( 0.3,1.5)0.4 ( 0.2 , 1.0)0.7 ( 0.4 , 1.5)0.6 ( 0.3 , 1.0)2025refrefrefref25301.6 ( 1.3 , 2.1)1.3 ( 1.0 , 1.7)1.5 ( 1.1 , 1.9)1.4 ( 1.1 , 1.7)302.0 ( 1.4 , 2.8)2.2 ( 1.6 , 3.1)2.2 ( 1.6 , 3.1)2.3 ( 1.7 , 3.2)smoking former smokingyes1.3 ( 1.0 , 1.8)1.2 ( 0.9 , 1.6)1.0 ( 0.8 , 1.4)1.1 ( 0.8 , 1.4)norefrefrefref current smokingyes1.0 ( 0.8,1.4)1.3 ( 1.0 , 1.7)1.3 ( 1.0 , 1.7)1.2 ( 1.0 , 1.6)norefrefrefrefpostmenopausal statusyes1.5 ( 1.2 , 2.0)1.6 ( 1.2 , 2.0)1.4 ( 1.1 , 1.8)1.3 ( 1.0 , 1.7)norefrefrefrefhormonal suppletion therapyyes1.2 ( 0.7 , 1.9)1.2 ( 0.7 , 1.8)0.7 ( 0.4 , 1.2)0.9 ( 0.6 , 1.4)norefrefrefrefprevious gynaecological surgery prolapse surgeryyes2.5 ( 1.7 , 3.8)2.6 ( 1.7 , 3.9)1.9 ( 1.3 , 2.9)2.9 ( 1.9 , 4.6)norefrefrefref hysterectomyyes1.5 ( 1.1 , 2.1)1.3 ( 0.9 , 1.7)1.3 ( 1.0 , 1.8)1.3 ( 1.0 , 1.7)norefrefrefref incontinence surgeryyes3.5 ( 1.9 , 6.5)3.4 ( 1.9 , 6.3)4.3 ( 2.3 , 8.0)6.5 ( 2.9 , 14.5)norefrefrefrefprolapse symptomsyes2.0 ( 1.4 , 2.7)2.1 ( 1.5 , 2.9)1.7(1.2 , 2.4)2.6 ( 1.7 , 3.9)norefrefrefrefall values with p < 0.05 are illustrated in boldref : referencedata on 16 women are missingdata on 33 women are missingdata on 15 women are missingdata on 13 women are missingdata on 30 women are missingdata on 25 women are missingdata on 18 women are missingdata on 14 women are missingdata on 24 women are missing factors of the univariate logistic regression analysis on the various oab symptoms all values with p < 0.05 are illustrated in bold data on 16 women are missing data on 33 women are missing data on 15 women are missing data on 13 women are missing data on 30 women are missing data on 25 women are missing data on 18 women are missing data on 14 women are missing data on 24 women are missing the or shows the chance of the presence of oab symptoms . an or > 1 indicates that the factor is positively correlated with the outcome variable ; an or < 1 means that the factor indicates a negative correlation . table 5 shows the multivariate analysis of the oab symptoms where all factors of the univariate analysis with a p < 0.3 are taken into account . table 5risk factors on oab symptoms after multivariate regression analysisfrequency or ( 95% ci)urgency or ( 95% ci)urge incontinence or ( 95% ci)any oab or ( 95% ci)age ( years)4555refrefref56651.0 ( 0.8 , 1.4)0.9 ( 0.6 , 1.1)0.9 ( 0.7 , 1.2)66751.4 ( 1.0 , 2.0)1.3 ( 0.9 , 1.8)1.3 ( 1.0 , 1.8)76852.7 ( 1.5 , 4.9)2.2 ( 1.3 , 3.8)2.1 ( 1.2 , 3.7)body mass index ( kg / m)<200.7 ( 0.3 , 1.6)0.4 ( 0.2 , 0.9)0.7 ( 0.3 , 1.5)0.5 ( 0.3 , 1.0)2025refrefrefref25301.5 ( 1.1 , 2.0)1.2 ( 0.9 , 1.6)1.3 ( 1.0 , 1.8)1.3 ( 1.0 , 1.6)301.7 ( 1.2 , 2.4)2.2 ( 1.5 , 3.1)2.0 ( 1.4 , 2.9)2.2((1.5 , 3.1)smoking smoking in the pastyes1.4 ( 1.1 , 1.9)noref current smokingyes1.7 ( 1.2 , 2.3)1.4 ( 1.0 , 1.8)norefrefpostmenopausal statusyes1.3 ( 1.0 , 1.8)norefprevious gynaecological surgery prolapse surgeryyes2.3 ( 1.4 , 3.6)noref incontinence surgeryyes2.9 ( 1.5 , 5.5)4.3 ( 2.3 , 8.2)6.7 ( 2.8 , 16.3)norefrefrefprolapse symptomsyes2.4 ( 1.6 , 3.5)2.2 ( 1.4 , 3.3)1.8 ( 1.2 , 2.6)2.3 ( 1.5 , 3.5)norefrefrefrefvariance explained by the model5.8%8.8%6.6%9.1%or odds rario , ref referencedata on 16 women are missingdata on 33 women are missingdata on 15 women are missingdata on 13 women are missingdata on 18 women are missingdata on 24 women are missingnagelkerke r risk factors on oab symptoms after multivariate regression analysis or odds rario , ref reference data on 16 women are missing data on 33 women are missing data on 15 women are missing data on 13 women are missing data on 18 women are missing data on 24 women are missing in this cross - sectional study , we looked at the prevalence of oab symptoms and specifically the relationship between oab symptoms and pop symptoms . we found a prevalence of urgency , frequency and urge urinary incontinence of 34% , 29% and 30% , respectively . this is comparable with other studies , where a prevalence of 16.849% was found in women [ 3 , 8 ] . pop symptoms were present in 11.4% , which is comparable with other studies where a prevalence of 430% was found [ 911 ] . the sparse cross - sectional studies who mentioned a relationship between pop symptoms and oab , showed a higher prevalence of oab symptoms with pop symptoms than without pop symptoms ( ! ) but were not controlled for other risk factors . the same relationship between pop and oab is found in hospital based studies , the prevalence of oab symptoms is greater in patients with pop than without pop . however , by the nature of these epidemiological studies a causal relationship can not be established . there are many possible theories regarding the pathophysiology of oab in relation to pop and it is likely that bladder obstruction plays an important role . nevertheless , several other mechanisms might be considered . the pathophysiological relationship between oab and pop needs to be studied further . important clinical implication of the relationship between pop and oab is that treatment of pop could give an improvement in oab symptoms . another important risk factor for oab symptoms was surgery for urinary incontinence in the past . many studies have shown a relationship between continence surgery and oab symptoms where the prevalence of de novo oab varied between 15% and 29% on the short term ( 13 months postoperatively ) [ 12 , 13 ] and 030% on the long term [ 1214 ] . as in other studies , we found the prevalence of oab symptoms increased with advancing age [ 3 , 8 ] . overweight ( body mass index [ bmi ] greater then 30 ) was another independent risk factor for oab symptoms . this is consistent with other studies , who found the same relationship [ 1519 ] . the study of cheung found a similar or for overweight , where the study of lawrence found a higher or ( 2.73 ) . on the other hand , choo et al . found that bmi was only a predictor for oab dry ( urgency with or without frequency or nocturia ) , but not for oab wet ( urgency with urge incontinence , with or without frequency or nocturia ) . other studies are not conclusive about the role of smoking in oab [ 17 , 20 , 21 ] . the study of bradley et al . found no relation between current smoking and urinary symptoms , while a large cross - sectional study showed that current and former smoking was associated with urgency . one study found that current smokers were 1.44 time more likely to develop oab , and the increased risk for former smokers was nearly significant . the induction of oab in smoking could be related to an anti - oestrogenic hormonal effect on the bladder and uretra and a nicotine induced phasic contraction of the detrusor muscle . this is consistent with another study which found postmenopausal status to be a predictor for oab symptoms . this can be explained because oestrogen has an important role in the urogenital tract through oestrogen receptors in urethra , bladder and pelvic floor , where deficiency causes atrophic changes , which is associated with oab symptoms . reversal of the atrophy by oestrogen treatment can have a positive influence on oab symptoms . surprisingly , we found previous prolapse surgery to be a predictor for the symptom urgency , this in contrast to practically all studies that showed an improvement of oab symptoms after prolapse surgery . a possible explanation for this finding is that women after prolapse surgery achieved de novo urgency , as was found in 5% of the patients in one study . the strong point about this study is that it is a large cross - sectional study in a homogenic female population , which made multivariate analysis possible ; as a result , however , genetic and racial factors could not be included , which is also an inherent weakness of the design . another weakness of this study is that not all factors of influence on oab are included , such as food and beverages ( coffee and alcoholic consumption ) and the use of oab therapies ( bladder training and pharmacotherapy ) . this study found pop symptoms to be an independent risk factor for oab symptoms . the sparse cross - sectional studies who mentioned a relationship between pop symptoms and oab , showed a higher prevalence of oab symptoms with pop symptoms than without pop symptoms ( ! ) but were not controlled for other risk factors . the same relationship between pop and oab is found in hospital based studies , the prevalence of oab symptoms is greater in patients with pop than without pop . however , by the nature of these epidemiological studies a causal relationship can not be established . there are many possible theories regarding the pathophysiology of oab in relation to pop and it is likely that bladder obstruction plays an important role . nevertheless , several other mechanisms might be considered . the pathophysiological relationship between oab and pop needs to be studied further . important clinical implication of the relationship between pop and oab is that treatment of pop could give an improvement in oab symptoms . another important risk factor for oab symptoms was surgery for urinary incontinence in the past . many studies have shown a relationship between continence surgery and oab symptoms where the prevalence of de novo oab varied between 15% and 29% on the short term ( 13 months postoperatively ) [ 12 , 13 ] and 030% on the long term [ 1214 ] . as in other studies , we found the prevalence of oab symptoms increased with advancing age [ 3 , 8 ] . overweight ( body mass index [ bmi ] greater then 30 ) was another independent risk factor for oab symptoms . this is consistent with other studies , who found the same relationship [ 1519 ] . the study of cheung found a similar or for overweight , where the study of lawrence found a higher or ( 2.73 ) . on the other hand , choo et al . found that bmi was only a predictor for oab dry ( urgency with or without frequency or nocturia ) , but not for oab wet ( urgency with urge incontinence , with or without frequency or nocturia ) . other studies are not conclusive about the role of smoking in oab [ 17 , 20 , 21 ] . the study of bradley et al . found no relation between current smoking and urinary symptoms , while a large cross - sectional study showed that current and former smoking was associated with urgency . one study found that current smokers were 1.44 time more likely to develop oab , and the increased risk for former smokers was nearly significant . the induction of oab in smoking could be related to an anti - oestrogenic hormonal effect on the bladder and uretra and a nicotine induced phasic contraction of the detrusor muscle . this is consistent with another study which found postmenopausal status to be a predictor for oab symptoms . this can be explained because oestrogen has an important role in the urogenital tract through oestrogen receptors in urethra , bladder and pelvic floor , where deficiency causes atrophic changes , which is associated with oab symptoms . reversal of the atrophy by oestrogen treatment can have a positive influence on oab symptoms . surprisingly , we found previous prolapse surgery to be a predictor for the symptom urgency , this in contrast to practically all studies that showed an improvement of oab symptoms after prolapse surgery . a possible explanation for this finding is that women after prolapse surgery achieved de novo urgency , as was found in 5% of the patients in one study . the strong point about this study is that it is a large cross - sectional study in a homogenic female population , which made multivariate analysis possible ; as a result , however , genetic and racial factors could not be included , which is also an inherent weakness of the design . another weakness of this study is that not all factors of influence on oab are included , such as food and beverages ( coffee and alcoholic consumption ) and the use of oab therapies ( bladder training and pharmacotherapy ) . in this study , we found a prevalence of urgency of 34% , as the core symptom of the oab spectrum , and of any oab symptoms of 49% . other risk factors are continence surgery in the past , age above 75 , overweight and smoking .

introduction and hypothesisto study the prevalence and risk factors of overactive bladder ( oab ) symptoms and its relationship with symptoms of pelvic organ prolapse ( pop).methodsthis is a cross - sectional study including women aged between 45 and 85 years , registered in eight general practices . all women were asked to self complete the validated dutch translated questionnaires . all symptoms were dichotomized as present or absent based on responses to each symptom and degree of bother.resultsforty-seven percent of the women filled out the questionnaire . prevalence of urgency was 34% and the prevalence of any oab symptoms 49% . prevalence of oab symptoms increased with advancing age . symptoms of pop were an independent risk factor for symptomatic oab . other risk factors were continence and prolapse surgery in the past , age above 75 , overweight , postmenopausal status and smoking.conclusionsthe prevalence of any oab symptoms was 49% . pop symptoms were an independent risk factor for symptomatic oab .

Introduction Methods Measurements Results Discussion Risk factors Strengths and weaknesses Conclusion

antitumor necrosis factor ( tnf ) biologics appeared over a decade ago in the armamentarium for inflammatory bowel disease ( ibd ) . originally evaluated in crohn 's disease ( cd ) and thereafter in ulcerative colitis ( uc ) , their efficacy was demonstrated in both diseases and has deeply modified the management of patients with ibd . although they are potentially able to change the natural course of ibd and to decrease the need for surgery , absence or loss of response is frequent and only one - third of patients remain in clinical remission at 1 year . clinical response , steroid - free remission , and mucosal healing have been correlated with drug trough levels [ 3 , 4 ] . however , anti - tnf pharmacokinetic is characterized by a considerable interindividual variability and antidrug antibodies ( adabs ) have been identified as one of the major factors impacting their clearance . thus , serum trough levels and adab measurement have been proposed for the monitoring of anti - tnf drugs and algorithms were defined for the management of patients with ibd . while the etiology of ibd is still unknown , it is thought to involve complex interactions between genetic disposition , environmental conditions , life style , and microbial and immune factors resulting in a deregulated and excessive immune response directed against components of the normal microflora . cd and uc have been associated with exaggerated t helper ( th ) type 1 and th2 responses , respectively . more recent studies demonstrated that tissue damages result from mucosal inflammation mainly mediated by proinflammatory th1 and th17 lymphocyte subpopulations and their respective proinflammatory effector cytokines . in the gut of cd patients , activated th1 and th17 cells produce ifn and il17 ( a and f ) , respectively , which stimulate macrophages and induce the production of other inflammatory cytokines such as il-1 and tnf that subsequently promote matrix metalloproteinases ( mmps ) production by stroma cells and mucosal damage . thus , it is now widely accepted that tnf plays a strategic role in ibd pathophysiology , at the cross talk of the different inflammatory pathways involved in gut mucosal inflammation . accordingly , most of the efficient biologic therapies developed so far in ibd aimed at neutralizing the proinflammatory activity of the tnf pathway . the effects of tnf are known to be mediated by tnf receptor i ( tnf - ri ) or tnf - rii . ligation of tnf - ri , which is expressed on a wide range of immune and nonimmune cells , results in nf-b activation , cytotoxicity , and induction of proinflammatory cytokines and chemokines as well as antiapoptotic peptides [ 9 , 10 ] . the effects on t lymphocytes are mainly mediated by interaction of tnf with tnf - rii inducing a costimulatory signal to tcr - mediated t cell activation , thereby increasing t cell proliferation , expression of t cell activation markers ( cd25 , human leukocyte antigen - dr , and tnf - rii ) , and secretion of inflammatory cytokines including ifn and tnf . accordingly , anti - tnf are able to inhibit t cell activation resulting in a decrease of proliferation and cytokine secretion ( ifn- , il-13 , il-17a , and tnf ) of both cd4 + and cd8 + t cell populations derived from uc patients . on the other hand , tnf and tnf - rii are also able to activate and expand protective cd4(+)foxp3(+ ) regulatory t cells ( tregs ) and seem critical for the stabilization of their phenotype and function in the inflammatory environment of the lamina propria in a mouse model of colitis . these contrasting effects of tnf on effector versus regulatory t cells may explain unexpected and disappointing results obtained with anti - tnf in some autoimmune diseases such as multiple sclerosis . altogether , these data underline the complexity of tnf function via tnf - ri or tnf - rii on the course of intestinal inflammation , due to different susceptibility of epithelial cells and effector or regulatory immune cells . as an illustration , in dextran sulfate sodium- ( dss- ) induced acute colitis in balb / c mice , tnf - ri ablation led to exacerbation of the disease with increased inflammation and intestinal damage , while tnf - rii deficiency had opposite effects . nonetheless , studies in patients with ibd have extensively demonstrated the efficiency of anti - tnf therapies which directly inhibit activation of effector t cells and sensitize them to treg - mediated inhibition with final restoration of immune homeostasis , resolution of inflammation , and mucosal healing . further studies are now required to better understand the respective protective and deleterious effects mediated by tnf on immune and nonimmune cells through tnf - ri and tnf - rii in order to develop more specific inhibitors with potentially an increased efficacy and/or safety . numerous randomized clinical trials and meta - analyses have demonstrated the efficacy of monoclonal antibodies against tnf for both induction and maintenance of remission in both cd and uc [ 1618 ] . infliximab ( ifx ) , a chimeric monoclonal antibody composed of human constant and murine variable regions , and adalimumab , a fully human monoclonal igg1 anti - tnf antibody , demonstrated their efficacy for the control of disease activity and the induction of clinical remission and mucosal healing in luminal cd and uc both in children and adult patients [ 1 , 1925 ] . several randomized clinical trials showed a better efficacy in inducing steroid - free clinical remission for a combination therapy with immunomodulators than anti - tnf monotherapy in cd and uc . moreover several studies established the use of infliximab and adalimumab in active fistulizing cd in adult patients [ 27 , 28 ] . certolizumab , a polyethylene - glycolated fab ' fragment of anti - tnf ab , also produced significant clinical benefit and mucosal healing in adult patients with cd . recently , golimumab , a fully human monoclonal antibody to tnf , was shown to induce and maintain clinical response in patients with active moderate - to - severe uc [ 30 , 31 ] . however , although 60 to 80 percent of patients exhibit a good initial response to anti - tnf treatments ( defined as a crohn 's disease activity index ( cdai ) decrease from baseline > 70 points for cd and a decrease in the mayo score of at least 3 points and at least 30 percent for uc ) , only one - third of patients are in clinical remission without steroids at one year ( defined as a cdai < 150 for cd and a total mayo score of 2 points or lower , with no individual subscore exceeding 1 point for uc ) . consequently , 20 to 30 percent of patients require dose intensification or interval adjustment in order to maintain long - term clinical benefit and an average of 10 to 20 percent per year lose response [ 3236 ] . despite the high effectiveness of anti - tnf in patients with ibd , more than one - third of patients present primary resistance , and another one - third become resistant over time . optimal clinical response required the maintenance of clinically effective drug concentrations , but the pharmacokinetic of anti - tnf is highly variable among patients and could be influenced by numerous factors including gender , body weight , associated treatments ( immunosuppressants are known to increase anti - tnf trough levels ) , route of administration , serum albumin concentration , and systemic inflammation with a markedly decreased half - life in patients with severe disease [ 3840 ] . however , the main factor impacting anti - tnf pharmacokinetic and efficacy over time is immunogenicity whereby antidrug antibodies ( adabs ) accelerate anti - tnf monoclonal abs clearance and shorten their half - life [ 41 , 42 ] . although humanized ( e.g. , certolizumab ) and fully human abs ( e.g. , adalimumab and golimumab ) are logically less immunogenic as compared with chimeric ones ( e.g. , ifx ) , they can all induce adabs targeting murine and/or variable domains of the monoclonal ab . other factors may promote immunogenicity such as genotype in a minority of patients and drug agitation or freeze - thaw cycles that can induce immunogenic protein aggregates ( for review ) . contrastingly , prescription of maintenance therapy with concomitant immunomodulators and achievement of suitable trough drug levels have been shown to reduce the risk of adabs . several studies assessed ifx trough levels after induction treatment or during maintenance therapy as predictors of sustained clinical response and showed a significant correlation between low ifx trough levels and decreased clinical response in cd and uc adult patients [ 3 , 4 , 34 , 4547 ] and in children with uc . in a recent prospective study of ibd patients who have developed secondary failure to ifx , paul et al . have shown that the only factor associated with mucosal healing after ifx optimization was a significant increase in ifx trough levels . antibodies to ifx ( atis ) were described in up to 60% when ifx was used on an ad hoc basis in practice and in 10 to 20% of patients in randomized controlled trials of maintenance therapy . atis were associated with loss of clinical response , deterioration of endoscopic activity , infusion reactions , and low serum ifx concentrations [ 5 , 41 , 44 , 46 , 5052 ] . however , some studies did not observe significant correlation between trough levels of ifx and cd activity or between positivity of atis and loss of clinical response or deterioration of endoscopic activity [ 3 , 4 , 5355 ] . these discrepancies could be explained by different methods of measurements for atis and ifx concentrations , by the short follow - up time in some studies , and by the lack of consensual optimal levels of ifx for prediction of efficacy . there are fewer data for adalimumab , but some studies also described a positive association between serum adalimumab concentration and clinical remission in cd [ 5658 ] . furthermore , while fully human , antiadalimumab antibodies were described in 2.6 to 17 percent of patients treated for cd or rheumatoid arthritis and significantly associated with low serum adalimumab trough levels and decreased clinical response [ 56 , 5961 ] . the relationship between pharmacokinetic data and efficacy is less clear for adalimumab than ifx with considerable variability and overlap in serum concentrations between patients with and without remission . however , in an observational study evaluating the efficacy of adalimumab in 168 active cd patients who failed to respond to ifx , long - term clinical benefit was significantly associated with higher serum trough concentrations and absence of adab . a recent study using adalimumab maintenance therapy in 40 adult patients with cd or uc showed a significant association of high trough levels of adalimumab with clinical remission and mucosal healing . antiadalimumab antibodies were associated with low trough levels of adalimumab and lack of mucosal healing . there is so far no data concerning trough levels and antidrug antibodies for adalimumab in children and in all patients for certolizumab and golimumab . serum trough levels measurement to detect subtherapeutic drug concentrations and identification of adab ( therapeutic drug monitoring or tdm ) are the most relevant and useful parameters for the monitoring of anti - tnf drugs to facilitate informed decision making in ibd patients with secondary loss of response to tnf antagonists . the clinical utility of the immunomonitoring was evaluated in a retrospective study conducted on 155 patients with ibd and loss of response to ifx . when adabs were detected , the switch to another anti - tnf molecule allowed a partial or complete response in 92% versus 17% for dose escalation whereas drug escalation was the most efficient strategy in patients with subtherapeutic ifx concentration ( 86% versus 33% of partial or complete response , resp . ) . they concluded that increasing anti - tnf doses is ineffective in patients with adab but appropriate in case of subtherapeutic drug concentration and proposed an algorithm for optimization of therapeutic strategy in ibd patients with loss of response to ifx based on adab and trough drug measurement . interestingly , in a prospective study examining the course of adab formation and the clinical relevance of its assessment in the followup of patients with rheumatoid arthritis , bartelds et al . showed that , among patients positive for antiadalimumab abs , 67% developed adabs during the first 28 weeks and almost one - third during the first month of treatment . however and despite a poor clinical response , patients with adabs discontinued treatment only after 52 weeks of therapy indicating an important delay between adab appearance and treatment adjustment . furthermore , early trough level measurement after induction might also have a prognostic value with ifx trough levels above 3.5 g / ml at 14 weeks being associated with a sustained therapeutic response . on the other hand , supratherapeutic anti - tnf trough levels might also be associated with paradoxical inflammatory side effects such as psoriasiform eczema or arthralgia . in such patients , lowering doses could be beneficial in terms of not only safety but also decrease of the cost for the healthcare payer . altogether , these data plead for the clinical and economical utility of early therapeutic drug monitoring in the management of patients receiving tnf inhibitors . in case of a loss of response with low trough level without adab , an intensified therapy with the same drug when low trough level is related to the presence of adabs , therapy should be switched within the anti - tnf class and if necessary to a drug with another mode of action . the addition of an immunomodulator might also be able to induce a decrease in adab level and to restore clinical response . of note , clinical response can occur despite the presence of adabs as described recently in a retrospective study . continued maintenance therapy with ifx induced adab disappearance in two - thirds of these patients after a median of 4 infusions suggesting that continued anti - tnf treatment could be considered in patients with clinical response and first adab detection . indeed , recent studies investigating the kinetics of ati formation confirmed that adab secretion may be transient and disappeared over time in almost one - third of patients [ 67 , 68 ] . compared to nontransient ati that appeared usually within the first 12months of therapy , transient ati was detected throughout the duration of ifx therapy . patients with sustained ati were more likely to discontinue ifx treatment compared with patients with transient ati . in a very recent study using a decision analytic model that simulated 2 cohorts of patients with cd who become resistant to anti - tnf inhibitors , velayos et al . compared the effectiveness of empiric dose escalation versus testing - based strategy over a 1-year time period . although both strategies yielded similar rates of remission ( 66% versus 63% , resp . ) and quality - adjusted life year ( 0.800 versus 0.801 ) , the testing - based strategy was less expensive than empiric dose escalation ( $ 31,870 versus $ 34,266 , resp . ) . similarly , steenholdt et al . showed in a randomized controlled trial that a testing - based strategy using an algorithm designed to identify the mechanism leading to secondary loss of response to ifx is more cost effective than empiric dose escalation in patients with cd . in the monitored arm , patients with low serum ifx and atis were switched to adalimumab , patients with low serum ifx without atis underwent dose intensification , and patients with high ifx trough levels with or without atis were switched to an out - of - class therapy or screened for an alternate cause of their symptoms . compared to the current dose intensification strategy , individualized therapy substantially reduced average treatment costs per patient with similar clinical response rates . large prospective and randomized studies are still required to validate all these approaches in patients with ibd and clear dose toxicity / efficacy relationships have yet to be established for anti - tnf inhibitors . finally , we have to keep in mind that , in the absence of standardization , the numerous assays developed for serum trough levels and adab measurement ( table 1 ) exhibit variable performances that could explain discrepancies between studies and difficulties in establishing clear cutoff values . there are currently no defined gold standard assays for quantification of anti - tnf drugs and adabs . a recent study compared three in house or commercially available assays ( elisa , bridging elisa , and ria ) developed for the analysis of ifx levels and atis . there was a good correlation between ifx and ati levels measured with all 3 tests . the sensitivity of the three assays to detect atis was comparable with a slight advantage for the ria test which is less sensitive than elisa to drug interference caused by the presence of ifx in the serum impeding the detection of low ati concentrations . nevertheless , discrepancies between the three assays were not rare and conclusions of the study were highly debated highlighting the high need for standardization [ 72 , 73 ] . since the advent of anti - tnf biologics more than a decade ago , they have demonstrated beneficial activity in induction and maintenance of clinical responses , mucosal healing , improvement in quality of life , reduction in surgeries and hospitalizations , and the treatment of extraintestinal manifestations of ibd . however , despite good overall initial effectiveness , a significant proportion of patients lose response over time mainly because of adab production and accelerated drug clearance . although optimal treatment strategies remain controversial , therapeutic algorithms were proposed based on serum trough levels and adab monitoring in order to rationalize drug adjustment . for the future , a better understanding of the ambivalent protective and deleterious effects mediated by tnf and its receptors on immune and nonimmune cells during ibd might be crucial for the development of more efficient and safe biological inhibitors .

since their appearance in the armamentarium for inflammatory bowel disease ( ibd ) more than a decade ago , antitumor necrosis factor ( tnf ) inhibitors have demonstrated beneficial activity in induction and maintenance of clinical remission , mucosal healing , improvement in quality of life , and reduction in surgeries and hospitalizations . however , more than one - third of patients present primary resistance , and another one - third become resistant over time . one of the main factors associated with loss of response is the immunogenicity of anti - tnf biologics leading to the production of antidrug antibodies ( adabs ) accelerating their clearance . in this review we present the current state of the literature on the place of tnf and its blockage in the treatment of patients with ibd and discuss the usefulness of serum trough levels and adab monitoring in the optimization of anti - tnf therapies .

1. Introduction 2. Role of TNF in IBD Pathophysiology 3. Anti-TNF Therapies in Patients with IBD 4. Drug Monitoring of Anti-TNF Biologics 5. Conclusion

a major task for evolutionary biology has been to develop and test theories for the origin of novelty that are consistent with the fundamental genetic principles of gradual populational change . novelty , however , is a loaded term with many different definitions that include or exclude a variety of morphological characters ( brigandt and love 2012 ) . following pigliucci ( 2008 ) , we prefer a more inclusive definition of evolutionary novelty : new traits , or novel combinations of traits within a lineage that perform a new ecological function and may result in the establishment of new evolutionary lineages . more narrowly focused definitions might be desirable for some purposes ( muller and wagner 1991 ; wagner and lynch 2010 ) . however , our goal in this essay is to elaborate one mechanism for the sudden origin and evolutionary success of new variants that applies just as well to exceptional size and shape , new color patterns , use of new habitats , and new exons . some theorists have invoked special phenomena such as genome - wide macromutations ( goldschmidt 1940 ) or genetic revolutions ( mayr 1954 ) to get around perceived difficulties with the emergence of profound change as the accumulation of subtle changes by the conventional dynamics of mutation , gene flow , drift and selection . however , modern evolutionary theory and empirical research in genetics have consistently reaffirmed the ability of conventional population genetics to explain the origin of new species and phenotypes , and simultaneously exposed flaws in the alternatives ( charlesworth et al . for example , goldschmidt ( 1933 , 1940 ) proposed that a novel phenotype ( such as insect wings , a character associated with higher level taxonomy ) must first arise as an instantaneous product of a single macromutation or systemic mutation . individuals bearing such macromutations were characterized as hopeful monsters by goldschmidt ( 1933 , 1940 ) to emphasize that their appearance is neither purposeful nor gradual , and their prospects for success are a matter of luck . a hopeful monster is an individual phenotypically discontinuous from the range of phenotypes of its population , and whose hopes of establishing a new lineage lie in finding a novel niche for which its monstrosity happens to be preadapted . such a mechanism of speciation was criticized early for being so improbable as to overtax one s credulity ( dobzhansky 1937 , p. 53 ) because of the rarity of the initial mutation of large effect , and the resulting improbability of finding an equally monstrous mate ( dobzhansky 1937 ) . recent empirical and theoretical research on hybrid speciation might have revived the hopeful monster in a new , more credible form ( mallet 2007 ) . recombination of parental chromosomes in the f2 and later generations during hybridization can generate genotypes that express phenotypes outside the normal range of variation observed in either parental gene pool , a phenomenon termed transgressive segregation ; rosenthal et al . 2005 ; johnson et al . 2010 ; parsons et al . 2011 ) . often , transgressive hybrids have higher fitness in novel environments , increasing the likelihood of divergence from parental populations ( arnold and hodges 1995 ; buerkle et al . 2007 ; shahid et al . 2008 ; abbott et al . 2010 ; fitzpatrick et al . 2010 ) . a few examples of new phenotypes inferred to arise from hybridization include ( see arnold 1997 ; arnold 2006 ; stelkens and seehausen 2009 for more exhaustive reviews ) : extreme size of tiger x lion f1 hybrids ( gray 1954 ) ; unique shapes and colors of hybrid orchids ( rolfe and hurst 1909 ) ; ability of recombinant sunflowers to thrive in extreme habits ( lexer et al . 2003 , 2007 ) ; specialization on a novel host plant in lonicera flies ( schwarz et al . 2005 ) ; and expression of novel gene transcripts ( including new exons ) via alternative splicing in hybrid poplars ( scascitelli et al . not all specific examples are relevant in nature , and not all would qualify as evolutionary novelty under certain definitions ( muller and wagner 1991 ; pigliucci 2008 ; wagner and lynch 2010 ) , but this small selection of cases serves to illustrate sudden appearance of profound differences between parents and hybrid offspring reminiscent of goldschmidt s hopeful monsters.fig . 1recently metamorphosed juvenile tiger salamanders representative of ambystoma mavortium ( bts ) , a. californiense ( cts ) and transgressive later generation hybrid . the late generation hybrid has both a transgressive coloration and body size ( mass and snout - vent length ) beyond the range of parental populations recently metamorphosed juvenile tiger salamanders representative of ambystoma mavortium ( bts ) , a. californiense ( cts ) and transgressive later generation hybrid . the late generation hybrid has both a transgressive coloration and body size ( mass and snout - vent length ) beyond the range of parental populations arnold and colleagues have promoted the importance of transgressive segregation as the evolutionary novelty model of hybridization ( arnold 1997 ; arnold et al . mallet ( 2007 ) even referred to transgressive hybrids as hopeful monsters , and p. bateson ( 1984 , 2002 ) proposed a simple model for the sudden appearance and successful spread of a novel phenotype via hybridization as a mechanism of saltational evolution . we expand and make genetically explicit the haploid , diploid and polyploid cases of his model ( fig . 2 ) . 2003 ) and hybrid fitness ( dobzhansky 1937 ; muller 1942 ; turelli and orr 2000 ) . all are special cases of a general multilocus model ( fitzpatrick 2008 ) which can give rise to the evolution of novelty or discontinuity as the cumulative or combined outcome of conventional population genetic change . indeed , recombination has always been recognized as an important source of variation ( mendel 1866 ) ; whether such variation is perceived as profound or 2genetically explicit versions of bateson s model . a the haploid case , b the diploid case , c allopolyploidy . genotypes with asterisks are novel recombinant , true - breeding genotypes genetically explicit versions of bateson s model . a the haploid case , b the diploid case , c allopolyploidy . bateson s ( 1984 , 2002 ) proposal for how recombination can generate sudden change is a straightforward narrative . two different mutations ( a and b ) appear and become fixed in different populations with similar phenotypes ( circles in his diagram ) . when the populations merge , recombinant individuals with both a and b express a new phenotype ( diamonds in his diagram ) , which is more successful and becomes fixed . aside from mutation , bateson did not use genetically explicit vocabulary , but his diagram suggests a haploid genome , with mutations a and b occurring in different loci such that recombination can place them together in the same individual . we show a version of bateson s model with explicit haploid , diploid , and allopolyploid cases in fig . 2 . the key feature is that the new phenotype depends on the interaction between alleles a and b at different loci . if both a and b alleles are common in the admixed population , the new phenotype will be expressed by a large number of individuals who can interbreed with each other , rather than a single mutant monster with no prospect for a mate . moreover , even if interactions at other loci render some hybrids ( even f1 hybrids ) partly or mostly sterile , recombination could produce transgressive hybrids with restored fertility in the f2 and later generations ( fig . . 3a schematic representation of the process by which two fixed allelic differences ( a and b ) at unlinked loci might recombine during meiosis in two f1 hybrids to create a novel homozygous genotype ( aabb ) in the f2 hybrid . note that the two novel recombinant chromosomes in the f2 are the result of independent recombinational events a schematic representation of the process by which two fixed allelic differences ( a and b ) at unlinked loci might recombine during meiosis in two f1 hybrids to create a novel homozygous genotype ( aabb ) in the f2 hybrid . note that the two novel recombinant chromosomes in the f2 are the result of independent recombinational events bateson ( 2002 ) went on to note that his idea had points of similarity with the dobzhansky - muller model of hybrid dysfunction ( dobzhansky 1937 ; muller 1942 ; turelli and orr 2000 ) and the earlier verbal model of w. bateson ( 1909 ) . in fact , the explicit diploid version of bateson s model differs from the dobzhansky - muller model only in the sign of the interaction : the bateson model supposes the interaction between a and b increases fitness , while the dobzhansky - muller model specifies a decrease in fitness of recombinant hybrids ( table 1a , b ) . both models describe gene interaction ( epistasis ) causing a hybrid phenotype to fall outside the range for either parental population . that is , they are special cases of transgressive segregation.table 1diploid , two - locus models for hybrid phenotypesaaaaaa(a ) epistatic hybrid dysfunction bb111 bb11 h01 h1 bb11 h11 h2(b ) epistatic hybrid vigor bb111 bb11 + s01 + s1 bb11 + s11 + s2(c ) additive complementation bb1 2x1 x1 bb1 x11 + x bb11 + x1 + 2xin each case , parental genotypes are aabb aabb . epistatic hybrid dysfunction ( a : the dobzhansky - muller model ) and epistatic hybrid vigor ( b : the bateson model ) differ only in whether effects are assumed to be deleterious or beneficial . the additive complementation model ( c ) shows how recombinants can be phenotypically extreme relative to parentals ( aabb and aabb ) even without gene interaction ( each a or b allele contributes an amount x to the phenotypic value , regardless of the other locus ) . all can be written as special cases of a general quantitative genetic model ( hill 1984 ; lynch and walsh 1997 ; fitzpatrick 2008 ) diploid , two - locus models for hybrid phenotypes in each case , parental genotypes are aabb aabb . epistatic hybrid dysfunction ( a : the dobzhansky - muller model ) and epistatic hybrid vigor ( b : the bateson model ) differ only in whether effects are assumed to be deleterious or beneficial . the additive complementation model ( c ) shows how recombinants can be phenotypically extreme relative to parentals ( aabb and aabb ) even without gene interaction ( each a or b allele contributes an amount x to the phenotypic value , regardless of the other locus ) . all can be written as special cases of a general quantitative genetic model ( hill 1984 ; lynch and walsh 1997 ; fitzpatrick 2008 ) transgressive segregation can also be caused by strictly additive effects of multiple genes ( table 1c ; nilsson - ehle 1911 ; grant 1975 ) . ( 2003 ) because in qtl studies of transgressive hybridization in plants , additive effects are detected more often than epistatic or dominance interactions ( rieseberg et al . strictly additive and strictly epistatic models are special cases of the general quantitative genetic model allowing phenotypes to be affected by additive , dominance , and epistatic effects ( hill 1984 ; lynch and walsh 1997 ; fitzpatrick 2008 ) . extending these basic ideas to many loci and multivariate phenotypes leads to the very general conclusion that recombination between disparate genomes has great potential to produce novel phenotypes ( gavrilets 1999 ) . the primary prediction characterizing many years of speciation research is that hybridization between disparate genomes will often generate novel phenotypes that are inviable or sterile ( hopeless monsters ) , and this becomes ever more likely with increasing differentiation ( dobzhansky 1937 ; mayr 1942 ; muller 1942 ; orr and turelli 2001 ; coyne and orr 2004 ; gavrilets 2004 ) . at the same time , the number of potentially beneficial interactions might increase ( stelkens and seehausen 2009 ; stelkens et al . 2009 ) , leading to a race between the potential for hybrid speciation and the evolution of complete reproductive isolation . here , as in the case of mutations of large effect , there is probably an inverse relationship between the magnitude of a transgressive beneficial phenotype and the likelihood that it will actually be generated in nature . the most important prediction arising from hybridization as a source of novelty is that admixed populations with many recombinant individuals repeatedly bring together many genetic differences in many unique combinations . first , instead of a single genetic difference , the diversity of recombinant genotypes after the f1 generation provides a wide field for selection of beneficial versus deleterious interactions ( lexer et al . 2003 ; parsons et al . 2011 ) . as pointed out by arnold and hodges ( 1995 ) , this means that even if most hybrid interactions are deleterious , there is still a good chance for the rare beneficial recombinant to appear , unless f1 hybrids are completely sterile or inviable . second , segregating hybrid populations will repeatedly produce recombinant genotypes with transgressive phenotypes ( figs . 2 , 3 ) , instead of only producing a single unique mutant or rare variant likely to be lost , even if advantageous ( gillespie 2004 ) . this means hopeful monsters produced by transgressive segregation have a good chance of finding suitably monstrous mates in a hybrid population and can establish a true - breeding population derived from many independent interspecific matings ( bateson 2002 ) . although speciation by transgressive hybridization is expected to be rapid in diploids ( ungerer et al . 1998 ) , we predict fixation of novel transgressive hybrids to be more rapid and perhaps more common in haploid and allopolyploid hybrids . all of the recombinant hybrids in haploid and allopolyploid populations will be true - breeding , compared to just a fraction of diploid recombinant hybrids ( fig . 2 ) . in the case of complete or incomplete dominance of a and b , all four diploid recombinant genotypes will exhibit a transgressive phenotype , but only the double homozygote will be true - breeding . this might lead to lower average fitness of a diploid hybrid population that contains some high - fitness transgressive phenotypes for several generations after hybridization is initiated ( johnson et al . finally , other more subtle predictions might arise from variation in genomic structure and development . for example , the dobzhansky - muller model helps explain empirical generalizations including haldane s rule and the large - x effect in hybrid dysfunction . by extension , the expression of beneficial transgressive phenotypes might differ between sex chromosomes and autosomes , with differential consequences for males and females in lineages with chromosomal sex determination . specifically , if transgressive phenotypes are often recessive ( s0 < s1 < s2 in table 1b ) and one or more of the interacting genes is on the sex chromosome , then the phenotype is more likely to be expressed in the heterogametic sex , even in the f1 generation . whether such rules might exist for transgressive phenotypes depends largely on whether dominance is a consistent effect in trait expression . the only broad generalization emerging from reviews of the empirical literature so far appears to be that the additive complementation model is often adequate to explain the data ( rieseberg et al . 1999 ; burke and arnold 2001 ) . however , epistasis and dominance are not infrequently detected , and the difference might reflect lower statistical power to detect non - additive effects . the idea that hybridization can rapidly produce novel forms is familiar among botanists , but rarely appeared in mainstream discussions of speciation until recently thanks to several case studies of homoploid hybrid speciation ( for reviews see : arnold 1997 ; rieseberg et al . recombination of fixed genetic differences between two populations in the f2 and later generations can produce hybrids with phenotypes novel to both parental populations ( fig . 3 ) . when these recombinant phenotypes have fitness beyond the range of parental phenotypes they are transgressive ( fig . 1 ) . bateson s model of hybridogenic hopeful monsters and the dobzhansky - muller incompatibility model of hybrid inviability are both cases of transgressive segregation . the dobzhansky - muller model produces a hopeless monster : hopeless because sterility and inviability make finding a mate and/or novel niche moot and monstrous because sterility and inviability are both phenotypes outside the parental range of phenotypes ( table 1a ) . the bateson model produces a hopeful monster : hopeful because it has a good chance of finding a mate given continued hybridization and greater fitness than parental phenotypes in some environments , and monstrous because of its transgressive phenotype ( table 1b ) . all three models are special cases of the general quantitative genetic model , thus reconciling sudden and gradual origins of novelty without requiring a special class of mutations or population dynamics . transgressive segregation might be an important mechanism promoting sudden phenotypic changes and ecological transitions in evolution . even if most of the variation produced is deleterious , a rare transgressive hybrid genotype could rapidly fix in a population or establish a novel lineage . it is even possible that regularities in the distribution of dominance effects could lead to general predictions ( such as the large x effect and haldane s rule ) for transgressive trait expression , but more research on the genetic architecture of transgressive traits is needed . regardless of those details , admixture can simultaneously bring together many new combinations of alleles , generating multilocus novelties that might never have appeared via gradual accumulation of new mutations in a single population . gene exchange is not the sole , nor even necessarily most likely , source of evolutionary novelty ( meyer 2002 ; moczek 2008 ) , but is perhaps the most likely mechanism of sudden , population level change .

the origin of novelty is a critical subject for evolutionary biologists . early geneticists speculated about the sudden appearance of new species via special macromutations , epitomized by goldschmidt s infamous hopeful monster . although these ideas were easily dismissed by the insights of the modern synthesis , a lingering fascination with the possibility of sudden , dramatic change has persisted . recent work on hybridization and gene exchange suggests an underappreciated mechanism for the sudden appearance of evolutionary novelty that is entirely consistent with the principles of modern population genetics . genetic recombination in hybrids can produce transgressive phenotypes , monstrous phenotypes beyond the range of parental populations . transgressive phenotypes can be products of epistatic interactions or additive effects of multiple recombined loci . we compare several epistatic and additive models of transgressive segregation in hybrids and find that they are special cases of a general , classic quantitative genetic model . the dobzhansky - muller model predicts hopeless monsters , sterile and inviable transgressive phenotypes . the bateson model predicts hopeful monsters with fitness greater than either parental population . the complementation model predicts both . transgressive segregation after hybridization can rapidly produce novel phenotypes by recombining multiple loci simultaneously . admixed populations will also produce many similar recombinant phenotypes at the same time , increasing the probability that recombinant hopeful monsters will establish true - breeding evolutionary lineages . recombination is not the only ( or even most common ) process generating evolutionary novelty , but might be the most credible mechanism for sudden appearance of new forms .

Revival of the Hopeful Monster The Bateson Model The General Model Predictions Conclusions

caries activity usually causes tooth decay or cavities and can even lead to the loss of afflicted teeth , which is particularly harmful to children 's growth and development . the world health organization ( who ) reported that 6090% of schoolchildren worldwide experience caries , with the disease being most prevalent in asian and latin american countries . surveys among the us population showed an incidence of 45.3% in children with either past or present coronal caries , and the rate of caries has been increasing in developing countries with the increasing consumption of highly refined sugars in the diet . according to the third national epidemiological survey of dental health in china in 2007 , the prevalence of dental caries among 5-year - old children in china was 66.0% , with a mean number of 3.5 per capita of caries teeth . therefore , caries disease has been ranked by the who as the third most important disease that requires worldwide attention for its prevention and treatment . studies on the etiology of dental caries have identified some risk factors , such as consuming foods and beverages with a high content of refined carbohydrates , receiving bottle - feed for a prolonged period , having deficient hygiene , etc . given the strong evidence supporting the relationship of dental caries with irregular dietary patterns and the link between abnormal dietary intake and obesity several reports described the link between caries and weight , indicating that obese children had more caries than children in the normal - weight group . however , others concluded that there was no significant difference in the dmft / dmft score among different body mass index ( bmi ) groups in 38-year - old children . therefore , no consensus has been reached on the relationship between obesity and dental caries in children . qingdao is a key economic center and a port city of china . yet , till now , there is no national data concerning caries status among school children native of this area , and less is known about the relationship of caries and weight status . in this study , a large - scale population - based oral health survey among 8-year - old children was conducted from january to july in 2012 at qingdao , shandong province , china , supported by the qingdao government . the aims of the present study were : ( 1 ) to assess the caries prevalence and severity in 8-year - old children of qingdao and ( 2 ) to assess the relationship between caries disease and the weight status in 8-year - old children of qingdao . in this cross - sectional study , oral health survey was conducted from january to july in 2012 . eight - year - old children from 28 public elementary schools in qingdao were invited to participate in this survey . all children and their parents were informed about the nature of the experiment and they gave their written informed consent . the study was conducted after obtaining the approval of the ethical committee of the qingdao municipal hospital . the oral examination , mainly visual inspection plus probing , was undertaken according to the who criteria for the diagnosis of caries . a dental mirror and exploring probe generally , dental caries was diagnosed only if there was a prominent color , shape , or caries - like change in pit , fissure , or smooth surface . caries experience was evaluated on all the subjects teeth included in the deciduous ( dmft ) or mixed ( dmft + dmft ) dentition . dmft ( number of decayed , missing , and filled teeth in permanent dentition ; in this study , of 8-year - old children ; permanent teeth included central incisors , lateral incisors , and first molars in mandibular and maxillary bone ) and dmft ( number of decayed , missing , and filled teeth in deciduous dentition ) indices were recorded from the dental charts . all examinations were performed by eight calibrated professional dentists who were trained for the assessment of the dmft/(dmft + dmft ) index prior to the oral survey . in an initial evaluation on caries detection , the inter - examiner kappa values were above 0.80 , indicating good agreement among the examiners . weight was evaluated using a single calibrated scale ( tanita ultimate series 2204 ; tanita corporation inc . , height was measured using harpenden stadiometer ( holtain ltd , dyved , uk ) by having the subject standing straight without shoes ( corrected to the nearest 0.1 cm ) . bmi was calculated using the following formula : weight in kg/(height in meters squared ) . weight status was defined by gender - related bmi according to the centers for disease control and prevention ( cdc ) guidelines as follows : underweight ( bmi 5 percentile ) , normal weight ( bmi > 5 and < 85 percentile ) , at risk of overweight ( bmi 85 and < 95 percentile ) , and overweight ( bmi 95 percentile ) . all data were collected on standardized forms , and a database was created with all the collected information , including age and gender . to protect confidentiality , the database was password secured and accessible to only one data analyst . descriptive data were obtained for all outcome variables and reported as mean sd . the student 's t - test , chi - square test , and one - way analysis of variance ( anova ) were applied for the statistical evaluation of means and comparisons of proportions . the overall caries disease status in each bmi group was compared based on dmft , ( dmft + dmft ) , and caries prevalence . pearson 's correlation was applied between dmft/(dmft + dmft ) and bmi value to assess the relationship between caries and weight status . in this cross - sectional study , oral health survey was conducted from january to july in 2012 . eight - year - old children from 28 public elementary schools in qingdao were invited to participate in this survey . all children and their parents were informed about the nature of the experiment and they gave their written informed consent . the study was conducted after obtaining the approval of the ethical committee of the qingdao municipal hospital . the oral examination , mainly visual inspection plus probing , was undertaken according to the who criteria for the diagnosis of caries . a dental mirror and exploring probe generally , dental caries was diagnosed only if there was a prominent color , shape , or caries - like change in pit , fissure , or smooth surface . caries experience was evaluated on all the subjects teeth included in the deciduous ( dmft ) or mixed ( dmft + dmft ) dentition . dmft ( number of decayed , missing , and filled teeth in permanent dentition ; in this study , of 8-year - old children ; permanent teeth included central incisors , lateral incisors , and first molars in mandibular and maxillary bone ) and dmft ( number of decayed , missing , and filled teeth in deciduous dentition ) indices were recorded from the dental charts . all examinations were performed by eight calibrated professional dentists who were trained for the assessment of the dmft/(dmft + dmft ) index prior to the oral survey . in an initial evaluation on caries detection , the inter - examiner kappa values were above 0.80 , indicating good agreement among the examiners . weight was evaluated using a single calibrated scale ( tanita ultimate series 2204 ; tanita corporation inc . , height was measured using harpenden stadiometer ( holtain ltd , dyved , uk ) by having the subject standing straight without shoes ( corrected to the nearest 0.1 cm ) . bmi was calculated using the following formula : weight in kg/(height in meters squared ) . weight status was defined by gender - related bmi according to the centers for disease control and prevention ( cdc ) guidelines as follows : underweight ( bmi 5 percentile ) , normal weight ( bmi > 5 and < 85 percentile ) , at risk of overweight ( bmi 85 and < 95 percentile ) , and overweight ( bmi 95 percentile ) . all data were collected on standardized forms , and a database was created with all the collected information , including age and gender . to protect confidentiality , the database was password secured and accessible to only one data analyst . descriptive data were obtained for all outcome variables and reported as mean sd . the student 's t - test , chi - square test , and one - way analysis of variance ( anova ) were applied for the statistical evaluation of means and comparisons of proportions . the overall caries disease status in each bmi group was compared based on dmft , ( dmft + dmft ) , and caries prevalence . pearson 's correlation was applied between dmft/(dmft + dmft ) and bmi value to assess the relationship between caries and weight status . a total of 758 children from 28 primary schools in qingdao were initially included in this study . the withdrawal of 14 children was due to the missing values and it resulted in the final group of 744 children that consisted of 384 males and 360 females . subjects fell in the age range of 88.5 years , with a nearly symmetric gender distribution ( 51.6% males and 48.4% females ) . the total number of teeth that went through inspection was 17,139 ( averaging 23.0 per capita ) and the decayed teeth numbered 3008 ( averaging 4.0 per capita ) . results showed that 642 ( 86.3% of the total ; 327 males and 315 females ) children suffered from dental caries and only 102 of them ( 13.1% of the total ; 57 males and 45 females ) were caries - free . approximately 6.0% of the children in the study ( n = 45 ; 16 males and 29 females ) were underweight , 73.9% ( n = 550 ; 264 males and 286 females ) had normal weight , while 9.8% ( n = 73 ; 38 males and 35 females ) and 10.2% ( n = 96 ; 76 males and 10 females ) were at risk of overweight and overweight , respectively . no significant difference was found between the two genders ( p > 0.05 ) with respect to caries prevalence . the mean dmft values were 4.29 in males and 4.34 in females in deciduous dentition . among the permanent teeth , except for the first permanent molar the mean dmft values were 0.43 in males and 0.61 in females in permanent dentition . the constitution of d , m , dmft , and dmft index between the two genders was similar ( p > 0.05 ) , except for f value which was significantly higher in males than in females ( p < 0.1 ) . similarly , significantly higher restoration rates were found in male children ( 2.9% ) than in female children ( 1.2% ) ( p < 0.01 ) table 1 . caries experience in children teeth with the highest caries prevalence of 51.6% were the right mandibular second deciduous molar ( teeth 85 ) ( 52.6% in females and 50.7% in males ) , followed by teeth 75 , 74 , and 84 with caries prevalence of 50.2 , 47.2 , and 47.0% , respectively [ figure 1 ] . significantly higher caries prevalence was found in mandibular deciduous molars ( 49.0% ) as compared with maxilla deciduous molars ( 38.1% ) [ figure 1 ] . among the newly erupted permanent first molars , teeth 36 and 46 were found to have a high caries prevalence of 15.8 and 15.0% , respectively , in contrast to teeth 16 and 26 that had relatively low caries prevalence of 5.7 and 5.0% , respectively [ figure 1 ] . the mandibular second deciduous molars ( teeth 85 ) accounted for 51.6% of caries prevalence among the children . there was a significantly higher rate of caries prevalence in mandibular deciduous molars ( 49.0% ) than in maxillary deciduous molars ( 38.1% ) . among the newly erupted permanent first molars , teeth 36 and 46 were found with the highest caries prevalence of 15.8 and 15.0% , respectively among the female children , 8.1% fell into the underweight category and 79.4% fell into the healthy category , while 9.7 and 2.8% were considered as at risk of overweight and overweight , respectively . among the male children , 14.2% were underweight and 68.6% were healthy , while 9.9 and 17.2% were considered to be at risk of overweight and overweight , respectively . considering all the male and female children , the mean height was 132.1 cm and the mean weight was 19.0 kg . significant difference was detected in the weight and height between the two genders ( p < 0.01 ) [ table 2 ] . classification of body weight status according to cdc guidelines values of dmft/(dmft + dmft ) in each bmi group were compared and the results showed statistically significant differences between different bmi groups [ p < 0.1 for ( dmft + dmft ) , p < 0.01 for dmft ] [ table 3 ] , with the highest dmft/(dmft + dmft ) values in the underweight group . no significant difference was found in the caries prevalence between different bmi groups [ table 3 ] . pearson 's correlation between dmft/(dmft + dmft ) and bmi was significant [ p = 0.04 for dmft , p = 0.004 for ( dmft + dmft ) ] , with r values of 0.075 and 0.104 for dmft and ( dmft + dmft ) , respectively . these results indicated an inverse relationship between bmi and caries severity [ figure 2 ] . comparison of caries status according to bmi classification pearson 's correlation of bmi and caries experience . ( a ) caries severity was evaluated by ( dmft + dmft ) values ( b ) dmft values . pearson 's correlation between caries severity and bmi index was significant ( p = 0.004 for ( a ) and p = 0.04 for ( b ) ) no significant difference was found between the two genders ( p > 0.05 ) with respect to caries prevalence . the mean dmft values were 4.29 in males and 4.34 in females in deciduous dentition . among the permanent teeth , except for the first permanent molar the mean dmft values were 0.43 in males and 0.61 in females in permanent dentition . the constitution of d , m , dmft , and dmft index between the two genders was similar ( p > 0.05 ) , except for f value which was significantly higher in males than in females ( p < 0.1 ) . similarly , significantly higher restoration rates were found in male children ( 2.9% ) than in female children ( 1.2% ) ( p < 0.01 ) table 1 . caries experience in children teeth with the highest caries prevalence of 51.6% were the right mandibular second deciduous molar ( teeth 85 ) ( 52.6% in females and 50.7% in males ) , followed by teeth 75 , 74 , and 84 with caries prevalence of 50.2 , 47.2 , and 47.0% , respectively [ figure 1 ] . significantly higher caries prevalence was found in mandibular deciduous molars ( 49.0% ) as compared with maxilla deciduous molars ( 38.1% ) [ figure 1 ] . among the newly erupted permanent first molars , teeth 36 and 46 were found to have a high caries prevalence of 15.8 and 15.0% , respectively , in contrast to teeth 16 and 26 that had relatively low caries prevalence of 5.7 and 5.0% , respectively [ figure 1 ] . the mandibular second deciduous molars ( teeth 85 ) accounted for 51.6% of caries prevalence among the children . there was a significantly higher rate of caries prevalence in mandibular deciduous molars ( 49.0% ) than in maxillary deciduous molars ( 38.1% ) . among the newly erupted permanent first molars , teeth 36 and 46 were found with the highest caries prevalence of 15.8 and 15.0% , respectively among the female children , 8.1% fell into the underweight category and 79.4% fell into the healthy category , while 9.7 and 2.8% were considered as at risk of overweight and overweight , respectively . among the male children , 14.2% were underweight and 68.6% were healthy , while 9.9 and 17.2% were considered to be at risk of overweight and overweight , respectively . considering all the male and female children , the mean height was 132.1 cm and the mean weight was 19.0 kg . significant difference was detected in the weight and height between the two genders ( p < 0.01 ) [ table 2 ] . values of dmft/(dmft + dmft ) in each bmi group were compared and the results showed statistically significant differences between different bmi groups [ p < 0.1 for ( dmft + dmft ) , p < 0.01 for dmft ] [ table 3 ] , with the highest dmft/(dmft + dmft ) values in the underweight group . no significant difference was found in the caries prevalence between different bmi groups [ table 3 ] . pearson 's correlation between dmft/(dmft + dmft ) and bmi was significant [ p = 0.04 for dmft , p = 0.004 for ( dmft + dmft ) ] , with r values of 0.075 and 0.104 for dmft and ( dmft + dmft ) , respectively . these results indicated an inverse relationship between bmi and caries severity [ figure 2 ] . comparison of caries status according to bmi classification pearson 's correlation of bmi and caries experience . ( a ) caries severity was evaluated by ( dmft + dmft ) values ( b ) dmft values . pearson 's correlation between caries severity and bmi index was significant ( p = 0.004 for ( a ) and p = 0.04 for ( b ) ) qingdao is located in the southern part of shandong peninsula , facing the yellow sea in the east and south . the qingdao government initiated a public welfare project of oral health examination , pit and fissure sealant , and caries filling treatment in public primary school students , which provided an opportunity to systematically assess the caries status in children of qingdao . for assessment of dental caries , we used the dmft / dmft index , as it is one of the most commonly used indices in epidemiological studies for evaluating caries experience . we found that firstly , the 744 children aged 8 years in qingdao were generally in poor oral health status . although this rate was similar to that reported in other domestic studies ( 85.4% of caries prevalence in children of similar age group in guangzhou ) , the average dmft index ( 4.31 ) in our study was significantly higher than that reported ( dmft index : 3.11 ) in the study of guangzhou . furthermore , caries prevalence and dmft value in these 8-year - old children from qingdao were significantly higher than those reported from other countries such as sudan . the reason might be the changes in dietary habit that happened during the last decade . changes in dietary content of the children of qingdao have been observed in the past few years , with them consuming significantly more desserts than ever before ; yet , their oral health is at a low level . secondly , it was noted that averagely , only 2.1% of the decayed teeth were treated with filling . this finding was consistent with the restoration rate of children in a previous regional study , supporting the widespread negligence for the oral health of children in china . therefore , dental treatment and dental health education of children , parents , teachers , and even the whole society are urgently needed . thirdly , the significantly higher restoration rate in males might partially reflect that male children have been receiving more oral care than female children . our findings might add some support to the notion that the restoration rate correlated with the regional economy , culture , and education . lastly , our data showed that the mandibular second deciduous molars accounted for over 50% of all the decayed teeth , which suggested the necessity to pay more attention to protecting the particular teeth from caries at an early stage . weight status in children was measured by bmi , according to gender and age - ranked percentages . our study showed that 10% of children were overweight among all the 8-year - old children , which is slightly lower than the corresponding values previously reported by other analyses conducted in other chinese regions . according to the studies conducted in chengdu and shanghai , the prevalence of obesity was about 3% , respectively , which was similar to our data on female children but significantly lower than our data on male children ( 17.2% ) . methodological differences ( e.g. criteria recommended by cdc guidelines , international obesity task force , or world health organization ) might have contributed to such differences , causing the difficulty in comparing the results of the studies . in other recent studies , based on the same criteria recommended by cdc guidelines , 31.0% of us children were at risk of overweight and 16.0% were overweight ; both percentages are obviously higher than those obtained in our survey . caries is usually regarded as a consequence of frequent ingestion of fermentable sugars , which can also lead to obesity . as a result however , findings of obesity caries relationship in children from different countries have actually been mixed and inconclusive . conclusions on positive links , no links , and inverse links are all present . for example , the sole study from china reported no correlation between obesity and caries among 280 children . this can be due to the confounding factors such as age , gender , dentition , race / ethnicity , and socioeconomic status , which might affect the final conclusion . to eliminate these potential confounding factors , we conducted our study on subjects from a relatively homogeneous environment . they were all children from a single specific age group from the native population with similar living environment and eating habits , from public primary schools with similar socioeconomic background , and with a nearly symmetric gender distribution . also , we utilized two indices [ dmft and ( dmft + dmft ) ] to assess the caries severity in children both in deciduous and mixed dentition . regardless of the index used in the analysis , instead of a previously suspected positive link , a weak inverse relationship of body weight and caries severity was found and the comparison also showed underweight individuals to have significantly more severe caries disease . this finding suggested that the relationship of obesity and caries was not as straightforward as was earlier suspected . moreover , although not focusing on an identical age group , our result is consistent with a nationally representative survey based on a dataset of 10,800 american adolescents national health and nutrition examination survey ( nhanes ) iii 19881994 and nhanes 19992002 ) which suggested that overweight status may be associated with a somewhat decreased risk for caries . our results showed that underweight children were more likely found with more severe caries disease , regardless of primary or mixed dentition . this result was consistent with mojarad 's , werner 's , and roswitha 's reports . nowadays , in china , the living standard has improved significantly along with the rapid progress in economy . in addition , china 's one child policy has turned many children into the little emperor of their families , which argues against the fact that underweight status of the children is due to poverty . thus , it is more plausible that caries might be one cause behind children being underweight . the following mechanisms might account for the observed inverse relationship between bmi and dmft/(dmft + dmft ) index . firstly , untreated caries might have caused severe pain and discomfort in children and , thus , reduced food intake . secondly , caries can also lead to infection , irritability , and disturbed sleeping habits , which might reduce the quality of life and affect the growth . one limitation of our study is the use of cross - sectional data to examine the link between caries experience and weight status . to test the cause however , the strength of our study is the use of localized and relatively simple representative samples of chinese children to explore and control for multiple confounding factors , and our results yielded consistent findings with deciduous and mixed dentition . for assessment of dental caries , we used the dmft / dmft index , as it is one of the most commonly used indices in epidemiological studies for evaluating caries experience . we found that firstly , the 744 children aged 8 years in qingdao were generally in poor oral health status . although this rate was similar to that reported in other domestic studies ( 85.4% of caries prevalence in children of similar age group in guangzhou ) , the average dmft index ( 4.31 ) in our study was significantly higher than that reported ( dmft index : 3.11 ) in the study of guangzhou . furthermore , caries prevalence and dmft value in these 8-year - old children from qingdao were significantly higher than those reported from other countries such as sudan . the reason might be the changes in dietary habit that happened during the last decade . changes in dietary content of the children of qingdao have been observed in the past few years , with them consuming significantly more desserts than ever before ; yet , their oral health is at a low level . secondly , it was noted that averagely , only 2.1% of the decayed teeth were treated with filling . this finding was consistent with the restoration rate of children in a previous regional study , supporting the widespread negligence for the oral health of children in china . therefore , dental treatment and dental health education of children , parents , teachers , and even the whole society are urgently needed . thirdly , the significantly higher restoration rate in males might partially reflect that male children have been receiving more oral care than female children . our findings might add some support to the notion that the restoration rate correlated with the regional economy , culture , and education . lastly , our data showed that the mandibular second deciduous molars accounted for over 50% of all the decayed teeth , which suggested the necessity to pay more attention to protecting the particular teeth from caries at an early stage . weight status in children was measured by bmi , according to gender and age - ranked percentages . our study showed that 10% of children were overweight among all the 8-year - old children , which is slightly lower than the corresponding values previously reported by other analyses conducted in other chinese regions . according to the studies conducted in chengdu and shanghai , the prevalence of obesity was about 3% , respectively , which was similar to our data on female children but significantly lower than our data on male children ( 17.2% ) . methodological differences ( e.g. criteria recommended by cdc guidelines , international obesity task force , or world health organization ) might have contributed to such differences , causing the difficulty in comparing the results of the studies . in other recent studies , based on the same criteria recommended by cdc guidelines , 31.0% of us children were at risk of overweight and 16.0% were overweight ; both percentages are obviously higher than those obtained in our survey . caries is usually regarded as a consequence of frequent ingestion of fermentable sugars , which can also lead to obesity . as a result however , findings of obesity caries relationship in children from different countries have actually been mixed and inconclusive . conclusions on positive links , no links , and inverse links are all present . for example , the sole study from china reported no correlation between obesity and caries among 280 children . this can be due to the confounding factors such as age , gender , dentition , race / ethnicity , and socioeconomic status , which might affect the final conclusion . to eliminate these potential confounding factors , we conducted our study on subjects from a relatively homogeneous environment . they were all children from a single specific age group from the native population with similar living environment and eating habits , from public primary schools with similar socioeconomic background , and with a nearly symmetric gender distribution . also , we utilized two indices [ dmft and ( dmft + dmft ) ] to assess the caries severity in children both in deciduous and mixed dentition . regardless of the index used in the analysis , instead of a previously suspected positive link , a weak inverse relationship of body weight and caries severity was found and the comparison also showed underweight individuals to have significantly more severe caries disease . this finding suggested that the relationship of obesity and caries was not as straightforward as was earlier suspected . moreover , although not focusing on an identical age group , our result is consistent with a nationally representative survey based on a dataset of 10,800 american adolescents national health and nutrition examination survey ( nhanes ) iii 19881994 and nhanes 19992002 ) which suggested that overweight status may be associated with a somewhat decreased risk for caries . our results showed that underweight children were more likely found with more severe caries disease , regardless of primary or mixed dentition . this result was consistent with mojarad 's , werner 's , and roswitha 's reports . nowadays , in china , the living standard has improved significantly along with the rapid progress in economy . in addition , china 's one child policy has turned many children into the little emperor of their families , which argues against the fact that underweight status of the children is due to poverty . thus , it is more plausible that caries might be one cause behind children being underweight . the following mechanisms might account for the observed inverse relationship between bmi and dmft/(dmft + dmft ) index . firstly , untreated caries might have caused severe pain and discomfort in children and , thus , reduced food intake . secondly , caries can also lead to infection , irritability , and disturbed sleeping habits , which might reduce the quality of life and affect the growth . one limitation of our study is the use of cross - sectional data to examine the link between caries experience and weight status . to test the cause however , the strength of our study is the use of localized and relatively simple representative samples of chinese children to explore and control for multiple confounding factors , and our results yielded consistent findings with deciduous and mixed dentition . this study was a population - based epidemiological survey on caries and body weight status in 8-year - old children in qingdao , china . a severe state of caries disease was revealed in this survey of 744 children aged 8 years , for whom urgent dental intervention and treatment were needed . our study identified a weak negative association between caries severity and weight status in this pilot population , indicating caries lesions might be a reason affecting the growth and development of children . our finding suggested that until more evidence is available , obesity should not be considered as a risk factor for caries diagnosis and treatment planning in children .

objectives : childhood obesity / underweight status and caries are both important public health problems . this study aims to investigate the caries status and its association with body weight in 8-year - old children in qingdao , china.materials and methods : we initiated a cross - sectional investigation on 744 children aged 8 years during the oral health survey in 2012 . dental caries assessments were carried out and weight status was recorded accordingly . the resulting caries status including caries prevalence , dmft ( deciduous dentition ) , and ( dmft + dmft ) ( mixed dentition ) , as well as bmi indices were analyzed for comparison and correlation.results:the prevalence of dental caries among the 744 children aged 8 years participating in this survey was 86.3% . the caries status represented by dmft ( deciduous dentition ) and ( dmft + dmft ) ( mixed dentition ) values was 4.31 and 4.85 , respectively , and the restoration rate was extremely low , which was no more than 3.0% . significant difference was found in dmft/(dmft + dmft ) values between different bmi groups , and underweight individuals were found to have the highest dmft/(dmft + dmft ) value . an inverse relationship between body bmi and dmft/(dmft + dmft ) index was identified based on pearson 's correlation.conclusions:a severe state of caries disease was revealed in 8-year - old children in the chinese city of qingdao , for whom urgent dental intervention and treatment were needed . furthermore , underweight individuals were found with the most severe caries experience , indicating caries may affect the development and growth of the afflicted children . thus , more emphasis should be placed on improving their dental health , with caries prevention being given the priority .

INTRODUCTION MATERIALS AND METHODS Study design Clinical examination Weight evaluation Statistical analysis RESULTS Caries status in 8-year-old children in Qingdao Weight status in 8-year-old children in Qingdao Relationship between caries disease and weight status DISCUSSION The status of dental caries in 8-year-old children in Qingdao The status of body weight in 8-year-old children in Qingdao Relationship between caries severity and weight status CONCLUSIONS

the development and function of mammals , like that of any multicellular organism , depends on intercellular communication . classically , this occurs either through direct cell - to - cell interaction bringing together cell surface proteins or , at a distance , through secreted soluble molecules binding to cell surface receptors . these interactions lead to transduction of intracellular signals from the cell surface to the nucleus , where regulation of gene expression might occur . a breach of this dogma , which is based on the impermeable property of biological membranes , came from the demonstration that lipid vesicles containing rnas and proteins released by mammalian cells , can modify the biological activity of non - contacting cells ( simons and raposo , 2009 ) . they can also form through budding into the lumen of endosomes and be released after fusion of the limiting membrane of endosomes to the plasma membrane . once secreted in extracellular milieu the endosomal intraluminal vesicles ( ilvs ) are referred to as exosomes . our review will stick to this strict definition of exosomes to discuss the most recent findings indicating the potential role of neuronal exosomes in intercellular communication within the normal and pathological central nervous system . it is now widely accepted that exosomes represent a way of intercellular exchange of effector molecules , which allows emitting cells to modify gene and protein expression in receiving cells . they allow transfer of membrane and cytoplasmic proteins ( thery et al . , 2002 ; morelli et al . , 2004 ) , as well as lipids involved in signal transduction ( laulagnier et al . , 2004 ; subra et al . , exosomal mrnas can be translated ( valadi et al . , 2007 ) , and small rnas , including micrornas ( mirnas ) mediate gene silencing in receiving cells ( kosaka et al . functioning of the brain relies on the capacity of neurons to locally modulate each other at the level of synapses . chemical synapses are made of a presynaptic part filled with neurotransmitter ( nt ) containing vesicles and a post - synaptic part in which nt receptors are anchored at the level of the post - synaptic density ( psd ) . specific patterns of stimulation of the presynaptic cell can durably increase or decrease the strength of synaptic responses , thereby reinforcing circuits underlying associations and memory . changes in synaptic efficacy are based on modifications of the number of post - synaptic nt - receptors or of the amount of nt released pre - synaptically for a given stimulus . changes of one neuron driven by another , have so far been explained by ways of classical signal transduction : nts , lipids , or proteins secreted from one side of the synapse bind to receptors of the opposite surface . pre - synaptic - activity substances can also be released by cell bodies and dendrites ( regehr et al . , 2009 ) . this leads to modulations of second messengers and enzymatic activities acting on effectors of the synaptic changes ( adhesion molecules , neurotransmitter receptors , cytoskeleton anchors ; malenka and bear , 2004 ) . signal transduction also leads to changes in gene expression and translation , which are needed for long - lasting synaptic modifications ( bullmore and sporns , 2009 ) . indeed , control of transcription occurs in the nucleus far away from synapses undergoing plastic changes . transcripts can be specifically transported along dendrites to synapses undergoing specific patterns of activation , where they are translated into proteins modifying synaptic strength . translation of targets mediating dendritic growth can also be regulated by mirnas , which are expressed within dendrites ( schratt et al . , 2006 ; we have recently observed that exosomes secreted by neurons contain mirnas ( unpublished observations ) . given that single mirnas have multiple targets , the impact of exosome - mediated local transfer of mirna on the pattern of translated mrnas in receiving neurons may be quite extensive . confined exchange of rnas at synapses would thus certainly represent an efficient mechanism for long - term modifications of specific synapses . therefore , the exosomal pathway may constitute a well designed mechanism for local and systemic inter - neuronal transfer of information within functional brain networks , with a complexity superior to that of direct cell - to - cell contacts or secreted soluble factors ( belting and wittrup , 2008 ) . the dark side would be that exosome transfer might also represent a privileged way for propagating pathological alterations throughout the brain ( fevrier et al . , 2005 ; aguzzi and rajendran , 2009 ) . endosomes are intracellular compartments collecting plasma membrane proteins , which are constantly renewed by constitutive or selective endocytosis ( figure 1 ) . other proteins , classically those meant for degradation , are selectively entrapped in vesicles budding from the endosomal membrane into the lumen of endosomes . maturation of endosomes leads to individualization of multivesicular bodies ( mvbs ) , which are large vacuoles delimited by a single membrane and containing a varying number of 5080 nm membrane vesicles ( figure 2 ; gruenberg and stenmark , 2004 ; van der goot and gruenberg , 2006 ) . invagination of the endosomal membrane leading to the formation of mvbs also allows selective microautophagy of cytoplasmic proteins ( sahu et al . , 2011 ) . membrane and cytoplasmic proteins entrapped in vesicles will be hydrolyzed after fusion of mvbs with lysosomes . they can also be expelled from cells after fusion of mvbs with the plasma membrane leading to the release of exosomes into the extracellular milieu ( figure 1 ; simons and raposo , 2009 ) . after endocytosis ( 1 ) the endocytic vesicle fuses to early endosomes ( 2 ) . proteins can be concentrated into recycling endosomes , which fuse to the plasma membrane and allow re - expression at the cell surface ( 3 ) . alternatively proteins can be entrapped in vesicles budding from the limiting membrane of the endosome ( 4 ) . maturation of the endosome leads to the individualization of a multivesicular body containing intraluminal vesicles ( ilv ) ( 5 ) . the multivesicular body can fuse with lysosomes in which the ilvs and their cargoes are hydrolyzed ( 6 ) . the multivesicular body can also fuse with the plasma membrane ( 7 ) thereby releasing ilvs . once in the extracellular milieu ilvs exosomes released by cell a , can bind to and be endocytosed by a receiving cell [ cell b , 8 ] . once inside the endosome , the exosome undergoes back - fusion with the endosomal membrane ( 10 ) . fusion of recycling endosomes to the plasma membrane allows expression of protein of the cell a at the surface of cell b. back - fusion also allows the release of the intraluminal content of exosomes [ proteins and rnas of cell a ] into the cytosol of cell b. it is important to note that steps 9 , 10 , and 11 remain speculative . ( a ) electron micrograph of a multivesicular body present in a neuron of the ca1 region of the adult rat hippocampus . note the budding of a vesicle from the limiting membrane of the mvb ( upper right ; fiona hemming , unpublished ) . ( b ) electron micrograph of a multivesicular body in a dendrite ( colored ; ca1 region of an adult rat hippocampus ) . the protrusion of the dendrite , called dendritic spine , corresponds to the post - synaptic part of a glutamatergic synapse . two post - synaptic densities , which anchor ionotropic glutamate receptors , are visible . in this case the multivesicular body is present within the dendritic shaft at the base of the spine neck ( fiona hemming , unpublished ) . in neurons , electron microscopy ( em ) observations of the adult hippocampus revealed the presence of mvbs and sorting endosomes in dendritic shafts and inside a limited number of spines , which represent post - synaptic parts of glutamatergic synapses ( figure 2b ; cooney et al . , 2002 ) . noteworthy , is that mvbs are about 50 times more represented in somatodendritic compartments than in axons ( von bartheld and altick , 2011 ) . endosome - containing spines are mostly mushroom - like spines i.e. , with the most active synapses ( kasai et al . , 2003 ) . enhancement of synaptic activity after injection of peptides known to improve cognitive functions or during kindling , significantly increased the proportion of mvbs inside spines in the dentate gyrus ( popov et al . similarly , water maze training of rats led to the migration of mvbs to the vicinity of psds in dendrites of ca3 pyramidal cells , while chronic restraint stress diminished the number of mvbs associated with psds ( stewart et al . , 2005 ) . similarly neutrotrophic factors ( bdnf , gdnf ) induced a relocalization of dendritic mvbs very near psds of hypoglossal motoneurons ( rind et al . , 2005 ) . thus , in the cns , movements of mvbs to synapses are tightly linked to synaptic plasticity ( von bartheld and altick , 2011 ) . studies of the trafficking of synaptic ampa type - receptors , which represent the major mediators of fast synaptic transmission among glutamate receptors of the cns , led to the demonstration that dendritic endosomes act as stores and sorting platforms for synaptic receptors ( kennedy and ehlers , 2006 ) . during long - term potentiation ( ltp ) , a form of synaptic plasticity now widely accepted as a model of learning and memory processes ( ehlers , 2000 ) , membrane insertion of new post - synaptic ampa receptors increases excitatory post - synaptic currents thereby potentiating the synapses . live cell imaging of dissociated hippocampal neurons demonstrated that glycine stimulation , a protocol used to induce chemical ltp through activation of synaptic nmda - receptors , leads to the recruitment of endosomes into , or near spines and their fusion with the plasma membrane ( correia et al . thereby , ampa - rs present in the limiting membrane of endosomes become inserted at the neuronal surface and diffuse laterally to synaptic sites where they accumulate through interaction with proteins of psds . live imaging of the insertion at the plasma membrane of transferrin receptors ( tfr ) contained in endosomes , showed the requirement of rab11 or syntaxin 13 for the endosomal fusion at the dendritic surface ( park et al . accordingly , expression of a dominant negative form of rab11 was found to inhibit ltp in slice cultures ( brown et al . , 2007 ; wang et al . , 2008 ) , demonstrating that endosomal fusion to the dendritic membrane is a necessary step for synaptic potentiation . the compartments fusing at the plasma membrane of cultured neurons were identified as recycling endosomes because tfr is usually detected in recycling endosomes , and rab11 or syntaxin 13 are known regulators of recycling endosomes . however , the strict separation between recycling endosomes and mvbs , considered as late endosomes , needs to be made with caution . indeed , in reticulocytes , tfr is present in exosomes ( geminard et al . , 2004 ) and colombo and collaborators found that rab11 is required for mvb fusion to the plasma membrane in an erythroleukemic cell line ( savina et al . , 2005 ) . furthermore , neep21 , known to regulate recycling of ampa receptors at the synapse , has been localized by immunofluorescence inside tfr - containing endosomes of cultured hippocampal neurons ( steiner et al . , 2005 ) . however , em observations of rat brain sections demonstrated that the protein is expressed at psds as well as in intralumenal , but not limiting , membranes of mvbs ( utvik et al . , 2009 ) . thus , even if fluorescence data suggest that endosomes fusing to the plasma membrane during synaptic plasticity are recycling endosomes , one can not yet exclude that some of these endosomes are mvbs . the final proof that mvbs fuse to the dendritic surface awaited the visualization of this process by em and the demonstration that exosome release is modified by synaptic glutamate receptor activity . we made the first demonstration that cortical neurons in culture release exosomes ( faure et al . , 2006 ) . as in the case of other cells , exosomes isolated from neuron culture media floated on sucrose gradients at a density of 1.11.2 g / ml and contained both tsg101 and alix . tsg101 belongs to the endosomal sorting complex required for transport ( escrt-0 to iii ) , necessary for the making of ilvs accumulating inside mvbs ( babst , 2011 ) . alix is acytoplasmic protein binding to tsg101 of escrt - i and chmp4b of escrt - iii ( missotten et al . , 1999 ; endophilin a , which also interacts with alix ( chatellard - causse et al . , 2002 ) , was not detected in exosomes , demonstrating that entry of cargoes into ilvs is regulated . other cytoplasmic proteins and enzymes were present inside exosomes , including gadph , ubiquitin , and hsc70 . this is in good agreement with the recent finding thathsc70 binding to gadph drives its escrt - dependent engulfment into mvb - ilvs ( sahu et al . , 2011 ) . exosomes also contained ampa- , but not nmda - receptors and the cell adhesion molecule l1/ngcam , which , in the central nervous system is expressed only by neurons ( maness and schachner , 2007 ) , thus demonstrating that exosomes are secreted by neurons . we also observed that electrical activity regulates exosomal secretion since long - term depolarization of neurons with 25 mm potassium strongly increased the release of ampa - r containing exosomes . three other studies reported secretion of exosomes by neurons ( vingtdeux et al . , 2007 ; these studies , as well as our initial one , used embryonic neurons cultured for only 38 days . in short term cultures , neurons make only few synapses and neurite outgrowth is still on - going . thus , exosome release could simply reflect the fusion of late endosomes / lysosomes at growth cones necessary for neurite elongation ( arantes and andrews , 2006 ) . more recently , we have studied exosome release from fully differentiated cultures ( 15 div ; lachenal et al . , 2010 ) . dissociated cortical cells contain both glutamatergic and gabaergic neurons , which make functional networks within the second week in culture . thus , incubation with gaba receptor antagonists , such as picrotoxin or bicuculline , alleviates inhibitory activities within the networks and increases synaptic glutamatergic activity . picrotoxin or bicucullin rapidly ( 1015 min ) and massively augmented the secretion of exosomes in a way dependent on ampa- and nmda - receptors ( lachenal et al . , 2010 ) . we also found that increasing cytosolic calcium , using the calcium ionophore ionomycin , drastically elevated exosome secretion . em examination of cultures treated for 1 min with ionomycin revealed clusters of exosomes at the surface of dendrites visualizing the fusion of mvbs with the plasma membrane . altogether , our data suggest that calcium entry through synaptic nmda - receptors is a potent activator of mvb fusion to the plasma membrane and thereby of exosome secretion . the enhanced secretion of ampa - r - containing exosomes following glutamatergic synaptic activation , underlines exosomal release as a way of local elimination of receptors at synapses undergoing plastic changes . the loss of ampa receptors upon extensive synaptic activation could be a mechanism of homeostatic synaptic scaling , necessary for adjusting the strength of all of a neuron s excitatory synapses to stabilize firing ( turrigiano , 2008 ) . thus , while fusion of endosomes leads to an increase of receptors at synapses undergoing potentiation , sustained synaptic activation would lead to calcium increase within the dendritic shaft triggering fusion of mvbs at the base of nearby synapses to allow the local elimination of the intracellular pool of ampa receptors and thereby synaptic down - scaling . in this scenario , regulation of the pool of surface synaptic receptors by exosome secretion would be a local event , avoiding retrograde transport of mvbs necessary to hydrolyze the receptors in lysosomes , which are only present in proximal dendrites and soma . exosomes were first shown to be endocytosed by dendritic cells of the immune system ( skokos et al . , 2003 ) . those released by neurons into the cns parenchyma could potentially be endocytosed by nearby cells as shown for oligodendrocyte derived exosomes which are endocytosed by microglial cells ( fitzner et al . , 2011 ) . astrocyte end feet , which enwrap a number of glutamatergic synapses , can also endocytose / phagocytose cellular debris ( haydon and carmignoto , 2006 ) and could thus capture exosomes released at synapses . transfer of exosomes could also occur between spines of the same neuron or across synapses to end up in afferent neurons . indeed , the diameter of neuronal exosomes is compatible with possible endocytosis in neuronal clathrin coated pits occurring in presynaptic boutons , in spines , or dendritic shafts ( lu et al . , 2007 ) . we have recently obtained evidence that exosomes bind to and are endocytosed by hippocampal neurons ( unpublished observations ) and that they allow the inter - neuronal transfer of tetanus toxin , which is known to cross synapses in vivo ( lachenal et al . , 2010 ) . em observations are now needed to characterize the site of entry of exosomes and their fate inside endosomes . in non - neuronal cells , the fact that exosomal rnas can act on receiving cells demonstrates that exosome intralumenal cargoes are released into the cytosol i.e. , that the membrane of exosomes fuses with the plasma membrane or with endosomal membranes after their endocytosis ( figure 1 ) . back - fusion of intralumenal vesicles has been demonstrated to occur in mvbs ( falguieres et al . , 2009 ) and could thus concern exosomes , which have the same origin . such a process would lead to the entry of exosomal membrane proteins into the endosomal protein pool and possibly re - expression at the cell surface ( e.g. , ampa receptors ) . it would also allow the release into the cytosol of the exosome content , including signal transduction molecules and mirnas . exosomes can contain pathogenic proteins such as alpha - synuclein , prpsc , amyloid precursor protein ( app ) , and phosphorylated tau , which are involved in parkinson s , prion , and alzheimer s diseases respectively . the scrapie form of the prion protein ( prpsc ) contained in exosomes is secreted via exosomes and remains infectious under this form ( fevrier et al . , 2004 ) . thus , trans - synaptic exchange could be one way for the propagation of prion diseases from the periphery to the cns . alpha - synuclein secreted together with exosomes released by neuroblastoma cells causes cell death of recipient neuronal cells suggesting that alpha - synuclein secretion via exosomes serves to amplify and propagate parkinson s disease - related pathology ( emmanouilidou et al . , the catabolism of app giving rise to the amyloidogenic c - terminal app fragment occurs in endosomes and this fragment as well as a amyloid peptides , are released by way of exosomes ( rajendran et al . , 2006 ; vingtdeux et al . these puzzling hypotheses require in vivo work ( i ) to show that exosomal released from mvbs occurs in situ ( ii ) to find out the privileged site of this release ( iii ) to demonstrate transynaptic exchange of exosomes . furthermore , even though the activity dependent - release of exosomes suggests a genuine function of exosomes in synaptic plasticity , molecular tools to specifically block mvb fusion with the plasma membrane must be developed to test this hypothesis . nevertheless , studies on exosomes in the cns are bound to shed new light on intercellular exchanges within the brain and to open new avenues toward understanding how neurodegenerative diseases spread over time throughout the nervous system . 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 .

exosomes are small extracellular vesicles , which stem from endosomes fusing with the plasma membrane , and can be recaptured by receiving cells . they contain lipids , proteins , and rnas able to modify the physiology of receiving cells . functioning of the brain relies on intercellular communication between neural cells . these communications can modulate the strength of responses at sparse groups of specific synapses , to modulate circuits underlying associations and memory . expression of new genes must then follow to stabilize the long - term modifications of the synaptic response . local changes of the physiology of synapses from one neuron driven by another , have so far been explained by classical signal transduction to modulate transcription , translation , and posttranslational modifications . in vitro evidence now demonstrates that exosomes are released by neurons in a way depending on synaptic activity ; these exosomes can be retaken by other neurons suggesting a novel way for inter - neuronal communication . the efficacy of inter - neuronal transfer of biochemical information allowed by exosomes would be far superior to that of direct cell - to - cell contacts or secreted soluble factors . indeed , lipids , proteins , and rnas contained in exosomes secreted by emitting neurons could directly modify signal transduction and protein expression in receiving cells . exosomes could thus represent an ideal mechanism for inter - neuronal transfer of information allowing anterograde and retrograde signaling across synapses necessary for plasticity . they might also allow spreading across the nervous system of pathological proteins like prpsc , app fragments , phosphorylated tau , or alpha - synuclein .

Introduction Why Neurons Could Make Good Use of Exosomes Endosomes in Neurons Control Synaptic Plasticity Regulated Secretion of Exosomes by Neurons The Fate of Exosomes Released by Neurons Relevance of Neuronal Exosomes for Neurodegenerative Diseases Conflict of Interest Statement

patients with symptomatic sciatic hernias commonly present with flank , abdominal , pelvic , lower back or thigh pain . most asymptomatic patients have been treated conservatively , while surgery has been reserved for symptomatic patients . we describe a case revealed by left hydronephrosis with severe left back pain in a 72-year - old female . this case shows that minimally invasive endourological techniques may provide a novel method for relieving this cause of obstruction . a 72-year - old female accidentally struck her left forechest 2 months before she was referred to our hospital , and lower left rib fracture was observed on x - ray examination . she was suffering from severe left - sided back pain , which was thought to be apart from the rib fracture site , and the pain was intermittent . the ultrasound sonography demonstrated left hydronephrosis and a simple renal cyst on the lower pole of the left kidney , and urinalysis showed microhematuria . routine blood tests were not remarkable except for a slightly elevated serum glucose level of 179 mg / dl . intravenous ureterography ( ivu ) showed findings compatible with a left sciatic ureter , a dilated ureter with a fixed kinking , which is known as the abdominal ct showed marked hydronephrosis and hydroureter on the level of the pelvic inlet and revealed the presence of a sciatic herniation of the ureter ( fig . 2 ) . the ureter was seen to course behind the left hip and through the greater sciatic foramen , anterior to the piriformis muscle . no evidence of inflammatory lesions , compressive tumor or herniation of another pelvic organ with the ureter was seen . subsequently , the placement of ureteral stent was performed to decompress the dilated upper urinary tract . interestingly , the ureterosciatic hernia was relieved with the passage of a flexible guide wire and a double - pigtail stent ( fig . however , the patient refused continuing to have an indwelling stent and the stent was removed . ivu obtained 3 months after the removal of the ureteral stent revealed the recurrence of the ureterosciatic hernia ; however , there was no evident recurrence of hydroureter , hydronephrosis or ureteral obstruction . since then six years after ureteral stenting , the patient continues to be without hydronephrosis , symptoms or the usage of the indwelling ureteral stent . most ureteral herniations occur in the inguinal area but they have also been noted in femoral , sciatic , thoracic , and parailiac locations . among them , ureterosciatic herniation is extremely uncommon ; only 31 cases have been reported since 1947 [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 ] . the clinical symptoms are nonspecific , consisting of ill - defined abdominal pain . patients may have nonurologic complaints including vague abdominal pain or symptoms more typical of small - bowel obstruction . when obstructive uropathy develops , patients with ureterosciatic hernias may or may not experience symptoms of renal colic . the diagnosis is difficult to establish by physical examination because ureterosciatic hernias are deep to the gluteal muscles . however , these hernias may be detected as a palpable mass located underneath the gluteal muscles in a thin patient [ 4 , 9 ] . anatomically , most ureteral herniations such as ureteroinguinal and ureterofemoral hernias are paraperitoneal , since they are adherent to the posterior parietal peritoneum and hence associated with a herniating peritoneal sac . in contrast , ureterosciatic hernias are always extraperitoneal . structures such as small intestine , meckel 's diverticulum , omentum , colon , ovary , fallopian tube , bladder , and ureter may be found in sciatic hernia sacs [ 3 , 4 , 5 , 6 ] . in ureterosciatic herniation , prolapse of the ureter occurs through the sciatic notch , which is divided by the sacrospinous ligament into the greater and lesser sciatic foramina . the greater sciatic foramen is considered a potential space , as the piriformis muscle completely occupies the greater sciatic foramen . the greater sciatic foramen is bound by the ilium laterally , the sacrum and sacrotuberous ligament medially , and the sacrospinous ligament inferiorly . the greater sciatic foramen is further subdivided into the suprapiriformis and infrapiriformis compartments by the piriformis muscle . ureterosciatic herniations usually occur through the suprapiriformis compartment of the greater sciatic foramen [ 5 , 10 ] . previous reports have suggested that atrophy of the piriformis muscle is the predisposing factor for the development of ureterosciatic herniation , creating a potential space through which the ureter could migrate . it has been speculated that the main predisposing factor could be piriformis muscle atrophy , related to hip joint disease , neuromuscular disorders or other locomotor disturbances of the lower extremities [ 4 , 5 , 6 , 7 , 9 ] . although most hernias occur through the greater sciatic foramen , several ones have been reported to herniate through the lesser sciatic foramen [ 2 , 5 , 8 , 9 ] . these cases are usually undiagnosed clinically because the gluteus maximus muscle overlies the sciatic foramen . sciatic ureter has a characteristic radiologic appearance in which a loop of ureter is displaced laterally , inferiorly , and posteriorly through the sciatic notch on ivu and retrograde urography . the diagnosis was confirmed by this image , known as the curlicue sign , which is considered pathognomonic for ureterosciatic herniation . ct may be helpful in diagnosis with the appearance of an ectopically positioned ureter posterior and lateral to the ischial spine , it can demonstrate an ureter with more anatomic information , and it may be useful in detecting the involvement of other structures [ 6 , 12 ] . moreover , ct with three - dimensional reconstruction might be useful for defining the exact location and extent of the herniation . in previous reports , treatments for symptomatic patients included relief of the hernia by excision and reimplantation , ureteroureterostomy or stent placement and observation [ 2 , 8 ] . in our patient , conservative endourological correction was performed . after the spontaneous removal of the ureteral stent , recurrent ureterosciatic hernia occurred . placement of an internal stent possibly provides the rigidity to the ureter , thereby recovering the hernia and urinary obstruction . observation should be used judiciously because the consequences of long - term obstruction can be devastating . indeed , ureterosciatic hernia causing intermittent ureteral obstruction and producing significant morbidity should be repaired . furthermore , when the ureter shows signs of inflammation or trophic disturbances , ureterolysis , reimplantation , and hernioplasty should be carried out [ 1 , 2 , 3 , 4 , 8 , 10 ] . surgical options include excision of the hernia with reimplantation of the remaining ureter , reduction of the ureter length , and transabdominal or transgluteal surgical reduction of the hernia plus fixation of the ureter [ 2 , 4 , 8 ] . recently , laparoscopic and robotic repair surgery has also been reported [ 10 , 15 ] . however , surgical repair should not be performed in asymptomatic patients . for the patient who is elderly or a poor surgical candidate sugimoto et al . also reported a case successfully treated by ureteral stent placement . it is advisable to place a stent for few months and then remove it to see whether the obstruction recurs . this approach provides a minimally invasive means for the treatment of what can be a devastating problem .

ureterosciatic herniation , the protrusion of the hernia sac through the sciatic foramen , is an extremely rare cause of ureteral obstruction . we describe a case revealed by severe left back pain in a 72-year - old female . she was referred to our hospital for urological assessment of left hydronephrosis observed by ultrasonography . intravenous ureterography ( ivu ) showed findings compatible with a left sciatic ureter , a dilated ureter with a fixed kinking , which is known as the curlicue sign . we decided to attempt recovery of the herniated ureter using a retrograde approach . ureteral stent placement was performed to decompress the dilated upper urinary tract . the ureterosciatic hernia was relieved with the passage of a flexible guide wire and a double - pigtail stent . three months after ureteral stenting , she refused continuing to have an indwelling stent and the stent was removed . thereafter , ivu revealed recurrent ureterosciatic hernia ; however , there was no hydroureter or hydronephrosis . the patient is currently being under observation for 6 years after stenting and continues to be without hydronephrosis or symptoms . placement of an internal stent possibly provides the rigidity to the ureter , thereby reducing the hernia and urinary obstruction . in the previous reports , most symptomatic patients have been treated surgically , with conservative therapy reserved for asymptomatic patients . for the patient who is elderly or a poor surgical candidate , retrograde stenting may provide safe reduction and efficacious treatment . this endourological approach provides a minimally invasive means for the management of urinary obstruction caused by ureterosciatic herniation .

Introduction Case Presentation Discussion

the randomized , controlled clinical trial is an attempt to minimize bias introduced by the knowledge of treatment allocation and , thereby , come to reliable conclusions regarding the hypothesis tested . many interventional , randomized , controlled clinical trials need to assume that randomization distributes baseline characteristics equally , and that patients enter the clinical trial in a stable state . however , inclusion and exclusion criteria can lead to biases which are not necessarily anticipated . when the intervention examined is already used by any portion of the population , while this may be drug - specific , the withdrawal of previously prescribed medication is often based on the pharmacologic class or mechanism of action of the study drug . most trials assume that patients are medically stable at randomization , but withdrawal of medication may worsen the underlying disease . three groups of investigators13 have highlighted issues introduced by withdrawal of inhaled corticosteroids at randomization into clinical trials examining inhaled corticosteroids in chronic obstructive pulmonary disease ( copd ) . suissa et al1 concluded that the effects of withdrawal of medication can not be assumed to be the same as addition of the medication . while the authors demonstrated this to be the case with inhaled corticosteroids , they considered whether withdrawal of other copd medication , including inhaled anticholinergics , may lead to a similar phenomenon . our access to the tiotropium clinical trial database allowed us to undertake a thorough analysis of whether withdrawal of inhaled anticholinergics influenced the results observed with tiotropium on reducing copd exacerbations . to our knowledge , this is the first analysis that has examined the issue of medication withdrawal at the onset of clinical trials of anticholinergic therapy . a pooled analysis of 10 randomized , double - blind , placebo - controlled , parallel - group studies of at least six months duration with tiotropium 18 g administered once daily via the handihaler device ( boehringer ingelheim gmbh , ingelheim , germany ) was performed ( trial numbers 205.114/117,4 205.115/128,5 205.130,6 205.137,6 205.266,7 205.270,8 205.235 [ understanding potential long - term impacts on function with tiotropium ( uplift)],9 205.214,10 205.256,11 205.25912 ) , as shown in figure 1 . one study was longer than one year ( uplift , which included 5993 copd patients followed over four years).9 all trials included evaluation of exacerbations and spirometry . seven trials ( trial numbers 205.114/117 , 205.115/128 , 205.130 , 205.137 , 205.235 [ uplift ] , 205.256 , and 205.259 ) included measurement using the st george s respiratory questionnaire ( sgrq).13 all trial protocols were approved by independent ethics committees , and patients in all trials provided written informed consent . all trials included in this pooled analysis had common entry criteria , ie , a clinical diagnosis of copd , age > 40 years , smoking history 10 pack - years , postbronchodilator forced expiratory volume in one second ( fev1)/forced vital capacity ratio < 0.7 , and fev1 either 65% or 70% of predicted . whilst the patients participating in these studies were classified as to the severity of their copd on the basis of post - bronchodilator fev1 measurements , exclusion criteria included a history of asthma , the need for continuous supplemental oxygen , a copd exacerbation within the previous six weeks , recent myocardial infarction or hospitalization for congestive heart failure , other unstable medical conditions that may preclude participation or interpretation of the results , and use of systemic corticosteroids in doses greater than the equivalent of prednisone 10 mg daily . we believe that this is the most dependable method for excluding patients with asthma , given that it is now well known that bronchodilator responsiveness does not discriminate between asthma and copd . the following standardized definition was used to characterize copd exacerbations in the current analysis : an exacerbation is defined by two or more ( increased or new - onset ) respiratory symptoms such as cough , sputum , wheezing , dyspnea , or chest tightness , lasting at least three days , and requiring treatment with antibiotics and/or steroids , and/or hospitalization.14 patients were grouped according to inhaled anticholinergic discontinuation , ie , d ( anticholinergic prescribed prior to participation and discontinued at randomization ) and nd ( anticholinergic not prescribed prior to participation and therefore not discontinued ) . kaplan meier curves of the probability of no exacerbation and no hospitalization due to exacerbations were displayed . cox regression was used to compute hazard ratios ( hr ) of tiotropium to placebo using trial as stratum . the analysis of number of exacerbations and number of hospitalizations due to exacerbations was performed using poisson regression , with correction for treatment exposure and overdispersion with terms for treatment subgroup and treatment by subgroup interaction . the effects of tiotropium or placebo on the sgrq total score in trials where the sgrq was measured was examined according to whether or not subjects discontinued anticholinergic therapy . these data are displayed as mean ( standard error of the mean ) values and have been compared with unpaired t - tests . a pooled analysis of 10 randomized , double - blind , placebo - controlled , parallel - group studies of at least six months duration with tiotropium 18 g administered once daily via the handihaler device ( boehringer ingelheim gmbh , ingelheim , germany ) was performed ( trial numbers 205.114/117,4 205.115/128,5 205.130,6 205.137,6 205.266,7 205.270,8 205.235 [ understanding potential long - term impacts on function with tiotropium ( uplift)],9 205.214,10 205.256,11 205.25912 ) , as shown in figure 1 . one study was longer than one year ( uplift , which included 5993 copd patients followed over four years).9 all trials included evaluation of exacerbations and spirometry . seven trials ( trial numbers 205.114/117 , 205.115/128 , 205.130 , 205.137 , 205.235 [ uplift ] , 205.256 , and 205.259 ) included measurement using the st george s respiratory questionnaire ( sgrq).13 all trial protocols were approved by independent ethics committees , and patients in all trials provided written informed consent . all trials included in this pooled analysis had common entry criteria , ie , a clinical diagnosis of copd , age > 40 years , smoking history 10 pack - years , postbronchodilator forced expiratory volume in one second ( fev1)/forced vital capacity ratio < 0.7 , and fev1 either 65% or 70% of predicted . whilst the patients participating in these studies were classified as to the severity of their copd on the basis of post - bronchodilator fev1 measurements , the data reported in this paper were prebronchodilator values . exclusion criteria included a history of asthma , the need for continuous supplemental oxygen , a copd exacerbation within the previous six weeks , recent myocardial infarction or hospitalization for congestive heart failure , other unstable medical conditions that may preclude participation or interpretation of the results , and use of systemic corticosteroids in doses greater than the equivalent of prednisone 10 mg daily . we believe that this is the most dependable method for excluding patients with asthma , given that it is now well known that bronchodilator responsiveness does not discriminate between asthma and copd . the following standardized definition was used to characterize copd exacerbations in the current analysis : an exacerbation is defined by two or more ( increased or new - onset ) respiratory symptoms such as cough , sputum , wheezing , dyspnea , or chest tightness , lasting at least three days , and requiring treatment with antibiotics and/or steroids , and/or hospitalization.14 patients were grouped according to inhaled anticholinergic discontinuation , ie , d ( anticholinergic prescribed prior to participation and discontinued at randomization ) and nd ( anticholinergic not prescribed prior to participation and therefore not discontinued ) . kaplan meier curves of the probability of no exacerbation and no hospitalization due to exacerbations were displayed . cox regression was used to compute hazard ratios ( hr ) of tiotropium to placebo using trial as stratum . the analysis of number of exacerbations and number of hospitalizations due to exacerbations was performed using poisson regression , with correction for treatment exposure and overdispersion with terms for treatment subgroup and treatment by subgroup interaction . the effects of tiotropium or placebo on the sgrq total score in trials where the sgrq was measured was examined according to whether or not subjects discontinued anticholinergic therapy . these data are displayed as mean ( standard error of the mean ) values and have been compared with unpaired t - tests . of the 12,163 patients who were randomized into these clinical trials , 5846 were receiving inhaled anticholinergics that were discontinued at randomization ( d group ) and 6317 were not receiving inhaled anticholinergics at the time of randomization ( nd group ) . the inhaled anticholinergic was ipratropium bromide in the vast majority of patients who were already receiving inhaled anticholinergics . at time of initiation of all of the aforementioned trials , there were a few patients who did receive tiotropium prior to randomization ( 109 of 12,164 patients ) , with 49 being withdrawn from tiotropium . for group d , the mean age was 66.1 years compared with 64.5 years for the nd group . patients who had previously been prescribed anticholinergics had lower lung function and longer smoking histories . overall , within the d and nd groups , the treatment subgroups ( tiotropium and placebo ) were reasonably well balanced with respect to their baseline characteristics . there was a significantly reduced risk of an exacerbation in patients receiving tiotropium compared with those receiving placebo in the d group ( hr [ 95% confidence interval ( ci ) ] = 0.83 [ 0.770.90 ] ) and in the nd groups ( hr [ 95% ci ] = 0.79 [ 0.730.85 ] ) . similar findings were observed for exacerbations leading to hospitalizations with an hr ( 95% ci ) of 0.85 ( 0.731.00 ) in the d group and 0.79 ( 0.650.94 ) in the nd group . tiotropium delayed the time to first exacerbation and first hospitalized exacerbation in both the d and nd groups , as observed in the cumulative incidence displays ( figures 2 and 3 ) . the number of patients having at least one exacerbation and at least one exacerbation leading to hospitalization was reduced with tiotropium relative to placebo in both d and nd groups , as reflected by lower rate ratios ( tables 2 and 3 ) . for the d group , the mean ( standard error ) total sgrq score at six months was improved with tiotropium ( 41.4 [ 0.31 ] ) relative to those receiving placebo ( 44.7 [ 0.34 ] , difference = 3.33 [ 0.40 ] , p < 0.0001 ) . for the nd group , the total scores were also improved with tiotropium versus placebo ( 40.3 compared with 43.1 , difference = 2.78 , p < 0.0001 ) . of the 12,163 patients who were randomized into these clinical trials , 5846 were receiving inhaled anticholinergics that were discontinued at randomization ( d group ) and 6317 were not receiving inhaled anticholinergics at the time of randomization ( nd group ) . the inhaled anticholinergic was ipratropium bromide in the vast majority of patients who were already receiving inhaled anticholinergics . at time of initiation of all of the aforementioned trials , there were a few patients who did receive tiotropium prior to randomization ( 109 of 12,164 patients ) , with 49 being withdrawn from tiotropium . for group d , the mean age was 66.1 years compared with 64.5 years for the nd group . patients who had previously been prescribed anticholinergics had lower lung function and longer smoking histories . overall , within the d and nd groups , the treatment subgroups ( tiotropium and placebo ) were reasonably well balanced with respect to their baseline characteristics . there was a significantly reduced risk of an exacerbation in patients receiving tiotropium compared with those receiving placebo in the d group ( hr [ 95% confidence interval ( ci ) ] = 0.83 [ 0.770.90 ] ) and in the nd groups ( hr [ 95% ci ] = 0.79 [ 0.730.85 ] ) . similar findings were observed for exacerbations leading to hospitalizations with an hr ( 95% ci ) of 0.85 ( 0.731.00 ) in the d group and 0.79 ( 0.650.94 ) in the nd group . tiotropium delayed the time to first exacerbation and first hospitalized exacerbation in both the d and nd groups , as observed in the cumulative incidence displays ( figures 2 and 3 ) . the number of patients having at least one exacerbation and at least one exacerbation leading to hospitalization was reduced with tiotropium relative to placebo in both d and nd groups , as reflected by lower rate ratios ( tables 2 and 3 ) . for the d group , the mean ( standard error ) total sgrq score at six months was improved with tiotropium ( 41.4 [ 0.31 ] ) relative to those receiving placebo ( 44.7 [ 0.34 ] , difference = 3.33 [ 0.40 ] , p < 0.0001 ) . for the nd group , the total scores were also improved with tiotropium versus placebo ( 40.3 compared with 43.1 , difference = 2.78 , p < 0.0001 ) . the pooled analysis of 10 randomized , double - blind , placebo - controlled clinical trials involving 12,163 patients with copd demonstrates that tiotropium was effective in reducing exacerbations in patients who did and also those who did not have inhaled anticholinergics discontinued upon randomization . in patients who discontinued anticholinergic therapy , there was a 17% risk reduction for an exacerbation when treated with tiotropium compared with placebo . in patients who did not discontinue anticholinergic therapy , the risk reduction was 21% with tiotropium compared with placebo . in a similar comparison , the risk for a hospitalized exacerbation was reduced by 15% and 21% , respectively . the reduced risk was also associated with reduced rates of exacerbations and hospitalized exacerbations , regardless of previous anticholinergic use . thus , withdrawal of inhaled anticholinergics upon entry into tiotropium trials did not appear to influence the effect of tiotropium on the clinically important patient outcome of exacerbations of copd . in addition , in the seven trials where the sgrq was measured , tiotropium significantly reduced the sgrq total score in both groups of patients . exacerbations are a significant component of the clinical course in copd.15,16 furthermore , as copd progresses , exacerbations become more frequent.16 some investigators have suggested that more frequent exacerbations are associated with more rapid decline of fev1.17,18 certainly , exacerbations have profound effects on quality of life.19 for example , sgrq scores have been shown to be decreased ( worsened ) below baseline for up to six months following an exacerbation.20 finally , exacerbations are associated with significant mortality.21 more widespread clinical use of tiotropium in copd patients might predictably ameliorate the effects of recurrent exacerbations on the clinical course of the disease and impact the design of future clinical trials . recently , there have been increasing concerns about the effects of withdrawal of medications on the entry into clinical trials . van der valk et al3 treated patients with inhaled fluticasone 1000 g / day for four months and then randomized the patients to continue receiving fluticasone or to switch to placebo . during the subsequent six months of follow - up , 47% of patients in the fluticasone group developed a copd exacerbation compared with 57% in the placebo group . significant differences were seen in the hr for first exacerbation , as well as differences in health - related quality of life as measured by the sgrq , which favored those patients continuing on fluticasone . however , the question arises as to whether this is a safety concern due to sudden withdrawal of a high dose of systemically absorbed inhaled corticosteroids or if the observations are surrogate measures of efficacy . suissa et al1 described methodological issues in therapeutic trials of copd , and drew attention to the problem of medication withdrawal using inhaled corticosteroids prior to randomization as an example . in the case of the towards a revolution in copd health ( torch ) study , this phenomenon led in effect to two comparisons , ie , withdrawal of inhaled corticosteroids compared with continuation of inhaled corticosteroids in patients previously taking these agents , and introduction of inhaled corticosteroids compared with placebo in patients not previously taking inhaled corticosteroids . suissa et al1 also analyzed data from the canadian optimal therapy of copd trial22 and found that the hr ( inhaled corticosteroids relative to bronchodilators only ) for the first exacerbation among previous inhaled corticosteroids users was 0.71 ( 95% ci 0.530.96 ) , while among those not using inhaled corticosteroids prior to randomization , the hr ( inhaled corticosteroids relative to bronchodilators only ) was 1.11 ( 95% ci 0.691.79 ) . in addition , the ratio for exacerbations in patients prior to discontinuing inhaled corticosteroids was 0.78 ( 95% ci 0.610.99 ) compared with 1.23 ( 95% ci 0.781.95 ) thereafter . these concerns prompted a detailed retrospective analysis of data from torch.23 in this paper , keene et al argued that a negative binomial model was the best approach to statistical analysis of exacerbation rates and that a similar reduction in exacerbations could be calculated regardless of whether or not subjects had discontinued inhaled corticosteroids at randomization . suissa et al1 suggested in their paper that withdrawal of inhaled anticholinergic therapy might cause similar problems to those they described related to withdrawal of inhaled corticosteroids . furthermore , we have demonstrated a similar reduction in the number of exacerbations per patient year in those who continued and those who discontinued inhaled anticholinergic therapy upon randomization into these tiotropium clinical trials . this significant benefit argues that the clinical trial results , in terms of the difference between tiotropium and placebo , favor a positive effect from tiotropium rather than a negative effect from inhaled anticholinergic withdrawal in the placebo group . as well as demonstrating that tiotropium reduced exacerbations regardless of changes in prior therapy , we were also able to demonstrate that tiotropium , when compared with placebo , improved health - related quality of life ( as measured by sgrq ) , regardless of whether or not patients had been taking inhaled anticholinergic therapy prior to randomization . the improvement in sgrq was statistically significant for both the d and nd groups , although numerically greater for those patients who discontinued previous anticholinergic therapy . we can speculate why withdrawal of inhaled anticholinergic therapy does not affect the outcome of placebo - controlled clinical trials of tiotropium , whereas withdrawal of inhaled corticosteroids seems to be problematic . the effect of ipratropium , which is the short - acting anticholinergic taken by many patients prior to entry into the tiotropium trials , is well understood to be restricted to antimuscarinic smooth muscle relaxation and presumably wears off relatively quickly , although there is some evidence that the effects of extended therapy with ipratropium persist longer than the conventional 68-hour duration of acute bronchodilation.24 although considered a short - acting bronchodilator , ipratropium alone or in combination with albuterol has been shown to reduce exacerbations compared with albuterol alone.25 this observation suggests that one should consider whether a rebound effect ( ie , an increase in exacerbation rate ) might be a consequence of ipratropium withdrawal . in a study of nine subjects with mild asthma , newcomb et al26 described a short - term increase in airway hyperresponsiveness to methacholine upon withdrawal of regular use of ipratropium , which could represent upregulation of muscarinic receptors on the surface of airway smooth muscle cells . however , these findings have not been replicated , and the current analysis demonstrates that a rebound worsening of copd is unlikely to occur upon withdrawal of ipratropium . the corollary is not necessarily true for inhaled corticosteroids , because there are systemic effects of inhaled corticosteroids and both beneficial effects and adverse events are likely to occur over longer periods of time , which may also delay the onset of safety issues . as with any meta - analysis , our study has certain limitations.27 we acknowledge that this is a retrospective analysis and includes trials of different size and duration . however , the strengths of the study are the prospective nature of the clinical trials selected for the pooled analysis and the consistency of their entry criteria . furthermore , these studies were rigorously conducted , randomized , double - blind , controlled clinical trials that provided large numbers of patients , all of whom were included in the analysis . in summary , we have demonstrated that tiotropium reduced exacerbations and hospitalizations in a large number of copd patients , regardless of whether or not they discontinued inhaled anticholinergic therapy prior to randomization into the placebo - controlled clinical trials . these findings strengthen the validity of conclusions previously reported from these clinical trials that treatment with tiotropium reduces exacerbations , and that the beneficial effect is equally observed whether or not patients had previously received inhaled anticholinergic therapy .

background : data have highlighted the potential bias introduced by withdrawal of inhaled corticosteroids at randomization in chronic obstructive pulmonary disease trials examining inhaled corticosteroids . analyses were conducted to determine whether this was true of inhaled anticholinergic withdrawal in tiotropium trials.methods:a pooled analysis of randomized , double - blind , placebo - controlled , parallel - group tiotropium trials of at least six months duration was performed . trials had similar inclusion and exclusion criteria . exacerbation definition was standardized . patients were divided into two groups , ie , d ( anticholinergics discontinued at randomization , previously prescribed ) and nd ( anticholinergics not discontinued , not previously prescribed).results : demographics were balanced between the d ( n = 5846 ) and nd ( n = 6317 ) groups , except for higher cumulative smoking ( 56 pack - years versus 48 pack - years ) , lower forced expiratory volume in one second ( fev1)/forced vital capacity ( 43% versus 48% ) , and lower baseline fev1 ( 35.8% predicted versus 42.4% predicted ) in the d group . in both groups , tiotropium reduced the risk for an exacerbation ( hazard ratio [ hr ] = 0.83 , p < 0.0001 [ d ] versus 0.79 , p < 0.0001 [ nd ] ) and a hospitalized exacerbation ( hr = 0.85 , p = 0.0467 versus 0.79 , p = 0.0094 ) . tiotropium reduced the number of exacerbations per patient - year ( rate ratio [ rr ] = 0.82 , p < 0.0001 [ d ] versus rr = 0.80 , p < 0.0001 [ nd ] ) and associated hospitalizations per patient - year ( rr = 0.88 , p = 0.015 [ d ] versus rr = 0.74 , p < 0.0001 [ nd]).conclusion : tiotropium reduced exacerbations in patients who did and did not have anticholinergics discontinued upon randomization in clinical trials .

Introduction Methods Study design Study population COPD exacerbations Data analysis Results Demographics Exacerbations St. Georges respiratory questionnaire Discussion Conclusion

telepathology is defined as the practice of pathology at a distance , transmitting macroscopic and/or microscopic images via telecommunication links for ( 1 ) remote interpretations ( telediagnosis ) , second opinions or consultations ( teleconsultation ) , and educational purposes ( teleconferencing ) . with the widespread availability of imaging technology and telecommunication , access to global expert pathologists telepathology has been shown to be applicable for : ( i ) anatomical pathology including intra - operative consultation ( frozen sections ) , surgical pathology ( second opinions , immunostains ) , telecytology ( e.g. , on - site evaluation ) , and ultrastructural pathology , as well as ( ii ) clinical pathology including telehematology , microbiology ( e.g. , parasitology ) , and chemistry ( e.g. , interpretation of gels ) . the first is static ( store - and - forward ) telepathology that involves the examination of pre - captured still images transmitted via e - mail or stored on a shared server . the second mode of telepathology involves dynamic ( live ) examination of images in real - time , employing video or robotic microscopy . finally , hybrid technology involving whole slide imaging ( wsi ) has emerged that utilizes both dynamic viewing of a digitized ( scanned ) slide as well as viewing of selected areas of the saved image at higher magnification . the university of pittsburgh medical center ( upmc ) health system operates 20 geographically diverse hospitals within and around the city of pittsburgh , and also partners with hospitals located in distant states ( e.g. , indianapolis , indiana ) and other countries ( e.g. , italy , china ) . the anatomical pathology department employs an academic centers of excellence ( coe ) subspecialty model , where pathologists in their respective specialty tend to all be located in the same hospital . with this infrastructure , telepathology has been employed at upmc for over a decade to provide remote subspecialty expertise at local , national , and international sites . the aim of this article is to review the different modes of telepathology and share the experience garnered at upmc with respect to each teleconsultation method as well as describe future aspects of practicing telepathology . static image telepathology involves capturing , storing , and forwarding individual digital images , or galleries of static images , for remote diagnosis . advantages of this form of telepathology are the low cost involved , vendor independence , technical simplicity , the fact that the recipient does not require special software to view images , small manageable files are involved ( easy to retrieve , send , review , store , and share ) , and that these systems are easy to maintain . however , disadvantages of static telepathology relate to the fact that the consulting telepathologist has no remote control of the microscope or imaging device / camera , their interpretation is limited to only captured field of views , the host capturing images needs to have some expertise , acquiring images is labor intensive and may cause sampling error if the incorrect images are captured , there is frequent lack of clarity with low power magnification images , and not all still images are in focus . at upmc , , consultations were received from ismett ( mediterranean institute for transplantation and high specialization therapies ) located in palermo , italy . transplant - related biopsies are usually challenging and hence best interpreted by experts in the field . access to transplant pathologists is highly desirable for second opinions when dealing with difficult cases . teleconsultation in this setting also needs to be performed in a timely manner , since rapid and accurate interpretation of allograft biopsies influences outcome after organ transplantation . moreover , histopathologic interpretation determines whether a donor organ should be used for transplantation or disposed . the transplant telepathology system was , therefore , developed to support coverage 24 hours a day , 7 days a week . initially , this employed static images run in a store - and - forward mode . communication was limited to a private network using a thick client - server architecture between the host ( e.g. , italy ) and upmc consulting pathologists . analysis of early ( 14-month period ) accrued data for 102 transmitted cases showed full agreement with the original diagnosis in 86% of cases . for cases with disagreement ( 14% ) , 8 resulted in minor and 3 clinically significant differences in opinion . subsequently , during the 12-year partnership between upmc and ismett , approximately 3000 cases have been reviewed by telepathology . teleconsultation using static images improved with respect to workflow with only infrequent discrepancies being noted . this first generation home - grown static telepathology system has since been replaced by second generation dynamic robotic microscopy ( nikon coolscope streaming ) , third generation hybrid rapid virtual microscopy ( trestle live viewing ) , and most recently , in 2009 , with fourth generation wsi ultra - rapid virtual microscopy ( mirax midi ) [ figure 1 ] . with better technology allowing pathologists to view entire slides at high resolution , the performance of telepathology has improved . however , we did not change to these newer technologies until these approaches were proven to be technically feasible . although static images were quicker to read than robotic microscopy , being limited to specific field of views forced evolution . the evolution from static imaging to a wsi scanning system was necessitated due to the lag time required for robotic objective magnification changes and positional adjustments of the slide . with wsi scanning , these magnifications are digitally incorporated into the resultant image , as such the time required for biopsy interpretation was greatly reduced . secondly , configuring the wsi system with a high numerical aperture ( na ) objective ( e.g. , 40x , 95na ) enabled an image resolution that maximized image clarity ( detail ) , therefore , reducing eye fatigue of the reviewing pathologist , enabling longer review sessions . lower resolution imagery ( e.g. , 25 micron ) forces the human eye to continually focus trying to pull out acute pathology details , which leads to fatigue . lastly , high resolution scanning enabled subtle details pertaining to tissue rejection to be clearly identified , such as visual confirmation of splitting of the glomerular basement membranes . combined , these advancements improved the performance of the system such that routine consultation was practical and efficient . early and continued adoption of telepathology has provided invaluable experience with digital pathology , improved workflow , and accumulated resources ( facilitated funding for equipment , it infrastructure , and staffing ) within the transplant pathology division . today , digital pathology has evolved whereby multiplex - stained wsi is being used for microscopy and image analytics . multiplexing is defined as the application and analysis of multiple fluorescence markers ( typically greater than 3 ) to a single histologically prepared section . these markers can then be analyzed in the tissue context , quantifying spatial relationships between multiple markers as related to surrounding morphology expression patterns . diagram showing the information technology and server components for the upmc / ismett telepathology platform . cases are accessioned in the mirax digital slide desktop ( mdsd ) image repository ( blue box ) , individual slides are then scanned on the mirax midi wsi scanner and transferred via network file share to the mdsd repository . access to the images is direct via secure ( username / password ) connection to the mdsd system from client pc / mac using a java applet for viewing , or via affiliated / specific workflow applications for the purpose of transplant immunology assessment . the java applet viewer can be embedded in any website or client / server application for flexibility of workflow integration the division of hematopathology at our institution is another area that employed static telepathology . in several of the academic hospitals , without a hematopathologist on site , telehematology has ensured that interpretation of peripheral blood smears and differentials continue to be performed rapidly and accurately . expert hematopathologist interpretation is often required for difficult cases ( e.g. , blasts in leukemia ) . to accomplish this , the hematology laboratory relied on the cellavision automated digital system to digitize glass slides containing peripheral blood smears . the instrument was used to automatically locate and pre - classify digital images of white blood cells , red blood cells , and platelets . using this system , a technologist in the laboratory was able to email static images of concerning blood cells to a hematopathologist for interpretation [ figure 2 ] , while , in turn , the hematopathologist could access the system and the entire stored differential of a patient remotely from their office computer . analysis of our experience has shown that this device correctly classified up to 94% of cells , with some reduced accuracy for immature granulocytes . . this will also allow clinicians to view digitized blood smears in real time , instead of having to come to the laboratory to physically review the blood smear . ( a ) cellavision dm96 instrument ; ( b ) review monitor displaying captured images of different blood cells ; ( c ) e - mail with embedded selected static images generated using the systems remote review software the host driving ( navigating and focusing ) the slide on the microscope , to be viewed by a remote pathologist on a monitor , requires some expertise to perform this task . the remote consulting pathologist is usually in communication ( e.g. , via telephone or teleconferencing ) with the host . however , in order to transmit the image over the internet , the analog video signal needs to be converted ( using an ad converter ) to a digital signal . analog video signals unfortunately are subject to noise and degradation , which may affect the image quality . while many prior publications regarding telecytology have predominantly utilized static images , in practice , today , most cytology laboratories that use telecytology employ video streaming . at upmc , live video streaming has remained the mode of choice for providing immediate adequacy assessment of cytology specimens by telecytology . other methods , such as robotic microscopy , have nevertheless been investigated and shown to be effective . validation studies conducted in our cytopathology division using web - based streaming have shown that adequacy assessment and diagnostic accuracy with telecytology are acceptable . nevertheless , cytopathologists found that the quality of these digital images were inferior to viewing these cases on a conventional light microscope [ figure 3 ] . moreover , cytopathologists documented spending more time reviewing cases examined by telecytology [ figure 4 ] . these factors may explain , in part , why our cytopathologists only infrequently employ telecytology , despite the fact the necessary equipment to perform telepathology for immediate on - site evaluation of fine needle aspirations is available . for remote evaluations of fine needle aspirations , cytopathologists demonstrated similar performance using a conventional microscope ( blue bars ) and the telecytology system ( yellow bars ) for providing tissue adequacy ( far left bars ) and the correct diagnosis ( far right bars ) . however , the middle bars in this graph indicate that pathologists found reviewing cases remotely to appear more complex , with inferior image quality and more obscured features . consequently , they were less confident with their telecytology diagnoses average time ( seconds ) taken to review glass and telecytology cases . cytopathologists took longer to review a cytology slide using telecytology ( 94 seconds ) than they did when examining the same slide with a conventional light microscope ( 70 seconds ) robotic ( real - time , dynamic ) telepathology differs from video microscopy because the telepathologist ( consultant ) is now actively involved in selecting fields on the glass slide to be viewed . with a robotic telepathology system , the operator has remote control of critical motorized microscope functions , including glass slide movements in the x- and y - axes on the motorized microscope stage , focus of the glass slide , and selection of various microscope objective lenses by a robotic turret . this provides the pathologist with access to the entire slide with good image quality . a trained host ( e.g. , pathology assistant ) these devices are expensive and because of their point - to - point network connectivity requirements ( i.e. not a web - based protocol ) are often not desirable to implement within a hospital it infrastructure . additional disadvantages are the relatively slow scanning of slides ( approximately 10 min / slide ) , the fact that both the host and recipient require integrated software , and a required high level of system maintenance for routine optimization , cleaning , and adjustment . robotic telepathology has been extensively used at upmc to remotely interpret intra - operative neuropathology consultations ( frozen sections ) . a considerable volume of neurosurgery is performed within our organization . however , in keeping with our coe model , neuropathologists are all housed at one hospital location . the steady demand for neuropathology frozen sections in different hospitals has been met by utilizing robotic telepathology [ figure 5 ] . several zeiss ( previously trestle ) robotic microscopes have been strategically placed within frozen section rooms at the different facilities [ figure 6 ] . pathology staff , including trainees , have received instruction on how to handle tissue received for frozen section and load prepared slides onto these microscopes . the importance of such training is a key element to the success of our teleneuropathology service and should not be overlooked . since only small portions of brain / neural tissue are usually procured and entirely submitted for frozen section , in our experience , we found that a brief gross description of the tissues received to the remote neuropathologist over the telephone by a pathology resident or assistant was sufficient . at one site , a surgical pathologist does the sampling with guidance from a consulting neuropathologist . nevertheless , a secure webcam in the frozen section room is available to facilitate gross telepathology if required . also , investigations of newer technologies ( e.g. , high definition digital cameras and teleconferencing software ) are underway in an attempt to improve our macroscopic teleneuropathology capabilities [ figure 7 ] . software ( medmicroscopy and mirax navigator ) to access scanned images and remotely control robotic microscopes has been made readily available on the internet accessible from any workstation . the diagnostic outcome from 2002 to 2006 ( comparing 1227 conventional frozen sections to 402 performed by telepathology ) demonstrated that the telepathology deferral rate ( 19.7% ) varied , and was higher than the deferral rate for conventional frozen section cases ( 10% ) . however , with the adoption of newer technology ( trestle scanner replaced the nikon coolscope ) and increased pathologist experience , in 2007 to 2008 ( comparing 547 conventional frozen sections to 262 by telepathology ) , there was acceptable concordance ( 78.2% ) between telepathology and conventional frozen sections . in addition , discrepancy and deferral rate differences during this later time period were statistically insignificant . intra - operative final discrepancies were uncommon , occurring in only 2.7% of these neuropathology cases . discrepant events were more common in non - neoplastic ( reactive ) cases , lymphomas , and with rare tumors . on account of our neuropathology division 's unique intra - institutional experience , they were able to successfully expand their teleconsultation practice across state lines ( inter - institutional ) with a financially separate medical center in indianapolis , indiana . implementation of technology during this venture proved to be easier than the accompanying administrative ( e.g. , contracts ) and legal ( e.g. , licensing ) issues involved . 2007 ) information technology infrastructure used to support intra - institutional neuropathology frozen section telepathology gross teleneuropathology showing close - up views of two portions of tissue submitted for frozen section from a glioblastoma multiforme tumor wsi telepathology systems are being increasingly employed for clinical , education , and research applications . wsi systems provide users with access to the entire case ( including sets of slides ) , offers automated scanning , and rapidly produces high resolution images . wsi scanners often have added software available for teleconferencing , image management , and image analysis . however , current impediments to their widespread adoption are related to their high expense and limited vendor interoperability . newer wsi scanners designed to hold one to four slides are smaller and less expensive . with wsi , there is a minor scan failure rate ( 2 - 5% ) . our scan failure rate is particularly low ( below 1% ) , probably because we train our staff to recognize problems before presenting images to pathologists . furthermore , pathologists maintain a close working relationship with histology to optimize slides for scanning ( e.g. , care taken to avoid folds , bubbles , etc . ) . our it staff also adjusts scanning technique with the vendor to optimize scanner device settings . such optimizations included vendor provided focus point algorithm changes to account for tissue artifact ( edge effects , folds ) . nevertheless , occasional difficulties can be experienced when scanning slides ( e.g. , misplaced cover slips and sticky wet slides ) . small tissue fragments , faint tissue or material at the slide edge or even outside the coverslip may not be recognized . users may also have to contend with long scan times , especially for thick tissue sections and if high - resolution images are desired ( e.g. , native high numerical aperture 40 scanning ) . this web - based tool was designed to facilitate digital pathology second opinion consults , especially for wsi . a general portal was developed to be used by all clients , and another that required customization for individual clients [ table 1 ] . with these portals , wsi can either be uploaded for transmission to upmc consultants ( general portal ) or accessed on a specific client 's server ( client - specific portal ) [ figure 8 ] . with the client - specific portal , we are able to avoid lengthy transfers of large wsi files over the internet as well as automate the process for image association to cases . despite distance , however , with this client - specific model image files are largely outside our control , we can not always quickly resolve problems with the client 's image server , and any network / hardware changes on the client 's side may cause unexpected instability . the general portal allows supported image file types ( static and wsi formats of major vendors ) , as well as pertinent accompanying clinical information , to be uploaded . all clients are offered secure login to submit their patient 's data and upload images , as well as to check on the status of their case and view or print second opinion reports . using this tool , upmc consultants are able to view digital images using a java applet . they have the ability to annotate and capture static image snapshots to be embedded into their reports . workflow is handled by managers who triage requests , monitor cases , and maintain personnel data . streamlined workflow , as well as training of users , has ensured prompt turn - around time and buy - in by institute pathologists . post - launch feedback from pathologists has resulted in customization to incorporate transcription services , peer - to - peer review for consultants , provision for issuing addenda and amendments , and improved wsi viewing experience . all cases received via the portal thus far have been surgical pathology cases . on average , 11 wsi per case have been received including h and e , histochemical and immunohistochemistry slides . the mean turnaround time for 22 consulting pathologists was 40 hours ( range 2 - 152 hours ) . different upmc telepathology portals data workflow with the upmc portal and one of their clients ( kingmed in china ) . as scans are transferred from the scanner to the ndp . serve database , this system allows for automated notification of the upmc assigned pathologist that the case is ready for consult . perhaps more importantly , this automated system eliminates a person having to email or notify , which scan filenames are associated with a case , thereby removing the possibility of human error of assigning the wrong slide image to a case digital pathology consultation at upmc has spanned more than a decade . during this time , all modes of telepathology have been successfully utilized to exploit our subspecialty expertise and to compete for pathology services . the practice of telepathology at our institution has evolved in concert with advances in technology , which has become more cost - effective . although several of the aforementioned modes of practicing telepathology may be outdated , we believe it is nevertheless important to share our experiences and point out that very often technology is not the limiting issue , but rather the people and processes involved . of note , diagnostic accuracy and turnaround time telecytology has been accomplished largely using real - time video streaming , and for wsi of cytology slides , the low volume of cases received for teleconsultation have been interpretable without the need for scanning slides with z - stacking . early and continued adoption has promoted several digital pathology resources ( e.g. , facilitated funding for equipment , it infrastructure , and staff ) that are now being leveraged for other clinical , educational , and research purposes . table 2 lists many of the other factors that may need to be considered when practicing telepathology . a key aspect of a successful digital teleconsultation program is integration of digital images ( including wsi ) into the laboratory information system ( lis ) and electronic health record . such integration , for example , was one of the main objectives of the cost action efforts towards integrating the lis with wsi are underway at our institution in partnership with omnyx . telepathology requirements are different for developed and developing countries . based on our experience with china , some bottlenecks to telepathology included the high price of wsi devices and regulatory issues . technological issues , however , are often easier to overcome than administrative , contractual , and legal challenges our institution is currently evaluating newer approaches such as grid technology , open access forums , and mobile solutions to determine how they can be leveraged to enhance telepathology at our institution . grid technology , which uses open standards to access distributed information , has been suggested by some authors as a way to improve quality in image - based diagnosis . we have provided our pathologists with access to a suite of cloud - based applications through a web browser [ figure 9 ] . since the computer programs that support these various telepathology devices are stored on servers at a remote location , the hardware and software demands placed on the end users are greatly diminished . while the use of commercial web conferencing systems ( e.g. , skype ) has been shown to be feasible for telepathology , in our institutional setting , reliance on public servers raises potential security issues , which has proven to be a barrier for telepathology . more recently , the field of telepathology is witnessing the emergence of specific open access forums ( e.g. , medical electronic expert communication system [ meces ] with embedded virtual slide technology ) . these new generation forums , which employ browser - friendly w3c conform standards , offer additional benefits such as acoustic information transfer and assistance in image screening . finally , taking advantage of mobile devices ( e.g. , cellular phones ) , which are almost ubiquitous today , provides another opportunity to perform telepathology almost anywhere and anytime .

several modes of telepathology exist including static ( store - and - forward ) , dynamic ( live video streaming or robotic microscopy ) , and hybrid technology involving whole slide imaging ( wsi ) . telepathology has been employed at the university of pittsburgh medical center ( upmc ) for over a decade at local , national , and international sites . all modes of telepathology have been successfully utilized to exploit our institutions subspecialty expertise and to compete for pathology services . this article discusses the experience garnered at upmc with each of these teleconsultation methods . static and wsi telepathology systems have been utilized for many years in transplant pathology using a private network and client - server architecture . only minor clinically significant differences of opinion were documented . in hematopathology , the cellavision system is used to transmit , via email , static images of blood cells in peripheral blood smears for remote interpretation . while live video streaming has remained the mode of choice for providing immediate adequacy assessment of cytology specimens by telecytology , other methods such as robotic microscopy have been validated and shown to be effective . robotic telepathology has been extensively used to remotely interpret intra - operative neuropathology consultations ( frozen sections ) . adoption of newer technology and increased pathologist experience has improved accuracy and deferral rates in teleneuropathology . a digital pathology consultation portal ( https://pathconsult.upmc.com/ ) was recently created at our institution to facilitate digital pathology second opinion consults , especially for wsi . the success of this web - based tool is the ability to handle vendor agnostic , large image files of digitized slides , and ongoing user - friendly customization for clients and teleconsultants . it is evident that the practice of telepathology at our institution has evolved in concert with advances in technology and user experience . early and continued adoption of telepathology has promoted additional digital pathology resources that are now being leveraged for other clinical , educational , and research purposes .

INTRODUCTION STATIC TELEPATHOLOGY DYNAMIC VIDEO MICROSCOPY ROBOTIC TELEPATHOLOGY WSI TELEPATHOLOGY CONCLUSIONS POTENTIAL FUTURE SOLUTIONS

obstructive sleep apnea ( osa ) is a public - health problem as it affects at least 10% of the middle aged men and represents a main cause of cardiovascular morbidity and mortality . an independent association between osa , insulin resistance , and type 2 diabetes has been consistently demonstrated by a number of cross - sectional , observational , and large population - based studies [ 2 , 3 ] . moreover , osa patients have increased carotid intima - media thickness ( imt ) , an early sign of atherosclerosis , which correlates with nocturnal oxygen desaturation , independently of other cardiovascular risk factors [ 4 , 5 ] . osa severity may also predict occult coronary atherosclerosis in healthy overweight or obese male subjects . repetitive upper airway collapses during sleep result in intermittent hypoxia ( ih ) which is thought to be responsible for osa - associated cardiometabolic complications , including atherosclerosis and insulin resistance . data obtained from animals exposed to ih , a validated experimental model of sleep apnea , showed that activation of the sympathetic nervous system and systemic inflammation underlie ih - induced metabolic and vascular consequences [ 79 ] . we also recently demonstrated that ih - induced inflammatory changes of epididymal white adipose tissue ( ewat ) contributed to these outcomes . in obesity , it is increasingly recognized that chronic activation of inflammatory signaling pathways is causally linked to insulin resistance and vascular alterations . recent studies suggest that these deleterious effects could be mediated , at least in part , through the activation of toll - like receptors ( tlr ) , and in particular the tlr4 . tlrs are a family of pattern - recognition receptors that play a critical role in the innate immune system by activating proinflammatory signaling pathways in response to microbial pathogens . lipopolysaccharide ( lps ) binds to tlr4 , triggering a downstream cascade , which leads to the activation of the proinflammatory nuclear factor kappa - b ( nfb ) pathway and finally to the expression of numerous proinflammatory molecules , such as interleukin ( il)-6 and tumor necrosis factor ( tnf)- . furthermore , studies on different strains of mice showed that expression and activation of tlr4 are involved in aortic inflammation and atherogenesis . together these experimental data confirm the major role of tlr4/nfb pathway in the crosstalk between inflammation , atherosclerosis , and metabolism dysfunctions . in c57bl/6 mice , we previously found that ih - induced cardiovascular inflammation was characterized by an increased activity of nfb in aortic and cardiac tissues ( unpublished data ) . we also reported that , in lean animals , ewat exposed to ih became pathological , behaving like excess fat in obesity , as it exhibited increases in macrophage recruitment and secretion of il-6 and tnf- . collectively , these data suggest that cardiometabolic complications due to ih and obesity may share some pathophysiological mechanisms . therefore , we assessed whether metabolic and vascular consequences induced by ih involved the proinflammatory tlr4/nfb pathway activation . shizuo akira ( research institute for microbial diseases , osaka , japan ) and were obtained from the emma ( european mouse mutant archive ) network in orleans , france . seventeen - week - old male tlr4 mice and their control groups ( c57bl/6 mice ) fed on a standard - chow diet were used . the study was conducted in accordance with the european convention for the protection of vertebrate animals used for experimental and other scientific purposes ( council of europe , european treaties ets 123 , strasbourg , 18 march 1986 ) and with the guide for care and use of laboratory animals ( nih publication no . tlr4 and their control c57bl/6 mice were divided into 2 subgroups , exposed to either intermittent hypoxia ( ih ) or normoxia ( n ) . the four groups of animals were exposed to the ih stimulus during daytime ( n = 10 per cage , 8 h / day , cyclic 21 - 5% fio2 , 60 s cycle ( 60 events / h ) , lowest blood oxygen saturation up to 60% ) for 4 weeks . fio2 was measured with a gas analyzer ( ml206 , adinstruments ) throughout the experiment . control animals ( normoxic mice , n ) were exposed to air in similar cages to reproduce similar noise and turbulences to those of the ih stimulus . ambient temperature was maintained at 2022c . during the first week of ih exposure , intraperitoneal insulin tolerance test ( ipitt ) was performed to assess global insulin sensitivity and , on the day following the last exposure period , fasted animals were sacrificed under anesthesia with intraperitoneal injection of ketamine ( 100 mgkg ) and xylazine ( 10 mgkg ) for further analysis . mice were fasted for 5 hours and then weighted before blood was collected from the tail tip for baseline glucose determination ( t = 0 ) . insulin ( 0.5 iukg body weight , novo nordisk a / s , bagsvaerd , denmark ) was injected intraperitoneally , followed by further blood glucose measurements at 15 , 30 , 60 , and 90 minutes after the injection . the lowest blood glucose level ( nadir ) following insulin administration was calculated for each experimental group . at the time of sacrifice , blood was collected by cardiac puncture on edta tubes . the plasma fraction was collected after blood centrifugation during 10 minutes at 11000 rpm ( 4c ) . total cholesterol was measured in plasma by a colorimetric enzymatic reaction using the infinity kit ( thermoelectron corporation , massachusetts , usa ) according to the manufacturer 's guidelines . bilateral epididymal fat pads were collected , weighted , and either fixed in 90% ethanol for adipocyte morphology study or incubated for cytokine determinations . ethanol - fixed , paraffin - embedded ewat were sectioned ( 3.5 m ) , deparaffinized in toluene and rehydrated in descending ethanol series , and then stained with hematoxylin - eosin to assess tissue morphology . adipocyte size was measured from photographs ( 10 40 magnification ) using the nis - elements microscope imaging software ( nikon ) . each ewat pad was divided into two equal pieces and incubated at 37c with mild shaking in rpmi medium . after 120 minutes of incubation , il-6 and tnf- were measured in the supernatants using an elisa test according to manufacturer 's instructions ( r&d system europe , lille , france ) . cytokine concentrations were expressed as ng / ml for 1 g of adipose tissue . ih and n aortas were embedded in optimum cutting temperature ( oct ) compounds ( tissue - tek , sakura finetek europe bv , alphen aan den rijn , the netherlands ) , sectioned ( 10 m ) , and stained . hematoxylin - eosin staining was used to assess the intima - media thickness ( imt ) . morphometric analysis ( up to 15 measurements on 10 noncontiguous midthoracic descending aorta sections per animal ) was performed with a light microscope ( nikon eclipse 80i , nikon ) and the nis - elements microscope imaging software ( nikon instruments europe bv ) . we investigated whether ih could activate nfb by assessing the expression of its activated subunit nfb - p50 which has translocated into the nucleus . nuclear nfb - p50 was determined in thoracic aorta of mice exposed to n or ih . tissue homogenization and proteins extraction were performed according to manufacturer 's instructions using a nuclear extract kit ( active motif europe , rixensart , belgium ) . proteins concentration was evaluated using the bca assay ( thermoscientific , massachusetts , usa ) . nuclear proteins were assayed for the presence of the activated p50 by elisa using the transam nfb - p65/p50/p52 kit ( active motif europe ) . results were expressed as mean standard errors of the means ( sem ) and analyzed using 2-way anova and subsequent bonferroni 's multiple post hoc comparisons or mann - whitney u test . they had smaller fat pads with smaller adipocytes ( figures 1(a ) , 1(c ) , and 1(d ) ) , and ewat released more tnf- and il-6 compared to normoxic controls ( figures 1(e ) and 1(f ) ) . regarding body weight alterations , normoxic and hypoxic tlr4 mice were not different from their respective control animals ( figure 1(b ) ) . after one - week exposure , the 4 experimental groups were assessed for insulin tolerance test ( figure 2(a ) ) . c57bl/6 mice exposed to ih exhibited a decreased response to insulin as shown by a lower glucose decrement ( figure 2(b ) ) and a trend for a smaller glucose nadir compared to their normoxic controls ( figure 2(d ) ) . the insulin response was not affected in hypoxic tlr4 mice : the response curve was almost superposable with those of the normoxic tlr4 animals ( figures 2(c ) and 2(d ) ) and not significantly different from the curve of normoxic c57bl6 animals ( figure 2(a ) ) . hypoxic c57bl/6 mice had morphological and functional alterations of their aorta , as they exhibited larger intima - media thickness ( figures 3(a ) and 3(b ) ) and higher nfb - p50 activity ( figure 3(c ) ) . these alterations were not observed in hypoxic tlr4 mice ( figures 3(a ) , 3(b ) , and 3(c ) ) . plasma levels of total cholesterol were not different between the 4 experimental groups ( figure 3(d ) ) . there are some similarities with cardiometabolic complications due to obesity , the latter resulting from inflammation involving tlr4 signaling . here , we showed in nonobese c57bl/6 mice that ih induced morphological and inflammatory remodeling of vascular and white adipose tissues as well as insulin resistance . these alterations were prevented in hypoxic tlr4-deficient mice suggesting that ih - induced cardiometabolic consequences involve tlr4 signaling - mediated inflammation . as previously published by our group and others , we used a deep intermittent hypoxia which mimics severe sleep apnea whereas patients mainly suffer from mild to moderate sleep apnea [ 7 , 17 , 18 ] . indeed , in the absence of additional factors such as obesity , high fat diet , and genetic vulnerability , ih needs to be severe enough to induce measurable and reproducible vascular alterations , especially in c57bl/6 mice which are atheroresistant animals . in lean c57bl/6 mice , we showed that ih induced ewat alterations characterized by fat pad wasting and shrunken adipocytes . these results are in agreement with previous reports in lean c57bl/6 and apoe mice after 4 and 6 weeks of ih , respectively . shrunken adipocytes are suggestive of lipolysis , as elevated circulating free fatty acids ( ffas ) have been reported by us and others in ih - exposed animals [ 10 , 20 , 21 ] and in patients suffering from sleep apnea . in the latter , activation of beta - adrenergic receptors is a well - known mechanism of lipolysis , and both ih and osa are commonly associated with sympathoadrenergic activation [ 10 , 24 ] . adipose inflammation may have also contributed to lipolysis as we found an increased release of the inflammatory cytokines tnf- and il6 from fat pads of hypoxic c57bl/6 mice . this is again consistent with previous reports in lean apoe mice after 6 weeks of ih , and more recently in 3t3-l1 adipocytes exposed to fluctuating oxygen concentration . these inflammatory changes may be due to local hypoxia , which is known to be a leading cause of ewat dysregulation , as well as to systemic effects such as elevated circulating ffas and activation of the sympathoadrenergic system . we found that both ih - induced morphological and inflammatory alterations of ewat were prevented in tlr4-deficient mice . there is growing evidence from studies using murine models of obesity that activation of the proinflammatory tlr4/nfb pathway constitutes one mechanism that links inflammation and metabolic disorders [ 3032 ] . although tlr4 activation is known to enhance lipolysis [ 33 , 34 ] , we were surprised to find that ewat wasting and adipocyte hypotrophy were completely prevented in tlr4-deficient mice suggesting that tlr4 signaling could be the main mechanism of these consequences . as observed in mice models of diet - induced obesity [ 30 , 32 ] , tlr4 deficiency prevented the enhanced release of tnf- and il6 in our hypoxic mice . this strengthens the role of tlr4 in ih - induced inflammation , as well as the pathophysiological similarities with obesity , that is , a normal amount of fat under hypoxia behaving like excess fat in obesity . this is in agreement with previous findings obtained in various mouse strains ( genetically obese , lean c57bl/6 , and apoe mice ) and duration of ih exposure ( acute or chronic ih ) , using different methods to assess insulin sensitivity ( homa - ir , itt , hyperinsulinemic euglycemic clamp ) [ 10 , 19 , 35 , 36 ] . this confirms the role of ih in the alterations of glucose homeostasis observed in sleep apnea , the insulin resistance worsening with osa severity , independently of obesity [ 3739 ] . both chronic inflammation and elevated ffa levels [ 41 , 42 ] are established factors causing insulin resistance in obesity . indeed proinflammatory cytokines secreted from adipocytes are considered as a key step in obesity - induced insulin resistance , as the sole tnf- neutralization in obese rats is sufficient to improve insulin sensitivity . inversely , upstream activation of the inflammatory cytokine cascade using agonists of toll - like receptors leads to insulin resistance [ 33 , 34 ] . in the present study , we observed increased release of tnf- and il6 from ewat and decreased insulin sensitivity in hypoxic c57bl6 mice . both parameters were not impaired in tlr4-deficient mice suggesting that tlr4 mediated inflammation was involved in this metabolic consequence . body weight alterations did not contribute to these improvements , as hypoxic c57bl/6 and tlr4-deficient mice had similar body weight . we found that ih induced vascular remodeling , including morphological ( larger intima - media thickness ) and inflammatory changes ( higher nfb - p50 activity ) . this confirms our previous results and others regarding the detrimental remodeling effects of ih on preatherosclerotic [ 17 , 18 ] and atherosclerotic [ 21 , 44 , 45 ] processes . we confirmed in the present study that ih is indeed a powerful vascular stress , as only 28 days of exposure , which is very short compared to the duration of the human disease , induce early vascular alterations in the atheroresistant c57bl/6 mouse strain . besides the well - known role of the sympathoadrenergic system and the related hemodynamic alterations , accumulating evidence suggests that inflammation is involved early in the pathophysiology of ih - related atherosclerosis . regarding the aggravation of atherosclerosis by ih in apoe - deficient mice , we recently demonstrated that this deleterious effect involved inflammatory alterations of ewat , as ewat lipectomy prevented the proatherogenic effect of ih . interestingly , this beneficial effect occurred while insulin resistance ( a well - known risk factor for atherosclerosis ) was not improved by ewat lipectomy , suggesting that ewat inflammation could be the main determinant of ih - induced atherogenicity . here , we showed that ewat from hypoxic c57bl/6 mice released higher amounts of inflammatory cytokines and that this effect , as well as the morphological and inflammatory changes of aorta , was prevented in tlr4-deficient animals . given the role of tlr4 in adipose tissue inflammation and insulin resistance , and the relationship between adipose tissue inflammation , insulin resistance , and vascular dysfunction [ 12 , 47 ] , the beneficial effect of tlr4 deficiency on arterial remodeling could be explained by the prevention of adipose inflammation and insulin resistance . here , the role of ewat inflammation seems to be predominant compared to the light improvement of insulin response and the absence of cholesterol alterations in these animals . ih - induced dyslipidemic alterations are indeed inconstant in mice [ 45 , 4850 ] , suggesting that dyslipidemia contributes only in part to the first steps of vascular remodeling in this model . reduced ih - driven hemodynamic alterations could be a further explanation , as tlr4-deficient mice are less susceptible to hypertension [ 51 , 52 ] . finally , a direct effect on the vascular wall is also possible , as tlr4 has been evidenced in human and murine atherosclerotic plaques , and inhibition of tlr4 signaling pathway attenuated diet - induced atherosclerosis in apoe mice [ 16 , 55 ] . a recent study also identified tlr4 signaling pathway as a direct key mediator of vascular inflammation and impairment of endothelial insulin signaling in the setting of obesity . the direct effect on vasculature could even be predominant , as tlr4 deficiency prevented atherosclerosis in ldlr mice , with no effect on adipose tissue inflammation and whole - body insulin sensitivity . we showed in nonobese c57bl/6 mice that ih induced morphological and inflammatory remodeling of aorta and epididymal white adipose tissue , as well as insulin resistance . these alterations were prevented in tlr4-deficient mice suggesting that ih - induced cardiometabolic consequences involved inflammation mediated by tlr4 signaling . the precise mechanisms and the specific role of one type of tissue or cell ( e.g. , adipose tissue ) remain to be determined as tlr4 knockout used in the study was not cell specific . tlr4 is indeed expressed on many cell types , predominantly those of the immune system , but also on nonhematopoietic cell types ( e.g. , endothelial , epithelial cells , etc . ) . despite these limitations , as a practical point of view , the whole body is exposed to hypoxia during sleep apnea , and tlr4 in various cells is likely to be involved in the numerous complications of sleep apnea . moreover the available treatment that blocks tlr4 activation ( eritoran ) is not cell specific . however , one clinical study has recently investigated the activation of tlr4 signaling pathway in osa . the authors found increases in tlr4 expression , nfb nuclear binding , and release of ifn , tnf- , and il-6 in circulating monocytes . there are therefore similarities between these clinical findings and our experimental results suggesting that targeting tlr4/nfb pathway could provide further therapeutic options for sleep apnea patients .

objective . intermittent hypoxia ( ih ) is a major component of sleep apnea syndrome as its cardiometabolic complications have been mainly attributed to ih . the pathophysiology is still poorly understood but there are some similarities with the obesity - associated cardiometabolic complications . as the latter results from inflammation involving toll - like receptor-4 ( tlr4 ) signaling , we assessed this pathway in the cardiometabolic consequences of ih . methods . lean adult male tlr4-deficient ( tlr4/ ) mice and their controls ( c57bl/6 mice ) were exposed to either ih ( fio2 21 - 5% , 1 min cycle , 8 h / day ) or air ( normoxic mice ) for 4 weeks . animals were assessed at 1-week exposure for insulin tolerance test and after 4-week exposure for morphological and inflammatory changes of the epididymal fat and thoracic aorta . results . ih induced insulin resistance , morphological and inflammatory changes of the epididymal fat ( smaller pads and adipocytes , higher release of tnf- and il-6 ) and aorta ( larger intima - media thickness and higher nfb - p50 activity ) . all these alterations were prevented by tlr4 deletion . conclusion . ih induces metabolic and vascular alterations that involve tlr4 mediated inflammation . these results confirm the important role of inflammation in the cardiometabolic consequences of ih and suggest that targeting tlr4/nfb pathway could represent a further therapeutic option for sleep apnea patients .

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

there are different diagnostic methods but they have some drawbacks . to prepare mycobacterium culture , which is the golden standard for tb diagnosis , it may take 8 weeks . finding acid - fast bacilli is the quick screening method for pulmonary tb diagnosis ; nevertheless , its sensitivity is low . the polymerase chain reaction ( pcr ) test for tb diagnosis is expensive and it requires skilled personnel and lot of equipments . therefore , in recent years , there has been a great demand for finding new microbiological , genetic , immunological , and biomedical diagnostic methods to diagnosis tb quickly and accurately . ada is essential for proliferation and differentiation of lymphoid cells , especially t cells , and helps in the maturation of monocytes to macrophages . it seems ada is an index for cellular immunity and previous studies have proved its value in tb diagnosis , even for assessing tb effusions . , the level of ada in sputum and serum was used for diagnosis of tb , and it was monitored during tb treatment . however , previous studies used effusion fluids and a very limited number of studies used patients serum . it is not always possible to access effusion liquids everywhere in pulmonary and extra - pulmonary tb ; therefore , it would be helpful to take advantage of serum levels . the goal of this study is to assess the diagnostic value and cut - off point of serum ada levels in pulmonary tb patients . it was a cross - sectional study ( 2011 ) , which is conducted in tohid hospital ( university referral hospital ) in sanandaj in iran . in our study , case group included 40 sputum smear - positive tb patients who were admitted in infectious disease ward of the hospital . inclusion criteria for tb patients were : having 2 or 3 sputum positive smears , or one sputum positive smear and one positive sputum culture , or one positive tb microbe smear sputum and suspected chest radiography . in addition , 40 non - tb patients , referred to tohid hospital in sannandaj for surgeries , were selected as the control group . inclusion criteria for control group were : not having any tb patient in their family , not having history of close contact with tb patients , not having any infectious disease ( according to the interview and their records ) , not having fever or any symptom of illness or of being toxic , normal cell blood count , and normal chest radiography . after diagnosis of tb ( before initiation of treatment ) , blood samples and patients approval of consent form ( research project number : 1387/87 ) were taken from tb group ( at 7 am ) . then samples were centrifuged and serum ada levels were measured by ada kit manufactured by diazyme laboratories company . in the first step , adenosine was affected by ada and it becomes de - ammonized and shifted to inosine and then ammoniac was released . in the second reaction , because of glometat , released nh3 had become dehydrogenized and when it got close to allosteric activators , it combined with nicotinamide adenine dinucleotide phosphate hydrogen ( nadph ) and released nicotinamide adenine dinucleotide phosphate ( nadp ) . consequently , there was a direct relationship between activity ( density ) of ada enzyme and speed reduction of radiation absorption in 340 nanometer wavelength ( as nadph changed to nadp+ ) ; it was measured by diazyme adenosine deaminase assay kitt ( diazyme laboratory ; usa ) . then , data were analyzed using statistical package for the social sciences ( spss ) 11.5 ( chicago , usa ) software and roc curve was plotted . from all 40 tb patients who participated in this study , 16 were males and 24 were females . from 42 participants in control group , 22 were males and 20 were females . the most common age group for tb patients was 50s and for control group , it was 40s . age average was 59 ( 13.5 ) in tb patients and it was 49 ( 15.6 ) for non - tb patients . the average of serum ada in tb patients group and control group was 20.88 ( 5.97 ) and 10.69 ( 2.98 ) , respectively . u / l in which sensitivity and specificity were 92.7% ( 95% ci , 84.7 - 100 ) and 88.1% ( 95% ci , 78.3 - 97.8 ) , respectively . the positive and negative predicative values were 88.4% ( 95% ci , 75 - 95.1 ) and 92.5% ( 95% ci , 79.6 - 98.4 ) , respectively [ figure 1 ] . the calculated area under the roc curve was 0.955 ( 95% ci , 0.914 - 0.995 ) ( p < 0.001 ) . in this study , serum ada level was an appropriate index for diagnosing smear - positive tb . therefore , serum ada could be also used for diagnosis of pulmonary tb . moreover , in this study , all tb patients had a serum level of more than 22.5 u / l . hence , for suspicious cases of tb , increased levels of ada could facilitate diagnosis . diagnostic value of serum ada in pulmonary tb has been assessed only in few numbers of studies . pairs et al . reported an increase in ada level in tb pleural effusion ; other studies have also confirmed such an increase in tb pericardial effusions , peritoneum , and central nervous system ( cns ) . the main reason for the increased ada levels in pleural effusion is the movement of t lymphocytes toward this area . increase in ada level is the result of a tropical inflammatory reaction caused by monocytes and macrophages . when alveolar macrophages are infected by mycobacterium , this enzyme could be found in serum during active pulmonary disease . when tb infection is controlled , growth - markers of lymphocytes decrease ; leucocytes will decrease in serum ada levels concurrent with the decrease in lymphocytes . because of this , serum ada level could be utilized as a treatment response index . in agarwal 's study , ada level was 15.3 ( 0.23 ) in healthy people , 19 ( 0.68 ) in non - pulmonary tb cases , and 38.48 ( 1.56 ) in pulmonary tb patients . in jhamaria et al . 's study , the average of serum ada level was 19.9 u / l ( 2.99 ) in control group , 43.95 u / l ( 2.48 ) in sputum smear - positive people with typical or progressive disease , and 42.09 u / l ( 1.46 ) and 40.02 u / l ( 2.58 ) in negative sputum patients with mild or typical disease . in their study , in the cut - off point of 33 it seems that as the disease progresses , ada levels increase ; this subject was not considered in our study . in another study with an ada level of 26.2 u / l , sensitivity , specificity , and positive predictive value were 95% , 83.3% , and 79.2% , respectively . in gupta 's study , sensitivity , specificity , positive predictive value , and negative predictive value were 92.8% , 90% , 92.8% , and 90% , respectively , for diagnosis of tb in pleural effusion with an ada level of more than 40 . in conde 's study , ada level of 14 u / l was chosen as cut - off point . stevanovic et al . assessed serums of extra - pulmonary tb patients and in a cut - off point of 24 , sensitivity and specificity were 94.3% and 92.2% , respectively ; in their study , serum ada level decreased as treatment started . in dilmac 's study , serum ada level in pulmonary tb patients was reported as 27.5 ( 11 ) and it was 23.9 ( 24 ) in chronic obstructive pulmonary disease ( copd ) patients . in rasolinejad 's study , serum ada level was 21.51 in pulmonary tb patients and 11.47 in healthy people ; in cut - off point of 14.5 u / u / l in negative smear and negative tuberculin patients , 33.52 ( 15.22 ) in smear - positive and purified protein derivative ( ppd ) positive patients , and 16.5 ( 3.18 ) in volunteer healthy people . such differences may be due to tb severity , age groups , genetic differences , and dissimilarities in control groups . therefore , further studies for identifying normal ada levels in different societies may be useful . fortunately , in some autoimmune patients like rheumatoid arthritis , synovial ada level is normal and it is similar to control group . thus , in autoimmune diseases that involve lung , ada level could be used for tb differentiation . in some studies , ada2 was also considered a useful tool for diagnosis ; it needs further studies . according to this study , serum ada level is proposed as a proper index for tb diagnosis ; in a cut - off point of 14 , its sensitivity and specificity are calculated as 92.7% and 88.1% , respectively .

background : in some studies , the level of adenosine deaminase ( ada ) in sputum and effusion liquids was used for the diagnosis of tuberculosis ( tb ) . but it is not always possible to access these materials . the goal of this study is to assess the diagnostic value of serum ada levels in pulmonary tb patients.materials and methods : in this study , 40 sputum smear - positive tb patients who were hospitalized and 40 non - tb patients who referred for surgeries were selected . a serum sample was collected and serum ada level was measured by ada kit.results:the average ( sd ) of serum ada in tb and non - tb patients were 20.88 ( 5.97 ) and 10.69 ( 2.98 ) u / l , respectively ( p value < 0.05 ) . the best cut - off point was 14 u / l . the calculated area under the receiver operating characteristic ( roc ) curve was 0.955 ( 95% ci , 0.914 - 0.995 ) ; sensitivity was 92.7% ( 95% ci , 84.7 - 100 ) and specificity was 88.1% ( 95% ci , 78.3 - 97.8 ) ( p < 0.001).conclusion : serum ada level may be proposed as a proper index for tb diagnosis .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

androgen deprivation therapy ( adt ) by bilateral orchiectomy or gonadotropin - releasing hormone analogs , with or without antiandrogens , is indicated as front - line treatment in metastatic prostate cancer , as well as in the adjuvant setting following radical prostatectomy with nodal metastasis or radiation therapy , and occasionally in patients with localized disease.1 adt has influence on many metabolic pathways but the most common side effect is reduction of bone density.26 prostate cancer usually spreads to the bone in metastatic disease , and treatment with intravenous bisphosphonates is commonly prescribed at this stage in order to reduce skeletal - related events.7,8 loss of bmd correlates with the duration of adt but is more pronounced during the first year of therapy.710 men initiating adt are recommended to have an assessment of risk factors for osteoporosis , calcium and vitamin d intake , lifestyle modifications , and baseline and serial bmd assessment while on adt , along with bisphosphonate therapy.1 there is evidence that loss of bmd is a strong predictor of fracture risk.11 treatment with bisphosphonates used to prevent osteoporotic fractures has been shown to increase bone mineral density ( bmd).12 although the national comprehensive cancer network and american society of clinical oncology guidelines propose baseline bmd assessment and bisphosphonate therapy , there has been no systematic review of the utilization of these interventions by practicing physicians in the community . the current study evaluated bone densitometry ( bd ) and bisphosphonate therapy utilization patterns to prevent osteoporosis in this patient population . a retrospective cohort study was conducted using a surveillance epidemiology end results ( seer)-medicare linked database of men aged 65 years and older with a diagnosis of non - bony metastatic prostate cancer between 2004 and 2007 . individuals with a diagnosis of non - metastatic prostate cancer were identified . only those treated with adt patient data were obtained from the seer patient entitlement and diagnosis summary file ( pedsf ) , and medicare inpatient treatment claims were identified using national claims history noninstitutional physician / supplier part b files ( claims for preventive services and coverage for outpatient prescription services ) and outpatient part b claims ( outpatient services , prescriptions , and durable medical devices ) from hospital facilities . seer - linked patient data from the pedsf file were unavailable for the year 2008 at the time of the study ; so prostate cancer patient data were analyzed for 20042007 with linked medicare treatment claims from 2004 to 2008 . in order to limit our analysis to patients with no metastatic disease to bone , only prostate tumors coded as no metastasis and only localized tumors in the pedsf file medicare claims from the national claims history physician and outpatient files were matched with pedsf patient data by seer case i d number to select prostate cancer cases that had ever received adt as part of treatment . adt was identified using healthcare common procedure coding system ( hcpcs ) and current procedural terminology codes ( cpt ) in the medicare physician and outpatient claims files . coding used for orchiectomy included 54520 , 54521 , 54522 , 54530 , and 54535 or icd-9 code 624 , and coding for goserelin , leuprolide , leuprolide implant , or triptorelin were identified as codes j1950 , j9202 , j9217 , j9218 , or j9219.13 bmd assessment by dual - energy x - ray absorptiometry was identified using hcpcs or cpt codes 77080 or 77081 . treatment with intravenous bisphosphonates such as pamidronate or zoledronic acid were identified using medicare hcpcs codes j2430 and j3487 during the study period 20042008 . since the medicare part d data for outpatient medications including oral bisphosphonates were only available from 2007 in the seer medicare files , they could not be utilized for the analysis . oral bisphosphonates are generally poorly absorbed and have gastrointestinal side effects , leading to low patient adherence with these drugs.14,15 due to lack of adherence , intravenous bisphosphonates are preferred to oral bisphosphonates in this patient population . oral bisphosphonate use was not captured because the medicare database does not collect details of oral therapy . the one - sided exact binomial test of proportion was used to determine if physician compliance with serial bmd assessment and bisphosphonate treatment in patients undergoing adt is consistently 80% . a survey of practicing physicians with a response rate of 63% found that physician s self - reported adherence to the clinical guidelines was 77%.16 therefore , 80% adherence to the clinical guidelines was used in this study . logistic regression analysis was used to determine treatment differences by patient characteristics such as age group , race , clinical stage , and seer registry geographic region . sas 9.3 ( sas institute , cary , nc , usa ) was used to analyze differences in treatment practices . further logistic regression analysis was also done to explore the relationship between history of bone fractures and bisphosphonate treatment , age , race , tumor stage at diagnosis , and duration of adt . the study was approved by the institutional review board at the university of arkansas for medical sciences . among 157,974 newly diagnosed prostate cancer cases , 100,865 were aged 65 years and older and had no bone metastases from 2004 to 2007 . subjects who did not have adt claims were excluded from the analysis . in total , 30,846 prostate cancer patients aged 65 years and older with no bone metastases receiving adt were eligible for analysis ( table 1 ) . neither bd nor parenteral bisphosphonate therapy were utilized in 87.3% ( n=26,935 ) of subjects on adt ( table 2 ) . the age group , race , t stage , and geographic distribution are shown in table 1 , and for every covariate ( age , race , t stage , and geographic location ) , the percentage of bd utilization and parenteral bisphosphonate administration was markedly low . in our study , 8.8% ( n=2,707 ) of subjects on adt received bd assessments without ever receiving intravenous bisphosphonates . three percent ( n=931 ) of the cases on adt received bisphosphonate treatment without ever receiving a bd assessment . only 0.9% ( n=278 ) of the subjects on adt received both bd and bisphosphonates . a compliance rate of nearly 1.0% ( 0.9% ) , those subjects receiving both bd to screen for bone loss and preventive bisphosphonate therapy , was well below the expected rate of 80% ( table 2 ) . figure 1 shows the trend of bd and intravenous bisphosphonate utilization during the study period ( 20042008 ) . bisphosphonates were prescribed more often from 2006 but utilization remained low at below 10% for all years studied . odds ratios ( ors ) show differences in preventive osteoporotic therapy by treatment category ( table 1 ) . bd utilization increased with advancing age , except for those older than 85 years . bd utilization differed by race . black men were less likely to receive bd than white men ( or 0.6 ; 95% confidence interval [ ci ] 0.540.72 ) , and asian men were more likely to have bd measurement performed than white men ( or 1.6 ; 95% ci 1.361.88 ) . patients diagnosed at later stages of disease ( t3 ) received bd more often than men diagnosed with earlier stages of disease ( t0t1 ; or 1.7 ; 95% ci 1.491.96 ) . men with prostate cancer living in the west region were more likely to receive bd than men living in any other region ( table 1 ) . patients in the midwest , northeast , and south regions were significantly less likely to receive bd than patients in the west , ( or 0.7 ; 95% ci 0.640.81 , or 0.8 ; 95% ci 0.760.92 , and or 0.5 ; 95% ci 0.460.58 respectively ) . using the midwest region as the reference group , patients in the west had 20% greater use of bd ( or 1.2 ; 95% ci 1.021.33 ) . intravenous bisphosphonate therapy was prescribed more often to older men ( 8084 years ) than to younger men ( aged 6569 years ; or 1.3 ; 95% ci 1.071.54 ) . men diagnosed at later stages of disease ( t2t4 ) were more likely to receive bisphosphonate treatment than men diagnosed at earlier stages ( t0t1 ; table 1 ) . men living in the northeast and south regions were less likely to receive bisphosphonates than men living in the west region ( table 1 ) . among men receiving the recommended treatment of bd and bisphosphonates , there were differences by race , tumor stage , and region . black men were less likely to receive both bd and bisphosphonates compared with white men ( or 0.6 ; 95% ci 0.360.93 ) . men with prostate cancer in the northwest and south regions were less likely to receive bd and bisphosphonates than men in the west region ( table 1 ) . only 25 of the 30,846 subjects in this study were identified to have a diagnosis of fracture based on icd code in the seer - medicare database . diagnosed bone fractures were identified using icd-9 codes of 733.1 for osteoporotic fractures , and 808809 for fractures to the spine or trunk . logistic regression analysis was performed using bone fractures as the outcome variable . when controlling for age ( a continuous variable ) , bisphosphonate treatment ( a dichotomous variable ) , non - white race ( a dichotomous variable with white race as the reference group ) , tumor stage ( a dichotomous variable with t4 stage as the reference group ) , and duration of adt ( a continuous variable ) , an association between length of therapy and history of bone fractures was found . however , bone fracture was not associated with bisphosphonate therapy in this study population . in multiple studies , it has been demonstrated that adt results in bone loss which could be a surrogate for increased skeletal - related events.1721 although the duration of follow - up after diagnosis was short ( 5 years ) , continuous adt increased the risk of bone fracture in this study cohort ( p0.05 ) . this population - level finding supports the clinical evidence of accelerated bone loss in patients receiving adt . in our study , 8.8% ( n=2,707 ) of subjects on adt received bd assessments without ever receiving intravenous bisphosphonates . three percent ( n=931 ) of the cases on adt received bisphosphonate treatment without ever receiving a bd assessment . only 0.9% ( n=278 ) of the subjects on adt received both bd and bisphosphonates . a compliance rate of nearly 1.0% ( 0.9% ) , those subjects receiving both bd to screen for bone loss and preventive bisphosphonate therapy , was well below the expected rate of 80% ( table 2 ) . figure 1 shows the trend of bd and intravenous bisphosphonate utilization during the study period ( 20042008 ) . bisphosphonates were prescribed more often from 2006 but utilization remained low at below 10% for all years studied . odds ratios ( ors ) show differences in preventive osteoporotic therapy by treatment category ( table 1 ) . bd utilization increased with advancing age , except for those older than 85 years . black men were less likely to receive bd than white men ( or 0.6 ; 95% confidence interval [ ci ] 0.540.72 ) , and asian men were more likely to have bd measurement performed than white men ( or 1.6 ; 95% ci 1.361.88 ) . patients diagnosed at later stages of disease ( t3 ) received bd more often than men diagnosed with earlier stages of disease ( t0t1 ; or 1.7 ; 95% ci 1.491.96 ) . men with prostate cancer living in the west region were more likely to receive bd than men living in any other region ( table 1 ) . patients in the midwest , northeast , and south regions were significantly less likely to receive bd than patients in the west , ( or 0.7 ; 95% ci 0.640.81 , or 0.8 ; 95% ci 0.760.92 , and or 0.5 ; 95% ci 0.460.58 respectively ) . using the midwest region as the reference group , patients in the west had 20% greater use of bd ( or 1.2 ; 95% ci 1.021.33 ) . intravenous bisphosphonate therapy was prescribed more often to older men ( 8084 years ) than to younger men ( aged 6569 years ; or 1.3 ; 95% ci 1.071.54 ) . men diagnosed at later stages of disease ( t2t4 ) were more likely to receive bisphosphonate treatment than men diagnosed at earlier stages ( t0t1 ; table 1 ) . men living in the northeast and south regions were less likely to receive bisphosphonates than men living in the west region ( table 1 ) . among men receiving the recommended treatment of bd and bisphosphonates , there were differences by race , tumor stage , and region . black men were less likely to receive both bd and bisphosphonates compared with white men ( or 0.6 ; 95% ci 0.360.93 ) . men with prostate cancer in the northwest and south regions were less likely to receive bd and bisphosphonates than men in the west region ( table 1 ) . only 25 of the 30,846 subjects in this study were identified to have a diagnosis of fracture based on icd code in the seer - medicare database . diagnosed bone fractures were identified using icd-9 codes of 733.1 for osteoporotic fractures , and 808809 for fractures to the spine or trunk . logistic regression analysis was performed using bone fractures as the outcome variable . when controlling for age ( a continuous variable ) , bisphosphonate treatment ( a dichotomous variable ) , non - white race ( a dichotomous variable with white race as the reference group ) , tumor stage ( a dichotomous variable with t4 stage as the reference group ) , and duration of adt ( a continuous variable ) , an association between length of therapy and history of bone fractures was found . however , bone fracture was not associated with bisphosphonate therapy in this study population . in multiple studies , it has been demonstrated that adt results in bone loss which could be a surrogate for increased skeletal - related events.1721 although the duration of follow - up after diagnosis was short ( 5 years ) , continuous adt increased the risk of bone fracture in this study cohort ( p0.05 ) . this population - level finding supports the clinical evidence of accelerated bone loss in patients receiving adt . adt has been shown to reduce disease progression and increase survival in subjects with prostate cancer.22 bmd loss and osteoporosis is one of the main adverse effects of adt in this population.26,23 in this study , only a small proportion of prostate cancer patients on adt were evaluated for bone loss , and an even smaller proportion ever received intravenous bisphosphonate therapy . among the patient characteristics investigated , approximately 12% of men in the general population have osteoporosis.24 osteoporosis may be asymptomatic , but 20% of men on adt have skeletal - related events such as fracture.24 adt has been associated with accelerated bone loss of up to 4.5% per year , and loss of bmd is considered a strong surrogate for increased risk of skeletal - related events.2,4,5,1922,25 the rate of bone loss is greatest in the first year of adt , and osteoporosis is prevalent in nearly 50% patients on adt by 4 years and 80% by 8 years.8 early bmd assessment and treatment may potentially prevent fragility fractures , as recommended by national organizations such as the national comprehensive cancer network and american society of clinical oncology.1,21,22,25 prevention of bone loss , resulting in a reduction of skeletal - related events , may maintain good quality of life for patients with prostate cancer on adt . concurrent administration of a bisphosphonate or a selective estrogen receptor modulator has been shown to stabilize or increase bmd . in randomized studies , bisphosphonate therapy , including pamidronate , zoledronic acid , or alendronate , has been shown to improve bmd and decrease markers of bone metabolism in men on adt.4,5,7,2029 this community - level study supports evidence that bd utilization and bisphosphonate therapy reduce fracture risk in men with prostate cancer receiving adt . intravenous bisphosphonate therapy , ie , zoledronic acid , has been evaluated in several studies , and was determined to be the best treatment to prevent bone loss in prostate cancer patients undergoing adt.30 treatment with denosumab , which is currently recommended as an alternative to zoledronic acid for patients with nonmetastatic prostate cancer , was approved by the us food and drug administration in 2011 , and was not a treatment option during the study years examined.31 therefore , analysis of guideline compliance using intravenous bisphosphonates as a measure was appropriate for this study . in addition , the cost of osteoporosis - related fractures among men in the usa is approximately $ 4.1 billion per year , and the effect on rising health care costs could potentially have an impact on the survival benefit of adt in patients with metastatic prostate cancer . subjects on adt are recommended to be screened for fracture risk , with bmd testing at baseline , after 1 year of adt , and then every 2 years or as clinically indicated.32,33 in addition , counseling patients for fall risk and applying interventions to reduce falls may consequently reduce fracture risk in this population . most evidence supporting the use of bisphosphonates for prevention of adt - related bone loss in men with prostate cancer is derived from studies evaluating intravenous bisphosphonates.4,5,20,21 this is due to the lack of data available to analyze oral bisphosphonate use in large population studies . greenspan et al recently demonstrated a significant increase in bmd in men with prostate cancer receiving adt and once - weekly oral alendronate 70 mg.22 denosumab , another current treatment and unavailable during the study years examined , is recommended as an alternative to zoledronic acid for nonmetastatic prostate cancer patients.31 the long - term use of oral bisphosphonate therapy could not be evaluated in this study because medicare part d data are not available during the study period . however , due to lack of patient adherence , oral bisphosphonates have not been the preferred treatment over intravenous bisphosphonates in this patient population , so likely did not significantly affect the outcome.14,15 our study re - emphasizes the importance of using bd measures to evaluate skeletal integrity and prevent osteoporosis with the utilization of bisphosphonates among patients who are on adt . there seems to be a lack of understanding regarding the implications of adt for prostate cancer . although the follow - up time was short , continuous adt was associated with an increased risk of bone fracture , which supports previous clinical studies concerning accelerated bone loss in patients receiving adt . in this community population study , we demonstrated that a very small proportion of patients underwent evaluation for bone loss and an even smaller proportion of patients received bisphosphonates . in addition , even when overall treatment utilization is low , black men were less likely to receive the recommended treatment of bd and bisphosphonates compared with white men . further , utilization practices differed by region , with men residing in the west and midwest regions receiving optimum treatment of bd and bisphosphonates when compared with men in the northeast and south . contrary to the recommendations , screening for bone loss and preventive treatment practices among this community population was markedly low for every age group , race , stage at diagnosis ( t0t4 ) , and seer registries by us geographic region .

backgroundprostate cancer subjects with prostate - specific antigen ( psa ) relapse who are treated with androgen deprivation therapy ( adt ) are recommended to have baseline and serial bone densitometry and receive bisphosphonates . the purpose of this community population study was to assess the utilization of bone densitometry and bisphosphonate therapy in men receiving adt for non - metastatic prostate cancer.methodsa cohort study of men aged 65 years or older with non - metastatic incident diagnoses of prostate cancer was obtained from the surveillance epidemiology end results ( seer)-linked medicare claims between 2004 and 2008 . claims were used to assess prescribed treatment of adt , bone densitometry , and bisphosphonates.resultsa total of 30,846 incident prostate cancer cases receiving adt and aged 65 years or older had no bone metastases ; 87.3% ( n=26,935 ) on adt did not receive either bone densitometry or bisphosphonate therapy . three percent ( n=931 ) of the cases on adt received bisphosphonate therapy without ever receiving bone densitometry , 8.8% ( n=2,702 ) of the cases on adt received bone densitometry without receiving intravenous bisphosphonates , while nearly 1% ( 0.90% , n=278 ) of the cases on adt received both bone densitometry and bisphosphonates . analysis showed treatment differed by patient characteristics.conclusioncontrary to the recommendations , bone densitometry and bisphosphonate therapy are underutilized in men receiving adt for non - metastatic prostate cancer .

Introduction Subjects and methods Results Utilization of BD and intravenous bisphosphonate therapy Utilization of BD and intravenous bisphosphonates by patient characteristics Characteristics of study subjects with bone fractures Discussion Conclusion

all adults had bmi > 40 kg / m , and all obese children had a bmi > 97th percentile for sex and age ( table 1 ) . three hundred forty - five french morbidly obese adults were recruited by the cnrs umr8090 at lille and the department of nutrition of paris hotel dieu hospital . we also sequenced 287 swiss morbidly obese adults , all of them recruited after gastric surgery in zurich , switzerland . three hundred ninety - nine french obese children were recruited in cnrs umr8090 , and 281 obese children were recruited as part of the genetics of obesity study ( goos ) cohort ( 28 ) . a total of 1,221 lean adult subjects were part of the french desir ( data from an epidemiological study on the insulin resistance syndrome ) general prospective study ( 29 ) . inclusion criteria was bmi z score ( zbmi ) < 90th percentile for sex and age according to the eastern cooperative oncology group ( ecog ) ( 31 ) . the study protocol was approved by all local ethics committees , and informed consent was obtained from each subject before participating in the study . clinical characteristics for obese and lean subjects data are means sd unless otherwise indicated . genotyping of the fto rs9939609 snp was performed using a taqman snp genotyping assay ( applied biosystems ) on an abi 7900ht fast real - time pcr system ( applied biosystems ) according to the manufacturer 's instructions . we amplified overlapping pcr fragments according to primers and pcr - optimized conditions ( available from the authors upon request ) . pcr amplifications were inspected for single bands of expected sizes on agarose gels before purification with agencourt ampure on biomek nx ( beckman coulter ) . sequencing was achieved using the automated abi prism 3730xl dna sequencer in combination with the big dye terminator cycle sequencing ready reaction kit 3.1 ( applied biosystems ) , and purification of sequencing reaction was performed with agencourt cleanseq on biomek nx ( beckman coulter ) . the comparison of prevalence has been tested with fisher exact test , and the reported p values are two - sided . p values of < 0.05 were considered to indicate statistical significance . wild - type human fto cdna was amplified ( expand dna polymerase ; roche ) using xhoi - tagged primers with the first strand of the reverse - transcribed cdna ( superscript ii ; invitrogen ) from human brain total rna as a template . using the xhoi sites , the resulting pcr product was cloned into an nh2-terminal 6 his - tagged bacterial expression vector ( pet302/nt - his ; invitrogen ) . the fto mutants were generated from cloned wild - type fto using the quikchange site - directed mutagenesis kit ( stratagene ) according to manufacturer 's protocols . the mutations were confirmed by dna sequencing using big dye terminator chemistry ( applied biosystems ) and electrophoresis on an abi 3730 automated dna sequencer . human wild - type and mutant fto protein production was carried out as previously described ( 32 ) . briefly , expression plasmids were transformed into escherichia coli bl21-gold ( de3 ; stratagene ) and cultured in 2 l luria - bertani broth and 50 g / ml carbenicillin to a600 1.0 at 37c . expression of the cloned gene was induced by addition of 0.5 mmol / l isopropyl--d-1-thiogalactopyranoside ( iptg ) at 15c for 4 h. the cells were harvested and washed in pbs , and pellets were stored at 80c . cells were resuspended in 40 ml of lysis buffer ( 50 mmol / l hepes - koh , ph 8 , 2 mmol / l -mercaptoethanol , 5% glycerol , and 300 mmol / l nacl ) , prior to addition of lysozyme ( 2 mg / ml ) . after 30 min of incubation on ice , cells were sonicated and the lysate was clarified by centrifugation at 15000 g for 20 min . the lysate was supplemented with imidazole ( 10 mmol / l ) and loaded onto a 1.5-ml ni - nta ( nickel nitrilotriacetic acid)agarose column ( qiagen ) previously equilibrated in lysis buffer containing 10 mmol / l imidazole . the column was washed with 30 ml of lysis buffer containing 10 mmol / l imidazole , followed by 7.5 ml of lysis buffer containing 40 mmol / l imidazole . the eluate was then loaded onto a 30-kda concentrator ( sartorius stedium biotech ) and centrifuged at 2500 g for 30 min . then 2 ml of buffer ( 20 mmol / l hepes , ph 8 , 5% glycerol , 50 mmol / l nacl ) was added to the concentrated fraction , which was concentrated again down to 100 l by centrifugation at 2500 g . conversion of c-2-oxoglutarate to c - succinate by 2-og fe - dependent dna dioxygenases has been assayed previously ( 33 ) . this method was modified and optimized to assay fto as previously described ( 32 ) . to measure the uncoupled reaction ( no prime substrate present ) , 1.5 mol / l fto was assayed in 10 l reaction mixture containing 50 mmol / l hepes - koh , ph 7 , 50 g / ml bsa , 4 mmol / l ascorbate , 75 mol / l fe(nh4)2(so4)2 , and 20 mol / l [ 5-c]-2-oxoglutarate ( 30 mci / mmol from moravek biochemicals ) and incubated at 37c for various times . to measure stimulation of this activity by 3-methylthymidine , 1 the reaction was stopped by adding 5 l stop solution containing 20 mmol / l succinate , 20 mmol / l 2-og , followed by 5 l 160 mmol / l dinitrophenylhydrazine , which precipitates 2-og . an additional 10 l 1 m 2-og was added and incubated for a further 30 min . clear supernatant ( 10 l ) was scintillation counted to monitor the c - succinate generated . genotyping of the fto rs9939609 snp was performed using a taqman snp genotyping assay ( applied biosystems ) on an abi 7900ht fast real - time pcr system ( applied biosystems ) according to the manufacturer 's instructions . we amplified overlapping pcr fragments according to primers and pcr - optimized conditions ( available from the authors upon request ) . pcr amplifications were inspected for single bands of expected sizes on agarose gels before purification with agencourt ampure on biomek nx ( beckman coulter ) . sequencing was achieved using the automated abi prism 3730xl dna sequencer in combination with the big dye terminator cycle sequencing ready reaction kit 3.1 ( applied biosystems ) , and purification of sequencing reaction was performed with agencourt cleanseq on biomek nx ( beckman coulter ) . the comparison of prevalence has been tested with fisher exact test , and the reported p values are two - sided . wild - type human fto cdna was amplified ( expand dna polymerase ; roche ) using xhoi - tagged primers with the first strand of the reverse - transcribed cdna ( superscript ii ; invitrogen ) from human brain total rna as a template . using the xhoi sites , the resulting pcr product was cloned into an nh2-terminal 6 his - tagged bacterial expression vector ( pet302/nt - his ; invitrogen ) . the fto mutants were generated from cloned wild - type fto using the quikchange site - directed mutagenesis kit ( stratagene ) according to manufacturer 's protocols . the mutations were confirmed by dna sequencing using big dye terminator chemistry ( applied biosystems ) and electrophoresis on an abi 3730 automated dna sequencer . human wild - type and mutant fto protein production was carried out as previously described ( 32 ) . briefly , expression plasmids were transformed into escherichia coli bl21-gold ( de3 ; stratagene ) and cultured in 2 l luria - bertani broth and 50 g / ml carbenicillin to a600 1.0 at 37c . expression of the cloned gene was induced by addition of 0.5 mmol / l isopropyl--d-1-thiogalactopyranoside ( iptg ) at 15c for 4 h. the cells were harvested and washed in pbs , and pellets were stored at 80c . cells were resuspended in 40 ml of lysis buffer ( 50 mmol / l hepes - koh , ph 8 , 2 mmol / l -mercaptoethanol , 5% glycerol , and 300 mmol / l nacl ) , prior to addition of lysozyme ( 2 mg / ml ) . after 30 min of incubation on ice , cells were sonicated and the lysate was clarified by centrifugation at 15000 g for 20 min . the lysate was supplemented with imidazole ( 10 mmol / l ) and loaded onto a 1.5-ml ni - nta ( nickel nitrilotriacetic acid)agarose column ( qiagen ) previously equilibrated in lysis buffer containing 10 mmol / l imidazole . the column was washed with 30 ml of lysis buffer containing 10 mmol / l imidazole , followed by 7.5 ml of lysis buffer containing 40 mmol / l imidazole . the eluate was then loaded onto a 30-kda concentrator ( sartorius stedium biotech ) and centrifuged at 2500 g for 30 min . then 2 ml of buffer ( 20 mmol / l hepes , ph 8 , 5% glycerol , 50 mmol / l nacl ) was added to the concentrated fraction , which was concentrated again down to 100 l by centrifugation at 2500 g . conversion of c-2-oxoglutarate to c - succinate by 2-og fe - dependent dna dioxygenases has been assayed previously ( 33 ) . this method was modified and optimized to assay fto as previously described ( 32 ) . to measure the uncoupled reaction ( no prime substrate present ) , 1.5 mol / l fto was assayed in 10 l reaction mixture containing 50 mmol / l hepes - koh , ph 7 , 50 g / ml bsa , 4 mmol / l ascorbate , 75 mol / l fe(nh4)2(so4)2 , and 20 mol / l [ 5-c]-2-oxoglutarate ( 30 mci / mmol from moravek biochemicals ) and incubated at 37c for various times . to measure stimulation of this activity by 3-methylthymidine , 1 the reaction was stopped by adding 5 l stop solution containing 20 mmol / l succinate , 20 mmol / l 2-og , followed by 5 l 160 mmol / l dinitrophenylhydrazine , which precipitates 2-og . an additional 10 l 1 m 2-og was added and incubated for a further 30 min . clear supernatant ( 10 l ) was scintillation counted to monitor the c - succinate generated . to identify potential loss - of - function mutations , we screened the nine coding exons of fto by direct nucleotide sequencing in 1,433 extremely obese subjects ( 753 adults with a bmi > 40 kg / m and 680 children with a bmi > 97th percentile ) and in 1,433 lean subjects ( 1,221 adults with a bmi < 23 kg / m and 212 children recruited with a zbmi < 90th percentile ) according to the ecog ( 31 ) , with an average zbmi close to the 50th percentile ( zbmi = 0.1 0.98 ) ( table 1 ) . additionally , we also genotyped the fto rs9939609 snp ( 2 ) in all of the patients . genotype call rate was > 99% in both lean and obese subjects , and genotypic distribution obeyed the hardy - weinberg equilibrium in the lean control subgroup ( p = 0.58 ) . we genotyped 363 duplicated dna samples and observed a concordance rate of 100% . in the lean subgroup , genotype counts were 532 ( tt ) , 668 ( ta ) , and 224 ( aa ) . in the obese subgroup , genotype counts were 367 ( tt ) , 665 ( ta ) , and 390 ( aa ) . as expected , the obesity risk a allele frequency was 11.6% higher in obese versus lean subjects ( obese : 50.8% ; lean : 39.2% ; or 1.60 [ 1.441.78 ] ; p = 1.2 10 ) . a total of 26 different rare ( frequency < 1% in the present cohort ) nonsynonymous variants were identified in fto ( table 2 ) . the prevalence of nonsynonymous variants was similar between the two groups , with 33 lean ( 2.3% ) and 35 obese ( 2.4% ) individuals carrying mutations , and no significant differences in prevalence were observed between children and adults ( table 2 ) . eight ( a134 t , g187a , m223v , a241 t , h419r , e471 g , i492v , and v493f ) nonsynonymous mutations were identified uniquely in the obese cohort , whereas 11 ( p5l , e24k , r80p , p93r , v94i , n143s , i148r , d189n , e234d , r316q , and p399h ) were identified uniquely in the lean cohort ( table 3 ) . g187a ( n = 2 ) , a241 t ( n = 2 ) , and v493f ( n = 4 ) were identified in multiple obese subjects ( table 3 ) . seven variants ( r96h , l146 m , a163 t , v201i , s256n , r322q , and a405v ) were present in both cohorts . the prevalence of synonymous mutations was not significantly different in obese compared with lean subjects ( 0.8% vs. 0.4% , p > 0.05 ; table 4 ) . all mutations were identified in heterozygous form , and no nonsense variants were reported in the studied populations . thus , there is no obvious enrichment of nonsynonymous mutations in lean or obese individuals . summary of all nonsynonymous mutations found in obese and lean subjects data in boldface indicate combined adult and children numbers . summary of nonsynonymous mutations unique to the lean or the obese group summary of all synonymous mutations found in lean and obese subjects fto is a member of the alkb homologue ( abh ) protein family , which numbers nine homologues in humans ( 27 ) . most 2-og and fe - dependent dioxygenases slowly catalyze the conversion of 2-og to succinate even in the absence of their prime substrate ( 33 ) , and this so - called uncoupled reaction may be stimulated by substrates or their analogues . we have previously reported that murine fto is capable of demethylating the base 3-methylthymine in the context of single - stranded dna ( 27 ) , but that reaction proceeds rather slowly . human fto can also accomplish this reaction , but again at a very slow turnover rate ( 34 ) . we have previously exploited the ability of wild - type human fto to efficiently catalyze the conversion of 2-og to succinate in a manner that is stimulated by the presence of the free nucleoside 3-methylthymidine ( 32 ) , and have used this assay throughout this study . analysis based on a three - dimensional ( 3d ) homology model ( 27 ) comparing fto with the known 3d structure of three members of the family ( abh2 , abh3 , and alkb ) ( 35 ) suggests that fto can be divided in several structural and functional regions ( fig . the more conserved nh2-terminal segment contains 1 ) a putative nuclear localization signal present only in fto ( 36 ) , 2 ) a double - stranded -helix in a jellyroll fold containing all the catalytic apparatus that is well conserved in all abhs , 3 ) a substrate recognition lid that is very different in each abh member but phylogenetically well conserved among species within each paralog , and 4 ) two long insertions , absent in other abh members , that are of variable lengths in different species and are substantially less conserved than the rest of the protein . the cooh - terminal domain , whose structure and function remain unknown , is absent in all other abh members but contains several residues that are absolutely conserved among highly diverse species . predicted structural and functional regions of fto based on a 3d homology model of fto ( 27 ) . two of the naturally occurring mutations that we detected , namely r316q ( found in one lean subject ) and r322q ( found in one lean and one obese ) , replace absolutely conserved residues in the catalytic domain ( fig . 2 ) . the model predicts r316 and r322 to be involved in 2-og coordination , by forming stabilizing salt bridges with the carboxylates of this cosubstrate . we have previously studied the enzymatic property of r316q ( 32 ) , and in this study we have examined r322q compared with wild - type fto ( fig . as predicted , r322q , like r316q ( 32 ) , was completely unable to convert 2-og to succinate in either the absence or presence of 3-methylthymidine . mutants are those that have been assayed in the uncoupled reaction in the absence and presence of 3-methylthymidine . mutants that were inactive in the uncoupled reaction assay with 3-methylthymidine are shown in red , whereas those that did not affect this reaction are shown in blue . decarboxylation of c-2-oxoglutarate to c - succinate by 1.5 mol / l fto in the absence or presence of 3-methylthymidine was measured at various time intervals . a : wt1 and r322q fto proteins . wt1 , 2 , and 3 are three different wild - type fto preparations made at the same time as the mutant proteins with which they are assayed in a , b , c , or d. open symbols indicate without 3-methylthymidine ; closed symbols indicate with 1 mmol / l 3-methylthymidine ( also indicated by and + ) . e : sds10% page of fto protein preparations stained with coomassie blue . per lane , 3 g total protein was loaded . one mutation , r96h ( detected in one lean and one obese subject ) occurs at an absolutely conserved residue within the putative substrate recognition lid of the protein ( fig . 1 ) . in other members of the abh family , this region of the molecule is known to be required for binding of the primary substrate but is not involved in interactions with the cosubstrate 2-og . notably , in contrast to r322q , r96h retained some ability to convert 2-og to succinate , but this activity was not enhanced by the presence of 3-methylthymidine ( fig . 3b ) , a finding consistent with the hypothesis that r96 functions as part of the primary substrate recognition lid . we also tested the enzymatic activity of several other variants representing the different regions of fto ( p5l , v94i , i148r , m223v , e234d , a241 t , a405v , i492v , and v493f ) ( fig . 2 ) . none of these variants , all of which are located in less conserved positions , had any significant impact on enzymatic activity ( fig . bioinformatic analysis suggests that a sequence of 18 amino acid residues in the nh2-terminal of fto encodes a putative nuclear localization signal ( fig . cos-7 cells were transfected with a construct expressing wt fto - gfp ( wild - type fto green fluorescent protein ) , p5l fto - gfp mutant protein , or gfp alone ( mock - gfp ) . wt fto - gfp and p5l fto - gfp colocalized with dap1 , whereas gfp alone was seen in both the cytoplasm and the nucleus ( supplementary fig . to identify potential loss - of - function mutations , we screened the nine coding exons of fto by direct nucleotide sequencing in 1,433 extremely obese subjects ( 753 adults with a bmi > 40 kg / m and 680 children with a bmi > 97th percentile ) and in 1,433 lean subjects ( 1,221 adults with a bmi < 23 kg / m and 212 children recruited with a zbmi < 90th percentile ) according to the ecog ( 31 ) , with an average zbmi close to the 50th percentile ( zbmi = 0.1 0.98 ) ( table 1 ) . additionally , we also genotyped the fto rs9939609 snp ( 2 ) in all of the patients . genotype call rate was > 99% in both lean and obese subjects , and genotypic distribution obeyed the hardy - weinberg equilibrium in the lean control subgroup ( p = 0.58 ) . we genotyped 363 duplicated dna samples and observed a concordance rate of 100% . in the lean subgroup , genotype counts were 532 ( tt ) , 668 ( ta ) , and 224 ( aa ) . in the obese subgroup , genotype counts were 367 ( tt ) , 665 ( ta ) , and 390 ( aa ) . as expected , the obesity risk a allele frequency was 11.6% higher in obese versus lean subjects ( obese : 50.8% ; lean : 39.2% ; or 1.60 [ 1.441.78 ] ; p = 1.2 10 ) . a total of 26 different rare ( frequency < 1% in the present cohort ) nonsynonymous variants were identified in fto ( table 2 ) . the prevalence of nonsynonymous variants was similar between the two groups , with 33 lean ( 2.3% ) and 35 obese ( 2.4% ) individuals carrying mutations , and no significant differences in prevalence were observed between children and adults ( table 2 ) . eight ( a134 t , g187a , m223v , a241 t , h419r , e471 g , i492v , and v493f ) nonsynonymous mutations were identified uniquely in the obese cohort , whereas 11 ( p5l , e24k , r80p , p93r , v94i , n143s , i148r , d189n , e234d , r316q , and p399h ) were identified uniquely in the lean cohort ( table 3 ) . g187a ( n = 2 ) , a241 t ( n = 2 ) , and v493f ( n = 4 ) were identified in multiple obese subjects ( table 3 ) . seven variants ( r96h , l146 m , a163 t , v201i , s256n , r322q , and a405v ) were present in both cohorts . the prevalence of synonymous mutations was not significantly different in obese compared with lean subjects ( 0.8% vs. 0.4% , p > 0.05 ; table 4 ) . all mutations were identified in heterozygous form , and no nonsense variants were reported in the studied populations . thus , there is no obvious enrichment of nonsynonymous mutations in lean or obese individuals . summary of all nonsynonymous mutations found in obese and lean subjects data in boldface indicate combined adult and children numbers . summary of nonsynonymous mutations unique to the lean or the obese group summary of all synonymous mutations found in lean and obese subjects fto is a member of the alkb homologue ( abh ) protein family , which numbers nine homologues in humans ( 27 ) . most 2-og and fe - dependent dioxygenases slowly catalyze the conversion of 2-og to succinate even in the absence of their prime substrate ( 33 ) , and this so - called uncoupled reaction may be stimulated by substrates or their analogues . we have previously reported that murine fto is capable of demethylating the base 3-methylthymine in the context of single - stranded dna ( 27 ) , but that reaction proceeds rather slowly . human fto can also accomplish this reaction , but again at a very slow turnover rate ( 34 ) . we have previously exploited the ability of wild - type human fto to efficiently catalyze the conversion of 2-og to succinate in a manner that is stimulated by the presence of the free nucleoside 3-methylthymidine ( 32 ) , and have used this assay throughout this study . analysis based on a three - dimensional ( 3d ) homology model ( 27 ) comparing fto with the known 3d structure of three members of the family ( abh2 , abh3 , and alkb ) ( 35 ) suggests that fto can be divided in several structural and functional regions ( fig . the more conserved nh2-terminal segment contains 1 ) a putative nuclear localization signal present only in fto ( 36 ) , 2 ) a double - stranded -helix in a jellyroll fold containing all the catalytic apparatus that is well conserved in all abhs , 3 ) a substrate recognition lid that is very different in each abh member but phylogenetically well conserved among species within each paralog , and 4 ) two long insertions , absent in other abh members , that are of variable lengths in different species and are substantially less conserved than the rest of the protein . the cooh - terminal domain , whose structure and function remain unknown , is absent in all other abh members but contains several residues that are absolutely conserved among highly diverse species . predicted structural and functional regions of fto based on a 3d homology model of fto ( 27 ) . two of the naturally occurring mutations that we detected , namely r316q ( found in one lean subject ) and r322q ( found in one lean and one obese ) , replace absolutely conserved residues in the catalytic domain ( fig . 2 ) . the model predicts r316 and r322 to be involved in 2-og coordination , by forming stabilizing salt bridges with the carboxylates of this cosubstrate . we have previously studied the enzymatic property of r316q ( 32 ) , and in this study we have examined r322q compared with wild - type fto ( fig . as predicted , r322q , like r316q ( 32 ) , was completely unable to convert 2-og to succinate in either the absence or presence of 3-methylthymidine . mutants are those that have been assayed in the uncoupled reaction in the absence and presence of 3-methylthymidine . mutants that were inactive in the uncoupled reaction assay with 3-methylthymidine are shown in red , whereas those that did not affect this reaction are shown in blue . decarboxylation of c-2-oxoglutarate to c - succinate by 1.5 mol / l fto in the absence or presence of 3-methylthymidine was measured at various time intervals . wt1 , 2 , and 3 are three different wild - type fto preparations made at the same time as the mutant proteins with which they are assayed in a , b , c , or d. open symbols indicate without 3-methylthymidine ; closed symbols indicate with 1 mmol / l 3-methylthymidine ( also indicated by and + ) . e : sds10% page of fto protein preparations stained with coomassie blue . per lane , 3 g total protein was loaded . one mutation , r96h ( detected in one lean and one obese subject ) occurs at an absolutely conserved residue within the putative substrate recognition lid of the protein ( fig . 1 ) . in other members of the abh family , this region of the molecule is known to be required for binding of the primary substrate but is not involved in interactions with the cosubstrate 2-og . notably , in contrast to r322q , r96h retained some ability to convert 2-og to succinate , but this activity was not enhanced by the presence of 3-methylthymidine ( fig . 3b ) , a finding consistent with the hypothesis that r96 functions as part of the primary substrate recognition lid . we also tested the enzymatic activity of several other variants representing the different regions of fto ( p5l , v94i , i148r , m223v , e234d , a241 t , a405v , i492v , and v493f ) ( fig . 2 ) . none of these variants , all of which are located in less conserved positions , had any significant impact on enzymatic activity ( fig . bioinformatic analysis suggests that a sequence of 18 amino acid residues in the nh2-terminal of fto encodes a putative nuclear localization signal ( fig . cos-7 cells were transfected with a construct expressing wt fto - gfp ( wild - type fto green fluorescent protein ) , p5l fto - gfp mutant protein , or gfp alone ( mock - gfp ) . wt fto - gfp and p5l fto - gfp colocalized with dap1 , whereas gfp alone was seen in both the cytoplasm and the nucleus ( supplementary fig . recent genome - wide association efforts have been highly successful in identifying a large number of common genetic variants reliably associated with important human diseases and quantitative traits ( 2,32,37 ) . understanding the precise biological mechanisms underlying such associations will provide a major scientific challenge that will require a multiplicity of approaches in humans and in model organisms . although snps in intron 1 of fto are unequivocally associated with adiposity in multiple populations , there is still no evidence that those snps influence the expression or splicing of fto itself . fto is located adjacent to other genes , the expression or function of which could conceivably influence energy balance ( 10,36,38 ) . recent findings in mice rendered null for fto support the notion that fto itself has an important influence on energy balance . fto - null mice are small , are lean , have an increased metabolic rate , and are hyperphagic , whereas fto mice are resistant to diet - induced obesity ( 39 ) . given that information , it is reasonable to speculate that loss - of - function mutations in fto might be more common in lean rather than obese humans . to test this hypothesis , we sequenced fto in a large number of lean and obese subjects and found that 1 ) nonsynonymous mutations were equally common in both the obese and lean cohorts and 2 ) heterozygous mutations that severely impaired enzymatic activity of fto were found in both lean and obese individuals with no other obvious major clinical phenotypes . genes whose candidacy derives largely from genome - wide association studies in extreme phenotypes , of studying both ends of the spectrum of a quantitative trait . ( if we had considered fto to be an obesity gene and sequenced only obese subjects , the finding of loss - of - function mutations would have been misleading . ) from our human genetic data , we can conclude that heterozygosity for a severely dysfunctional fto allele is compatible with being either lean or obese in humans . understanding the relationship between snps in intron 1 of fto with human adiposity will require other approaches . in collaboration with dr . laurence colleaux from hopital necker paris and others , we have recently found a consanguineous israeli - arab family in which nine siblings were homozygous for the r316q mutation in fto ( 32 ) . all homozygous carriers were severely growth retarded , had multiple congenital malformations , and died in infancy . although heterozygous parents of these children had no obvious metabolic phenotype , ongoing efforts are directed at establishing measures of adiposity in heterozygous versus wild - type relatives ( 32 ) . our studies have brought new insights into aspects of the structure - function relationship of the human fto enzyme . human fto can catalyze the uncoupled reaction ( 2-og to succinate ) and is stimulated by 3-methylthymidine ( 32 ) , and we have used this as a robust test of fto 's catalytic activity . as predicted , we found that the arginine at position 322 is essential for the catalytic activity of human fto , and we have made similar observations for r316q ( 32 ) . mutation of the arginine corresponding to human 316 in mouse fto ( r313a ) also generated an inactive enzyme ( 27 ) . perhaps more interestingly , we have demonstrated that mutation of r96 to histidine in human fto produces an enzyme that is capable of some basal conversion of 2-og to succinate but is insensitive to the effects of 3-methylthymidine . these findings are consistent with r96 being part of the so - called substrate recognition lid that is responsible for substrate fixation and selectivity . thus , the lid is well conserved among species orthologues , but has diverged substantially in the various paralogs of abh to accommodate different substrates . for instance , r131 at this position in the lid of abh3 is positioned to interact with 1-mea n3 , and r131a in abh3 abolishes the activity toward 1-me - a containing single - stranded dna ( 27 ) . although all of the other nonsynonymous variants we tested appeared to have normal enzymatic activity , we should express caution about categorically stating that these are fully wild type in function . first , we do not know what the true natural primary substrate(s ) of fto is ( are ) , and it is entirely possible that mutations that affect fto 's actions on this elusive substrate will not necessarily impair the uncoupled reaction . second , although we have demonstrated that fto , in vitro , possesses dioxygenase activity , whether it has any other biological role , enzymatic or otherwise , is yet to be determined . this could potentially be of relevance to v493f , a cooh - terminal mutation that was found only in the obese population but was found in four different individuals ( fig . 2 ) , and yet did not impact on the ability of fto to convert 2-og to succinate or its stimulation by 3-methylthymidine ( fig . the cooh - terminal region of fto , although highly conserved across species , is not shared with other members of the abh family , and its structure and function are unknown . we noted two potentially interesting observations regarding the cooh - terminal region of the protein . first , on a mutations per nucleotide basis , nonsynonymous mutations are found 3 times less frequently in the cooh - terminus than in the rest of the molecule . second , although nonsynonymous variants elsewhere in the molecule are found equally in obese and lean subjects , eight such variants found in the cooh - terminus were detected in obese subjects and only two in lean ( fig . these preliminary observations are of potential interest and might lead to a better understanding of fto function and its role in energy homeostasis localization .

objectivesingle nucleotide polymorphisms ( snps ) in intron 1 of fat mass and obesity - associated gene ( fto ) are strongly associated with human adiposity , whereas fto/ mice are lean and fto+/ mice are resistant to diet - induced obesity . we aimed to determine whether fto mutations are disproportionately represented in lean or obese humans and to use these mutations to understand structure - function relationships within fto.research design and methodswe sequenced all coding exons of fto in 1,433 severely obese and 1,433 lean individuals . we studied the enzymatic activity of selected nonsynonymous variants.resultswe identified 33 heterozygous nonsynonymous variants in lean ( 2.3% ) and 35 in obese ( 2.4% ) individuals , with 8 mutations unique to the obese and 11 unique to the lean . two novel mutations replace absolutely conserved residues : r322q in the catalytic domain and r96h in the predicted substrate recognition lid . r322q was unable to catalyze the conversion of 2-oxoglutarate to succinate in the presence or absence of 3-methylthymidine . r96h retained some basal activity , which was not enhanced by 3-methylthymidine . however , both were found in lean and obese individuals.conclusionsheterozygous , loss - of - function mutations in fto exist but are found in both lean and obese subjects . although intron 1 snps are unequivocally associated with obesity in multiple populations and murine studies strongly suggest that fto has a role in energy balance , it appears that loss of one functional copy of fto in humans is compatible with being either lean or obese . functional analyses of fto mutations have given novel insights into structure - function relationships in this enzyme .

RESEARCH DESIGN AND METHODS Genotyping of the FTO rs9939609 SNP. Sequencing. Statistical analysis. Cloning of human FTO. Human wild-type and mutant FTO protein production. Assays of FTO activity. RESULTS Prevalence of nonsynonymous mutations in lean and obese subjects. Functional properties of nonsynonymous mutations. DISCUSSION Supplementary Material

in the last 30 years , we have made great strides in the treatment of ms . we have developed and brought to market seven disease - modifying medications to reduce the frequency of ms relapses and slow the accumulation of irreversible disability , and many therapies are available to control ms - related symptoms . however , one of the significant challenges for developing new and better therapies for ms is that current measures , including the neurological examination , the expanded disability status scale ( edss ) , the multiple sclerosis functional composite ( msfc ) , mri measures , and relapse rates , have limited ability to capture between - relapse and ongoing disease progression in the absence of relapses . as dennis bourdette , m.d . , chairman of the department of neurology at oregon health & science university ( ohsu ) said , what we need [ in ms ] are measures that are quick , easy , accurate , and sensitive to change both worsening and improvement and are understandable [ to clinicians] gait and balance measures have the potential to meet these requirements . he emphasized the need for communication between neurologists and others caring for people with ms and conducting clinical trials and nonneurologists engaged in developing and testing measures of gait and balance . clinician - administered measures of gait and balance are the measures most commonly used in clinical practice . these measures are generally inexpensive and easy to administer , and many have been validated in people with ms . however , as highlighted by rebecca spain , m.d . , msph , a neurologist from ohsu , because of the diverse goals and perspectives of different clinicians , one measure is not likely to meet every clinician 's needs . neurologists localize neuroanatomically and seek guidance for recommending medications that globally effect ms - related health . in contrast , physical therapists and engineering - based researchers evaluate biomechanical details that underlie gait and balance abnormalities and seek improved methods to direct targeted rehabilitation interventions . , a physiatrist from the cleveland clinic , noted that many of the currently used clinician - administered measures of gait , including the timed 25-foot walk ( t25fw ) , the 6-minute and 2-minute walk tests ( 2mwt , 6mwt ) , and the timed up and go ( tug ) test , can detect changes in gait speed or endurance but do not detect visually obvious improvements in gait quality , or assess important contributors to gait changes such as reduced cognitive function , visual function , upper body trunk control , spasticity , or sensation . although this limits their utility for guiding selection of specific interventions , these simple clinician - administered measures are still recommended for screening individuals for gait impairment and for standardized assessment of performance over time . susan bennett , pt , ed d from the university of buffalo , described some of the more sophisticated clinician - administered measures of balance , such as the berg balance scale ( bbs ) , the functional reach test , and the recently developed 4-square - step test and mini - best test . these measures can differentiate between people with ms who fall from those who do not ( bbs , 4-square - step test ) and can provide information to guide rehabilitation . dr . bennett also emphasized the utility of the trunk control test [ 12 , 13 ] for assessing balance in nonambulatory patients for whom postural control of the trunk is essential for safe and independent transfers . , from the university of illinois at chicago , noted that self - reported measures , also known as patient reported outcome measures ( proms ) , have recently attracted attention from government agencies , including the national institutes of health and food and drug administration , because these measures capture directly from patients how they feel or function without interpretation by others . self - reported measures of gait and balance can ascertain patients ' quality of life , balance confidence , fear of falling , circumstances surrounding fall events , and other important information that can not be determined by clinical testing or physical instrumentation . these data are usually best captured with diaries , questionnaires , surveys , or web - based methods . , from saint louis university , discussed the ms walking scale-12 ( msws-12 ) , the only prom specific to walking in ms . msws-12 scores correlate with clinician - administered and instrumented measures of walking , including the edss ( r = 0.65 to 0.84 ) [ 14 , 15 ] , the t25fw ( r = 0.46 to 0.65 ) [ 14 , 15 ] , the 6mwt ( r = 0.81 ) , the metabolic cost of walking ( r = 0.64 ) , and free - living accelerometry ( r = 0.48 to 0.79 ) [ 17 , 18 ] . the msws-12 also has high test - retest reliability ( icc 0.75 to 0.87 ) [ 14 , 19 ] , was responsive to change in two medication trials in ms , and has acceptable floor and ceiling effects . a second version of the msws-12 , adapted from the first version based on rasch analysis , has been developed and submitted for publication ( hobart , personal communication ) . , pt , a neurologist from ohsu , discussed the activities - specific balance confidence ( abc ) scale and the dizziness handicap inventory ( dhi ) , the two self - administered measures of balance measures frequently used in people with ms which , although originally developed for other populations , have high test - retest reliability in people with ms ( abc 0.92 , dhi 0.90 ) . they also have fair concurrent validity with each other and identify individuals who fall better than a number of clinician - administered measures including the bbs , the tug test , the ambulation index , and the dynamic gait index . both the abc and dhi have also been found to be responsive to the change in trials of rehabilitation interventions . , from orebro university in sweden , focused on using self - reported measures to identify people with ms at increased risk for falling and to determine predictors of falls in people with ms . in a recent study by dr . nilsagrd , close to 2/3 of ms subjects recorded falls prospectively in a 3-month period . factors associated with falls were imbalance , weakness , fatigue , and environmental or task - specific factors including being bumped by others , walking in crowds , on ramps , or while carrying objects . self - administered measures of gait and balance in ms provide insight into patients ' perspectives about their abilities , are inexpensive to administer , most have minimal floor and ceiling effects , and identify who is at increased risk for falls . however , because these measures do not provide information on neuroanatomical , biomechanical , environmental , or personal factors contributing to gait and balance impairment in ms , or shed light on the impact of activity limitations on an individual 's participation or quality of life , they give limited guidance for intervention . the speakers recommended using these measures , in addition to clinician - administered measures , for screening people with ms for gait dysfunction , imbalance , and fall risk , and as outcome measures in trials of interventions expected to have a positive impact on mobility and quality of life . when precise information about gait and balance are needed , instrumented measures are recommended . as related by fay horak , pt , ph.d . , and jessie huisinga , ph.d . from ohsu , and robert motl , ph.d . , from the university of illinois at urbana - champaign , until recently , sophisticated and bulky equipment installed in specialized motion - analysis laboratories was needed to obtain precise , accurate quantitative gait and balance data . now , however , wearable sensors housing triaxial accelerometers , gyroscopes , and magnetometers are increasingly used in research settings , and a variety of pedometers and accelerometers are readily available for clinical and home use . these portable devices are also less expensive , require less technical expertise than laboratory - based instruments , and can be used to assess gait and balance in various environments including at home and other real world situations . wearable sensors may have the sensitivity to detect ms - related disability when the clinician can not and may provide more precise long - term monitoring of disability progression , thus reducing the sample size and trial duration required to determine treatment efficacy . despite their great potential , currently , integration of novel instrumented measures of gait and balance into ms clinical practice and research is limited by lack of test protocol and device standardization . we also need to know if these devices are sensitive to changes associated with ms progression and treatment . however , their validation is challenging because it is not clear what measures should be used as a gold standard for comparison . in addition , we do not know how acceptable , feasible , and affordable these new instrumented measures will be for clinical practice and large - scale clinical trials . the final speaker of the day , kathleen zackowski , ph.d . , otr , from kennedy krieger institute and johns hopkins university , discussed current research on the relationships between conventional as well as novel mri measures such as magnetization transfer ( mt ) and diffusion tensor imaging ( dti ) with instrumented measures of specific impairments as well as balance and gait changes . these methods allow for a noninvasive evaluation of white matter that is especially useful in diseases where myelin and axonal integrity are disrupted [ 2630 ] . mt indirectly assesses the status of water protons associated with macromolecular structures in tissues such as myelin and is especially useful for studying white matter integrity because white matter has such a high myelin content . however , mt can sensitively quantify white and gray matter abnormalities in both brain and spinal cord [ 3234 ] . dti - derived indices are sensitive to macroscopic and microscopic tissue disruption and appear to have higher specificity than conventional imaging for areas affected by ms pathology . using tractography , dti can define the approximate anatomy of individual white matter tracts within the brain and spinal cord . dti can identify abnormal values of mri indices in specific white matter tracts that may underlie clinical disability in ms , and the integration of information derived from other imaging sequences , such as mt , can increase pathologic specificity [ 34 , 37 ] . dti abnormalities in the corticospinal tract have been shown to correlate with weakness in ms . these advanced mri measures may be able to detect and quantify subtle subclinical changes in function impacted by neurorehabilitation and link these with disease pathology . for example , zackowski et al . demonstrated relationships between instrumented walking velocity measures and dti and mt indices of the spinal cord in ms . new mri techniques together with novel instrumented measures of gait and balance may also allow us to define the anatomic substrates of disability and to design specific innovative rehabilitation strategies for people with ms based on the relationships between structure and function . however , larger , more comprehensive studies using a combination of instrumented measures of walking and clinical rating scales , along with more specific mri indices , such as those from dti and mt , are needed . the panelists concurred that measures of gait and balance dysfunction should be used throughout the course of ms;currently , available simple clinician and self - administered measures are recommended for screening for imbalance , gait dysfunction , and fall risk ; self - administered measures are important for capturing the patients ' perspective of their abilities and the effectiveness of interventions ; more complex clinician - administered measures and instrumented measures are recommended to guide the selection and development of tailored interventions for walking and balance problems in people with ms and for precise monitoring of change in clinical trials ; remaining critical research needs are to identify and standardize the most scientifically valid and practically useful measures to capture the continuum of gait and balance deficits in ms and to understand the relationships between these measures.abstracts of posters of primary research presented at this symposium were published in winter 2011 international journal of ms care . the second international gait and balance symposium in ms is scheduled to be held in portland , oregon on october 19th and 20th 2012 and will focus on interventions for gait impairment , imbalance , and falls in people with ms . measures of gait and balance dysfunction should be used throughout the course of ms ; currently , available simple clinician and self - administered measures are recommended for screening for imbalance , gait dysfunction , and fall risk ; self - administered measures are important for capturing the patients ' perspective of their abilities and the effectiveness of interventions ; more complex clinician - administered measures and instrumented measures are recommended to guide the selection and development of tailored interventions for walking and balance problems in people with ms and for precise monitoring of change in clinical trials ; remaining critical research needs are to identify and standardize the most scientifically valid and practically useful measures to capture the continuum of gait and balance deficits in ms and to understand the relationships between these measures .

gait and balance measures have particular potential as outcome measures in multiple sclerosis ( ms ) because , of the many hallmarks of ms disability , gait and balance dysfunction are present throughout the course of the disease , impact many aspects of a person 's life , and progress over time . to highlight the importance and relevance of gait and balance measures in ms , explore novel measurements of gait and balance in ms , and discuss how gait , balance , and fall measures can best be used and developed in clinical and research settings , the 1st international symposium on gait and balance in multiple sclerosis was held in portland , oregon , usa on october 1 , 2011 . this meeting brought together nearly 100 neurologists , physiatrists , physical therapists , occupational therapists , nurses , engineers , and others to discuss the current status and recent advances in the measurement of gait and balance in ms . presentations focused on clinician - administered , self - administered , and instrumented measures of gait , balance , and falls in ms .

1. Statement of Need 2. Clinician-Administered Measures 3. Self-Administered Measures 4. Instrumented Measures 5. Relationships between Novel MRI Measures and Instrumented Measures of Gait Impairment 6. Discussion

harmony among the skeletal , dental , and soft - tissue structures is a prerequisite for good occlusion . disharmony in these structures results in malocclusion.1 for proper intercuspation , the teeth must be proportional in size . if teeth are mismatched , with unusually large teeth in one arch compared to the other , then an ideal occlusion can not be attained , which is not uncommon and defined as tooth size discrepancy ( tsd).1 a significant variation in this harmony will lead to malocclusion and difficulties in obtaining an occlusion with optimal overjet , overbite , and class i canine and molar relationships . the dental cast facilitates the analysis of tooth size and shape , alignment and rotations of the teeth , presence or absence of teeth , arch width , length , form and symmetry ; and the occlusal relationship . tsd can be assessed using diagnostic setup or using a mathematical formula like the bolton analysis.1 before treatment , it is necessary to identify total bolton index ( tbi ) because teeth removal has a direct influence on upper and lower tsd , also on upper and lower incisors position.2 bolton stated that after four premolars are removed , normal tbi value was between 87% and 89% when upper teeth sizes suited lower teeth sizes . when teeth are too wide in the upper jaw - tbi is lower than 87% ( low tbi ) and when teeth are too wide in the lower jaw - tbi value is higher than 89% ( high tbi).3 when malocclusion requires extraction , tooth size differences and spaces are often seen at the end of treatment.4 many investigators have expressed the opinion that premolar extraction is responsible for tsd in some cases but none of them have reported on the percentage of cases in which this occurs . the purpose of this study was to determine the prevalence of tooth size discrepancies among orthodontic patients in general and also between different malocclusion groups , to analyze the change in the degree of severity in bolton discrepancy before and after the hypothetical extraction and to identify any incidence in bolton discrepancy taking place after hypothetical extractions in normal or control groups without any bolton discrepancy . this study was conducted on 100 pre - treatment diagnostic casts collected on randomized clinical trials from the department of orthodontics in bapuji dental college , davangere . sufficiently erupted permanent teeth to allow measurement of their widest mesiodistal dimensionsstudy models without any mutilated teeth . sufficiently erupted permanent teeth to allow measurement of their widest mesiodistal dimensions study models without any mutilated teeth . the teeth were measured with a fine point mitutoyo dial calipers ( made in japan , model no . -633 -50 d15 ) to the nearest of 0.02 mm ( figures 1 and 2 ) . the mesiodistal widths of 12 maxillary teeth and 12 mandibular teeth from right to left first permanent molar are totaled and compared . these mesio - distal crown measurements were taken from mesial and distal contact areas , respectively ( figure 3 ) . the dividend of two is the percentage relationship of mandibular tooth size to maxillary which is called as to over - all ratio . subsequent to calculated pre - treatment bolton s value ( bv ) , hypothetical extraction of four premolars was accomplished by substituting these combinations were : ( 1 ) removal of all first premolars , ( 2 ) removal of all second premolars , ( 3 ) removal of upper first and lower second premolars , ( 4 ) removal of upper second and lower first premolars . the resultant measurements were again subjected to bolton s analysis to see whether a tooth size discrepancy had been created . finally , the pre - treatment and postextraction tooth size ratios and bv were evaluated statistically by the use of paired student s t - test . incidence of tooth discrepancy in different groups of malocclusion and its relation to extraction patients name : _ _ _ _ _ _ _ _ _ _ _ _ _ _ date : sex : _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ type of malocclusion : _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ mesiodistal tooth sizes ( mm ) if overall ratio exceeds 91.3%if overall ratio is < 91.3% if overall ratio exceeds 91.3% if overall ratio is < 91.3% similar proforma was followed for different extraction patterns as mentioned earlier . sufficiently erupted permanent teeth to allow measurement of their widest mesiodistal dimensionsstudy models without any mutilated teeth . sufficiently erupted permanent teeth to allow measurement of their widest mesiodistal dimensions study models without any mutilated teeth . the teeth were measured with a fine point mitutoyo dial calipers ( made in japan , model no . -633 -50 d15 ) to the nearest of 0.02 mm ( figures 1 and 2 ) . the mesiodistal widths of 12 maxillary teeth and 12 mandibular teeth from right to left first permanent molar are totaled and compared . these mesio - distal crown measurements were taken from mesial and distal contact areas , respectively ( figure 3 ) . the dividend of two is the percentage relationship of mandibular tooth size to maxillary which is called as to over - all ratio . subsequent to calculated pre - treatment bolton s value ( bv ) , hypothetical extraction of four premolars was accomplished by substituting these combinations were : ( 1 ) removal of all first premolars , ( 2 ) removal of all second premolars , ( 3 ) removal of upper first and lower second premolars , ( 4 ) removal of upper second and lower first premolars . the resultant measurements were again subjected to bolton s analysis to see whether a tooth size discrepancy had been created . finally , the pre - treatment and postextraction tooth size ratios and bv were evaluated statistically by the use of paired student s t - test . incidence of tooth discrepancy in different groups of malocclusion and its relation to extraction patients name : _ _ _ _ _ _ _ _ _ _ _ _ _ _ date : sex : _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ type of malocclusion : _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ mesiodistal tooth sizes ( mm ) if overall ratio exceeds 91.3%if overall ratio is < 91.3% if overall ratio exceeds 91.3% if overall ratio is < 91.3% similar proforma was followed for different extraction patterns as mentioned earlier . in this study , 100 pre - treatment casts were randomly collected , of these samples 71 were falling within bv of 91.3% 1 standard deviation ( sd ) , 24 were falling within bv of 91.3% 2 sd , and 5 were above bv 2 sd ( table 1 and graph 1 ) . , there was an anterior discrepancy with a mean value of 78.5% with a sd of 2.9 mm , showing no statistical significance ( table 2 ) . furthermore , in the overall discrepancy with a value of 91.9% with a sd of 2.1 mm , showed no statistical significance ( table 3 ) . the f value obtained from anova test was 1.33 for the anterior ratio for different malocclusion groups , class i , class ii division 1 , class ii division 2 , and class iii malocclusion . the p value was 0.27 ( > 0.05 ) suggesting that it is clinically insignificant ( table 4 ) . the mean values for class i , class ii division 1 , class ii division 2 , and class iii malocclusions for anterior ratio are indicated in graph 2 . f value of 3.34 and a p value 0.05 indicating statistically significant but clinically insignificant ( table 5 ) . the mean value for class i , class ii division 1 , class ii division 2 , and class iii malocclusion for the overall ratio are shown in graph 3 . hypothetical extractions were conducted for different combinations and the values are subjected again to bolton s analysis . all second premolar extraction exhibits the minimum t value of 2.96 and a p value of 0.01 suggesting that it is statistically significant but clinically insignificant ( tables 6a - d ) . the graph 4 shows a comparison of mean bv before and after various patterns of extraction for bolton group . comparison of mean bolton value between before and after all first premolar extraction pattern in bolton group . comparison of mean bolton value between before and after all second premolar extraction pattern in bolton group . comparison of mean bolton value between before and after upper 4 first premolar lower second premolar extraction patterns in bolton group . comparison of mean bolton value between before and after upper second premolar lower first premolar extraction pattern in bolton group . comparison of mean bolton value between before and after various premolar extractions in bolton group . for an orthodontist , the most gratifying realization of balance in the denture is a treated case , which remains unaltered for a long period after removal of retaining appliance . if the teeth are mismatched with unusually large teeth in one arch compared with the other , then an ideal occlusion can not be attained then a tsd develops . many investigators have expressed the opinion that removal of premolars is responsible for creating a tsd in some cases but none of them have reported on the percentage of cases in which this occurs.5 the present study conducted demonstrates the prevalence of tooth size discrepancies among orthodontic patients in various malocclusions . bolton s analysis in 1958 included comparisons of total mesiodistal widths of dental arches up to the distal surfaces of the first molars and gave the ideal anterior and overall ratio . from bolton s results , it can be seen that there is a relatively small range in which tooth size ratios should fall , to achieve optimal occlusal relationship . anterior coefficient to be used as a guide to the finished relationship of the anterior segments.6 comparison of anterior discrepancy and overall discrepancy of the present study sample ( local population ) and bolton s sample was done which showed that overall ratio was almost matching but the anterior ratio was slightly higher than the anterior ratio of bolton sample . this difference could be because the present study was done on local population sample which is randomly collected and the bolton s study was on the caucasian population sample selected with a criterion of good occlusion.7 the mean , sd , and range for class i , class ii division 1 , class ii division 2 , and class iii malocclusion8 were calculated for both anterior ratio and overall ratio . in the present study , no statistical significant difference is seen between findings indicating that the tooth discrepancy is not related to jaw relationship . this is in confirmation with study done by alkofide and hashim9 who found that there is no statistical significant difference between the different classes of malocclusion . there was a maximum increase in the mean bv following all first premolar extraction and minimum increase was noticed in all second premolar extraction indicating that again following all first premolar premolar extractions mandibular discrepancy is increasing significantly and this is in confirmation with the with study done by saatci and yukay,10 showing that all second premolar extraction is favorable compared to other patterns of premolar extraction especially with all first premolar extraction in this group . in this study the same procedure has been followed for mesio - distal measurements as suggested by bolton3 in 1958 . it was found that the difference between the pre - treatment bv and the values after removal of first premolars was statistically significant and not significant after second premolar removal . conversely , the extraction of all second premolars did not increase discrepancies that had existed before treatment and instead it reduced in some subjects . it was also noted in the study that , if we extract premolars of equal mesiodistal dimension from upper and lower dental arches , more severe tooth - size discrepancies were created when compared with the removal of greater mandibular premolars . it appeared that , because most discrepancies created by extraction occurred as a mandibular excess , removal of the mandibular second premolars , which usually has wider mesio - distal dimensions , was likely to result in discrepancies of a smaller size than the mandibular first premolars.11 therefore this result is in agreement with the opinion expressed by bolton that the removal of mandibular second premolars often crates the potential for a better occlusion than the removal of the first premolars , as the mandibular molars are allowed more mesial movement . however , bolton also cautioned , as we do , that this statement should not be interpreted as a broad recommendation for extraction of mandibular second premolars . the results obtained in this study suggest a new point of view to the question of which teeth to extract when evaluated from a tooth - size discrepancy12 standpoint only . the question of the reduction of tooth structure as a treatment procedure in orthodontics has always been a controversial one . controversy centers on how far we should go and what the dividing line between extraction and non - extraction is . the decision to extract must be preceded by a great deal of thought and study . there was a significant difference in the anterior ratio between the present study and bolton s sample and the overall ratio did not show any significant differencea specific malocclusion group was not shown to contain a larger percentage of tooth size discrepanciesthe results obtained in the present study are in agreement with the removal of all second premolars in mandibular discrepancy cases and removal of all first premolars in maxillary discrepancy cases . there was a significant difference in the anterior ratio between the present study and bolton s sample and the overall ratio did not show any significant difference a specific malocclusion group was not shown to contain a larger percentage of tooth size discrepancies the results obtained in the present study are in agreement with the removal of all second premolars in mandibular discrepancy cases and removal of all first premolars in maxillary discrepancy cases . the inference of this study suggests a new point of view of the question of which teeth to extract when evaluated from a tsd standpoint only . the present study accordingly concludes that clinicians should always remember to look on each patient individually and be aware of other factors in determining what teeth , if any ? should be removed and use these findings only as one factor to be considered together with many others .

background : for proper intercuspation , the teeth must be proportional in size . if teeth are mismatched , with unusually large teeth in one arch compared to the other , then an ideal occlusion can not be attained . this study has been done to determine the prevalence of tooth size discrepancies among orthodontic patients in general and also between different malocclusion groups , sex , and to analyze the change in the degree of severity in bolton discrepancy before and after the hypothetical premolar extraction.methods:the study was carried out on randomly collected 100 pre - treatment dental casts . tooth size analyses were performed on these pre - treatment models and mesio distal tooth size ratios were measured as described by bolton before and after various patterns of hypothetical extraction.result:the results were statistically evaluated using anova and paired samples t - test . 5 out of 100 patients are seen with severe bolton discrepancy with bolton values ( bv ) ranging above and below 2 standard deviation . statistically insignificant difference is seen between males and females and also between various groups of malocclusion . the difference between the pre - treatment and post extraction bv was found statistically significant for the first premolar extraction and insignificant for others.conclusion:the results of this study indicate a new point of view to the question of which teeth to extract when evaluated for tooth size aspect only .

Introduction Methods Inclusion criteria Proforma Results Discussion Conclusion

anti--tubulin mouse monoclonal antibody ( sigma - aldrich corp ) , anti - arf6 mouse monoclonal antibody ( santa cruz biotechnology , inc ) , anti - rab11 rabbit polyclonal antibody , anti - tfr mouse monoclonal antibody ( invitrogen corp ) , anti - eea-1 mouse monoclonal antibody and anti - gm130 mouse monoclonal antibody ( bd biosciences ) were obtained commercially . s2 was performed with a fluorophore - conjugated anti - tfr antibody that was generated by using the zenon antibody labeling kit ( invitrogen corp ) . using this antibody slightly enhanced the tfr signals at the plasma membrane ( figs . s1 and s2 ) . all of the procedures used to perform cell culture , immunoblotting and the immunofluorescence analyses have been described elsewhere . anti--tubulin mouse monoclonal antibody ( sigma - aldrich corp ) , anti - arf6 mouse monoclonal antibody ( santa cruz biotechnology , inc ) , anti - rab11 rabbit polyclonal antibody , anti - tfr mouse monoclonal antibody ( invitrogen corp ) , anti - eea-1 mouse monoclonal antibody and anti - gm130 mouse monoclonal antibody ( bd biosciences ) were obtained commercially . s2 was performed with a fluorophore - conjugated anti - tfr antibody that was generated by using the zenon antibody labeling kit ( invitrogen corp ) . using this antibody slightly enhanced the tfr signals at the plasma membrane ( figs . s1 and s2 ) . all of the procedures used to perform cell culture , immunoblotting and the immunofluorescence analyses have been described elsewhere .

recycling endosomes are key platforms for endocytic recycling that return internalized molecules back to the plasma membrane . to determine how recycling endosomes perform their functions , searching for proteins and lipids that specifically localized at recycling endosomes has often been performed by colocalization analyses between candidate molecules and conventional recycling endosome markers . however , it remains unclear whether all the conventional markers have identical localizations . here we report finding that three well - known recycling endosome markers , i.e. , arf6 , rab11 and transferrin receptor ( tfr ) , have different intracellular localizations in pc12 cells . the results of immunofluorescence analyses showed that the signals of endogenous arf6 , rab11 and tfr in nerve growth factor - stimulated pc12 cells generally differed , although there was some overlapping . our findings provide new information about recycling endosome markers , and they highlight the heterogeneity of recycling endosomes .

Materials and Methods Antibodies Cell culture, immunoblotting and immunofluorescence analyses Supplementary Material

low back and neck pain are commonly seen in the workplace , and are the two most common spinal problems encountered in clinical practice . back and neck pain are important clinical conditions that can limit the ability to perform routine daily activities , decrease productivity , and negatively affect quality of life . it is reported that 80% of individuals have complained of back pain in some period of their lives1 . although neck pain does not occur as frequently as low back pain , it is also a common problem , with a 10% lifetime prevalence2 . in turkey , as in other countries1 , low back and neck pain are major health problems that affect individual motivation , and result in physical and psychological problems . kinesiophobia is among the most extreme forms of fear of pain due to movement or re - injury . in chronic cases , pain severity and cognitive responses to pain . a person with chronic pain may develop negative beliefs about their experience of pain or negative thoughts about themselves3 . patients with kinesiophobia believe that movement will cause re - injury and additional pain ; therefore , kinesiophobia is a risk factor for persistent pain . in the long term , kinesiophobia causes physical deconditioning , avoidance of physical activity , functional disability , and symptoms of depression . it is reported that psychological factors play an important role in the process of chronicity of the disease3,4,5,6 . several scoring systems have been reported , such as the tampa scale for kinesiophobia ( tsk ) , fear avoidance beliefs questionnaire ( fabq ) , and pictorial fear of activity scale - cervical ( pfacts - c ) , and are used to assess fear of movement - related pain / re - injury in patients with low back and neck pain7 . in a study on patients with neck and shoulder pain , a strong relationship between kinesiophobia , disability , and musculoskeletal system injuries was reported8 . in addition , patients who have chronic low back pain with high levels of fear of movement - related pain have high pain and disability scores8 . pain and restricted movement in patients with chronic low back and neck pain result in boredom , anxiety , and depression , negatively affecting the quality of life and causing disability9,10,11,12 . pain results in varying degrees of kinesiophobia , affective problems , and ultimately negatively affects the quality of life . the present study aimed to compare pain , kinesiophobia , and quality of life scores in patients with low back and neck pain . the study was performed at hacettepe university hospital , department of physical medicine and rehabilitation , ankara , turkey . patients 2065 years old with various diagnoses such as disc herniation , spondylosis , strain , sprain , or mechanical pain in the cervical ( neck , n=300 ) or lumbar ( low back , n=300 ) region with 6 months of pain were included in the study . patients who had any history of malignancy or a spinal fracture , had undergone any surgical procedure in the previous 6 months , or had orthopedic or neurological disease affecting ambulation were excluded from the study . patients who agreed to participate in this study signed informed consent forms , and the study was approved by the hacettepe university , non - invasive medical research and ethics committee , protocol number lut 09/41 - 56 . demographic data including gender , diagnosis , age ( years ) , height ( cm ) , weight ( kg ) , body mass index ( bmi , kg / cm ) , and duration of pain ( months ) were recorded . the turkish version of the short - form mcgill pain questionnaire ( sf - mpq ) was used to assess pain severity and properties13 . the main component of the sf - mpq consists of 15 descriptive adjectives for the sensation of pain ( 11 sensory and 4 affective ) that are self - rated according to their level of intensity on a 4-point likert - type scale ( 0=none , 1=mild , 2=moderate , 3=severe ) . the 3 pain scores are derived from the sum of the intensity scores for sensory , affective , and total description . the sensory and affective scores are calculated by adding the sensory and affective intensity scores . sf - mpq also includes a visual analogue scale ( vas ) for measuring the severity of pain . the total pain severity score was evaluated using a 6-point likert - type scale ( 0=no pain , 1=mild , 2=uncomfortable , 3=distressing , 4=horrible , 5=intolerable)14 . the nottingham health profile ( nhp ) this scale is preferred for its detailed dimensions including many items used to elicit quality of life impressions in patients with kinesiophobia . part i of the scale includes 38 yes / no questions across 6 dimensions of health : pain , physical mobility , affective reactions , energy , social isolation , and sleep . the 2 parts can be used independently and part ii was not used in the present study . part i is scored using weighted values that provide a range of possible scores from zero ( no problems at all ) to 100 ( presence of all problems within a dimension ) . in this study , the validated and reliable turkish version of nhp developed by kkdeveci et al . kinesiophobia was assessed by using the tampa scale for kinesiophobia - turkish version ( tsk ) . the tsk is used to assess patients with diseases associated with acute and chronic low back pain , fibromyalgia , and musculoskeletal and whiplash injuries . each item is scored by using a 4-point likert - type scale ranging from strongly disagree to strongly agree . a total score is calculated after inversion of the individual scores of items 4 , 8 , 12 , and 16 . vlaeyen et al . defined a cut - off score of 37 as a high degree of kinesiophobia4 . in this study , the validated and reliable turkish version of the tsk reported by ylmaz et al . was used to assess kinesiophobia in patients with low back and neck pain16 , 17 . in statistical analysis , mean standard deviation ( sd ) values were calculated for the quantitative variables and percentages were calculated for qualitative variables . the scores of the above - mentioned tests were compared using the test for qualitative and student s t - test and mann whitney - u test for quantitative variables in low back and neck pain groups . the diagnostic distribution of 600 patients is shown in table 1table 1.distribution of diagnosis in the low back pain ( n=300 ) and neck pain ( n=300 ) groupsdistribution of diagnosisn%low back pain grouplumbar disc herniation27391lumbar spondylosis144.7low back strain134.3total300100neck pain groupcervical disc herniation21471.3cervical spondylosis6822.7neck strain186total300100 . of 300 patients in the low back pain group , 273 ( 91% ) were diagnosed with lumbar disc herniation , while 214 ( 71.3% ) of 300 in the neck pain group had cervical disc herniation ( table 1 ) . demographic data and comparison of the groups are shown in table 2table 2.demographic characteristics of the patients and comparison between low back ( n=300 ) and neck pain ( n=300 ) groupsdemographiccharacteristicslow back pain groupneck pain groupx sdx sdage ( years)43.2 10.942.8 10.2height ( cm)166.2 9.3164.9 7.8weight ( kg)73.6 13.870.4 12.8bmi ( kg / cm)*26.7 4.525.9 4.5duration of pain ( months)66.8 78.146 63.1p<0.05 , p<0.01 . mean age ( t=0.473 ; p>0.05 ) and height ( t=1.844 ; p>0.05 ) in both groups were similar ; weight ( t=2.992 ; p<0.05 ) and bmi ( t=2.162 ; p<0.05 ) were significantly greater and duration of pain ( t=3.586 ; p<0.05 ) was longer in the low back pain group ( table 2 ) . in the low back pain group , 208 ( 69.3% ) were female and 92 ( 30.7% ) were male ; 230 ( 76.7% ) were female and 70 ( 23.3% ) were male in the neck pain group . the results related to pain severity and other pain characteristics of the sf - mpq such as sensory , affective , total pain , vas pain , and total density scores are shown in table 3table 3.short form - mcgill pain questionnaire [ sf - mpq ] scores and their comparison in low back ( n=300 ) and neck pain ( n=300 ) groupspain characteristics of short form - mcgill pain questionnairelow back pain groupneck pain groupx sdx sdsf - mpq sensory pain ( 033)7.9 5.97.8 6.1sf - mpq affective pain ( 012)3.2 3.13.2 3sf - mpq total score ( 045)11.2 8.311 8.3sf - mpq vas score ( 010)6.7 2.16.8 2sf - mpq total pain severity ( 05)2.5 1.12.5 1.1 . the subscores of the sf - mpq were similar in both groups ( p>0.05 ) . pain ( z=4.132 ; p<0.01 ) and physical activity ( z=5.640 ; p<0.01 ) sub - scores of the nhp were significantly higher in the low back pain group than the neck pain group while other nhp domain scores were similar between groups ( p>0.05 ) ( table 4table 4.nottingham health profile and tampa scale for kinesiophobia scores and their comparison in low back ( n=300 ) and neck pain ( n=300 ) groupsnottingham health profile domains and kinesiophobialow back pain groupneck pain groupx sdx sdenergy level ( 0100)50.2 36.452.2 35.3pain ( 0100)*54.9 26.245.1 29.5affective reactions ( 0100)26.4 27.228.7 26.4sleep ( 0100)18.2 25.119.8 26.4social isolation ( 0100)24.7 27.326.6 28.3physical activity ( 0100)*33.4 16.225.7 17.5nottingham health profile total score ( 0600)208 113.2199.3 113.2tampa scale for kinesiophobia ( 1768)*42.1 5.939.7 6p<0.01 ) . kinesiophobia scores measured by the tsk were significantly more severe in the low back pain group ( z=4.732 ; p<0.01 ) ( table 4 ) . our study was performed to evaluate and compare the characteristics of pain , kinesiophobia , and quality of life in patients with low back and neck pain . pain duration was longer , body weight was greater , kinesiophobia was more severe , and pain and physical activity domains of quality of life scales were worse in the low back pain group , without any difference in pain severity or other pain characteristics between the groups . kinesiophobia was defined by kori et al . as excessive , irrational , and debilitating fear of physical movement that limits physical activity , and is a result of a painful injury19 . patients with kinesiophobia believe that physical movement will cause additional pain . in the long term , kinesiophobia is reported to be associated with decreased physical fitness , avoidance of physical activity , functional disability , inability to fulfill social roles , and depression4 , 20 , 21 . many studies have examined fear of movement - related pain / re - injury in patients with low back and neck pain . used tsk in their research on patients with neck and shoulder pain and reported that there was a strong relationship between kinesiophobia and musculoskeletal disorders7 . studies that assessed patients with chronic low back and neck pain reported that pain and disability scores increased as the fear of movement - related pain increased20,21,22,23,24 . wilgen et al.21 used the tsk and pain disability index to evaluate fear of movement and disability in patients with low back pain . their findings showed that there was a significant relationship between kinesiophobia , leg pain , and disability21 . a relationship between kinesiophobia , disability , and quality of life in patients with chronic low back pain gheldof et al . reported that both pain severity and pain - related fear were factors negatively affecting routine daily and social activities in individuals with low back pain24 . in patients with neck pain because of whiplash syndrome , vangronsveld et al . found a relationship between kinesiophobia ( based on tsk ) and the severity of pain , difficulty in concentrating , and falling asleep23 . thompson et al . studied chronic whiplash patients and reported that the severity of pain was associated with the severity of kinesiophobia20 . according to our findings , although the severity of pain was moderate and similar in the low back and neck pain groups , kinesiophobia was more severe in the low back pain group . the decreased physical activity levels in the low back pain group derived from quality of life assessment also support this movement - related fear ; patients with lumbar problems are thought to limit physical activities that require great muscle effort such as walking because of kinesiophobia . this finding may highlight an important difference between patients with lumbar and cervical problems in terms of fear of movement or re - injury . clinicians and therapists should consider the tendency toward inactivity in patients with lumbar problems rather than in those with cervical problems , and institute measures to prevent complications caused by inactivity . it is well known that there is an interaction between pain and quality of life at different levels in patients with low back and neck pain . dndar et al . showed that physical activity and quality of life were decreased and depression was more severe in patients with chronic low back pain compared with healthy controls5 . in a study by yazc et al . , all quality of life parameters were statistically significantly decreased in patients with back pain25 . studied 1,100 neck pain patients and healthy controls aged 2069 , and used the chronic pain questionnaire to assess pain and the sf-36 to evaluate health - related quality of life . they reported that there was a weak correlation between neck pain and the physical component of health - related quality of life26 . ay et al . evaluated the severity of pain based on a vas ( 010 cm ) , quality of life based on the nhp , and the severity of depression using the beck depression inventory . the quality of life decreased as the severity of pain and symptoms of depression increased9 . lin et al . studied 52 chronic neck pain patients , and assessed health - related quality of life and psychological factors . the researchers concluded that health - related quality of life in patients with chronic neck pain was lower than in healthy controls , and that patients with neck pain had many physical and mental health symptoms . in addition , the majority of patients had psychiatric disorders , including psychosomatic symptoms , depression , and anxiety27 . in another study on 195 patients with low back pain by kovacs et al . , a weak correlation was found between the severity of pain , disability , and quality of life28 . in our study , nhp pain and physical activity scores significantly differed between the low back and neck pain groups . our nhp findings showed that the perception of pain was lower and the level of physical activity was higher in patients with neck pain ; consequently , quality of life was higher in the neck pain group than in the low back pain group . the severity of pain in both groups did not differ according to sf - mpq scores , but when the quality of life is considered , the negative effect of pain perception in the low back pain group calls attention to another difference between the groups . the longer duration of pain and higher levels of kinesiophobia in the low back pain group may also be associated with lower quality of life . bener et al . reported that there was a moderate positive relationship between obesity and low back pain29 . webb et al . reported that there was a significant relationship between obesity and low back pain . they also showed that the severity of pain and disability in patients with low back pain increased as the bmi increased30 . in our study , greater body weight may also be a predisposing factor for increased pain perception and may indirectly affect kinesiophobia in patients with low back pain . the primary goal of treatment in patients with low back and neck pain is to reduce the severity of pain ; however , our findings indicate that the differences observed between the groups in terms of pain perception , body weight , kinesiophobia , and physical activity should be taken into consideration when planning preventive and curative physical therapy programs for patients with low back and neck pain . patients should be educated about kinesiophobia , and the long - term goals of treatment should include increasing patient confidence and reducing the severity of kinesiophobia . the primary aim of this study was not to determine the factors that cause kinesiophobia . therefore , the lack of evaluation for the causes of kinesiophobia in patients with neck and low back pain is a limitation of our study .

[ purpose ] the purpose of this study was to compare patients with low back and neck pain with respect to kinesiophobia , pain , and quality of life . [ subjects and methods ] three - hundred patients with low back ( mean age 43.211 years ) and 300 with neck pain ( mean age 42.810.2 years ) were included in this study . pain severity was evaluated by using the short - form mcgill pain questionnaire , which includes a visual analogue scale , quality of life by the nottingham health profile , and kinesiophobia by the tampa scale for kinesiophobia . [ results ] pain severity was similar in both groups , with a visual analogue scale score of 6.72 in the low back pain and 6.82 in the neck pain group . nottingham health profile pain [ z=4.132 ] and physical activity scores [ z=5.640 ] in the low back pain group were significantly higher . kinesiophobia was also more severe in the low back pain group , with a mean 42.055.91 versus 39.76.0 tampa scale for kinesiophobia score [ z=4.732 ] . [ conclusion ] patients with low back pain developed more severe kinesiophobia , regardless of the pain severity , and had greater pain perception and lower physical activity levels . kinesiophobia adversely affects the quality of life and requires effective management of low back pain .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

current situation is the result of the increasing number of cases of patients being diagnosed in an extended state of the disease ( ca 70% of cases local extent ) and its frequent occurrence in elderly people ( 50% above 65 years old , 30% 70 years old ) , who are usually stressed with coexisting diseases . in these cases an important element of cancer treatment is palliative treatment , which mainly aims at improving the quality of life by mitigating the symptoms associated with the disease . the endobronchial location of the cancer can cause hemoptysis , atelectasis , and many other related symptoms , such as dyspnoea , cough , and chronic inflammation . the removal of the endobronchial obstruction often significantly improves the health status of the patient , which in turn affects the quality of the patient 's life . high - dose - rate ( hdr ) brachytherapy is one of the most effective methods used in palliative treatment [ 27 ] . in the application of treatment for endobronchial brachytherapy , the most commonly used orthogonal images are recorded using x - rays , which provide good visualization of the catheter and the bone tissue . in cases where one catheter is used , in order to illustrate the distribution of the isodoses around the guide , the dose can be defined by the reference points . these points are located at a constant distance from the guide or can be designated at different distances from the catheter . if many guides are used , the dose in the area of the envelope is calculated . the envelope is the area containing the catheters , in which the dose is specified at the same depth . the aim of this method is to choose a calculation point located between all of the guides , as well as those farthest from the nearest guide . the selection of such a point allows for the inclusion of the minimal dose within the area of interest . during the treatment plans , the use of a larger number of guides and handling these guides with different reference points allows for the reduction or increase of the dose in the area of the tumor , simultaneously with the least possible coverage of critical organs [ 8 , 9 ] . the paper present comparison of treatment plans made by using 2d and 3d methods in the oncentra planning system 4.1 sp2 version , brachy planning module ( nucletron , an elekta company , elekta ab , stockholm , sweden ) , as well as to assess the quality of treatment plans using the 2d and 3d methods . as such , the study is based on a group of patients treated in the brachytherapy department of the subcarpathian cancer center in brzozw . the studies involved a group of 31 patients with advanced lung cancer treated in the brachytherapy department of the subcarpathian cancer center in brzozw from 2011 to 2013 . in total , 31 patients and 76 treatment plans were analyzed . patients were treated with a microselectron unit ( nucletron , an elekta company , elekta ab , stockholm , sweden ) with an ir source , with a nominal activity of 10 ci . the treatment was applied using the oncentra masterplan brachy 4.0 system ( nucletron , an elekta company , elekta ab , stockholm , sweden ) . in brachytherapy , the lumencath applicator set was used ( nucletron , an elekta company , elekta ab , stockholm , sweden ) . all patient treatment was based on computed tomography of the thorax ( ct ge brightspeed , milwaukee , wi , usa ) . the endobronchial brachytherapy procedures were performed using local anesthesia under the control of a bronchovideoscope . during the bronchoscopy , guides were inserted in the bronchial tree and in the area of or near the tumor 's location . in the 3d method of treatment , three - dimensional images from computer tomography were used , so the physician could contour the volume of the tumor and critical structures . the physicist reconstructed the catheter or catheters in the treatment plan system , activated the source position near the ptv area and then carried out the optimization of the treatment plan . in this method , performed using the 2d method , images from the simulix oldelft simulator were used ( nucletron , an elekta company , elekta ab , stockholm , sweden ) . the dwell time was calculated in a different way : the catheter was created via virtual tomography , the length of the catheter , and the location of the source in it were the same as those in the 3d method , but the dose was specified to 1 cm from the catheter . the dwell time calculated in the 2d method was transferred to the treatment plan performed based on the 3d imaging method . after changing the dwell time , a comparison was made of the reference dose coverage of the target ( ptv ) by the isodose of 100% and 85% , and the calculation of the dose at 2 cm to the critical organ ( spinal cord ) was performer . comparison of ptv coverage in the 3d method to the 2d method highlighted the significant differences between these two methods . reference dose coverage of the target ( ptv ) by isodose of 100% and 85% , as well as the dose to the critical organ received at 2 cm ( in this case , the spinal cord ) , were compared . for the comparison of the variables in the 3d and the 2d methods , tests from two independent trials the tests assumed unequal variances , which were most suitable for comparing quantitative variables in the two groups . in the case of 100% ptv , the p value was 0.0001 , indicating that the statistical differences between the 3d and the 2d methods are significant . figure 1 depicts that the average method for the 3d and 2d was 75.82% and 45.05% , respectively . the standard deviation for the 3d method was 22.72% , and for the 2d method 25.63% , meaning there was a greater variation among the 2d results within the trial . however , the maximal values did not differ significantly between the 3d method and the 2d method ( the maximal value in the 2d method was 0.33% higher than the maximal value in the 3d method ) . the reverse was observed for the minimal values ; for the 3d method , the minimal value was 20.26% , and for the 2d method it was 8.16% . basic measures of outcomes : ptv100 in the 3d and the 2d methods in cases where ptv was 85% ( just as when ptv was 100% ) , the p value was 0.0001 , which means that the statistical differences between the 3d method and the 2d method are significant . figure 2 presents that the average in the 3d method is 36.31% higher than in the 2d method , and the median in the 3d method is 30.58% higher than in the 2d method . the minimal value in the 3d method was 36.1% and in the 2d method it was 14.82% . however , the maximal values did not differ significantly between the 3d and 2d methods . basic measures of outcomes : ptv85 in the 3d and 2d methods when the dose was used on the spinal cord every 2 cm , the p value was 0.009 , implying significant statistical differences between the 3d method and the 2d method . figure 3 shows that the average value of the results for the 3d method are 0.17 gy higher than in the 2d method . in the case of maximal values , the results for the 2d method are 0.44 gy higher than for the 3d method . the standard deviation is not significantly higher ( about 0.02 gy ) for the 2d method than for the 3d method , when the dose was used on the spinal cord every 2 cm . basic measures of outcomes : dose used on the spinal cord every 2 cm in the 3d and 2d methods for the comparison of the variables in the 3d and the 2d methods , tests from two independent trials were used . the tests assumed unequal variances , which were most suitable for comparing quantitative variables in the two groups . in the case of 100% ptv , the p value was 0.0001 , indicating that the statistical differences between the 3d and the 2d methods are significant . figure 1 depicts that the average method for the 3d and 2d was 75.82% and 45.05% , respectively . the standard deviation for the 3d method was 22.72% , and for the 2d method 25.63% , meaning there was a greater variation among the 2d results within the trial . however , the maximal values did not differ significantly between the 3d method and the 2d method ( the maximal value in the 2d method was 0.33% higher than the maximal value in the 3d method ) . the reverse was observed for the minimal values ; for the 3d method , the minimal value was 20.26% , and for the 2d method it was 8.16% . in cases where ptv was 85% ( just as when ptv was 100% ) , the p value was 0.0001 , which means that the statistical differences between the 3d method and the 2d method are significant . figure 2 presents that the average in the 3d method is 36.31% higher than in the 2d method , and the median in the 3d method is 30.58% higher than in the 2d method . the minimal value in the 3d method was 36.1% and in the 2d method it was 14.82% . however , the maximal values did not differ significantly between the 3d and 2d methods . when the dose was used on the spinal cord every 2 cm , the p value was 0.009 , implying significant statistical differences between the 3d method and the 2d method . figure 3 shows that the average value of the results for the 3d method are 0.17 gy higher than in the 2d method . in the case of maximal values , the results for the 2d method are 0.44 gy higher than for the 3d method . the standard deviation is not significantly higher ( about 0.02 gy ) for the 2d method than for the 3d method , when the dose was used on the spinal cord every 2 cm . basic measures of outcomes : dose used on the spinal cord every 2 cm in the 3d and 2d methods complications that can occur after endobronchial brachytherapy such as hemorrhages , shunts , radiation - induced pneumonia or bronchitis and radiation - induced stenosis have justified the search for new and better solutions that can reduce these side effects [ 10 , 11 ] . the planning of treatment with the use of two - dimensional images can not precisely determine where to find the area of the tumor . specifying the dose of the points that are located at a distance of 1 cm from the applicator , we have no possibility to optimize the treatment plan while considering critical organs . with the 2d method , the dose to critical organs often exceeds the limit values and simultaneously disqualifies the treatment plan . in the 3d method , we can precisely determine the position of the tumor and critical organs , which allows us to optimize the treatment and limit the dose to critical organs . figure 4 provides a comparison between the dvh for both methods , where it can be seen that a much better coverage of ptv dose is required in the 3d method than the 2d . at the same time , we can notice that the dose the critical organs receive is slightly different in the two methods . we can clearly state that a plan using 3d images gives better results in terms of treatment planning . technological progress and the utilization of computed tomography increases the possibility for treatment and reduces its toxicity . applying treatment with the 3d method , there is a possibility for dose optimization , decreasing the dose for the critical organs , and as a result , reducing the toxic effects of treatment on healthy tissue [ 6 , 12 , 13 ] . comparison of dvh for treatment plans in 3d ( purple line ) and 2d ( brown line ) methods and the spinal cord ( 3d green line , 2d blue line ) the main advantages of hdr brachytherapy include the simplicity of the procedure , short duration of treatment , and the possibility for patient to undergo the therapy in an outpatient setting . computed tomography allows for precise determination of the size of the tumor before the procedure , as well as determination of the changes in the bronchial tree that have occurred during therapy . in facilities that are ct - equipped , the 3d method should be routinely used in applying treatment in different localizations of tumors . the 3d method based on dynamic imaging the conclusions from the analysis and interpretation of the results of the research are as follows : reference doses with 100% coverage of the ptv in treatment applied with the 3d method are 31% higher than when applied with the 2d method.determination of the target area and the area of critical tissues is possible to a far greater extent by the application of the 3d method than is the case with the 2d method . the former method allows for the administration of high therapeutic doses , while minimizing any impact on critical organs.in the 2d method , application of dose is specified 1 cm from the catheter . hot points are those areas where the dose is much higher than specified . such problems do not occur in the 3d method , which allows for uniformity of high doses . reference doses with 100% coverage of the ptv in treatment applied with the 3d method are 31% higher than when applied with the 2d method . determination of the target area and the area of critical tissues is possible to a far greater extent by the application of the 3d method than is the case with the 2d method . the former method allows for the administration of high therapeutic doses , while minimizing any impact on critical organs . in the 2d method , such problems do not occur in the 3d method , which allows for uniformity of high doses .

purposeto present comparison of treatment plans made by using 2d and 3d methods in the planning system , as well as to assess the quality of treatment plans using the 2d and 3d methods.material and methodsthe studies involved a group of 31 patients with advanced lung cancer treated in the brachytherapy department of the subcarpathian cancer center in brzozw from 2011 to 2013 . in total , 31 patients and 76 treatment plans were analyzed . we compared coverage of ptv planned in 3d and 2d . in the 3d method of treatment , three - dimensional images from computer tomography were used . in treatment plans performed using the 2d method , images from the simulator were used.resultsthe comparison of treatment plans made by using 2d and 3d methods is described . this comparison highlighted the significant differences between these two methods assessing reference dose coverage of the ptv by 100% and 85% isodose.conclusionsreference doses with 100% coverage of the ptv in treatment applied with the 3d method are 31% higher than when applied with the 2d method .

Purpose Material and methods Results Dose coverage 100% PTV Dose coverage 85% PTV Dose for the spinal cord per every 2 cm Discussion Conclusions Disclosure

hip resurfacing arthroplasty ( hra ) is a conservative alternative to total hip arthroplasty in a young and active patient , with the midterm survival reported between 95% and 96% . clinical outcomes in hip resurfacing have been shown to be dependent on both patient selection and surgical technique . femoral neck fracture remains a common failure mode in hip resurfacing and mechanical error , while preparing the femoral head has been well established as a risk factor for catastrophic neck fracture . the use of computer navigation has been shown to improve the accuracy of femoral component placement , thus reducing the likelihood of preparatory error . compared to conventional instrumentation , imageless computer navigation increases component alignment accuracy and reduces outliers . there is a challenging learning curve associated with hip resurfacing , with many technical errors occurring early within a surgeon 's experience . the use of computer navigation has been demonstrated to reduce the length of the initial learning curve and improve the surgeon 's ability to perform the procedure safely . despite these demonstrated advantages , imageless computer navigation is sparsely used in many surgical centers . the lack of widespread use may be attributed to availability as well as cost of the navigation systems . considering the predisposition to technical error early on in hip resurfacing , it would be advantageous for the surgeon as a trainee to utilize computer - based methods to optimize the surgical technique and solidify component implantation methodology . evidence suggests that the use of computer navigation in the operating room may improve the accuracy of freehand component placement in the absence of navigation . thus , there may be a role for computer navigation as a training device for novice surgeons , particularly in the context of learning challenging orthopedic procedures , to improve component implantation once navigation is discontinued . the aim of this study was to examine whether femoral component alignment improved with conventional mechanical guidewire jig following experience with using imageless computer navigation in hsa . 213 consecutive hip resurfacings were performed by a single surgeon ( ehs ) between december 2004 and december 2008 . we retrospectively compared the first 17 ( cohort 1 ) and last 9 ( cohort 2 ) hip resurfacings performed using the conventional lateral pin guidewire alignment jig [ figure 1 ] . cohort 1 was the surgeon 's initial 17 cases of hip resurfacing , which were performed prior to our center 's acquisition of an imageless computer navigation system ( vectorvision sr , brainlab , feldkirchen , germany ) . after the center acquired the navigation system , the surgeon performed 187 hip resurfacings using the computer navigation . in december 2008 , the navigation unit required replacing . in the period pending replacement of the unit , the surgeon performed nine birmingham hip resurfacings ( bhrs ) using the conventional jig ; these nine patients comprise cohort 2 . thus , the hip resurfacings in cohort 2 were performed after the surgeon had gained significant experience with using imageless computer navigation [ figure 2 ] . the birmingham hip resurfacing conventional lateral pin femoral guidewire alignment jig ( smith and nephew inc . ) ( a ) anteroposterior ( ap ) and lateral radiographs of a right bhr implanted using a conventional guidewire alignment jig prior to any experience with imageless computer navigation . ( b ) ap and lateral radiographs of a right bhr implanted using conventional guidewire alignment jig after experience with imageless computer navigation . ( c ) ap and lateral radiographs of a right bhr implanted using imageless computer navigation of the 17 patients comprising cohort 1 , 16 patients had a preoperative diagnosis of osteoarthritis and one patient was diagnosed with avascular necrosis , who , as a result of this diagnosis was excluded from the analysis . the mean age of the patients was 48.7 years ( sd 6.6 , range 39 - 63 ) with a mean body mass index ( bmi ) of 30.4 kg / m ( sd 3.9 , range 23.3 - 40.4 ) . cohort 2 included nine patients all of whom were males with a preoperative diagnosis of osteoarthritis . the mean age of this group was 52.6 years ( sd 10.8 , range 29 - 71 years ) with a mean bmi of 28.5 kg / m ( sd 3.0 , range 25.1 - 34.3 kg / m2 ) . the differences in age and bmi between cohorts were not found to be significant ( p > 0.203 ) . the 3-month postoperative digital anteroposterior and cross - table lateral x - rays were used for comparison . images were obtained via a computed radiography system ( directview cr850/950 ; eastman kodak , rochester , ny , usa ) using a standardized imaging technique and positioning protocol , and were stored on our institutional picture archive and communication systems server ( sienet magicstore ve50 ; siemens medical , erlangen , germany ) . an observer ( zm ) experienced in using digital radiograph templating software ( magicview 300 , siemens medical ) analyzed the radiographs , and was blinded to all patient data and operative dates . the component positions in both the coronal and sagittal planes were measured . the coronal stem shaft angle ( ssa ) was defined as the angle subtended by the diaphyseal axis of the femur and a line drawn from the center of the prosthesis along the component stem toward the lateral cortex of the femur . the sagittal stem neck angle ( sna ) was defined as the angle subtended by the neck and component stem axis . measured values for component alignment were compared to the preoperatively planned position determined by the senior surgeon 's ( ehs ) surgical protocol . the preoperative plan in each case positioned the component in 10 of valgus relative to the native neck shaft angle ( nsa ) of the femur in the coronal plane and neutral to the neck axis in the sagittal plane . the component was considered neutral in the sagittal plane if the degree of component anteversion or retroversion was within 10 of the native neck version . descriptive statistics were calculated using microsoft excel ( microsoft inc . , redmond , wa , usa ) to determine differences between the final component placement and the target position . spss 16 ( spss inc . , chicago , il , usa ) was used to calculate two - sample t - tests for comparison of demographics as well as alignment values between the two cohorts . statistical power was determined to be 85.2% ( = 0.05 , effect size d = 1.02 ) for the comparison of ssa . descriptive statistics were calculated using microsoft excel ( microsoft inc . , redmond , wa , usa ) to determine differences between the final component placement and the target position . chicago , il , usa ) was used to calculate two - sample t - tests for comparison of demographics as well as alignment values between the two cohorts . statistical power was determined to be 85.2% ( = 0.05 , effect size d = 1.02 ) for the comparison of ssa . coronal alignment of the femoral component in cohort 2 was more accurate than cohort 1 . the mean deviation of the ssa from the target alignment was 2.2 ( sd 2.2 , 95% ci 0.8-3.7 ) in cohort 2 and 5.6 ( sd 4.3 , 95% ci 3.6-7.6 ) in cohort 1 [ figure 3 ] . the variance of cohort 2 ( 4.9 , range 4 varus to 7 valgus ) was threefold less than cohort 1 ( 17.6 , range 14 varus to 1 valgus ) . the mean coronal alignment in cohort 1 erred in varus relative to the planned ssa [ figure 3 ] . the component version in cohort 2 was also more accurate than cohort 1 [ figure 4 ] . the mean deviation from the target sna of cohort 2 had a mean difference of 4.0 ( sd 2.2 , 95% ci 2.6-5.4 ) , while that of cohort 1 was 7.3 ( sd 5.3 , 95% ci 4.8-9.9 ) . the variance in cohort 2 ( 27.7 , range 8.2 retroversion to 3.6 anteversion ) was half that of cohort 1 ( 4.7 , range 17.2 retroversion to 5.8 anteversion ) [ figure 4 ] . four implants in cohort 1 were considered to be retroverted ( > 10 ) [ figure 5 ] . box and whisker plot of stem shaft angle accuracy for the two conventional jig cohorts ( negative values denote relative varus and positive values denote relative valgus ) box and whisker plot of stem - neck angle accuracy of the two conventional jig cohorts ( negative values denote retroversion and positive values denote anteversion ) comparison of the accuracy of implant positioning using a conventional jig between the pre and post navigation cohorts hip resurfacing provides a viable bone conserving option for a young , active patient with end - stage hip disease . in addition to patient selection , surgical technique contributes greatly to the clinical outcomes of the procedure . in spite of many advances in surgical technique , femoral neck fracture remains a concern with hip resurfacing and continues to be the most common reason for revision . the etiology of femoral neck fracture in hip resurfacing has been studied thoroughly , and although the causes are often multifactorial , the biomechanics of implant alignment play a large role in resurfacing construct strength and resilience . previous biomechanical studies investigating implant alignment have shown that relative valgus alignment of the femoral component strengthens the proximal femur and may be protective against neck fracture . in addition , studies looking at femoral neck notching have demonstrated that as little as a 2-mm superior femoral neck notch may increase the risk of neck fracture . despite this knowledge , notching of the femoral neck and femoral components implanted in relative varus are still encountered , particularly during the surgical learning period for this procedure . these adverse events may be attributed to the difficulty of the procedure or the lack of experience of the surgeon . in this study , we found that the cohort of hip resurfacing patients following experience using computer navigation ( cohort 2 ) was more accurate and showed less variance in component positioning . the mean ssa for cohort 2 was 3.4 less than the mean ssa for cohort 1 . this decrease in mean ssa for component positioning results in a decrease of stress across the superior neck , potentially reducing the risk of femoral neck fracture . further , improved accuracy of positioning in the sagittal plane may theoretically reduce the risk of impingementn [ figure 5 ] . it has been well documented that computer - assisted surgery by way of imageless navigation functions to curtail femoral implant malalignment in hip resurfacing . however , the cost and availability of current navigation systems make ubiquitous use unrealistic , particularly for those centers that perform only small volumes of hip resurfacings . in order for surgeons new to hip resurfacing to perform optimally using conventional instrumentation , it may be necessary to first train using computer - assisted methods in order to enhance both surgical technique and component insertion protocol . a concern of using computer navigation for training purposes is reliance on the technology with poor retention performance following discontinuation . thus , this study looked to establish whether femoral component implantation accuracy utilizing a conventional guidewire alignment jig improves following the use of imageless computer navigation in hsa . in this study , a limitation of this study is the inability to account for the learning process that would occur normally after performing a series of hip resurfacings . in a study by seyler et al . , fellowship trained staff surgeons with experience in hip resurfacing ( > 75 cases ) exhibited a greater scatter of insertion angles when using conventional instrumentation than less experienced residents using imageless navigation as a surgical aid . this not only demonstrates the accuracy of computer navigation but also that experience alone may not prevent a greater degree of inaccuracy when using conventional manual instrumentation . a second limitation is that the number of resurfacings performed using a conventional jig is small relative to the number performed using computer navigation . the optimal number of procedures to achieve competency and a higher level of accuracy using conventional guidewire alignment jigs may be smaller than in the current study . further investigation is required to determine the ideal number of training cases required in order to obtain proficiency using conventional instrumentation in hip resurfacing . lastly , the lateral pin jig utilized in this study may not be representative of other guidewire alignment devices . the results of this study may not be extrapolated to other conventional guidewire alignment jigs in hip resurfacing . in this study , all femoral components implanted with a manual jig after acquiring experience using imageless navigation achieved the desired minimum of 10 of valgus relative to the native nsa and all were considered to have neutral sna angles . this is compared to three implants which were positioned more than 10 varus relative to the target ssa and another four which were considered retroverted in the group performed prior to experience using navigation . the improved use of the manual jig may be attributable to an increased familiarity with the location of the optimal guidewire insertion point . often the native anatomy of the end - stage hip disease patient is distorted with osteophytes and remodeled bone which can prove problematic when using a manual jig , as alignment and positioning depend largely on a visual assessment of the local anatomy for guidewire placement . the results from this study show improved accuracy and precision using a conventional guidewire alignment jig after training with computer navigation . this improvement conflicts with the literature on cognitive motor learning which suggests that the form of feedback that computer - assisted surgery provides may actually be detrimental to learning . according to motor learning theory , individuals learn new motor skills by evaluating available feedback to alter future performance . feedback can either be intrinsic ( as a natural consequence of the action ) or extrinsic ( from an external source such as an instructor or a computer ) . computer navigation provides a form of extrinsic feedback , or continuous concurrent feedback , in which continuous visual feedback guides the trainee to the correct position , thus minimizing errors and reinforcing proper technique . it has been hypothesized , however , that concurrent feedback does not contribute to retention of task performance as a result of the learner developing dependence on extrinsic feedback or being distracted from using intrinsic feedback . in contrast , a prospective randomized study by gofton et al . analyzing the effect of computer navigation on the learning of surgical skills by trainees demonstrated that concurrent feedback during the insertion of the acetabular cup in hip replacement did not compromise the learning process of trainees . this finding is supported by a systematic review by saithna and dekker looking at the influence of computer navigation in hip resurfacing training in which they concluded that there exists minimal evidence to support concerns regarding the detrimental impact of computer navigation on trainee learning and subsequent performance in hip resurfacing . the study demonstrates that femoral component placement utilizing conventional instrumentation may be more accurate following experience using imageless computer navigation . training or experience using computer navigation may provide the surgeon with appropriate feedback to facilitate adequate motor skill acquisition and spatial awareness that can be transferred in turn to conventional instrumentation . the success of hip resurfacing is particularly sensitive to surgical technique and component alignment ; training with computer navigation early in the learning curve may help optimize the subsequent use of conventional hip resurfacing instrumentation .

background : the use of computer navigation has been shown to improve the accuracy of femoral component placement compared to conventional instrumentation in hip resurfacing . whether exposure to computer navigation improves accuracy when the procedure is subsequently performed with conventional instrumentation without navigation has not been explored . we examined whether femoral component alignment utilizing a conventional jig improves following experience with the use of imageless computer navigation for hip resurfacing.materials and methods : between december 2004 and december 2008 , 213 consecutive hip resurfacings were performed by a single surgeon . the first 17 ( cohort 1 ) and the last 9 ( cohort 2 ) hip resurfacings were performed using a conventional guidewire alignment jig . in 187 cases , the femoral component was implanted using the imageless computer navigation . cohorts 1 and 2 were compared for femoral component alignment accuracy.results:all components in cohort 2 achieved the position determined by the preoperative plan . the mean deviation of the stem shaft angle ( ssa ) from the preoperatively planned target position was 2.2 in cohort 2 and 5.6 in cohort 1 ( p = 0.01 ) . four implants in cohort 1 were positioned at least 10 varus compared to the target ssa position and another four were retroverted.conclusions:femoral component placement utilizing conventional instrumentation may be more accurate following experience using imageless computer navigation .

I M Statistical analysis R D

continuous efforts by researchers and constant addition of literature minimized the aura of one of the deadliest disease , that is , oral malignant melanoma ( omm ) . as a result , the new innovative approaches came to existence and this became the first step to successful treatment against this disease . the disease is known since decades , but proper management is affected due to insufficient understanding regarding its pathophysiology and marked variation in its clinical findings . the mucosal melanoma was discussed first , by weber in germany , and later on , the valuable information of head and neck mucosal melanoma was reported by lincoln in 1885 . in late 19 century , further studies were carried out , that focused to minimize the mortality and enhance public awareness towards the disease . hence , an early detection and timely treatment is a key factor for its better prognosis . unlike omm , there is a marked variation in the etiology and incidence rate of generalized cutaneous melanomas . the continuous global environmental changes result in over - exposure to ultraviolet ( uv ) radiation . furthermore , the growing trends of sunbeds or tanning beds increased the risk of this disease . a sunbed is a device , which emits uv radiation ( typically 97% uva and 3% uvb , 3% ) to produce a cosmetic tan . today the worldwide incidence of newly diagnosed melanomas range between 3% and 8% . around 50% of mucosal melanomas affect the head and neck region , almost representing approximately 9% of all malignant head and neck tumors . comparatively , mucosal melanomas show an aggressive biological behavior resulting in a 5 years survival rate of < 25% . the etiology of mucosal melanoma differs from its cutaneous counterpart , as there is no direct role of uv radiation . primary causes are : ill - fitting dentures , betel nuts , tobacco , formaldehyde , amalgam tattoo , nevi at traumatic regions , racial pigmentation , etc . some literatures explain that during embryologic development , melanocytes migrate from the neural crest to epithelial lining , which later show reactive changes by cytotoxic stimulant in the basal epithelial layer . though dendritic cells , derived from neural crest , produces the melanocytes but the exact mechanism of proliferation of these cells in melanoma particularly , mucosal melanomas are asymptomatic in their initial phase , resulting in late diagnosis , thus allowing them to invade the deeper regions . the clinical characteristics include , dark brown / black in color , asymmetrical margins , and irregular surface , etc . , hence , the pigmented lesions of oral cavity ; which does not possess clinical specificity need to be carefully evaluated for possibility of omm . differential diagnosis include : smoke related melanosis , drug pigments , physiologic or racial pigmentation , melanotic macule , kaposi 's sarcoma , nevus or melanoacanthoma , chronic leukemic , etc . pigmentation is not the only criteria , as around 15% of the melanomas are nonpigmented . the omm has distinct gender variation with majority of them showing male predominance . among the oral melanomas ; hard palate and maxillary gingiva , we report a case of omm , in a 45-year - old female patient , who is conscious about the discoloration and growing bulk over mandibular anterior mucosa . a 42-year - old indian female with average height and moderate built reported to the dental office , complaining of blackish discoloration on the lower jaw since 6 months and difficulty while eating ; especially with the lower front teeth . intraoral examination revealed nontender and painless bluish - black growth with rough and irregular surface extending over the gingiva of 3544 regions , which revealed no findings of ulceration and bleeding [ figure 1 ] . there was major involvement over the labial aspect of mandibular gingiva followed by anteroposterior extension into the vestibule and oral aspect of lip mucosa ; but with minimal extension of the lesion on lingual aspect . the patient noticed a small blackish patch approximately 1 cm 1 cm which gradually increased to present size with associated mobility of teeth . the overall general examination of the neck , back , extremities , and chest was insignificant except for nevi . submandibular lymph nodes were enlarged but nontender approximately 1.5 cm 1.5 cm in dimension . after evaluating hematological parameters , an incisional biopsy was performed under local anesthesia , for a confirmatory diagnosis . clinical photograph showing bluish - black irregularly spreading growth extending from mandibular left second premolar to right first premolar in microscopic examination , the h and e stained section revealed in situ melanotic pigments [ figure 2 ] . the section showed large cells with pleomorphic vesicular nucleus and brown pigment , few abnormal mitoses , and altered nucleocytoplasmic ratio invading into the connective tissue in the form of sheets , cords , and islands [ figure 3 ] . the tissue was also immunohistochemically stained for human melanoma black-45 , a specific marker for melanocytes that also revealed cytoplasmic positivity of malignant melanocytes for the antibody [ figures 4 and 5 ] . after confirming the diagnosis as malignant melanoma , segmental resection , and bone grafting follow - up , patient reported with some other problems such as difficulty in eating and speech . the intraoral examination revealed uneven healing of resected part and further development of the bluish - black patch on left posterior mandible . due to severe trismus and discomfort wide excision of the growth and node biopsy was done and the patient is being recalled for follow - ups every 6 months . h and e stained section reveals malignant melanocytes with hyperchromatic nuclei ; infiltrating the connective tissue as sheets ( h and e , 10 ) h and e stained section reveals pigmented cells with abnormal mitosis ( h and e , 10 ) immunohistochemistry stained slide shows positive expression ( ihc , 10 ) immunohistochemistry stained slide shows cytoplasmic positivity by the melanocytes ( ihc , 40 ) the oral mucosal melanoma is a rare entity with incidence rates of < 1% of all melanomas and among head neck tumors , the head and neck mucosal melanomas accounted for 0.8% of all melanomas and 8% of head and neck melanomas . they reviewed 2.5 million individuals in denmark over a 30 years period and found that omm mostly occur between the fourth and seventh decades of life , with a mean age of 5557 years . melanocytes are dendritic cells that have migrated as neuroectodermal derivatives in the ectodermally derived mucosa . some studies also suggest familial inheritance specially , along with dysplastic nevus syndrome where ; p16 and differences in dna repair impairments contributes to carcinogenesis of malignant melanoma . in such circumstances , the damaged dna , activates the proto - oncogenes or inactivates the tumor suppressor genes . majority of patients show history of preexisting oral pigmentation before the diagnosis of oral melanoma . moreover , common primary sites include ; the nasal cavity , paranasal sinuses and in the oral cavity ; most commonly in the maxillary alveolar ridge and hard palate , whereas it is rarely seen on the mandibular gingiva . so far some studies have documented , relationship between free radicals , and melanoma cells , resulting into increased levels of reactive oxygen species . this is due to the metal binding properties of melanin and loss of structural integrity of melanosomes . at present it is accepted that , omm is very aggressive tumor and various factors contribute to its aggressiveness , such as late detection , poor resectability , and early metastasis . this not only limits the 5 years survival rate up to 1025% , but also affects prognosis . melanoma is notoriously resistant to chemotherapy , but the other approaches can be carried out during its treatment . according to oncosurgeons ; treatment of choice for malignant melanoma is surgery . stage 1 , melanomas are excised along with 1 mm margins and t2an0m0 ( stage 1b ) needs sentinel lymph node biopsy . stage 2 cases are treated with wide excision and node biposy , whereas for stage 3 it needs wide excision with 2 mm margins and node dissection and the radiation or chemotherapy is given post - operatively . stage 4 is really the challenge , it is treated with surgery and chemotherapy ; interferons ( ifns ) , interleukins ( ils ) , vaccines , and different biochemotherapeutic agents serves as adjuncts . today 's new technology has opened new hopes . as a result , biotherapies including ifns and il-2 provide intriguing avenues for further evaluation and treatment . the mechanism of clinically effective il-2 therapy may be the direct action of il-2 on a biologically distinct subset of melanoma cells , leading to up - regulation of tumor suppressor il-24 . hence , to avoid future complications the suspected oral pigmentations should be planned for biopsy . moreover , in some extent ; criteria given by green et al . may be helpful to clinicians , that suggests ; demonstration of melanoma in the oral mucosa , presence of junctional activity and inability to demonstrate extra oral primary melanoma . further , some studies have classified the disease for prediction of its prognosis , that is , stage 1 : when the lesion is confined locally , stage 2 : have positive metastasis and stage 3 : with hematogenous spread . in the later stage ; distant metastases may be found in a variety of sites , including the lungs , bones , liver , brain , and skin . a detailed case history , thorough clinical examination , and suspicious eye to the irregular intraoral pigments can help the clinician for early diagnosis . sometimes patients ignore the symptoms that may reflect indirectly on health care provision and result in a poor prognosis . any physiologic pigmentation to hormones , medication or pigmentations other than amalgam tattoo should be biopsied . the treatment promoted by thorough oral examination and biopsy should be well planned to improve patient prognosis and avoid chances of recurrence .

according to the world health organization , oral malignant melanoma ( omm ) is a rare disease , accounting for only 0.8% of all melanomas , 8% of head and neck melanomas , and up to 0.5% of all oral malignancies . omm presents as a pigmented lesion with asymmetrical borders , irregular surface characteristics , and a distinct color . melanoma - associated pigmented lesion of the oral cavity does not possess clinical specificity and frequently divert the clinical diagnosis ; hence , differential diagnosis becomes mandatory . furthermore , the unpredictable pathophysiological behavior and delayed detection , contributes for poor prognosis of the disease . as a result , the 5 years survival rate is only 1025% . commonly omm is seen in maxillary gingiva of males . however , we report a rare case of a middle - aged female having pigmentations and growth over mandibular gingiva .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION

urinary tract infection ( uti ) is one of the most common clinical syndromes encountered in general and gynecological practices . adult women ( 4050% ) have a history of at least one episode of uti in their lifetime . community acquired infection is caused by escherichia coli , klebsiella pneumoniae , proteus mirabilis , staphylococcus saprophyticus or enterococcus faecalis , while the hospital acquired ones are escherichia coli , pseudomonas aeruginosa , proteus sp , enterobacter sp . uti is a broad term that encompasses asymptomatic bacteriuria and symptomatic infection with microbial invasion and inflammation of the urinary tract . while up to 90% of the patients with utis complain of urinary tract symptoms , one third or more of the patients with these symptoms do not have bacteriuria . dysuria and frequency together raise the probability of uti to more than 90% , effectively ruling in the diagnosis by history alone . in almost all cases of uti , empirical antimicrobial treatment initiates before the laboratory results of urine culture are available ; thus antibiotic resistance may increase in uropathogens due to frequent use of antibiotics . the prevalence of antimicrobial resistance in patients with uti is increasing and can vary according to geographical and regional location . for this reason , knowledge of the etiological agents of utis and their antimicrobial resistance patterns in specific geographical locations may aid clinicians in choosing the appropriate antimicrobial empirical treatment . the literature on prevalence of uti among females in lucknow was scarce in recent decades ; thereby the present study was undertaken to find out the prevalence of uti and to determine the antimicrobial susceptibility patterns of commonly used antibiotics among females in lucknow . this observational , prospective 12 months study was carried out among out patient departments of obstetrics and gynaecology and medicine of vivekananda polyclinic and institute of medical sciences , lucknow after ethical clearance from the institution review board . all females aged 15 years and above attending the respective outpatient departments clinically suspected for uti were selected for the purpose of the study during the study period . patients who had no symptoms suggestive of uti at the time of observation were excluded from the study . females suffering from diabetes mellitus , renal disorders , hiv positivity , or on corticosteroid therapy were also excluded . those females who did not give consent were noncooperative or refused to provide the necessary information were not included in the study . the necessary information was collected using the interview technique from each respondent after informed consent . a structured questionnaire was used to assess the study subjects self - reported information regarding socio - demographic characteristics and urinary symptoms . all study subjects were advised to collect the mid - stream urine sample in wide - mouthed sterile containers . they were instructed to clean the area around the urethral opening with clean water , dry the area , and collect the urine with the labia held apart . samples were processed within 1 hour of collection . for direct microscopy ( wet film preparation ) 50 l of well - mixed uncentrifuged urine was taken on a slide and a cover slip placed on it . at least 20 fields were examined and detection of one or more morphologically similar bacteria per oil immersion field was treated as significant . a mixture of e. coli and staphylococcus aureus was used as a positive control and uninoculated broth as negative control . the presence of more than two morphologically different organisms indicated the presence of mixed flora . for urine culture the urine sample ( 1 l ) was inoculated on cysteine lactose electrolyte deficient medium , using a standard loop of internal diameter 1.34 mm ( semiquantitative method ) . they were further incubated for another 24 hours before a negative report was issued . a single organism obtained in counts of > 100,000 cfu / ml five colonies of the test organisms were streaked on agar plates using a sterile inoculating wire loop . the appropriate multidisk depending on whether the test organism plated will be gram negative or gram positive was then placed firmly onto the surface of the dried plates , using sterile forceps . the plates were left at room temperature for 1 hour to allow diffusion of the different antibiotics from the disk into the medium . the zone of inhibition of greater than 10 mm was considered sensitive , 510 mm as moderately sensitive and no zone of inhibition as resistant . the antibiotics tested were amoxicillin , ampicillin , ampicillin - sulbactam , piperacillin , cloxacillin tazobactam , cephalexin , cefadroxil , cefaclor , cefuroxime , cefixime , cefotaxime , cefoperazone , ceftazidine , ceftizoxime , ceftriaxone , cefepime , nalidixic acid , ciprofloxacin , lomefloxacin , norfloxacin , pefloxacin , ofloxacin , nitrofurantoin , tetracycline , co - trimaxazole , clarithromycin , roxithromycin , erythromycin , amikacin , gentamicin , tobramycin , netilmicin , kanamycin . tests of significance like pearson 's chi - square test and fisher exact test were applied to find out the results . were put to use in the present study : microscopy findings of more than 10 wbc per high power field were considered significant.significant bacteriuria was defined as culture of a single bacterial species from the urine sample at a concentration of more than 100,000 cfu / ml . significant bacteriuria was defined as culture of a single bacterial species from the urine sample at a concentration of more than 100,000 cfu / ml . five females suffering from diabetes mellitus , renal disorders , hiv positivity , or on corticosteroid therapy were also excluded . two females who did not give consent , were noncooperative or refused to provide the necessary information were not included in the study . thus total the overall prevalence of uti was found to be 45.32% ( 95% confidence interval , 37.5253.12 ) . overall 46.87% of 96 urban respondents and 41.86% of 43 rural respondents were identified with uti . thirty - two ( 51.61% ) of 62 females aged between 25 and 34 years and 15 ( 46.87% ) of 32 females aged between 15 and 24 years were identified with uti . ten ( 40.0% ) of 25 females between 35 and 44 years and 6 ( 30.0% ) of 20 females above 45 years of age had uti . five ( 26.31% ) of 19 illiterate females were identified with uti and 58 ( 48.33% ) of 120 literate females had uti . fifty - six ( 44.09% ) homemakers and six ( 66.67% ) students were identified with uti [ table 1 ] . distribution of respondents according to their socio - demographic and clinical characteristics the most common urinary symptom presented was burning micturition ( 73.4% ) followed by frequency ( 43.9% ) , urgency ( 20.9% ) , painful voiding ( 20.1% ) , difficulty ( 5.0% ) , and nocturnal incontinence ( 1.4% ) . ten ( 50.0% ) of 20 pregnant females had uti compared to 53 ( 44.53% ) of 119 nonpregnant females . out of the 63 pathogens , e. coli ( 33.1% ) was the most common organism isolated followed by klebsiella pnuemoniae ( 7.9% ) , staphylococcus aureus ( 2.2% ) , streptococcus pnuemoniae ( 1.4% ) , and proteus mirabilis ( 0.7% ) , respectively . the most effective antibiotic for the e. coli isolates observed was nitrofurantoin ( 86.95% ) followed by amoxicillin ( 69.56% ) , nalidixic acid ( 65.21% ) , and cotrimoxazole ( 60.86% ) . high efficacy of nitrofurantoin ( 90.90% ) followed by cotrimoxazole and tetracycline ( 81.81% ) both was observed against the klebsiella isolates . cephalexin , cefaclor , nalidixic acid , and norfloxacin also showed similar higher efficacies ( 72.72% ) for klebsiella isolates . amoxicillin , ampicillin - sulbactam , cefixime , pefloxacin , and ciprofloxacin also showed similar susceptibilities ( 63.63% ) for klebsiella isolates . high susceptibility ( 100% ) for ampicillin , nitrofurantoin , and tetracycline was observed among the identified proteus isolates . high susceptibility patterns to nalidixic acid , clarithromycin , cotrimaxazole , cefixime , cephalexin , and cefaclor ( 100.0% ) followed by nitrofurantoin ( 66.66% ) among the streptococcus isolates identified were observed . ceftriaxone , cefepime , cefuroxime , ceftizoxime , gatifloxacin , gentamicin , tetracycline , and erythromycin showed similar efficacies ( 50.0% ) for streptococcus isolates [ tables 2a and b ] . antibiotic susceptibility trends observed among the identified bacterial species causing urinary tract infection antibiotic susceptibility trends observed among the identified bacterial species causing urinary tract infection it is stated that uti is predominantly a disease of the females due to a short urethra and proximity to vestibule and the anal opening . in our study too , out of the 139 females studied , 63 ( 45.32% ) females were found to be urine culture positive . forty five ( 46.87% ) out of 96 urban respondents and 18 ( 41.86% ) out of 43 rural respondents were identified with uti in our study . this may be attributed to the higher levels of awareness and treatment seeking behavior in the urban respondents compared to the rural respondents . in our study , the prevalence of uti was higher among the females aged between 15 and 44 years compared to those aged above 45 years of age . more cases of utis were recorded among young and middle age patients ( 2049 years , 51.04% ) by akram et al . in another study by farhat ullah et al . , middle - aged patients accounted for 54.3% of uti . a higher percentage of literate females were identified with uti compared to the literate ones in the current study . this may be due to the high treatment seeking behavior of the infected literates compared to illiterate ones . in the present study the most common urinary symptom presented was burning micturition followed by frequency , urgency , and painful voiding . the percentage of pregnant females with uti was found to be slightly higher than the nonpregnant infected females in our study . , where out of 61 infected young women , 52 ( 86.8% ) were found in their early or late phases of pregnancy . these findings agree with other recent indian reports which have indicated that gram - negative bacteria , mostly e. coli and klebsiella pneumoniae , are the most common pathogens isolated in patients with uti [ table 3 ] . distribution of pathogens isolated among uti patients the most effective antibiotic for e. coli in this study observed was nitrofurantoin followed by amoxicillin , nalidixic acid , and co - trimaxazole . high efficacy of nitrofurantoin followed by co - trimaxazole and tetracycline both was observed against klebsiella in this study . cephalexin , cefclor , nalidixic acid , norfloxacin , amoxicillin , ampicillin - sulbactum , cefexime , pefloxacin , and ciprofloxacin were also effective for klebsiella . however klebsiella isolates showed higher susceptibility against imipenem ( 88% ) and amikacin ( 59% ) in the study by akram et al . regional antibiotic susceptibilities of e. coli isolates high susceptibility for ampicillin , nitrofurantoin , and tetracycline was observed among the identified proteus isolates in our study , whereas proteus spp . showed the highest sensitivity to ciprofloxacin ( 71.2% ) in the study by kashef et al . the present study revealed that cefexime and nalidixic acid were highly effective for staphylococcus aureus . whereas , all staphylococcus aureus isolates were found to be susceptible against imipenem , ceftriaxone , and cefotaxime by akram et al . streptococcus was found susceptible to nalidixic acid , clarithromycin , cotrimaxazole , cephalosporins , nitrofurantoin gatifloxacin , gentamycin , tetracycline , and erythromycin in our study . , in yola . as we could not find similar studies , we used references of different field populations to compare our results . those who visited gynaecology consultation may have a different pretest probability of uti than those who attended other hospital services or general practice consultations , and microbiological results can be also different . this reflects the need for accurate and updated population surveillance data , particularly in light of concerns regarding variable regional antimicrobial susceptibility patterns . regular monitoring is required to establish reliable information about susceptibility pattern of urinary pathogens for optimal empirical therapy of patients with utis . we suggest that empirical antibiotic selection should be based on the knowledge of local prevalence of bacterial organisms and antibiotic sensitivities rather than on universal guidelines . iec messages for all the uti patients must include the additional information about the risk reduction methods and need to take regular supervised treatment .

background : urinary tract infection ( uti ) is among the most common infections described in outpatient setting and hospital patients . in almost all cases empirical antimicrobial treatment initiates before the laboratory results of urine culture are available ; thus antibiotic resistance may increase in uropathogens due to frequent use of antibiotics.aims:the study was designed to find the prevalence of uti in females with urinary tract symptoms , to determine the causative organism ( s ) of uti , and to determine the antibiotic susceptibility pattern of microbial agents isolated from urine culture ( antibiogram).materials and methods : the prospective , observational study involved 139 females , aged 15 years and above clinically suspected for uti attending outpatient departments of vivekananda polyclinic and institute of medical sciences , lucknow . a structured questionnaire was used to interview the study subjects . a chi - square test and fisher exact test were used to analyze data.results:the overall prevalence of uti was found to be 45.32% ( 63/139 ) . escherichia coli ( 33.1% ) and klebsiella pneumoniae ( 7.9% ) were the most common organisms isolated . the most effective antibiotic for both was nitrofurantoin.conclusions:regular monitoring is required to establish reliable information about susceptibility pattern of urinary pathogens for optimal empirical therapy of patients with uti .

Introduction Materials and Methods Results Discussion Conclusions

it is well established that high - risk ( hr ) human papillomavirus ( hpv ) types are causative for the development of cervical cancer [ 13 ] . the majority of hpv infections are cleared without further consequences for the host , but some infections with hr - hpv types may give rise to high - grade cervical intraepithelial neoplasia ( cin iii ) and cervical cancer [ 46 ] . there is evidence that cell - mediated immune responses of the host , both systemic and local , are important determinants for the course of the infection . cell - mediated immune responses are regulated by t lymphocytes [ t - helper ( th ) lymphocytes and cytotoxic lymphocytes ( ctls ) ] in cooperation with antigen - presenting cells ( apcs ) [ monocytes ( mcs ) and dendritic cells ( dcs ) ] . these cells all release cytokines that can influence one another 's synthesis and actions in the setting of an immuno - regulating cytokine network . cytokines in immune responses to infection are often classified as immuno - stimulating ( tumour - suppressing ) th1type cytokines and immuno - inhibitory ( tumour - promoting ) th2-type cytokines . th1-type cytokines such as interferon ( ifn ) , tumour necrosis factor ( tnf ) , interleukin 2 ( il-2 ) , and il-12 are produced mainly by lymphocytes , apcs , and natural killer cells ( nk - cells ) . th2-type cytokines ( il-4 , il-5 , il-6 , il-8 , il-10 ) , produced by lymphocytes and mcs , are immuno - inhibitory for cell - mediated responses and predominantly induce humoral immunity [ 8 , 9 ] . qualitative and quantitative analyses of cytokine profiles have been used to characterize the immune response in hpv - related cin . these were performed with peripheral blood mononuclear cells ( pbmcs ) [ 1012 ] or with t - cell fractions isolated from pbmcs [ 1316 ] and occasionally with whole blood cultures after stimulation with several antigens . selective cytokines , mostly ifn [ 11 , 12 , 1418 ] , il-2 [ 1014 ] ; and occasionally the apc - derived il-12 or tnf were measured together with one or two of the typical th2-type cytokines il-4 , il-5 , and il-10 [ 11 , 12 , 16 , 17 ] . generally a shift from a th1-type to a th2-type cytokine response was observed when healthy controls or women with low - grade squamous intraepithelial lesions ( lsil ) were compared with cases of high - grade sil ( hsil ) or cervical carcinoma [ 7 , 11 , 17 , 19 ] . we previously observed manifestation of a th2-type cytokine pattern in plasma of hr - hpv - positive women during carcinogenesis of cervical cancer at the stage of cin iii . recent studies with isolated t - cell fractions stimulated with hpv16-derived oncopeptides indicate a reactivation of an inflammatory response in patients with carcinoma [ 12 , 15 ] . these results let us assume that significant changes in the immunocompetence of circulating leukocytes are involved in the development from cervical dysplasia to cervical cancer . in the present study we used whole blood cultures from hr - hpv - negative controls , hr - hpv - positive women without cervical dysplasia and hr - hpv positive patients with different grades of cin and cervical cancer to investigate changes in immunocompetence expressed in the capacity of circulating leukocytes to release cytokines in response to a mitogenic challenge . of interest were the effect of hr - hpv infection without clinical manifestations , the special position of cin iii with a th2-type cytokine response , and a possible revival of inflammatory cytokine activity in cervical carcinoma . inclusion took place at the outpatient clinic of the obstetrics and gynaecology department of the erasmus university medical center ( rotterdam , the netherlands ) between july 2000 and august 2002 . our selection of patients for this study was based on the presence of hr - hpv and the grade of cervical intraepithelial neoplasia . histology results were defined as no dysplasia , mild dysplasia ( cin i ) , moderate dysplasia ( cin ii ) , severe dysplasia ( cin iii ) , or ( micro- ) invasive cancer . an experienced pathologist revised all histological samples . women with cin i lesions ( mild dysplasia ) were excluded since more than fifty percent of our patients with cin i turned out to be hr - hpv - negative . healthy women who attended the outpatient clinic for a regular sterilisation procedure were recruited as hr - hpv - negative controls after sampling for histology and hpv . exclusion criteria for all participants were ( anamnestic required ) : postmenopausal state , pregnancy at time of sampling , chronic diseases ( diabetes , allergy , auto - immune ) , presence of sexually transmitted diseases ( stds ) and infection with human immunodeficiency virus ( hiv ) , signs of acute infection at time of sampling , and an immune - compromised state . with the exception of oral contraceptives , no participant used medication on a regular base . no participant had used pain - medication ( including nsaids ) for at least two weeks prior to sampling in order to avoid the well - known influence of nsaids on cytokine release from pbmcs . the study protocol was approved by the ethics committee of the erasmus medical center and all women voluntarily gave signed informed consent . cervical scrapes for hpv detection and typing were taken using a cervical bio - sampler ( accellon combi medscand medical , malm , sweden ) . hpv testing was performed with the consensus gp5+/gp6 + pcr enzyme immunoassay ( eia ) using a cocktail probe covering 37 ( sub- ) types , including all ( probably ) hr - hpv types , as previously described . we used -globin pcr to identify sampling errors and to monitor for pcr inhibitors . additionally , reverse line blot ( rbl ) analysis was performed on pcr - eia - positive cases to identify individual hpv types . for the preparation of whole blood cultures , peripheral venous blood samples were collected between 812 am in sterile endotoxin - free vacutainers ( endo tubes chromogenix ab , mlndal , sweden ) coated with na - heparin as anticoagulant , and immediately processed . for a leukocyte count peripheral venous blood samples , collected between 8 and 12 am , were drawn into endotoxin - free vacutainers ( becton - dickinson , meylan , nj , usa ) with ethylene - diaminetetra - acetic acid ( edta ) as anticoagulant and leukocyte counts performed with a sysmex xe-2100 . for preparation of whole blood cultures , blood was diluted 1:10 with rpmi 1640 culture medium with 25 mm hepes , supplemented with 10 u / ml penicillin , 100 g / ml streptomycin , and 4 mm l - glutamine ( medium and supplements from life technologies bv , breda , the netherlands ) . diluted blood was distributed in cell culture plates and incubated with phytohemagglutinin ( pha ) ( sigma - aldrish , mo , usa ) dissolved in rpmi medium to a final concentration of 10 g / ml blood culture , for 96 hours at 37c and 5% co2 . all cultures were sampled at 0 , 24 , 48 , 72 , and 96 hours , centrifuged for 10 minutes at 4c and 1500 g , and culture supernatants kept at 80c until analysis . all samples were analysed by commercially available enzyme - linked immunoassays ( biosource europe , nivelle , belgium ) for the cytokines tnf , ifn , il-2 , il-4 , il-10 , and il-12 . the detecting antibody in the immunoassay for il-12 recognized the bioactive heterodimeric ( p40 + p35 ) cytokine as well as the subunit p40 monomer or homodimer . according to the manufacturer , the minimal detectable concentrations ( mdcs ) and intra- and interassay coefficients ( cvs ) of variation were as follows : tnf : mdc , 3 pg / ml ; cvs , < 6 and < 10% ; ifn : mdc , 2 pg / ml ; cvs , < 5 and < 10% ; il-2 : mdc , 7 pg / ml ; vcs , < 6 and < 10% ; il-4 : mdc , 2 pg / ml ; cvs , < 5 and < 7% ; il-10 : mdc , 1 pg / ml ; cvs , < 5 and < 10% ; il-12 + p40 : mdc , 1.5 pg / ml , cvs , < 10 and < 10% . preliminary komolgoroff - smirnov tests showed an abnor mal distribution of cytokine values in pha - stimulated whole blood cultures . accordingly , cytokine data are presented as medians with ranges unless stated otherwise . the nonparametric kruskal - wallis test ( k. w. test ) and mann - whitney 's u - test were used as appropriate to assess differences in cytokine levels between groups . levels of statistical significance were adjusted for the number of comparisons according to bonferroni 's method , as indicated in the graphics . differences in patient characteristics between groups were evaluated by one - way anova and unpaired two - tailed t - tests . spearman 's correlations were used to investigate possible relations between age at time of sampling and released cytokines . five of them were excluded because of diabetes ( n=1 ) , allergy ( n=2 ) , autoimmune disease ( n=1 ) , or acute infection at time of sampling ( n=1 ) , leaving 30 women eligible for inclusion : 10 women with moderate dysplasia ( cin ii ) , 10 women with severe dysplasia ( cin iii ) , and 10 women with cervical carcinoma ( 8 squamous cell carcinoma , 2 adenocarcinoma ) . all women of this group revealed a positive gp5+/6 + hr - hpv pcr test . three of them were excluded because of the presence of allergy ( n=2 ) or acute infection at time of sampling ( n=1 ) , leaving 19 healthy women without cervical dysplasia . nine women had a positive hr - hpv test , 10 women tested negative for hpv - dna , forming the control group . baseline characteristics of the study groups are summarized in table 1 . the mean age of hr - hpv - positive women without cervical dysplasia is significantly lower than in the other groups . this could be expected since first infection without clinical manifestation is frequently observed in young sexually active women . spearman 's correlations between age at time of sampling and released cytokines over the whole group of patients and controls were not significant ( data not shown ) . the changes in immune - competence in our study are not related to age . the results of cytokine assays were calculated per 10 leukocytes , in order to stratify for possible different numbers of cytokine - producing leukocytes between study subjects [ 20 , 24 ] . preliminary experiments were carried out on all investigated cytokines to determine the time of peak production in response to pha stimulation of our whole blood culture system ( data not shown ) . cytokine concentrations from 0 to 96 hours stimulation time were analysed in at least six randomly chosen study subjects for each stage of cin . peak time for tnf , ifn , and il-12 + p40 production was 72 hours , for il-2 48 hours of cultivation time . a typical sample for the time - course of cytokine release in our blood culture system is shown in figure 1 . in general our data of maximum cytokine release are in accordance with kinetic studies of pbmc 's . on the basis of these results , il-2 release was determined after 48 hours , release of tnf , ifn , and il-12 + p40 after 72 hours ; and of il-4 and il-10 after 48 and 72 hours of cultivation . for calculations of the latter two cytokines values of maximal release was observed between the two groups of women without dysplasia : with the exception of il-12 all investigated cytokines were significantly increased in hr - hpv - positive women . the results are summarized in table 2 . in hr - hpv - infected women , release of th1-type cytokines ifn , tnf , and il-2 decreased with increasing grades of cin . il-12 reached a maximum in cin ii and decreased in cin iii and carcinoma ; but the differences between groups were statistically not significant ( k. w. test : p=.068 for il-12 + p40 , p=.264 for ifn , p=.077 for tnf and p=.071 for il-2 ) . release reached a maximum for il-10 and il-4 in patients with cin iii and decreased significantly for both cytokines in patients with invasive carcinoma . the results are summarized in figure 2 . in order to characterize a possible th1-type / th2-type shift we calculated the ratios of th1-type cytokines il-12 , ifn , tnf , and il-2 to th2-type cytokines il-10 and il-4 in hr - hpv infected groups . ( results of k. w. tests : il-12/il-10 p=.005 , il-12/il-4 p=.01 , ifn/il-10 p=.013 , ifn/il-4 p=.015 , tnf/il-10 p=.303 , tnf/il-4 p=.096 , il-2/il-10 p=.642 , il-2/il-4 p=.251 ) . there was a significant decrease in th1-type / th2-type ratios between cin ii and cin iii for il-12/il-4 and il-12/il-10 . also , ifn/il-4 and ifn/il-10 showed a similar though statistically not significant trend as demonstrated in figure 3 . this increase was significant for il-12/il-4 , il-12/il-10 , ifn/il-4 , and ifn/il-10 . in order to characterize a possible th-1 type cytokine pattern after establishment of an invasive carcinoma we compared cytokine levels in pha - stimulated blood cultures of patients with invasive carcinoma with levels in hr - hpv - positive women without dysplasia . there was no difference between levels of il-12 + p40 and ifn in both groups , but release of tnf and il-2 as well as of il-10 and il-4 was significantly lower in patients with carcinoma . the significant increase in th1-type as well as th2type cytokines in our hr - hpv - positive women with normal histology suggests viral activation of the systemic cytokine network and induction of cell - mediated immunity after initial hr - hpv infection ( table 2 ) . to our knowledge this is the first description of activation of the systemic cytokine network in hr - hpv - positive women without dysplasia . cytokine release changed to an antiinflammatory , tumour - promoting pattern by increase in il-4 and il-10 expression at the stage of cin iii . this result confirms and extends our earlier observations of a change to a th2-type cytokine pattern in the circulation of patients with cin iii and is in agreement with earlier studies showing a shift from th1-type to th2-type cytokines during carcinogenesis . . observed decreased ifn and il-2 and increased il-4 and il-10 in mitogen - stimulated cultures of pbmcs isolated from women with cin iii when compared with cultures from hr - hpv - negative women . described increased il-10 and decreased il-12 release in whole blood cultures of patients with hsil when compared with hr - hpv - negative controls . they found decreasing il-2 release with increasing severity of the disease , which is in agreement with our results for il-2 . the observed minimium for ifn release in cin iii but not in invasive carcinoma differs from the observations of an earlier study by mori et al . where pha - stimulated ifn release from pbmcs in cases of invasive carcinoma was significantly decreased when compared with data from healthy women . in the study of mori et al . however , the presence of hr - hpv was not investigated , which might explain the difference in results with our study . a shift to a th2-type cytokine pattern in cin iii was more obvious when the ratios between th1-type and th2-type cytokines ( figure 2 ) are evaluated . they show a tumour - promoting change in cytokine balance , significant for il-12/il-4 and il-12/il-10 , and a trend for ifn/il-4 , ifn/il-10 , and tnf/il-4 . our study describes for the first time changes in the cytokine pattern within the cytokine network , developing from hr - hpv infection without clinical symptoms via cin ii and cin iii to carcinoma . il-12 is one of the first cytokines released during an innate immune reaction and stimulates a th-1 type cytokine response in cell - mediated immunity . our hr - hpv - positive women with normal histology demonstrated significantly increased th1- and th2-type cytokine release , with the exception of il-12 which was low . our observation of high secretion of il-12 in cin ii might be explained by an observation made by moscicki et al . . these authors reported high levels of il-12 in cervical mucous in hsil and hypothesized that high il-12 levels could represent a defence mechanism in turning on a th1-type antitumour response and , as il-12 is known to inhibit angiogenesis , preventing growth of a tumour . the significant increase of the four cytokine ratios between cin iii and carcinoma may indicate that the presence of a tumour with an inflammatory reaction and exposure of viral antigens ( high viral load ) eventually induces a certain t - cell response . this response remains incomplete as shown in our cytokine data presented in table 3 . values of ifn and il-12 release in cervical carcinoma are comparable to data obtained after initial hr - hpv infection ; all other cytokine levels remain significantly lower . these results suggest a second deregulated and incompetent immune response in cervical carcinoma , probably due to manifestation of an inflammatory effect of the tumour itself . this reaction is partly comparable to the inflammatory reaction on the initial hr - hpv infection , as expressed in the ratio 's of ifn and il-12 in figures 3(a ) , 3(b ) , 3(e ) , 3(f ) . these results are in agreement with observations of de jong et al . , and steele et al . studied t - cell responses to hpv 16 oncoproteins by measuring ifn release in women with low- and high - grade cin and cervical carcinoma and found higher levels of t - cell responses in carcinoma patients compared to high - grade cin cases . a similar observation was made by de jong et al . who investigated hpv16-positive women . this study reports a higher frequency of hpv16-specific cd4 + t - cell responses in patients with cervical carcinoma than in women with cin iii lesions . the increase in the ifn/il-4 ratio found in our study was not observed by de jong et al . when t - cell cultures were stimulated with pha . in part , this discrepancy might be owing to differences in hr - hpv types within the study groups since de jong et al . correlations between specific hpv types and ifn release , possibly influenced by ifn gene polymorphisms , are suspected but not yet fully investigated . it was our goal to study changes in the cytokine network in blood of hr - hpv - infected women at various stages of cin upto onset of cervical carcinoma . the use of whole blood cultures for determination of mitogen - stimulated cytokine release by immunocompetent leukocytes has distinct advantages over cultures of isolated leukocytes or lymphocytes . it permits interaction between different leukocytes , preserves concentrations of stimulatory and inhibitory mediators , and avoids activation and changes in cell ratios associated with procedures of isolation and purification . for stimulation of the cytokine network we chose the mitogen pha . pha activates mainly lymphocytes and induces rapid cell proliferation together with release of inflammatory and immune cytokines . endotoxin ( lps ) as used in jacobs ' study induces mainly inflammatory cytokines but almost no lymphocyte - derived interleukins . most studies dealing with cytokine patterns in hr - hpv - related cervical neoplasia and cancer concentrate on infections with hpv 16 ( the most frequently observed oncogenic hpv type in caucasian population ) . in contrast to these studies we did not select our patients for particular hr - hpv types . the small sample size of our study groups did not allow us to correlate cytokine response with specific hr - hpv - types . further studies with enlarged numbers of participants are needed to investigate the individual impact of different hr - hpv - types on the cytokine network . 1 . our study suggests that infection with hr - hpv in women without cervical dysplasia induces activation of the cytokine network . 2 . manifestation of a tumour induces a second deregulated and incompetent immune response . 3 . our results confirm and expand our earlier observations on circulating cytokines : significant changes in the kinetics of cytokine release to a th2-type immune response in blood of women with cervical dysplasia occur progressively from cin ii to cin iii . these immunological findings are supported by clinical observations : many cin i or ii lesions usually regress without treatment , whereas cin iii lesions mostly will develop into invasive cancer if not properly treated .

aims . we investigated the effect of hr - hpv infection on the capacity of the cytokine network in whole blood cultures during carcinogenesis of cervical carcinoma . methods . thirty - nine women with moderate dysplasia , severe dysplasia , cervical carcinoma , or without dysplasia formed the study group . the control group consisted of 10 hr - hpv - negative women without cin . whole blood cultures were stimulated with phytohemagglutinin ( pha ) and concentrations of tumour necrosis factor ( tnf ) , interferon ( ifn ) , interleukin 2 ( il-2 ) , interleukin 12 ( il-12 ) , interleukin 4 ( il-4 ) , and interleukin 10 ( il-10 ) were determined by elisas . results . a significant increase in cytokine release was detected in hr - hpv - positive women without dysplasia . in women with cervical cancer , release of ifn and il-12 was of the same magnitude as in hr - hpv - positive women without clinical manifestations . most th1-type / th2-type ratios decreased form cin ii to cin iii , and increased from cin iii to invasive carcinoma . conclusions . ( 1 ) infection with hr - hpv without expression of cervical dysplasia induces activation of the cytokine network . ( 2 ) increases in ratios of th1-type to th2-type cytokines at the stage of cervical carcinoma were found by comparison with stage cin iii . ( 3 ) significant changes in the kinetics of cytokine release to a th2-type immune response in blood of women with cervical dysplasia occurred progressively from cin ii to cin iii .

1. INTRODUCTION 2. MATERIALS AND METHODS 3. RESULTS 4. DISCUSSION 5. CONCLUSIONS

damage - associated molecular patterns ( damps ) are endogenous molecules that can perpetuate inflammatory responses during cell stress or injury . the ecm glycoprotein tn - c , is specifically induced upon tissue injury [ 1 , 2 ] and infection [ 3 , 4 ] and upregulated in septic patients . tlr4-mediated tn - c expression induces cytokine production in both human and murine macrophages and in rheumatoid arthritis synovial fibroblasts . importantly , glucocorticoids can inhibit the expression of tn - c in bone marrow stromal cells and fibroblasts . in addition , mice and bone marrow - derived macrophages ( bmdms ) deficient in tn - c display lower production of proinflammatory cytokines such as tnf- during lps - induced sepsis . thus , tn - c has been recognized as a regulator of the early immune response . il-10 is a vital anti - inflammatory cytokine which is required for dampening inflammatory signals and defending the host from excessive inflammation . mice lacking il-10 infected with bacterial pathogens display high mortality , associated with excessive inflammatory responses . low levels of il-10 expression were associated with various inflammatory diseases such as ulcerative colitis , crohn 's disease , and asthma in humans [ 11 , 12 ] . the anti - inflammatory effect of il-10 is mediated through the jak1-stat3 pathway which leads to the inhibition of proinflammatory proteins such as tnf- and il-6 [ 10 , 13 ] . higher expression of il-10 was found in bmdms from tn - c - deficient mice while there was lower expression of proinflammatory cytokines , indicating an anti - inflammatory role of il-10 in the tn - c - mediated inflammatory disease model . carbon monoxide ( co ) is generated as an end product of the oxidative degradation of heme by the enzymatic action of heme oxygenase , which converts heme into biliverdin , free iron , and co . anti - inflammatory effects of co have been evident in murine models of sepsis , postoperative ileus , and organ xenotransplantation [ 15 , 16 ] . in addition , co has been found to be an important regulator in the suppression of inflammatory cytokines and mediators including inducible nitric oxide synthase ( inos ) , tnf- , and il-6 [ 17 , 18 ] as well as induction of the anti - inflammatory cytokine il-10 . to date , there are no reports regarding the effects of co - mediated il-10 production on the regulation of tn - c - mediated inflammation . therefore , in the current study , we examined the effects of co - dependent il-10 generation on tn - c - mediated inflammation in macrophages and in the septic mice model . tenascin - c antibody was purchased from cell signaling technology ( ma , usa ) . -actin , anti - mouse and anti - goat antibodies conjugated to horseradish peroxidase were obtained from santa cruz biotechnology ( santa cruz , ca , usa ) . lipopolysaccharide ( lps ) and protease inhibitor cocktail sets were purchased from sigma - aldrich ( st . louis , mo , usa ) . dulbecco 's modified eagle medium ( dmem ) , fetal bovine serum ( fbs ) , penicillin - streptomycin , and sodium pyruvate were purchased from invitrogen ( grand island , ny , usa ) . raw 264.7 cells and peritoneal macrophages were cultured in dmem ( invitrogen ) containing 10% fetal bovine serum ( fbs ) and 1% penicillin streptomycin at 37c in 5% co2 until 7580% confluence . for preparation of peritoneal macrophages , mice were injected intraperitoneally with 3% thioglycolate for 3 days and cells were collected for culture . cells ( 5 10/ml ) were seeded in 6-well plates and incubated overnight for subsequent experiments . seven - week - old wild type male c57bl/6 mice were pretreated with corm-2 ( 30 mg / kg , i.p . ) or rucl3 ( 30 mg / kg , i.p . ) . and then , mice were injected with lps ( 10 mg / kg , i.p . ) . after 2 hours , blood serum and liver tissues were collected and stored at 80c for protein and rna analysis . all experiments with mice were approved by the animal care committee of the university of ulsan , ulsan , korea . raw 264.7 cells ( 5 10/ml ) were cultured in 6-well plates for 3 h and transfected with il-10 sirna ( 100 nm ) or tn - c sirna ( 100 nm ) from santa cruz biotechnology using lipofectamine 2000 according to the manufacturer 's instructions . after transfection , cells were incubated with or without corm-2 ( 20 m ) and then stimulated with or without lps ( 100 ng / ml ) . cell extracts were lysed using lysis buffer containing ripa buffer , protease inhibitor , and phosphatase inhibitors . after lysis , protein concentration was measured by bca assay ( pierce biotechnology inc . , samples containing equal amounts of protein were subjected to electrophoresis and proteins were transferred to polyvinylidene difluoride ( pvdf ) membranes . membranes were blocked with 5% skim milk for 20 min and then incubated at 4c overnight with primary antibodies , followed by secondary antibodies against tn - c and -actin conjugated with horseradish peroxidase . the enhanced chemiluminescence ( ecl ) western blotting detection system ( ge healthcare life sciences , buckinghamshire , uk ) was used to visualize the immunoreactive bands . total rna isolation was performed from raw 264.7 macrophages using trizol reagent ( invitrogen ) according to manufacturer 's instructions . briefly , total rna ( 2 g ) was used to prepare cdna by using m - mlv reverse transcriptase ( promega corporation , madison , wi , usa ) and oligo ( dt ) 15 primer ( promega ) . the resulted cdna was subjected to pcr for mouse gapdh ( 5-aggccggtgctgagtatgtc-3 , 5-tgcctgcttcaccttct-3 , 530 bp ) , ho-1 ( 5-tcccagacaccgctcctccag-3 , 5-ggatttggggctggtttc-3 , 313 bp ) , tn - c ( 5-caggtacttcttcacggagc-3 , 5-gcagtcttccccagtgaaac-3 , 834 bp ) , tnf- ( 5-agcccacgtcgtagcaaaccaccaa-3 , 5-acacccattcccttcacagagcaat-3 , 421 bp ) , il-6 ( 5-gtggaaatgagaaaagagttgt-3 , 5-cctcttggttgaagatatgaat-3 , 283 bp ) , and il-10 ( 5-gacaataactgcacccactt-3 , 5-tcaaatgctccttgatttct-3 , 250 bp ) , and gapdh was used as internal loading control . total rna was extracted from raw 264.7 peritoneal macrophages / liver tissues using trizol reagent ( invitrogen ) according to the manufacturer 's instructions . in addition , cdna was prepared by using m - mlv reverse transcriptase ( promega ) and oligo ( dt ) 15 primer ( promega ) . the formulated cdna was subjected to real time rt - pcr using sybr green qpcr master mix ( 2x ) ( usb products , affymetrix ) on an abi 7500 fast real - time pcr system ( applied biosystems ) for mouse gapdh ( 5-gggaagcccatcaccatct-3 , 5-cggcctcaccccatttg-3 ) , tn - c ( 5-accatgctgagatagatgttccaaa-3 , 5-cttgacagcagaaacaccaatcc-3 ) , tnf - a ( 5-agaccctcacactcagatcactttc-3 , 5-ttgctacgacgtgggctaca-3 ) , il-6 ( 5-cgatgatgcacttgcagaaa-3 , 5-tggaaattggggtaggaagg-3 ) , il-10 ( 5-actgctatgctgcctgctcttact-3 , 5-gaattcaaatgctccttgatttct-3 ) , and ho-1 ( 5-tcagtcccaaacctcgcggt-3 , 5-gctgtgcaggtgttgagcc-3 ) . macrophages on 6-well plates were incubated overnight and then pretreated with corm-2 for 1 h followed by stimulation with lps for 24 h. in addition , mice were administrated with corm-2 for 2 h and then sepsis was induced by lps injection . after 2 h , supernatants collected from various samples or blood serum collected from different mice were assayed for tnf- and il-6 by using a mouse elisa kit ( biolegend ) . statistical differences between groups were evaluated by one - way anova ( nonparametric ) or student 's t - test when multiple groups were compared . differences were considered to be significant when p < 0.05 , p < 0.01 , and p < 0.001 . stimulation of macrophages with gram - negative bacterial lps can enhance the expression of tn - c . tlr4 was involved in the induction of tn - c and subsequent cytokine synthesis in both human and murine macrophages and human chondrocytes . in the present study , we examined inflammatory responses in murine raw 264.7 macrophages treated with lps ( 100 ng / ml ) . tn - c mrna and protein expression increased at 4 and 8 h after lps treatment ( figures 1(a ) and 1(b ) ) , respectively . therefore , in subsequent experiments , we measured tn - c mrna and protein expression at 8 h. furthermore , lps dose - dependently increased tn - c mrna and protein expression at 8 h ( figures 1(c ) and 1(d ) ) . these results suggest that lps induces tn - c expression in a time- and dose - dependent manner in raw 264.7 macrophages . the anti - inflammatory , antiapoptotic , and cytoprotective properties of co are well known . furthermore , it has been reported that the generation of endogenous co was necessary for il-10-dependent inhibition of tnf- expression . co can be generated pharmacologically from co - releasing molecules ( corms ) , which consist of a heavy metal such as ruthenium surrounded by carbonyl groups [ 26 , 27 ] . in brain endothelial cells , the lps - induced activation of inflammatory signals such as nf-b ( p65 ) , cox-2 expression , and pge2 production was inhibited by corm-2 pretreatment . in the present study , we investigated the effects of co on tn - c - mediated inflammation . we found that corm-2 significantly and dose - dependently suppressed the expression of lps - stimulated tn - c expression ( figure 2(a ) ) . in addition , pretreatment with corm-2 significantly reduced the mrna and protein levels of proinflammatory cytokines such as tnf- and il-6 ( figures 2(b ) , 2(c ) , and 2(d ) ) , respectively . to confirm the effects of co on tn - c - meditated inflammation , cells were pretreated with or without corm-2 or rucl3 ( negative control for corm-2 ) and then stimulated with or without lps . interestingly , corm-2 significantly downregulated lps - induced tn - c as well as proinflammatory cytokines expression whereas rucl3 did not have any effect ( figures 2(e ) , 2(f ) , 2(g ) , and 2(h ) ) . to confirm the effects of co consistently , co gas dramatically reduced lps - stimulated expression of tn - c ( figure 2(i ) ) , as well as tnf- ( figure 2(j ) ) and il-6 ( figure 2(k ) ) . to further confirm the effects of co on lps - induced tn - c expression and proinflammatory cytokines , mouse peritoneal macrophages were pretreated with corm-2 at various concentrations and incubated with lps . we found that corm-2 dramatically decreased lps - induced tn - c ( figure 3(a ) ) and its downstream cytokines ( figure 3(b ) ) . in contrast , rucl3 did not reduce the expression of tn - c and proinflammatory cytokines ( figures 3(c ) and 3(d ) ) . low doses of co suppressed inflammatory responses in a murine model of sepsis through inhibition of inflammatory cytokines production [ 18 , 29 ] as well as increased lps - induced expression of the anti - inflammatory cytokine il-10 in various cell types [ 19 , 29 ] . in addition , mice deficient with tn - c displayed lower levels of tnf- and downstream cytokine production in lps - treated septic mice and bone marrow - derived macrophages ( bmdms ) . in our study , we investigated the effects of co on tn - c - induced proinflammatory cytokines expression and the expression of anti - inflammatory il-10 in raw 264.7 and peritoneal macrophages . corm-2 significantly and dose - dependently induced the expression of il-10 in lps - stimulated macrophages ( figures 4(a ) and 4(b ) ) . furthermore , treatment with co gas significantly increased levels of il-10 ( figure 4(c ) ) in lps - stimulated raw 264.7 macrophages . however , treatment with rucl3 did not have any effect on il-10 expression in these cells ( figures 4(d ) and 4(e ) ) . these observations indicate that the anti - inflammatory effects of co are mediated by il-10 in lps - stimulated macrophages . the incubation of raw 264.7 cells with tn - c sirna significantly suppressed the effects of lps on tn - c and proinflammatory cytokines production ( figures 4(f ) , 4(g ) , and 4(h ) ) , whereas it had no effect on il-10 expression ( figure 4(i ) ) , suggesting that il-10 is regulated independently of tn - c and its downstream cytokines . to confirm the function of co - induced il-10 on tn - c - mediated inflammation , macrophages were transfected with il-10 sirna and treated with corm-2 prior to lps - stimulation . we found that il-10 sirna reversed the inhibitory effect of corm-2 on tn - c expression and inflammatory cytokines production in lps - stimulated macrophages relative to control sirna ( figures 4(k ) , 4(l ) , and 4(m ) ) . pretreatment of recombinant il-10 with or without lps stimulation showed the same efficiency of corm-2 to significantly decrease tn - c expression ( figure 4(n ) ) . also , ho-1 increases il-10 production . according to inoue and colleagues , overexpressions of ho-1 thus , we examined the expression of anti - inflammatory gene ho-1 under these conditions . interestingly , we found that corm-2 significantly increased the level of ho-1 expression ( figure 4(o ) ) , whereas rucl3 did not have any effect on ho-1 expression ( figure 4(o ) ) and conversely decreased the expression levels of tn - c . the inhibition of ho activity using znppix , however , did not reverse the effects of corm-2 on tn - c expression ( figure 4(p ) ) indicating that corm-2 mediated suppression of tn - c is independent of ho activity in lps - stimulated raw 264.7 macrophages . based on these results , we conclude that co - induced il-10 inhibits tn - c - mediated inflammation . sepsis , a systemic inflammatory response , results from excessive production of proinflammatory cytokines by lps stimulation . in addition , proinflammatory cytokines such as tnf- , il-1 , and il-6 have been found at higher levels in septic patients [ 32 , 33 ] . administration of lps in mice revealed that tn - c expression is necessary for proinflammatory signaling . furthermore , application of exogenous co inhibits lps - induced production of tnf- while it increases il-10 production in vitro and in vivo . however , there are no reports regarding the effects of co - mediated il-10 production in relation to the regulation of tn - c and inflammation in a septic mouse model . in our study , to examine the in vivo effects of co using corm-2 on lps - induced endotoxemia and tn - c - mediated inflammatory cytokines expression , we pretreated mice with corm-2 ( 30 mg / kg , i.p . ) or rucl3 ( 30 mg / kg , i.p . ) for 2 h and lps ( 10 mg / kg , i.p . ) for 2 h. interestingly , corm-2 significantly decreased tn - c ( figure 5(a ) ) , tnf- and il-6 mrna expression ( figure 5(b ) ) , and protein secretion ( figure 5(c ) ) and simultaneously increased il-10 expression ( figure 5(d ) ) in liver tissue from lps - induced endotoxemic mice . also , the levels of il-10 ( figure 5(e ) ) were increased and reversely tn - c levels ( figure 5(f ) ) were decreased in the serum of mice treated with corm-2 . therefore , the results from in vivo experiments suggest that co inhibited tn - c and its downstream inflammatory cytokines whereby il-10 expression was upregulated in a septic mice model . tn - c is unique in its distinct pattern of expression . upon tissue injury tn - c is transiently expressed , whereas its expression is reduced after the tissue is repaired . moreover , persistent tn - c expression occurs during chronic inflammation . in addition , tn - c is absent in most healthy adult tissues whereas high levels are found during infection and in patients with sepsis . however , tn - c is expressed at sites of inflammation regardless of the location or type of causative insult , indicating its capability to participate in the global inflammatory response . tn - c can increase the synthesis of cytokines in human chondrocytes and myeloid cells in a tlr4-dependent manner and also activate murine myeloid cells [ 35 , 36 ] . additionally , tn - c expression is transiently induced by lps in innate immune cells in a nf-b - dependent manner [ 7 , 37 ] and its dysregulation is observed in both autoimmune and inflammatory diseases , such as sepsis . in the present study , we found that lps significantly increased tn - c and proinflammatory cytokines production in macrophages as well as in a mouse model . therefore , understanding which compounds can inhibit tlr - mediated tn - c expression and cytokines production may refine strategies to manipulate excessive inflammation . recently , researchers reported that co gas can exert beneficial effects in various cell and animal models . co plays an important role in preventing apoptosis in several cell types such as endothelial cells , fibroblasts , and pancreatic -cells and inhibits the proliferation of smooth muscle cells , thus preventing atherosclerotic lesions . in animal models , corm compounds provide a reliable source of co that can mimic co gas in many biological functions [ 26 , 45 ] . therefore , corms represent important tools to understand the biological significance of co in physiology and disease . corm-2 was the first compound used to deliver co in biological systems in a controlled manner . in the current study , to examine the effects of co , we pretreated macrophages and mice with corm-2 in an lps - stimulated inflammation model . interestingly , corm-2 significantly decreased lps - induced tn - c and proinflammatory cytokines production in vitro and in vivo . similarly , pretreatment with co gas significantly decreased lps - stimulated tn - c and cytokines production in macrophages , supporting the direct effects of co on tn - c - mediated inflammation . in addition , rucl3 , a negative control for corm-2 , did not affect lps - mediated tn - c , tnf- , and il-6 expression in macrophages or in septic mice . these results confirm that co inhibits lps - mediated tn - c and proinflammatory cytokines production . the anti - inflammatory cytokine il-10 plays a crucial role in dampening toll - like receptor ( tlr ) signaling - induced proinflammatory genes . interestingly , co was found to increase the levels of anti - inflammatory , il-10 , while the levels of proinflammatory cytokines were decreased in several in vitro systems [ 18 , 29 ] . additionally , corm-2 was also found to regulate inflammatory responses through decreasing il-1 expression and increasing il-10 expression . in a sepsis model , co - mediated activation of the mkk3/p38 mapk signaling pathway was involved in the induction of il-10 . in our investigation , we determined that the effects of co significantly increased il-10 expression under lps - stimulated conditions while rucl3 had no effect in vitro or in vivo . furthermore , il-10 sirna significantly reversed the effects of corm-2 on tn - c and proinflammatory cytokines production whereas tn - c sirna significantly decreased proinflammatory gene expression levels without having an effect on co - mediated il-10 expression . this evidence suggests that co - mediated il-10 expression was involved in inhibition of tn - c - mediated inflammation . in summary , we identified that co - induced il-10 was involved in the inhibition of tlr4 signaling - dependent tn - c expression and thus inhibited the inflammatory response in vitro and in vivo . this study describes a novel co - dependent il-10 signaling pathway responsible for the inhibition of tn - c - driven inflammation and potentially provides the rationale for novel therapeutic strategies for the treatment of inflammatory diseases .

tenascin - c ( tn - c ) , an extracellular matrix ( ecm ) glycoprotein , is specifically induced upon tissue injury and infection and during septic conditions . carbon monoxide ( co ) gas is known to exert various anti - inflammatory effects in various inflammatory diseases . however , the mechanisms underlying the effect of co on tn - c - mediated inflammation are unknown . in the present study , we found that treatment with lps significantly enhanced tn - c expression in macrophages . co gas , or treatment with the co - donor compound , corm-2 , dramatically reduced lps - induced expression of tn - c and proinflammatory cytokines while significantly increased the expression of il-10 . treatment with tn - c sirna significantly suppressed the effects of lps on proinflammatory cytokines production . tn - c sirna did not affect the corm-2-dependent increase of il-10 expression . in cells transfected with il-10 sirna , corm-2 had no effect on the lps - induced expression of tn - c and its downstream cytokines . these data suggest that il-10 mediates the inhibitory effect of co on tn - c and the downstream production of proinflammatory cytokines . additionally , administration of corm-2 dramatically reduced lps - induced tn - c and proinflammatory cytokines production while expression of il-10 was significantly increased . in conclusion , co regulated il-10 expression and thus inhibited tn - c - mediated inflammation in vitro and in vivo .

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

type 2 diabetes mellitus ( t2 dm ) is one of the most common noncommunicable diseases characterized by insulin resistance and impaired insulin secretion [ 1 , 2 ] . metabolic proinflammatory disorder including chronic hyperglycemia and increased levels of circulating cytokines suggests immunological disturbances [ 37 ] , which seriously affects the quality of life of the patients and imposes a large economic burden on the national health care system . genetic and environmental factors are blamed for t2 dm and up to 25% of first - degree relatives of t2 dm patients may develop this disease . the origin and development of t2 dm were involved in multiple risk factors . regulatory t cells ( treg ) and cytokines play important roles in the development of t2 dm . treg is a subset of cd4 t cells that maintain peripheral tolerance and suppress antigen specific immune responses by secreting transforming growth factor- ( tgf- ) , interleukin-10 ( il-10 ) , and il-4 to inhibit autoimmunity . it was found that the ratios of cd4cd25treg / th17 cells and cd4cd25treg / th1 cells were significantly decreased in t2 dm patients . expression of foxp3 , a key player for the development and function of treg , correlates well with regulatory activity and number of treg . indeed , foxp3 is exclusively expressed in cd4cd25treg [ 1316 ] . a positive correlation between cd4cd25foxp3treg and the enhanced expression of il-6 on cd4 t cells il-10 , as a multifunctional cytokine and secretion of treg , plays a key role in the inflammatory response that is associated with insulin resistant states and t2 dm . increased levels of il-17 were found to protect against autoimmune mediated t1 dm in nonobese diabetic mice . on the other hand , loss of il-17 has been associated with disease susceptibility in part because it has been suggested that the absence of il-17 results in enhanced production of other proinflammatory cytokines . tgf- is also a multifunctional cytokine circulating as a biologically inactive form in human plasma [ 21 , 22 ] . the tgf- family includes multifunctional molecules that exert specific effects on cell proliferation , differentiation , migration , development , tissue remodeling , and repair . tnf- inhibits the insulin signaling cascade through regulating several pivotal regulatory proteins , such as the insulin receptor substrate ( irs ) and akt substrate 160 in human skeletal muscle in vitro and in vivo . it has reported that polymorphism of immune genes such as tnf- and tgf- was associated with the development of t2 dm . intriguingly , increased renal production of tgf- was a distinct feature of diabetes [ 2831 ] . within the past few years , many clinical studies have been focusing on the association of treg with proinflammatory and immunosuppressive cytokines in t2 dm . despite intensive research efforts , therefore , we performed this meta - analysis synthesizing the data from case - control studies to evaluate changes of treg , il-6 , il-10 , il-17 , tgf- , and tnf- in t2 dm patients . our study was conducted according to the preferred reporting items for systematic reviews and meta - analyses ( prisma ) criteria . we identified relevant studies of treg , il-6 , il-10 , il-17 , tgf- , and tnf- in t2 dm patients by systematically searching pubmed , wanfang database , chinese - cqvip , and cnki databases from february 1 , 1991 , to july 15 , 2016 . il-17 ) or ( transforming growth factor beta or tgf- ) or ( tumor necrosis factor alpha or tnf- ) or ( regulatory t cells or treg or cd4cd25 t cell or cd4cd25foxp3 t cell ) and ( type 2 diabetes mellitus or type 2 diabetes or diabetes mellitus or in addition , we also conducted an extensive literature search and articles were further identified in reference lists . we reviewed all relevant articles using the following inclusion criteria : ( 1 ) the study should evaluate the relationship of cd4cd25foxp3treg , cd4cd25treg , il-6 , il-10 , il-17 , tgf- , or tnf- with t2 dm patients ; ( 2 ) the design had to be a case - control study ; ( 3 ) original data were displayed or could be converted to as mean sd ; and ( 4 ) original report showed no duplicated data . the data were extracted independently by two reviewers ( yong - chao qiao and jian shen ) by using predefined data extraction forms and the quality of all eligible studies was evaluated according to the newcastle - ottawa scale ( nos ) . the following information was extracted : ( 1 ) name of the first author ; ( 2 ) date of publication ; ( 3 ) country of the study ; ( 4 ) study design ; ( 5 ) sample size of patients and controls ; ( 6 ) mean age of the sample ; and ( 7 ) mean sd of patients and controls . in case of disagreement , a third investigator ( hai - lu zhao ) we presented the data ( sample size , mean sd ) to illustrate the changes of treg , il-6 , il-10 , il-17 , tgf- , and tnf- in t2 dm patients versus healthy controls , and chi - squared q test and i statistics were used to assess heterogeneity . when p < 0.1 or i > 50% , the heterogeneity was considered significant and a random effect model was used ; otherwise , a fixed - effect model was used . considering the influence of diabetic complications , patients were divided into two groups ( t2 dm with complication and t2 dm without complication ) for subgroup analysis . we performed sensitivity analysis by limiting the studies of nos score 7 or excluding studies with a high risk of bias . publication bias was examined graphically by constructing egger 's test and p < 0.05 was considered to be representative of statistically significant publication bias . the flow chart of the article search and inclusion process was displayed in figure 1 . based on the search strategy , a total of 5,064 articles were collected and 332 were removed after our initial screening . furthermore , 3,954 articles were excluded because they were not dm relevant , have no controls , or were animal studies or review articles . then , we excluded 687 studies because of duplicated data , no original data , or original data expressed with figures . eventually , this meta - analysis included 91 articles involving 138 case - control studies of 5642 t2 dm patients and 7378 healthy controls : 13 for il-6 [ 3446 ] , 22 for tgf- [ 23 , 4767 ] , 7 for tnf- [ 3436 , 38 , 45 , 68 , 69 ] , 6 for cd4cd25foxp3treg [ 7075 ] , 15 for il-10 [ 7690 ] , 18 for cd4cd25treg [ 70 , 72 , 74 , 75 , 91104 ] , and 10 for il-17 [ 105114 ] . t2 dm patients had significantly increased levels of serum il-6 ( smd , 1.28 ; 95% ci , 0.73 to 1.83 ; p < 0.001 ) ( figure 2 ) , tgf- ( smd , 2.88 ; 95% ci , 2.37 to 3.40 ; p < 0.001 ) ( figure 3 ) , and tnf- ( smd , 1.56 ; 95% ci , 1.10 to 2.02 ; p < 0.001 ) ( figure 4 ) but significantly decreased the percentage of cd4cd25foxp3treg ( smd , 0.47 ; 95% ci , 0.72 to 0.23 ; p < 0.001 ) ( figure 5 ) and the level of serum il-10 ( smd , 1.37 ; 95% ci , 2.32 to 0.42 ; p = 0.005 ) ( figure 6 ) . changes in the percentage of cd4cd25treg ( smd , 0.24 ; 95% ci , 0.76 to 0.28 ; p = 0.360 ) ( figure 7 ) and il-17 ( smd , 0.51 ; 95% ci , 1.87 to 0.84 ; p = 0.459 ) ( figure 8) were not significant . some but not all the results of the meta - analysis displayed significant heterogeneity . subgroup analysis was performed to explore the impact of diabetic complication on the changes in treg and cytokines . as shown in figures 28 , both t2 dm patients with complication and the patients without complication had significantly increased levels of serum il-6 ( figure 2 ) , tgf- ( figure 3 ) , and tnf- ( figure 4 ) , while not significant changes were found in the percentage of peripheral cd4cd25treg ( figure 7 ) and il-17 ( figure 8) . intriguingly , t2 dm patients with complication showed lower percentage of peripheral cd4cd25foxp3treg ( p < 0.001 ) ( figure 5 ) , whereas patients without complication had decreased levels of serum il-10 ( p = 0.033 ) ( figure 6 ) . the high heterogeneity existed in some subgroup analysis . in order to explore the source of heterogeneity , we further conducted regression analysis according to the complication as covariate . the results were as follows : tgf- ( t = 4.08 ; p < 0.001 ; 95% ci , 1.23 to 3.65 ) , il-6 ( t = 0.09 ; p = 0.929 ; 95% ci , 1.09 to 1.18 ) , tnf- ( t = 0.34 ; p = 0.740 ; 95% ci , 1.23 to 1.67 ) , cd4cd25foxp3treg ( t = 2.04 ; p = 0.097 ; 95% ci , 1.55 to 0.18 ) , il-10 ( t = 0.36 ; p = 0.723 ; 95% ci , 5.33 to 3.77 ) , cd4cd25treg ( t = 0.63 ; p = 0.534 ; 95% ci , 0.96 to 1.81 ) , and il-17 ( t = 0.56 ; p = 0.586 ; 95% ci , 4.84 to 2.86 ) . therefore , diabetic complication was a key influencing factor for the high heterogeneity in the meta - analysis of tgf- but not the others . sensitivity analysis was used to assess the stability of the results by excluding studies with high risk of bias and no significant changes in the results were found . we further conducted sensitivity analysis by including studies with high nos score ( 7 ) and found that all the results remained consistent . egger 's test showed significant publication bias in the meta - analysis of tnf- but not the others ( figure 9 ) . in this study , we found that the patients with t2 dm had increased serum levels of il-6 , tgf- , and tnf- but decreased percentage of peripheral cd4cd25foxp3treg and serum il-10 level . furthermore , the percentage of peripheral cd4cd25foxp3treg and serum il-10 level were influenced by diabetic complication . the expression of inflammatory and proinflammatory cytokines from peripheral blood t lymphocyte plays an important role in the development of diabetes and diabetic complications . many studies have proved the maintenance of immunological self - tolerance by cd4cd25treg and cd4cd25foxp3treg . the finding of decreased percentage of peripheral cd4cd25foxp3treg in t2 dm patients indicates that foxp3 might be a key player for the development and function of treg . some researchers also considered that the differentiation and function maintenance of treg were dependent on the expression of the foxp3 , and , consequently , foxp3 is considered as the key transcriptional factor in treg cells [ 117119 ] . il-10 and tgf- secreted by treg [ 116 , 120 ] are the biomarkers in t2 dm patients [ 2 , 116 ] . previous studies suggested that il-10 could suppress the proliferation of t leukomonocyte and the secretion of cytokines , whereas tgf- may sustain the expression of foxp3 in cd4cd25treg to enhance immunosuppressive function [ 122 , 123 ] . consistent with our findings , several studies have shown a significantly decreased level of serum il-10 in t2 dm patients [ 88 , 124 ] . a recent investigation has showed that il-6 could enhance treg in mice . in the present meta - analysis of t2 dm patients , increased levels of serum il-6 , tgf- , and tnf- coexisted with decreased levels of il-10 and decreased percentage of cd4cd25foxp3treg . this finding highlights that the cytokines and growth factors may originate from multiple sources such as macrophages , t cells , and other tissue cells rather than treg alone . furthermore , chronic persistent activation of innate immunity and il-6 secretion occurring in t2 dm might inhibit the development of inducible treg cells . th17 cells could produce il-17 , tnf- , and il-6 and induce inflammation in the pathogenesis of autoimmune diseases . th17 cells are a major t cell subset implicated in the pathogenesis of multiple sclerosis , rheumatoid arthritis , and psoriasis . a previous study has revealed that not only th1/th2 imbalance but also th17/treg imbalance can contribute to the pathogenesis of autoimmune diseases such as t1 dm as well as proinflammatory disorders and such as t2 dm . t2 dm patients have elevated serum levels of il-6 , il-1 , and tgf- , the cytokines known to induce th17 differentiation . enhanced production of il-6 and tnf- and decreased levels of serum il-10 that occurred in t2 dm patients may suppress treg cells and ratios of treg to th17 and th1 cells [ 132 , 133 ] . the immunocompromised effects on macrophages and lymphocytes likely drive an inflammatory state to contribute to the occurrence of diabetic complications . here , in this study , no significant changes of foxp3treg cells and serum il-17 levels were found in t2 dm subjects without complication . there is an intimate relationship of the differentiation of th17 cells with the relative abundance of peripheral cd4cd25foxp3treg cells and the serum levels of il-6 , il-10 , and tgf-. although changes of serum levels of il-17 were not significant in this meta - analysis of t2 dm patients versus controls , il-17 may be a clue to the possible involvement of th17 cells in t2 dm pathogenesis . firstly , a decrease of treg cells might be accompanied by an increase of th17 cells . the study by guan et al . has indicated the existence of a developmental switch between th1/th17 cells , on one hand , and th2/treg cells , on the other hand . secondly , in the presence of high serum levels of il-6 and tgf- , as we reported here , differentiation of th17 cells might be favoured . lastly , th17 cells might be , together with innate cells , a primary source of the increased il-6 levels and might be actively orchestrating the immunity - driven , chronic inflammation of target tissues and organs in t2 dm . in this systematic review , the studies examining the number of th17 cells in t2 dm were too scarce for being included in the meta - analysis . future studies are required to focus on the role of th17/treg and products of the th17 cells in the pathogenesis of t2 dm and associated complications . diabetic complications such as retinopathy , nephropathy , and cardiovascular disease affect immune cells and cytokines in type 2 diabetes [ 135 , 136 ] . actually , urinary tgf- levels are elevated in the presence of microalbuminuria and overt proteinuria . additionally , elevated plasma tgf- may reflect the state of hyperglycemia in t2 dm patients . systemic inflammation in t2 dm is linked to the development of diabetic complications [ 138 , 139 ] . yet , the mechanism of immune alteration in t2 dm and diabetic complication remains unclear . in this meta - analysis , diabetic complication indeed has an impact on the percentage of peripheral cd4cd25foxp3treg and level of serum il-10 . the percentage of treg cells and levels of cytokines in t2 dm may also depend on ethnicity , sex , weight , age , and disease duration . the results of egger 's tests explain that no publication bias existed in all comparisons except for tnf-. the publication bias in this meta - analysis might be attributed to studies of small samples and positive results published more easily than negative reports . firstly , we have selected random effect model to synthesize smd because of the high heterogeneity existing in some comparisons , but this selection may affect the accuracy of outcome . secondly , we could not conduct further subgroup analysis of gender , weight , and disease duration because most of the included studies lack sufficient original data . thirdly , articles published in chinese or english are included , while unpublished data and papers published in other languages are unknown . in summary , t2 dm patients and the patients with diabetic complication have decreased immunosuppressive cd4cd25foxp3treg cells and increased proinflammatory il-10 , tgf- , and tnf-. the presence of diabetic complication has an impact on the compromised immunosuppression .

objective . the aim of this study was to investigate the changes of regulatory t cells ( treg ) , interleukin-6 ( il-6 ) , il-10 , transforming growth factor- ( tgf- ) , and tumor necrosis factor - alpha ( tnf- ) in patients with type 2 diabetes mellitus ( t2 dm ) . methods . we performed a comprehensive search up to july 2016 for all clinical studies about the changes of treg , il-6 , il-10 , il-17 , tgf- , and tnf- in t2 dm patients versus healthy controls . results . a total of 91 articles ( 5642 cases and 7378 controls ) were included for this meta - analysis . compared with the controls ( all p < 0.001 ) , the patients had increased serum levels of il-6 , tgf- , and tnf- but decreased the percentage of peripheral cd4+cd25+foxp3+treg and serum il-10 level . furthermore , the percentage of peripheral cd4+cd25+foxp3+treg ( p < 0.001 ) and serum il-10 level ( p = 0.033 ) were significantly lower in the patients with complication and in the patients without complication , respectively . no significant changes about the percentage of cd4+cd25+treg ( p = 0.360 ) and serum il-17 level ( p = 0.459 ) were found in t2 dm patients . conclusions . t2 dm patients have decreased the percentage of peripheral cd4+cd25+foxp3+treg and levels of serum il-10 but elevated serum levels of il-6 , tgf- , and tnf-. presence of diabetic complications further lowers the peripheral cd4+cd25+foxp3+treg number .

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

antioxidant deficiency contributes to develop and aggravate various chronic diseases , including copd which is sensitive to oxidative stress . supplementation with vitamin c , as an antioxidant , improves antioxidant status in copd.1 several epidemiologic studies2,3 and prospective studies4,5 revealed protective effects of vitamin c intake on copd . vitamin c has symptom - relieving effects on the exacerbation of copd.6 in addition , it improves pulmonary function in copd.7,8 moreover , a recent animal study has revealed that supplementation with vitamin c not only prevents the development of copd but also restores the lung function in copd subjects.9 the korea national health and nutrition examination survey ( knhanes ) is a large - scale survey containing a vast amount of data on demographics , underlying disease , smoking history , lung function , and nutritional status , conducted by the korean centers for disease control and prevention ( kcdc ) . the sample in the present study was selected by a well - designed national program with a complex , multistage probability sample extraction . therefore , the results obtained by this survey using complex analysis can be generalized to the entire korean general population.10,11 thus , knhanes is the best tool for defining and confirming the correlation between vitamin c intake and copd . therefore , in this study , we aimed to confirm the protective effects of vitamin c against copd in the korean general population using knhanes data . this survey used stratification and multiple stages of cluster selection to represent the entire korean general population . we followed guidelines reporting the sample weight and stratification designated by the kcdc ; this information is available at the knhanes website to facilitate obtaining appropriate results , and all the raw data are open to the public ( https://knhanes.cdc.go.kr ) . the institutional review board of gangnam severance hospital approved this study ( approval number : 3 - 2016 - 0195 ) . we included 3,283 subjects aged 40 years or older , who responded to the health interview survey , underwent pulmonary function tests , and responded to questionnaires on smoking history and vitamin c intake from january 2012 to december 2012 . all interview items , including nutritional status , were assessed by questioning the subjects in face - to - face interviews . the resident district was classified as urban ( concordant with dong ) and suburban / rural ( concordant with eup / myun ) . household income was classified into 4 categories ( lowest quartile , q1 ; low - middle quartile , q2 ; high - middle quartile , q3 ; highest quartile , q4 ) . vitamin c intake was also classified into 4 categories ( q1 , < 48.50 mg ; q2 , 48.5084.38 mg ; q3 , 84.38141.63 mg ; q4 , > 141.63 mg ) . the amount smoked ( pack year ) was calculated based on the duration of smoking history ( year ) , the amount of cigarettes smoked ( pack / day ) , and the frequency of smoking ( day / month ) . smoking history was classified into 4 categories according to the amount smoked ( never smoker , 0 pack - years ; light smoker , 020 pack - years ; medium smoker , 2040 pack - years ; heavy smoker , > 40 pack - years ) . pulmonary function tests , including forced vital capacity ( fvc ) and forced expiratory volume in 1 second ( fev1 ) , were performed using commercially available equipment ( masterlab - ios ; erich jaeger co. , friedburg , germany ) on all the subjects . copd was defined as an fev1/fvc ratio of < 0.7.12 for complex sample survey data analysis , we used the knhanes stratification variables and sampling weight . between the non - copd and copd groups , differences in the mean age and nutritional status were assessed with the independent sample t - test , and differences in the categorized variables were tested using the chi - square test . the prevalence of copd according to smoking history and the amount of vitamin c ingested was compared using the chi - square test . we used spss 18.0 ( spss inc . , chicago , il , usa ) statistical software . this survey used stratification and multiple stages of cluster selection to represent the entire korean general population . we followed guidelines reporting the sample weight and stratification designated by the kcdc ; this information is available at the knhanes website to facilitate obtaining appropriate results , and all the raw data are open to the public ( https://knhanes.cdc.go.kr ) . the institutional review board of gangnam severance hospital approved this study ( approval number : 3 - 2016 - 0195 ) . we included 3,283 subjects aged 40 years or older , who responded to the health interview survey , underwent pulmonary function tests , and responded to questionnaires on smoking history and vitamin c intake from january 2012 to december 2012 . all interview items , including nutritional status , were assessed by questioning the subjects in face - to - face interviews . the resident district was classified as urban ( concordant with dong ) and suburban / rural ( concordant with eup / myun ) . household income was classified into 4 categories ( lowest quartile , q1 ; low - middle quartile , q2 ; high - middle quartile , q3 ; highest quartile , q4 ) . vitamin c intake was also classified into 4 categories ( q1 , < 48.50 mg ; q2 , 48.5084.38 mg ; q3 , 84.38141.63 mg ; q4 , > 141.63 mg ) . the amount smoked ( pack year ) was calculated based on the duration of smoking history ( year ) , the amount of cigarettes smoked ( pack / day ) , and the frequency of smoking ( day / month ) . smoking history was classified into 4 categories according to the amount smoked ( never smoker , 0 pack - years ; light smoker , 020 pack - years ; medium smoker , 2040 pack - years ; heavy smoker , > 40 pack - years ) . pulmonary function tests , including forced vital capacity ( fvc ) and forced expiratory volume in 1 second ( fev1 ) , were performed using commercially available equipment ( masterlab - ios ; erich jaeger co. , friedburg , germany ) on all the subjects . for complex sample survey data analysis , we used the knhanes stratification variables and sampling weight . between the non - copd and copd groups , differences in the mean age and nutritional status were assessed with the independent sample t - test , and differences in the categorized variables were tested using the chi - square test . the prevalence of copd according to smoking history and the amount of vitamin c ingested was compared using the chi - square test . we used spss 18.0 ( spss inc . , chicago , il , usa ) statistical software . we enrolled 3,283 subjects representing a total of 23,541,704 korean subjects . among all the subjects , we classified all subjects into 2 groups ( non - copd and copd ) , and compared the demographic characteristics between the 2 groups . in males , the prevalence of copd was significantly higher than that in females ( 23.5% vs 6.6% ; p<0.001 ) . the mean age ( standard deviation ) of the copd group ( 64.20.7 ) was significantly older than that in the non - copd group ( 54.50.4 ; p<0.001 ) . the prevalence of copd in subjects who live in suburban / rural areas was significantly higher than that in subjects living in urban areas ( 20.8% vs 12.9% ; p<0.001 ) . in subjects who lived in the general type of house , the prevalence of copd was higher ( 16.6% ) than that in subjects who lived in an apartment ( 9.3% ; p<0.001 ) . the prevalence of copd decreased according to the increase in household income ( p<0.001 ) . subjects who worked in agriculture or fisheries showed a higher prevalence of copd ( 25.0% ) than subjects who had a professional job ( 7.7% ) , or had a service and sales job ( 6.0% ; p<0.001 ) . among never smokers , the prevalence of copd was 7.9% , and the prevalence increased according to the increase in the amount smoked ( 16.7% in light smokers ; 24.2% in medium smokers ; we compared the nutritional status assessed by the self - reported questionnaire between the non - copd and copd group . the amount of food intake ( g ) in the copd group ( 1,320.437.9 ) was significantly less than that in the non - copd group ( 1,462.323.6 ; p=0.001 ) . the amount of potassium ( mg ) ingested in the copd group ( 2,915.485.9 ) was significantly less than that in the non - copd group ( 3,200.058.9 ; p=0.005 ) . the amount of vitamin a and carotene taken by the copd group was also less than that in the non - copd group ( p=0.040 and 0.015 , respectively ) . in the copd group , the retinol intake ( 63.84.9 g ) was significantly less than that in the non - copd group ( 93.46.5 g ; p<0.001 ) . the vitamin c intake ( mg ) was also significantly less than that in the non - copd group ( 93.23.9 vs 122.14.0 ; p<0.001 ; table 2 ) . the prevalence of copd was positively correlated with the amount of smoking , and negatively correlated with the amount of vitamin c ingested . in light smokers , the prevalence of copd in subjects with vitamin c intake in q1 ( 24.7% ) was significantly higher than that in subjects whose vitamin c intake fell in q4 ( 7.0% ; p=0.002 ) . in heavy smokers , the prevalence of copd in subjects with q1 ( 63.0% ) and q2 ( 56.4% ) vitamin c intakes was significantly higher than that in subjects with q3 ( 29.5% ) and q4 ( 32.6% ) vitamin c intakes ( p=0.015 ) . in addition , the prevalence of copd in heavy smokers with vitamin c intake in q3 ( 29.5% ) was lower than that in medium smokers with q1 vitamin c intake ( 36.8% ; figure 1 ) . we selected significant risk factors for copd using univariate analysis with logistic regression of all the variables used in tables 1 and 2 . sex , age , resident district , type of residence , household income , educational level , occupation , smoking history , and amount of retinol intake and vitamin c intake were significant risk factors for copd in the univariate analysis . in the multivariate analysis , sex , age , smoking history , and amount of vitamin c intake were independent risk factors for copd . female gender was a preventative factor ( odds ratio [ or ] , 0.234 ; 95% confidence interval [ ci ] , 0.1440.378 ; p<0.001 ) compared to male gender . aging was a significant risk factor ( or , 1.089 ; 95% ci , 1.0681.110 ; p<0.001 ) . compared to never smokers , medium smokers ( or , 2.192 ; 95% ci , 1.3053.680 ) and heavy smokers ( or , 2.894 ; 95% ci , 1.6415.102 ) were at high risk for copd ( p<0.001 ) . the amount of vitamin c intake was a preventative factor for copd independent of sex , age , and even smoking history ( or , 0.998 ; 95% ci , 0.9960.999 ; p=0.001 ) . a 1-mg increase of vitamin c intake reduced the risk of copd by 0.2% ( table 3 ) . we classified the subjects according to smoking history , and subclassified them according to the amount of vitamin c ingested , and confirmed that the amount of vitamin c is an independent preventative factor for copd when adjusted for age and sex . in light smokers , vitamin c intake in q4 was protective against copd , compared to vitamin c intake in q2 . in particular , in heavy smokers , the risk reduction rate in individuals with vitamin c intake falling in q3 was 76.7% , compared to those with vitamin c intake in q1 ( or , 0.233 ; 95% ci , 0.0940.576 ; table 4 ) . we enrolled 3,283 subjects representing a total of 23,541,704 korean subjects . among all the subjects , we classified all subjects into 2 groups ( non - copd and copd ) , and compared the demographic characteristics between the 2 groups . in males , the prevalence of copd was significantly higher than that in females ( 23.5% vs 6.6% ; p<0.001 ) . the mean age ( standard deviation ) of the copd group ( 64.20.7 ) was significantly older than that in the non - copd group ( 54.50.4 ; p<0.001 ) . the prevalence of copd in subjects who live in suburban / rural areas was significantly higher than that in subjects living in urban areas ( 20.8% vs 12.9% ; p<0.001 ) . in subjects who lived in the general type of house , the prevalence of copd was higher ( 16.6% ) than that in subjects who lived in an apartment ( 9.3% ; p<0.001 ) . the prevalence of copd decreased according to the increase in household income ( p<0.001 ) . subjects who worked in agriculture or fisheries showed a higher prevalence of copd ( 25.0% ) than subjects who had a professional job ( 7.7% ) , or had a service and sales job ( 6.0% ; p<0.001 ) . among never smokers , the prevalence of copd was 7.9% , and the prevalence increased according to the increase in the amount smoked ( 16.7% in light smokers ; 24.2% in medium smokers ; we compared the nutritional status assessed by the self - reported questionnaire between the non - copd and copd group . the amount of food intake ( g ) in the copd group ( 1,320.437.9 ) was significantly less than that in the non - copd group ( 1,462.323.6 ; p=0.001 ) . the amount of potassium ( mg ) ingested in the copd group ( 2,915.485.9 ) was significantly less than that in the non - copd group ( 3,200.058.9 ; p=0.005 ) . the amount of vitamin a and carotene taken by the copd group was also less than that in the non - copd group ( p=0.040 and 0.015 , respectively ) . in the copd group , the retinol intake ( 63.84.9 g ) was significantly less than that in the non - copd group ( 93.46.5 g ; p<0.001 ) . the vitamin c intake ( mg ) was also significantly less than that in the non - copd group ( 93.23.9 vs 122.14.0 ; p<0.001 ; table 2 ) . the prevalence of copd was positively correlated with the amount of smoking , and negatively correlated with the amount of vitamin c ingested . in light smokers , the prevalence of copd in subjects with vitamin c intake in q1 ( 24.7% ) was significantly higher than that in subjects whose vitamin c intake fell in q4 ( 7.0% ; p=0.002 ) . in heavy smokers , the prevalence of copd in subjects with q1 ( 63.0% ) and q2 ( 56.4% ) vitamin c intakes was significantly higher than that in subjects with q3 ( 29.5% ) and q4 ( 32.6% ) vitamin c intakes ( p=0.015 ) . in addition , the prevalence of copd in heavy smokers with vitamin c intake in q3 ( 29.5% ) was lower than that in medium smokers with q1 vitamin c intake ( 36.8% ; figure 1 ) . we selected significant risk factors for copd using univariate analysis with logistic regression of all the variables used in tables 1 and 2 . sex , age , resident district , type of residence , household income , educational level , occupation , smoking history , and amount of retinol intake and vitamin c intake were significant risk factors for copd in the univariate analysis . in the multivariate analysis , sex , age , smoking history , and amount of vitamin c intake were independent risk factors for copd . female gender was a preventative factor ( odds ratio [ or ] , 0.234 ; 95% confidence interval [ ci ] , 0.1440.378 ; p<0.001 ) compared to male gender . aging was a significant risk factor ( or , 1.089 ; 95% ci , 1.0681.110 ; p<0.001 ) . compared to never smokers , medium smokers ( or , 2.192 ; 95% ci , 1.3053.680 ) and heavy smokers ( or , 2.894 ; 95% ci , 1.6415.102 ) were at high risk for copd ( p<0.001 ) . the amount of vitamin c intake was a preventative factor for copd independent of sex , age , and even smoking history ( or , 0.998 ; 95% ci , 0.9960.999 ; p=0.001 ) . a 1-mg increase of vitamin c intake reduced the risk of copd by 0.2% ( table 3 ) . we classified the subjects according to smoking history , and subclassified them according to the amount of vitamin c ingested , and confirmed that the amount of vitamin c is an independent preventative factor for copd when adjusted for age and sex . in light smokers , vitamin c intake in q4 was protective against copd , compared to vitamin c intake in q2 . in particular , in heavy smokers , the risk reduction rate in individuals with vitamin c intake falling in q3 was 76.7% , compared to those with vitamin c intake in q1 ( or , 0.233 ; 95% ci , 0.0940.576 ; table 4 ) . various factors , including sex , age , resident district , type of residence , household income , educational level , occupation , and smoking history , affect the prevalence of copd . in addition , we found that subjects living in suburban / rural areas and general residence types , and subjects with a low income , low educational level , and agriculture or fisheries employment had a higher prevalence of copd . however , these factors may be related to male gender , old age , and a heavy smoking history , which are already well - known risk factors for copd . therefore , multivariate analysis should be performed to define if these factors are independent risk factors for copd . marital status was related to smoking history in a previous study,13 whereas the present study showed that marital status was not significantly associated with copd ; this result should be re - judged based on previous studies because the total number of unmarried subjects was small ( n=41 ; representing 419,691 subjects ; 1.3% of the total number of subjects ) in this study . the main contributing factors for the development of copd are genetic background , exposure to oxidizing agents , and reduced antioxidant capacity.14,15 dietary antioxidants are responsible for antioxidant defenses in the lungs.16 various nutrients , including vitamin c , retinol , tocopherol , and carotenoids , have been proven to be helpful in altering clinical outcomes of copd.3,1719 the present study showed a significant shortage of total nutritional , potassium , vitamin a , carotene , retinol , and vitamin c intake in the copd group compared to the non - copd group . however , the difference in nutritional status between the 2 groups may be due to the difference in total nutritional intake . therefore , again , multivariate analysis would be needed to identify independent risk factors . to define the independent risk factors for copd among the mentioned factors we revealed that male gender , old age , heavy smoking history , and vitamin c were independent risk factors for copd , whereas other factors were not independently significant . except for the well - known risk factors , vitamin c was the only independent risk factor . based on the multivariate analysis , as shown in table 3 , 100 mg of vitamin c intake resulted in a risk reduction of 20.0% . in heavy smokers , ~100 mg of vitamin c intake ( mean of 30.1 mg in q1 vs mean of 110.6 mg in q3 ) led to a marked risk reduction ( 76.7% ) for copd . one - and - a - half apples contains ~100 mg of vitamin c. most nutritional vitamin c supplements contain > 1,000 mg . however , abuse of vitamin c supplements may lead yet rarely to acute renal failure.20 therefore , one should carefully consider vitamin c supplements . vitamin c ( also known as ascorbate or l - ascorbic acid ) has antioxidant properties , and plays significant roles in the immune system including allergic reaction , maintenance of connective tissue , and even tumor suppression.2123 low levels of vitamin c have been associated with significantly more wheezing , dyspnea , and exacerbation of copd.2426 dietary vitamin c has also been shown to lower oxidative stress , increase collagen synthesis , and restore vascular endothelial growth factor levels and proliferation of alveolar cells in the lungs.9 extensive studies have shown that vitamin c intake provides protection against the development of copd.1,8,27,28 our results corroborate the findings of these studies . the results obtained by this study are extremely significant , as this study represents almost half of the total korean population ( n=23,541,704 ) . joshi et al have previously revealed the effect of dietary antioxidants on copd in korea.29 we strongly support this previous study with a vast amount of data . although many researchers have attempted to use knhanes data , many such studies ignore complex survey design , which leads to biased results , and overstated significance levels.10 however , we followed the guidelines suggested by the institute that conducted this complex survey , and therefore , the results of the positive effects of vitamin c on copd are deemed trustworthy . first , this is a cross - sectional cohort study . it is not possible to ascertain whether a low intake of vitamin c is the cause or the result of copd . however , we can assume that it is a causal factor , based on the many previous prospective studies mentioned in this paper . the nutritional status was also assessed using self - reported questionnaires , with assistance from an interviewer . measurement of vitamin c using blood sampling may be more helpful to confirm the nutritional status . third , we could not ascertain the optimal dose of vitamin c for preventing copd because the optimal dose differed according to the smoking history . for example , the association between q1 and q3 was stronger than that between q1 and q4 . hence , the or did not follow a linear trend . this might be due to the fact that vitamin c intake greater than the optimal level does not have any additional effects . lastly , height , body mass index , dietary fiber , and cured meat intake may be confounding factors in this study . this large - scale national study suggests that dietary vitamin c intake is protective against copd independent of smoking history in the korean general population .

backgroundvitamin c , as an antioxidant , has recently been suggested to provide protection against copd ; however , only few national cohort studies have investigated these effects . we aimed to confirm the protective effects of vitamin c against copd in korean patients.patients and methodswe analyzed the data of 3,283 adults aged 40 years ( representing 23,541,704 subjects ) who underwent pulmonary function tests and responded to questionnaires on smoking history and vitamin c intake , with stratification variables and sampling weight designated by the korea 2012 national health and nutrition examination survey.resultsamong all the subjects , 512 ( representing 3,459,679 subjects ; 15.6% ) were diagnosed as having copd based on pulmonary function test results . male gender , old age , residence in suburban / rural regions , low household income , low educational level , an occupation in agriculture or fisheries , and heavy smoking were significantly associated with copd . low intake of nutrients , including potassium , vitamin a , carotene , retinol , and vitamin c , was significantly associated with copd . the prevalence of copd in heavy smokers with the lowest quartile ( q1 , < 48.50 mg ; 63.0% ) and low - middle quartile ( q2 , 48.5084.38 mg ; 56.4% ) of vitamin c intake was significantly higher than that in subjects with the high - middle quartile ( q3 , 84.38141.63 mg ; 29.5% ) and highest quartile ( q4 , > 141.63 mg ; 32.6% ) of vitamin c intake ( p=0.015 ) . in multivariate analysis , male gender , old age , heavy smoking , and a low intake of vitamin c were significant independent risk factors for copd . a significant reduction of 76.7% in copd risk was observed with a q3 vitamin c intake compared to q1 vitamin c intake ( odds ratio , 0.233 ; 95% confidence interval , 0.0940.576 ) in heavy smokers.conclusionthis large - scale national study suggests that dietary vitamin c provides protection against copd , independent of smoking history , in the general korean population .

Introduction Patients and methods Study design and populations Interview items, including nutritional status Smoking history COPD diagnosis Statistical analyses Results Demographic characteristics according to COPD diagnosis Comparison of nutritional status according to COPD Prevalence of COPD according to smoking history and vitamin C intake Risk factors for COPD Vitamin C intake is protective against COPD in heavy smokers Discussion Conclusion

tobacco use has fallen in many high - income countries , at least in men , but is now rising rapidly in many low- and middle - income countries such as china . thirty - eight percent of the world 's smokers are chinese , especially chinese men who smoke one - third of the world 's cigarettes , with 52.9% smoking prevalence and 50.4% current cigarette use [ 2 , 3 ] . tobacco use has become the number one killer in china and is responsible for 1.2 million deaths annually , and this number is expected to rise to 3.5 million deaths annually by the year 2030 . diabetes is another major public health problem in china . according to 2010 china noncommunicable disease surveillance , the overall prevalence of diabetes and prediabetes was estimated to be 11.6% ( 12.1% among men ) and 50.1% ( 52.1% among men ) in chinese adults , respectively , representing up to 113.9 million adults with diabetes and 493.4 million with prediabetes . furthermore , the current prevalence of diabetes in the chinese population is very similar to the us population even though overweight and obesity are much more common in the latter . it is of urgent need to illuminate associated risk factors so as to develop effective strategies to prevent the development of diabetes in china . the association between smoking and increased risk for type 2 diabetes mellitus ( type 2 dm ) has been well documented [ 5 , 6 ] . both active and passive smoking have been demonstrated to be associated with an increased risk of type 2 dm [ 7 , 8 ] . recent study also reported that current smokers were dose - dependently associated with increased risk for incident metabolic syndrome in chinese men . insulin resistance ( ir ) and declining -cell function have proven to be potential mechanisms in the development of type 2 dm and can be demonstrated long before overt diabetes is diagnosed [ 10 , 11 ] . chronic smoking was reported to markedly and in a dose - dependent manner aggravate ir observed in type 2 dm patients . however , when the metabolic effects of smoking , especially chronic smoking , were studied in persons without diabetes , the findings were controversial . smoking was reported to acutely impair insulin action and led to ir in normal subjects . chronic smoking was also shown to be associated with ir and its degree [ 5 , 1417 ] . nevertheless , some studies found no difference in ir between smokers and nonsmokers in population without diabetes [ 1823 ] . on the other hand , few available studies failed to provide conclusive evidence [ 18 , 19 , 23 ] . even opposite effects on -cell function were reported in current and former smokers , when compared with nonsmokers . homeostatic model assessment ( homa ) using steady - state glucose and insulin is a method for assessing ir and -cell function and is more appropriate for use in large epidemiological studies . but few studies applying homa method are currently available for assessing the associations of chronic smoking in asians who exhibit lower levels of the homa of insulin resistance ( homa - ir ) than western populations [ 1719 ] . moreover , a dynamic evaluation of -cell function , such as oral glucose tolerance test ( ogtt ) , according to smoking status was rare . this population - based study aims to elucidate the effects of chronic smoking on ir and -cell function in a large sample of chinese men without diabetes using both homa analysis and ogtt . on the basis of china noncommunicable disease surveillance in 2010 , the study population in this cross - sectional study was a subset population recruited in jiangsu province , using a complex , multistage , probability sampling design . in brief , we first selected 6 counties from 106 counties using stratified random sampling according to the population , gross domestic product ( gdp ) , degree of urbanization , and geographic setting . secondly , 4 towns from each county and 3 villages from each town were selected with the probability proportional to the population size , using cluster random sampling . thirdly , 1 residential group including at least 50 households was selected from each village using simple random cluster sampling . random digit function in excel was applied in selection of these households from each residential group . finally , one family member aged 18 years or over was randomly selected from each household using the kish grid method [ 25 , 26 ] . the subjects who were diagnosed as diabetes according to the american diabetes association criteria the data collected during the face - to - face interview included demographic characteristics , lifestyle factors , and medical history . the investigated smoking history included age at smoking initiation , years of smoking , number of cigarettes smoked per day , and smoking cessation . briefly , an individual who never smoked or smoked less than 100 cigarettes in his lifetime was defined as a never smoker . an individual who smoked at least 100 cigarettes in his lifetime but quit smoking more than 12 months before the interview was considered as a former smoker . current smokers included those currently smoking and those who quit smoking less than 12 months before interview . the pack - year of smoking was calculated according to the number of packs of cigarettes smoked per day and smoking duration ( years ) . an individual who never consumed alcohol or consumed less than or equal to one drink per month was defined as a never drinker , otherwise as an ever drinker . the global physical activity questionnaire was used to evaluate physical activities by calculating the total weekly volume ( metabolic equivalents ( met ) min / wk ) across three separate domains ( work / home , during commuting , and during leisure time ) , by applying met values to the time variables according to the intensity of the activity [ 4 , 29 , 30 ] . height and weight were measured in light underclothes without shoes . in the standing position of participants in light clothing , waist circumference was measured at the midway between the lower edge of the costal arch and the upper edge of the iliac crest . body mass index ( bmi ) was calculated as weight ( kg)/height ( m ) . kg / m was considered as overweight , and waist circumference 90 cm as central obesity for chinese men [ 4 , 31 ] . blood pressure was measured by trained physicians using calibrated electronic sphygmomanometer ( hem-7071 , omron corporation , japan ) , on the nondominant arm in precordial level after at least 5 min of rest and with the subjects in sitting position . three readings were taken 2 min apart and the averaged values of systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) were calculated . the participants were identified as hypertensive subjects if they had high blood pressure values of sbp 140 mmhg and/or dbp 90 mmhg or if they had a history of hypertension or if they were under antihypertensive medications therapy . a venous blood sample ( 4 - 5 ml ) plasma samples for glucose test were collected with the tubes containing sodium fluoride at 0 h and 2 h after administration of a standard 75 g glucose solution during ogtt test . plasma glucose was measured using glucose oxidase or hexokinase methods within 24 h. capillary blood samples for glycated hemoglobin ( hba1c ) detection were collected by the hemoglobin capillary collection system . hba1c was measured within 4 w by high - performance liquid chromatography using the variant ii hemoglobin testing system ( bio - rad laboratories ) . serum insulin was measured by the electrochemiluminescence immunoassay on an automatic electrochemiluminescence analyzer ( cobas - e601 , roche company ) . serum total cholesterol ( tc ) , triglyceride ( tg ) , low density lipoprotein cholesterol ( ldl ) , and high density lipoprotein cholesterol ( hdl ) were detected with the enzymatic methods using an automatic biochemistry analyzer ( abbott laboratories ) . on the basis of fasting glucose and insulin levels , insulin resistance and secretion were evaluated using homa method with the formulas as homa - ir = fasting insulin ( mu / l ) fasting glucose ( mmol / l)/22.5 and -cell function ( homa- ) % = [ 20 fasting insulin ( mu / l)]/[fasting glucose ( mmol / l ) 3.5 ] . individuals with fasting glucose of 3.5 mmol / l or less were excluded from analysis . the cutoff points for ir and -cell deficiency were homa - ir 2.6 and homa- ( % ) < 50 , respectively [ 24 , 32 ] . dyslipidemia was defined by the presence of at least one abnormal serum lipid : tc 5.18 mmol / l , tg 1.7 mmol / l , ldl 3.37 mmol / l , and hdl in men < 1.04 mmol / l . all the data were double entered in the database and corrected for errors using epi - info 6.0 software . the statistical analyses were performed using the sas software version 9.2 ( sas institute , cary , nc ) . the differences in the distributions of demographic characteristics among never , former , and current smokers were compared by an unpaired student 's t - test for continuous variables and a test for categorical variables , using never smokers as references . to improve skewness , fasting insulin , glucose , hba1c , homa - ir , and homa- were logarithmically transformed for statistical analyses . unadjusted and adjusted geometric means ( adjustment for age , education level , drinking status , bmi , waist circumference , level of physical activity , hypertension , and dyslipidemia ) with 95% confidence interval ( ci ) were calculated by general linear model and back transformed to natural units for presentation . the associations between smoking status and ir and -cell deficiency were evaluated under univariate and multivariate unconditional logistic regression model after adjustment for covariates . odds ratio ( or ) with 95% ci was used to estimate the strength of association . dose - dependent effect of smoking intensity on -cell function was also analyzed as a continuous variable using fractional polynomial regression model . all the statistical tests were two - sided , and p < 0.05 was considered as statistically significant . the 1,568 chinese men without diabetes in this study included 598 never smokers , 120 former smokers , and 850 current smokers ( table 1 ) . there was no significant difference in age between current and never smokers ; however , the former smokers were younger than never smokers ( p = 0.0430 ) . higher percentages of ever drinkers were observed in both current ( 72.35% ) and former ( 75.83% ) smokers , compared with the percentage of 55.52% in never smokers ( p < 0.0001 ) . moreover , current smokers with a median equivalent combination of physical activity of 3360 met min / wk were more physically active , compared to never smokers ( 2520 met min / wk , p = 0.0063 ) . although there were more subjects with hypertension and dyslipidemia in current smokers , and more participants who had a higher level of education , bmi , and waist circumference in former smokers , the differences were not statistically significant . there was no statistical difference in fasting glucose ; however , the levels of 2 h glucose for ogtt were significantly higher in current smokers than in never smokers ( p = 0.0493 ) ( table 2 ) . compared to never smokers , current smokers had significantly decreased fasting insulin , after adjustment for covariates ( p = 0.0335 ) . the adjusted means with 95% ci for homa- ( % ) were 54.86 ( 52.1057.78 ) in current smokers and 58.81 ( 55.5762.24 ) in never smokers ( p = 0.0257 ) . no significant difference in hba1c and homa - ir was observed when comparing current or former smoker with never smokers . as shown in table 3 , there were 57.42% current smokers with a homa- value less than 50 . current smoking was associated with -cell deficiency ( or 1.23 , 95% ci 1.001.52 ) , compared to never smoking . the association was still statistically significant ( or 1.29 , 95% ci 1.011.64 ) after adjustment for age , education level , drinking status , bmi , waist circumference , level of physical activity , hypertension , and dyslipidemia . no association was observed between former smoking and -cell deficiency , as well as between smoking status and homa - ir . the strength of association with -cell deficiency was further analyzed after categorizing current smokers according to their pack - year of smoking and using never smokers as references . a dose - dependent effect of smoking intensity was observed to be associated with impaired -cell function , however , not with homa - ir . the -cell function gradually decreased with increasing smoking intensity ( ptrend < 0.0001 and 0.0026 after adjustment for covariates ) , and the differences were statistically significant when the pack - year of smoking was 20 or above ( figure 1 ) . to evaluate the observed association between smoking intensity and -cell function in a more dynamic manner , we further analyzed the dose - dependent effect of smoking intensity as a continuous variable on -cell deficiency using fractional polynomial regression model . a significant linear trend of increasing ors along with increasing smoking intensity was observed ( figure 2 , ptrend < 0.0001 ) , which was consistent with the results under logistic regression analysis of smoking intensity as a categorical variable . in addition , fasting glucose increased with cumulative intensity of smoking ; the adjusted means ( 95% ci ) were 5.65 ( 5.565.74 ) , 5.58 ( 5.445.73 ) ( p = 0.3857 ) , 5.55 ( 5.425.69 ) ( p = 0.1806 ) , 5.72 ( 5.595.86 ) ( p = 0.3271 ) , and 5.78 ( 5.665.91 ) ( p = 0.0384 ) , respectively , for never smokers and current smokers with the pack - year of smoking < 10 , 10~ , 20~ , and 30 ( ptrend = 0.2177 ) . in this population - based study , we evaluated the associations between chronic smoking and ir and -cell function in chinese men without diabetes . our results showed that current smoking was associated with decreased -cell function in a dose - dependent manner , and the levels of 2 h glucose were significantly higher in current smokers than in never smokers . the metabolic effects of smoking have generally been studied in persons without diabetes , in order to avoid the impacts from the medications or changed behaviors in patients with diabetes . ir and -cell function are usually determined by the hyperinsulinemic - euglycemic clamp and the hyperglycemic clamp . using the euglycemic clamp technique , eliasson et al . [ 14 , 33 ] observed ir in middle - aged men who chronically smoke and its normalization eight weeks after smoking cessation . however , in the insulin resistance atherosclerosis study , smoking was not associated with ir as assessed by a modified glucose tolerance test with minimal model analysis . although referred to as the gold standard tests , clamps are complex stress tests with insulin and glucose concentrations and flux well outside the normal range . moreover , clamp techniques are only suitable to be applied in studies with relatively small numbers of subjects . alternately , homa , a method for assessing ir and -cell function from basal glucose and insulin concentration , correlates well with estimates using other methods including clamps and is more appropriate for use in large epidemiological studies . the relationship between glucose and insulin in the basal state reflects the balance between hepatic glucose output and insulin secretion , which is maintained by a feedback loop between the liver and -cells . but recent studies using the homa method reported inconsistent effects of smoking in caucasians , such as high -cell values and ir in normoglycaemic persons , lower -cell function in men without hypertension and diabetes , or no association in men without diabetes . the study of ko et al . in chinese men without diabetes showed that no statistical difference in homa - ir or homa- was observed among current , former , and never smokers . but in this study smokers merely accounted for 22.53% ( 178 out of 790 ) of subjects without diabetes , and men with newly diagnosed impaired glucose tolerance ( igt ) were excluded . our study including 1,568 men without diabetes found impaired -cell secretion in current smokers , which was signified by both low values of homa- and fasting insulin . the inconsistency among these studies using homa method may be attributable to the differences in study design , subject recruit , ethnic origin , gender stratification , involved covariates , and sample size . animal experiments showed that both prenatal and postnatal exposure to nicotine could directly induce imbalance of metabolic control . the studies using rodent models demonstrated that nicotine exposure could cause -cell dysfunction , elevated pancreatic -cell apoptosis , and loss of -cell mass , which was mediated via the mitochondrial and/or death receptor pathway . smoking cessation could possibly reverse the unfavorable effects from nicotine . a recent study by stadler et al . reported that smoking cessation was associated with metabolic changes including increased -cell secretion in response to glucose . all these findings provided consistent evidence and biological plausibility for the decreased insulin secretion in smokers , especially heavy smokers in our study . in the dynamic evaluation of -cell function using ogtt test , we also found higher levels of 2 h glucose in current smokers , which may be attributed to long - term effects of nicotine exposure or may be from acute stress due to nicotine withdrawal . either acute or chronic nicotine exposure was reported to negatively affect insulin action in smokers preceded before type 2 dm . long - term use of nicotine - containing chewing gum in nonsmoking men was associated with ir and hyperinsulinemia . however , no association was observed between smoking and homa - ir in the current study . it is noteworthy that homa - ir , based on fasting insulin and glucose measurements to determine whole - body ir , primarily reflects hepatic ( central ) insulin sensitivity rather than impaired glucose uptake and consumption in skeletal muscle and adipose tissue ( peripheral ) , thus limiting its ability to assess ir , especially in individuals with igt [ 4043 ] . it was suggested that insulin responses during the ogtt may be a better surrogate measure for ir , particularly those that occurred in peripheral insulin - sensitive tissues such as muscle [ 42 , 44 , 45 ] . also pointed out that none of the surrogate estimates of insulin sensitivity in their study was superior to simple measurement of fasting plasma insulin concentrations in predicting insulin sensitivity . in our study , increased 2 h plasma glucose during ogtt test indicated probably insulin resistance in current smokers . in the present study based on the national survey , the subjects recruited using random sampling methods were representative . physical activities were investigated using standard questionnaires and evaluated by metabolic equivalent - minutes per week . first , we only detected 2 h plasma glucose after administration of a standard glucose solution . area under the curve which is more important in dynamic measurement could not be calculated and compared in this study . second , due to few former smokers available , we did not further analyze when -cell dysfunction in smokers would return to normal after smoking cessation . third , our study lacked information on other major confounders such as socioeconomic factors , psychosocial stress , and dietary patterns . fourth , the participants in this study were chinese men , which restricted the generalization of our results to other ethnicities and gender . last , cross - sectional design could not provide a causal relationship between smoking and -cell deficiency . therefore , large - scale prospective studies are warranted to confirm our findings . in conclusion , our study suggested that chronic smoking impaired insulin secretion and probably brought about insulin resistance in chinese men without diabetes . taking into consideration the increased risk of development of type 2 dm related to -cell dysfunction in current smokers , tobacco control should be considered the most urgent and immediate priority in china , especially in chinese men .

the aim of this study was to evaluate the associations between chronic smoking and insulin resistance and -cell function in chinese men without diabetes . a total of 1,568 participants were recruited by multistage sampling . using homeostatic model assessment ( homa ) , geometric means of insulin resistance ( homa - ir ) and -cell function ( homa- ) with 95% confidence interval ( ci ) were calculated by general linear model . odds ratios ( ors ) with 95% ci were estimated to evaluate the associations between smoking status and insulin resistance and -cell deficiency under a logistic regression model . current smokers had higher levels of 2 h glucose ( 6.66 versus 6.48 mmol / l ) for oral glucose tolerance test and lower levels of fasting insulin ( 5.68 versus 6.03 mu / l ) than never smokers . the adjusted means for homa- ( % ) were 54.86 in current smokers and 58.81 in never smokers ( p = 0.0257 ) . current smoking was associated with -cell deficiency ( or 1.29 , 95% ci 1.011.64 ) compared to never smoking . the -cell function gradually decreased with increasing smoking intensity ( ptrend = 0.0026 ) , and the differences were statistically significant when the pack - year of smoking was 20 or above . no association was observed between smoking status and homa - ir . our study suggested that chronic smoking may dose - dependently suppress insulin secretion in chinese men .

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

a stool sample was obtained from a healthy 2-year - old boy living in senegal . the stool was sent to and frozen in marseille at 80c until laboratory culture isolation . strain sit12 was isolated in may 2015 by cultivation on marine medium in anaerobic atmosphere after 21 days incubation . growth of the strain was tested under anaerobic and microaerophilic conditions using genbag anaer and genbag microaer systems respectively ( biomrieux , marcy ltoile , france ) , and in the presence of air , with or without 5% co2 , but growth was achieved only under anaerobic conditions . m. massiliensis grew at mesophilic temperatures between 25 and 42c after 48 hours incubation on columbia agar with 5% sheep s blood , chocolate agar and mller - hinton agar . growth occurred at ph 6 , 6.5 , 7 and 8.5 and exhibited tolerance for nacl until a concentration of 5 gram staining and electron microscopy of m. massiliensis were performed using a technaig cryo ( fei company , limeil - brevannes , france ) at an operating voltage of 200 kev . the cells were of coccus morphology , 0.5 m in diameter and occurred in pairs and short chains ( fig . 2 ) . the sporulation test was performed using a thermic shock ( 80c during 30 minutes ) , but no free spores were observed and no viable cells could be recovered from sporulating cultures . api zym and api 50 ch ( biomrieux ) gallery systems were used to perform biochemical assays . distinguishing results from biochemical tests between m. massiliensis sit 12 and murdochiella asaccharolitica are listed in table 1 . the antibiotic susceptibility was studied using antibiotics discs ( i2a , montpellier , france ) . murdochiella massiliensis sit12 was resistant to fosfomycin , tobramycin , naxidic acid and colistin but was susceptible to gentamicin , ciprofloxacin , trimethoprim sulfamethoxazole , teicoplanin , rifampicin , ceftazidime , erythromycin , imipenem , tazocillin and aztreonam . using 16s rrna phylogeny analyses , we demonstrated that strain sit12 exhibited a 97% 16s rrna sequence identity with murdochiella asaccharolytica ( eu483153 ) and levyella massiliensis ( hm587324 ) species ( fig . investigation of the most closely related described species revealed that the novel species strain was member of the phylum firmicutes . the major fatty acids for strain sit12 are mainly composed of 16 or 18 carbons : 16:0 ( 34% ) , 18:1n9 ( 28% ) , 18:2n6 ( 19% ) and 18:0 ( 12% ) . moreover , several fatty acids are described with unusual longer chains such as 20:4n6 , 20:5n3 and 22:6n3 ( < 1% ) ( table 2 ) . the sit12 spectra were imported into maldi biotyper 3.0 software ( bruker daltonics , leipzig , germany ) and analysed by standard pattern matching ( with default parameter settings ) against 7765 spectra of bacteria . a maximum of 100 peaks were compared with spectra in the database for every spectrum . the resulting score enabled the identification ( or not ) of tested species : a score of 2 with a validly published species enabled identification at the species level , a score of 1.7 but < 2 enabled identification at the genus level and a score of < 1.7 did not enable any identification . no significant maldi - tof ms score was obtained for strain sit12 against the bruker database , suggesting that our isolate was not a member of a known species . genomic dna of m. massiliensis was sequenced on the miseq technology ( illumina , san diego , ca , usa ) with the mate - pair strategy . automated cluster generation and sequencing run were performed in a single 39-hour run at a 2 151 bp read length . total information of 2.9 gb was obtained from a 297k / mm cluster density , with a cluster passing quality control filters of 97% ( 5 808 000 passing filter paired reads ) . within this run , the 440 495 paired reads were trimmed , then assembled in two scaffolds using the spades assembler . open reading frames ( orfs ) were predicted using prodigal with default parameters , but the predicted orfs were excluded if they spanned a sequencing gap region . the trnascanse tool was used to find trna genes , whereas ribosomal rnas were found by using rnammer . of the 1478 predicted genes , 1426 were protein - coding genes and 52 were rnas ( two genes were 5s rrna , two 16s rrna , one 23s rrna and 47 trna ) . a total of 1002 genes ( 70.27% ) were assigned as putative function ( by cogs or by nr blast ) . the resistome was analysed with the argannot ( antibiotic resistance gene - annotation ) database . the exhaustive bacteriocin database available in our laboratories ( bacteriocins from the urmite database ) was performed by collecting all currently available sequences from the databases and from the national center for biotechnology information . protein sequences from this database allowed putative bacteriocins from human gut microbiota to be identified using blastp methodology . analysis of presence of polyketide synthases nonribosomal peptide synthetases ( pks / nrps ) was performed by discriminating the gene with large size using a database realized in our laboratory ; predicted proteins were compared against nonredundant genbank database using blastp and then examined using antismash . murdochiella massiliensis did not contain bacteriocins or nrps , and the analysis of the resistome showed no resistance genes . the results indicated the presence of an incomplete phage with 48.2% g+c content and a complete phage with 49.5% g+c content ( fig . the draft gene sequence of m. massiliensis is smaller than those of anaerococcus hydrogenalis , peptoniphilus indolicus , parvimonas micra and helcococcus kunzii ( 1642 , 1889 , 2238 , 1704 and 2096 mb respectively ) , but larger than those of helcococcus sueciensis ( 1574 mb ) . the g+c content of m. massiliensis is larger than those of a. hydrogenalis , p. indolicus , h. sueciensis , p. micra and h. kunzii ( 48.97 , 29.64 , 31.69 , 28.4 , 28.66 and 29.35% respectively ) . the gene content of m. massiliensis is smaller than those of a. hydrogenalis , h. sueciensis , p. micra and h. kunzii ( 1.426 , 2.069 , 2.269 , 1.445 , 1.678 and 1.882 respectively ) . on the basis of taxonogenomic analyses , we formally propose the creation of murdochiella massiliensis sp . nov . that contains the strain sit12 . the murdochiella massiliensis name come from massilia , the ancient roman name for marseille , france , where the type strain was isolated . the strain was anaerobic , gram positive , non endospore forming , nonmotile and coccus shaped . the genome is 1 642 295 bp long , and g+c content is 48.9% . the 16s rrna gene sequence and whole - genome shotgun sequence of m. massiliensis strain sit12 are deposited in genbank under accession numbers ln866998 and fizw00000000.1 respectively . the type strain sit12 (= csur p1987 = dsm 29078 ) was isolated from the stool of a healthy 2-year - old senegalese boy .

murdochiella massiliensis strain sit12 (= csur p1987 = dsm 29078 ) is the type strain of m. massiliensis sp . nov . this bacterium was isolated from the stool of a healthy 2-year - old senegalese boy . m. massiliensis is an anaerobic , gram - positive coccus . the genome size of m. massiliensis strain sit12 is 1 642 295 bp with 48.9% g+c content and assembled into two scaffolds .

Organism Information Genome description Conclusion Description of

posterior vitreous detachment ( pvd ) is a common phenomenon frequently related with aging of ocular structures . the presence of persistent vitreomacular adhesions exerting tractional forces ( vitreomacular traction , vmt ) may be associated with the development of macular hole ( mh ) [ 2 , 3 ] . these alterations in the symptomatic phase may cause visual disturbances , including photopsia , metamorphopsia , blurred vision , and decreased visual acuity , which in addition of causing visual - related problems may affect negatively the patient 's health - related quality of life . the introduction of optical coherence tomography ( oct ) has allowed a more accurate visualization of the macular anatomy and better knowledge of the pathophysiology of the process , including measurement and assessment of mh characteristics [ 57 ] , facilitating treatment decision - making . the vitreous gel is responsible for the stabilization of the eyeball through collagen fibers ( mainly type ii collagen ) . collagen fibers are running in an anteroposterior direction through the vitreous center , convering in the anterior vitreous base , and inserting into the posterior vitreous cortex . spaces between the collagen fibrils are maintained by the protein opticin and the glycosaminoglycan chondroitin sulphate . spaces between the collagen fibrils are mostly filled with water ( 98% of the vitreous gel component ) and hyaluronic acid , which provides the gel - like consistency of the vitreous . the vitreoretinal interface is a complex anatomical structure composed by the union between the retina and the vitreous . densely packed collagen fibrils of the posterior vitreous cortex ( 100300 m in thickness ) lie over the macula and are superficially inserted into the internal limiting membrane ( ilm ) of the retina by means of adhesion molecules , such as laminin , fibronectin , and proteoglycans , which interact with opticin in the vitreous gel . adherences are more firmly attached to the retina at the vitreous base , optic disc , and fovea , as well as along the major retinal blood vessels . the vitreomacular junction has an annular shape , with a diameter of 3 - 4 mm . the set of events that occur as the eye ages are associated with a series of physiological changes in the vitreous gel , with progressive liquefaction ( at the age of 80 , around 50% of the vitreous gel has been liquefied ) and gradual destruction of the collagen - hyaluronic acid network . this occurs as a result of the development of fluid - filled pockets beginning in front of the macula , which over the time coalesce and enlarge , resulting in a weakened adhesion between the vitreous and the retina . this gradually predisposes to pvd , defined as separation of the posterior cortex from the ilm of the retina , which represents the final step of the normal vitreous aging process [ 11 , 12 ] . pvd is an insidious process that occurs over the course of months or years , being asymptomatic in many cases until complete separation of the vitreous from the macula and optic nerve , which is the final stage . however , the anterior attachment to the vitreous base is very strong and remains for a long time . acute symptoms of complete pvd include photopsia ( by vitreous traction on the peripheral retina ) and floaters by condensation of the vitreous collagen , glial tissue , or blood around the optic nerve . studies in healthy adults have shown that focal perifoveal pvd occurs in 50% of subjects aged between 30 and 39 , whereas complete pvd is found in 50% of subjects aged 70 years or older [ 13 , 14 ] . in addition to advanced age , pvd is more frequent in postmenopausal women by the effects of decreased estrogens on the connective tissue ( within the vitreous gel ) , as well as in the presence of myopia . the normal process of pvd due to vitreous aging may be complicated by the presence of vitreomacular adhesions between the cortex and the macular area , resulting from vitreous syneresis . these adherences may be focal or extensive , affecting the foveola only or a wide region of the macular area and the optic disc . simple vitreomacular adhesion ( vma ) is not associated with distortion of the macular architecture . however , these adherences may exert traction forces on the macula ( vmt ) , increasing secondarily during ocular saccades . this may cause retinal distortion and foveal detachment . on the other hand , continuous anteroposterior traction by vitreous contraction may cause alterations , such as cystoid macular edema . full - thickness mh is an anatomic defect in the fovea with interruption of all neural retinal layers . with the use of high - resolution oct , it has been shown that idiopathic mhs are initiated during perifoveal pvd as a consequence of the dynamic anteroposterior vmt process . this anteroposterior vmt may cause intraretinal cavitation with progression to dehiscence of the outer retinal layers and complete detachment of the cyst roof giving rise to a full - thickness defect . stages of the development of mh from focal vmt to complete aperture together with accompanying symptoms have been described by gass [ 18 , 19 ] . the introduction of enzymatic vitreolysis , which can result in the liberation of vmt , opens highly interesting new perspectives in this field . a few studies have been specifically addressed to the epidemiology of idiopathic vmt due to the overlapping of this condition with other ophthalmological diseases . a prevalence of isolated idiopathic vmt , without mh , has been estimated as approximately 22.5 cases per 100 000 of the general population , with an incidence of 0.6/100 000 persons - year . in different observational and intervention studies , the mean age of patients with vmt was around 6570 years ( range 4864 ) , with a predominance of females [ 4 , 15 ] . regarding the prevalence of mh , it has been reported around 0.1 to 0.8 in adults aged > 40 years , with an age - adjusted incidence of 7.8 cases per 100 000 of the general population per year . also , the risk of development of mh in the fellow eyes , without manifestations of pvd , has been estimated at around 712% after 5 years and 17% at 20 years . approximately two - thirds of patients with mh are women , and the disease is unilateral in 80% of cases . an increase in serum fibrinogen level has been reported as a risk factor for mh , whereas the use of estrogen replacement therapy in women decreases the risk . in subjects with myopia , now , nearly two decades since the introduction of oct , it is possible to assess and define the pathologic progression of disorders affecting vitreoretinal interface with a high level of accuracy and reproducibility . on the basis of oct - derived anatomic findings , a unified classification scheme for disease of the vitreomacular interface has been developed . with this purpose , a group of experts in diseases of the vitreoretinal interface ( international vitreomacular traction study group , ivts ) have proposed a classification system for diseases of the vitreomacular interface . this evidence - based classification is a clinically applicable system that is predictive of therapeutic outcomes and is useful for the execution and comparative analysis of clinical studies . vma represents a specific stage of partial vitreous detachment in the perifoveal area without retinal abnormalities . in previous classifications , vma is the equivalent of a stage 1 pvd [ 11 , 15 , 27 , 28 ] . vma is characterized by elevation of the cortical vitreous above the retinal surface , with the vitreous remaining attached within a 3 mm radius of the fovea ( as defined arbitrarily ) . the angle between the vitreous and the inner retinal surface is acute , and the retina displays no abnormalities in contour or morphological features of oct . vma is not accompanied by visual impairment and may be considered a normal finding in the natural course of pvd . also , vma may be subclassified by the size of the adhesion into focal ( 1500 m ) or broad ( > 1500 m ) . the cutoff of 1500 m corresponds to the area of increased vitreous adhesion to the fovea . vma usually resolves spontaneously as part of the normal process of pvd , although it may progress to vmt and , for this reason , periodic monitoring with oct is necessary . macular traction due to progression of pvd causes anatomic changes in contour of the foveal surface , intraretinal pseudocyst formation , and disappearance of foveolar depression , which typically results in reduced or distorted vision . the following anatomic criteria should be present at least in one oct image to classify an eye as having vmt : ( a ) evidence of perifoveal vitreous cortex detachment from the retinal surface , ( b ) attachment of the vitreous cortex to the macula within a 3 mm radius of the fovea , and ( c ) association of this attachment with distortion of the foveal surface , intraretinal structural changes , foveal detachment from the retinal pigment epithelium ( rpe ) , or a combination of these findings , without full - thickness interruption of all retinal layers . vmt can also be subclassified as focal or broad ( using the same cutoff of 1500 m ) depending on the width of the vitreous attachment . distortion of the foveal profile , formation of intraretinal cysts , intraretinal cavitation , subretinal fluid , and , even , rpe detachment can be observed . on the other hand , proliferation of residual of vitreous tissue provides the anatomic substrate to form an epiretinal membrane ( erm ) , which in turn may appear at any stage of vitreous separation . although spontaneous resolution of vmt may occur , traction on a large surface or the presence of erm is poor prognostic factor . in symptomatic patients , enzymatic vitreolysis or vitrectomy may be indicated . as stated above , full - thickness mh is an anatomic defect in the fovea featuring interruption of all neural retinal layers . the observation of the anatomic opening on several scans through the fovea is an unequivocal sign . according to the aperture size , mhs are considered small ( < 250 m ) , medium ( 250 to 400 m ) , and large ( diameter > 400 m ) . also , on the basis of oct findings , mh can be categorized according to the presence or absence of vmt . only patients with mh and concomitant vmt are candidates for pharmacologic vitreolysis . the correlations between mh stages commonly used in clinical practice and oct - based images proposed by the ivts group are shown in table 1 . primary mh results from vitreous traction on the fovea from anomalous pvd ( incomplete or inadequate separation of the vitreoretinal interface ) , whereas secondary mhs are caused by other pathologic conditions and do not have preexisting or concurrent vmt . secondary mhs have been reported in cases of blunt ocular trauma , lightning strike , high myopia [ 25 , 31 ] , macular schisis , macular telangiectasia type 2 , occlusion of the central retinal vein , diabetic macular edema , uveitis , and age - related macular degeneration . the availability of oct , particularly spectral domain oct ( sd - oct ) , has allowed a more accurate diagnosis and precise assessment of adhesion of the vitreous to the macula , differentiating vma from vmt . before the introduction of oct , only patients with advanced vma could have been diagnosed by biomicroscopy and , for this reason , the rates of spontaneous deterioration reported were high ( 64% ) . studies using sd - oct have shown that incomplete vitreous detachment with persistent vitreoretinal adhesions is more frequently observed than by clinical diagnosis . during the physiological process of pvd , the vitreous remains attached to the foveal region in the last stages ( figure 1 ) , so that , vma can be considered a normal stage in the natural history of pvd associated with vitreous aging [ 13 , 26 ] . only when symptoms are present or when foveal anatomic changes are observed , vma can be considered a pathological process . recently , john et al . investigated the spontaneous clinical course in 106 eyes of 81 patients identified as having vma by sd - oct and classified into three grades , with a mean follow - up of 18 months ( range 1 to 91 ) . the authors defined three grades to classify adherence : grade 1 ( 41% ) was incomplete cortical vitreous separation with attachment at the fovea , grade 2 ( 52% ) was the grade 1 findings and any intraretinal cysts , and grade 3 ( 7% ) was the grade 2 findings and the presence of subretinal fluid . by the last follow - up , spontaneous release of vma occurred in 32% of cases ( 34 eyes , in 30% , 30% , and 57% of grades 1 , 2 , and 3 , resp . ) . no changes were observed in 23 , 31 , and 2 eyes ( 52% of the total ) , and progression occurred in 7 , 8 , and 1 eye of grades 1 , 2 , and 3 , respectively ( 16% of the total ) . the authors conclude that the clinical course of patients with vma managed by initial observation was generally favourable in asymptomatic patients or with minimal symptoms of vmt . studying the retinal surface with sd - oct , it has been observed that pvd appears to begin in the perifoveal region , with a slow clinical course taking even years until complete separation of the vitreous from the papilla ( figure 2 ) . in most patients this process is asymptomatic but , in some cases , pvd may be complicated by macular pathology . in a oct study of eyes with macular edema secondary to vmt , published in 2012 , complete and spontaneous resolution of traction the clinical course of vma , particularly in asymptomatic patients , remains to be fully elucidated . systematic examination with sd - oct has been associated with an increase in diagnostic rates and has allowed assessing more accurately the course of this physiological process that may evolve into vmt , remain stable , or resolve spontaneously . therefore , in the presence of a vma syndrome , the first approach is to reexamine the patients using oct at a period of 3 months . even in cases of evolution to a vmt syndrome , observation still remains an option , given the possibility of spontaneous resolution of vmt . ocriplasmin is a truncated form of human plasmin that induces liquefaction of the vitreous and separation of the vitreous cortex from the retinal surface due to proteolytic activity against main components of the vitreomacular adhesion . the efficacy and safety of ocriplasmin have been evaluated in two pivotal , phase 3 clinical trials ( tg - mv-006 y tg - mv-007 ) carried out in the united states and europe . both studies were very similar except for the ratio of randomized assignments to ocriplasmin and placebo , which was 2 : 1 in the tg - mv-006 study and 3 : 1 in the tg - mv-007 . overall , 652 patients were randomized , 464 were assigned to treatment with a single intravitreal injection of ocriplasmin ( 125 g ) and 188 to a placebo intravitreal injection . the primary endpoint was the pharmacologic resolution of vma at day 28 , as determined by oct . secondary endpoints included the percentage of patients with complete pvd and nonsurgical closure of full - thickness mh at day 28 . eligible patients had symptomatic focal vma as seen on oct and a best - corrected visual acuity of 20/25 or less . exclusion criteria were high myopia ( more than 8 diopters or axial length > 26 mm ) , prior vitrectomy or prior laser photocoagulation of the macula , and other eye diseases that may affect visual acuity . patients with a mh > 400 m in diameter were also excluded . of note , the presence of an erm was not a criterion for exclusion . at day 28 , vma resolved in 26.5% of ocriplasmin - injected eyes and in 10.1% of placebo - injected eyes ( p < 0.001 ) . the between - group differences did not change substantially at 6 months ( 26.9% ocriplasmin versus 13.3% placebo , p = 0.001 ) . also , 72% of patients with resolution of vma showed the release during the first seven days . results of adhesion release were better in patients without erm ( 37.4% in the ocriplasmin group versus 14.3% in the placebo group , p < 0.001 ) . with regard to secondary variables ( day 28 ) , 13.4% of patients treated with ocriplasmin showed total pvd as compared to 3.7% of those treated with placebo ( p < 0.001 ) . also , nonsurgical closure of full - thickness mh was achieved in 40.6% of ocriplasmin - treated patients and in 10.6% of placebo - treated patients ( p < 0.001 ) . according to the investigator 's criteria , all patients could be treated with vitrectomy in the framework of the study if macular disease did not resolve . at 6 months , vitrectomy was performed in 17.7% of patients in the ocriplasmin group and in 26.6% of those in the placebo group ( p = 0.02 ) . at 6 months , there were statistically significant differences in favour of ocriplasmin in the gain of two or more lines ( 23.7% versus 11.2% , p < 0.001 ) or three or more lines ( 12.3% versus 6.4% , p = 0.02 ) . most adverse events were related to the development of pvd induced by ocriplasmin injection ( floaters and photopsia ) . there was a slightly higher incidence of retinal tears or detachments in the placebo group , which was attributed to the higher proportion of patients treated by means of vitrectomy in this group . the favourable results obtained in both clinical trials allowed approval of the use of intravitreal injection of ocriplasmin for the treatment of symptomatic vmt and mh by the food and drug administration ( fda ) in the united states , in november 2012 , and by the european medicines agency ( ema ) in may 2013 . outside the context of clinical trials , recent reports have provided data of the use of ocriplasmin in daily practice . in a retrospective study , 17 patients with symptomatic vmt were treated with a single intravitreal injection of ocriplasmin 0.125 mg . by day 28 , resolution of vmt was verified by sdoct in eight patients ( 47.1% ) , 7 of which ( 87.5% ) had already experienced release by day 7 . those who did not have traction release showed no statistically significant change in vma diameter . four of the five patients ( 80% ) with mh at baseline experienced resolution of their mh after injection . significant differences in visual acuity were not observed ( 20/49 at baseline and 20/46 at final follow - up ) . it should be noted that patients meeting the four positive predictor criteria ( younger than 65 years , no erm at baseline , traction < 1500 m , and phakic lens status ) showed a response rate of 75% ( three of four eyes ) . transient outer segment ellipsoid zone loss was documented in 7 cases ( 41.1% ) and subretinal fluid presence following injection was noted in 5 cases ( 29.4% ) . in another study of 19 patients with symptomatic vma treated with intravitreal ocriplasmin , resolution of vma results were significantly affected by lens status , with adhesion release in 53% of phakic patients , whereas no case of resolution of adhesions was observed in pseudophakic patients . also , closure of mhs after treatment was found in 3 of 6 patients ( 50% ) . visual acuity remains stable , with a slight tendency towards improvement in the majority of cases . only one patient showed an important loss of visual acuity ( from 20/70 to 20/200 ) due to progression of vmt to a full - thickness mh . the proportion of patients who had any ocular adverse event was 68.4% in the ocriplasmin group and 53.5% in the placebo group ( p < 0.001 ) . the most common complications included vitreous floaters ( ocriplasmin 16.8% versus placebo 7.5% , p = 0.002 ) , photopsia ( 11.8% versus 2.7% , p < 0.001 ) , blurred vision ( 8.6% versus 3.2% , p = 0.01 ) , and visual impairment ( 5.4% versus 1.6% , p = 0.02 ) . there were no differences between the groups in terms of severe ocular adverse events , including development of mh ( 5.2% versus 8.6% ) , retinal detachment ( 0% versus 1.6% ) , and reduced visual acuity ( 0.6% versus 0.5% ) . however , since the real - world use of the drug began , there have been some unfavourable reports of visual disturbances after ocriplasmin injection , including transient but profound visual decline , raising concerns regarding its safety . of 976 patients receiving ocriplasmin injection in clinical trials , 9 patients were reported to have experienced an acute decrease in vision , some to the hand motions level , within 24 hours of injection . in 8 of these 9 patients , in the clinical trials of ocriplasmin , dyschromatopsia , and electroretinographic ( erg ) changes occurred in a significantly greater number of eyes treated with ocriplasmin than in eyes receiving placebo [ 20 , 40 ] . freund et al . recently reported a single case report demonstrating changes seen in the outer photoreceptor segments by sd - oct . since the clinical trials used only time - domain oct with inferior resolution to sd - oct , it is possible that these cases may have been overlooked . in another study in which 17 patients were included , almost all the patients who responded to the treatment ( 7/8 ) had ellipsoid zone changes on the sd - oct ( figure 3 ) . these patients also had transient reduction of visual acuity and demonstrated subretinal fluid during the release process with almost the exact time course as the loss of the os ellipsoid zone . the loss of the os ellipsoid zone occurred after an average of 5 days after injection of ocriplasmin and the mean time of resolution on oct was 29.3 days . the occurrence and resolution of subretinal fluid occurred at an average of 4.8 days and 30 days after injection , respectively . however , in a retrospective review of 62 eyes with symptomatic vma treated with ocriplasmin , subretinal fluid appeared in 37% of cases , with persistence of fluid in 30% of cases after 5 months of follow - up . other studies have also shown resolution of the ellipsoid zone changes in most patients within weeks or months after ocriplasmin injection [ 43 , 44 ] . alteration of the ellipsoid zone on sd - oct and a significant decrease in erg amplitudes have been also reported in two patients with release of symptomatic vmt after ocriplasmin injection [ 45 , 46 ] . it is possible that this transient effect of the medication may be due to a diffuse enzymatic effect of the protease on the photoreceptors or the retinal pigment epithelium throughout the retina . the greater reduction in scotopic function compared with photopic function suggests that rod photoreceptors may be more susceptible than cone photoreceptors to the effects of ocriplasmin . if this transient affect occurs for both rods and cones , it may explain the dyschromatopsia , contrast sensitivity changes , dark adaptation issues , and erg changes reported in the ocriplasmin clinical trials . an ongoing phase 3b , 24-month randomized clinical trial which will evaluate erg and microperimetry in ocriplasmin - treated eyes compared to sham , will provide additional clarifications on the observed egr changes and dyschromatopsia events ( oasis study ; ntc01429441 ) already reported . surgery of idiopathic mh with ilm peeling is a very safe procedure , with good anatomic and functional results and scarce postoperative complications . data provided by clinical trials have shown that peeling of the ilm significantly increases mh closure rates and is also associated with significantly lower percentages of reoperation and reopening . therefore , ilm peeling is a cost - effective technique and the procedure of choice for all patients with idiopathic full - thickness mh susceptible to undergo surgical treatment [ 4853 ] . broad ilm peeling to the vascular arcades is recommended , so that tangential traction forces on the mh edges are removed facilitating approximation and closure . in cases of large mh ( > 400 m ) with increased risk of failure of primary surgery , alternative techniques have been proposed , such as the inverted ilm flap technique in which instead of completely removing the ilm , a remnant attached to the margins of the mh is left in place . this ilm remnant is then inverted upside down to cover the mh . with the use of this technique closure rates of 98% compared to 88% with the standard technique have been achieved . for refractory mh to the standard technique or for secondary mh after vitrectomy when peeling of the ilm has been already performed , an autologous transplantation of the ilm remnants introduced into the hole with subsequent gas tamponade contributes to the improvement of anatomic and visual outcomes . vital dyes have become effective and useful tools for identifying ocular tissues during vitrectomy , thereby facilitating ilm peeling and ensuring complete removal of this delicate membrane . the most frequently used vital dyes include triamcinolone acetonide suspension in balanced salt solution ( bss ) ( triesence ) , indocyanine green and infracyanine green , brilliant blue , and trypan blue with brilliant blue ( membrane blue - dual ) . triamcinolone suspension in bss is not a true dye but is very useful for the identification of vitreous remnants and the posterior hyaloid . deposition of crystals on the ilm surface helps the achievement of complete removal of the membrane , although it is less effective than vital dyes because triamcinolone does not increase the rigidity of ilm . indocyanine green and infracyanine green possess a great affinity for the matrix components of the ilm and produce intense staining of the ilm . besides the ability of indocyanine and infracyanine green to stain the ilm , they cause an increase in the biomechanical stiffness of the ilm , thereby facilitating its peeling . although in europe they are no longer used because of potential toxicity , they continue to be used in the united states [ 58 , 59 ] . brilliant blue has a remarkable affinity for the ilm and , although ilm staining is less intense than that achieved with indocyanine green , causes adequate staining of the ilm and may be used without fluid - air exchange . in europe the combination of trypan blue and brilliant blue allows staining of the erm , posterior hyaloid , and ilm simultaneously . this combination has a lower density than water and bss and circumvents the need for fluid - air exchange . there is controversy regarding posturing in mh surgery . although most authors recommend face - down posturing 90% of time for 10 days , different studies have reported successful hole closure in the absence of face - down positioning , given that isolation of the macula by gas tamponade maintaining the macula dried seems to be the most important factor for closure [ 6163 ] . in this respect , oct studies have shown that hole closure occurs during the first postoperative day independently of the types of gas tamponade and posturing , so that after vitrectomy with wide ilm peeling , gas tamponade would be sufficient ( preferably short - acting gases , such as sf6 ) at nonexpansible concentration , without the need of face - down posturing , avoiding the prone position during 3 to 5 days . this approach may be also indicated for phakic patients because it does not seem to increase the incidence of cataracts . combined phacovitrectomy or sequential vitrectomy and phacoemulsification are safe and effective for the treatment of mh , with equivalent anatomic and functional results . in most cases , idiopathic mh affects patients older than 50 years in which some degree of lens opacity is frequent . moreover , cataract develops in 75% to 95% of patients undergoing vitrectomy for mh within 3 years after surgery . for this reason , both cost and discomfort are lower with a single surgical procedure , and functional recovery is more rapid . combined phacovitrectomy may also decrease the risk of reopening after cataract extraction in the two - step surgical approach [ 66 , 67 ] . however , combined vitrectomy , phacoemulsification , and intraocular lens ( iol ) implantation may be associated with complications , including a high degree of postoperative anterior chamber inflammation and a higher risk of iol dislocation or papillary capture , generally as a result of excess gas tamponade and/or poor compliance to positioning . therefore , the decision of the combined versus the two - step procedure should be individualized according to the characteristics of each case and the patient 's and surgeon 's preferences . in the study of the moorfields macular hole ( mmhs ) group , an overall closure rate of 81% at 2 years was achieved in mhs stages 2 , 3 , and 4 as well as an improvement in visual acuity of 6/36 to 6/18 , which was clearly superior to results obtained in the observation group . in the vitrectomy for treatment of macular hole study ( vmhs ) , the rate of anatomic closure was 69% and the final visual acuity was higher in the operated than in nonoperated eyes ( 20/115 versus 20/166 ) . once peeling of the ilm has become popular , closure rates of 90% to 100% were reported [ 7175 ] . however , the use of indocyanine green was associated with potential toxicity in some cases and , for this reason , trypan blue and brilliant blue are in widespread use in some countries , with closure rates of 94% to 100% , without apparent severe side effects [ 7780 ] . despite its clear indication and safety in mh surgery , ilm peeling is a traumatic procedure that has acute effects on the underlying retinal nerve fiber layer . ilm peeling often results in temporary swelling of the arcuate nerve fiber layer ( sanfl ) which may be the earliest manifestation of dissociated nerve fiber layer ( donfl ) which occurs later in the postoperative period . however it is probably a transient feature that does not affect visual recovery . although peeling of the ilm has been widely adopted in mh surgery , the high percentages of hole closure obtained in the years prior to systematic ilm peeling add uncertainty about whether to use it in all cases . recently , spiteri cornish et al . [ 81 , 82 ] carried out a systematic review and meta - analysis to assess the success of hm surgery with ilm peeling compared with the nonpeeling technique . four randomized clinical trials comparing both techniques were identified [ 48 , 49 , 51 , 81 , 82 ] . there was no evidence of a difference in the primary outcome ( distance visual acuity at six months ) , nor in distance visual acuity at 12 months between randomized groups . overall , 66.2% achieved a visual acuity equal or greater than 69 letters on etdrs charts ( corresponding snellen visual acuity 20/40 ) and 77.9% gained more than three etdrs lines . improvement of visual acuity was higher in patients in which primary anatomic closure was achieved ( final visual acuity 72.8 7.6 letters and a mean improvement of 21.6 7.1 letters ) than in eyes in which further surgery was required ( 66.4 8.6 and 17.4 7.7 letters , resp . ) . however , visual improvement was obtained somewhat earlier in the ilm peeling group and , at 3 months , improvement was greater if ilm peeling was performed . in addition , the percentage of primary closure was higher in the ilm peeling as compared with no peeling ( 89.9% versus 50.3% , with an odds ratio ( or ) of 9.27 and 95% confidence interval [ ci ] of 4.9817.24 ) . when reoperations were excluded from the analysis , the ilm peeling group continued to have more favourable results ( or 3.99 , 95% ci 1.639.75 ) . also , in mh stage 2 , the efficacy rate was better for ilm peeling than no peeling ( 91.6% versus 61.3% , with an or of 6.19 ; 95% ci 1.6523.20 ) [ 48 , 49 , 83 ] . this higher success rate was not accompanied by an increase of perioperative complications , neither in the reports in which the ilm was stained with indocyanine green . in the meta - analysis , the rate of intraoperative complications was 19.32% for the ilm peeling group as compared with 21.1% for the nonpeeling group ( or 0.94 , 95% ci 0.471.87 ) . the most frequent intraoperative complications were small retinal hemorrhage ( 619% ) , retinal tears ( 5.432% ) , retinal detachment ( 26% ) , and choroidal hemorrhage ( 03% ) . according to these data , the authors conclude that ilm peeling offers more favourable cost - effectiveness compared with no peeling in mh surgery [ 81 , 82 ] . although anatomic closure in mh surgery is achieved in more than 90% of cases , sometimes it does not correlate well with improvement in visual acuity . multiple studies using oct have assessed hole configuration in an attempt to establish a correlation with postoperative visual acuity [ 8491 ] , emphasizing the importance of changes in the outer retina . defined a macular hole index ( mhi ) as a ratio of hole height to base diameter of hole , calculated from oct transverse images of the macular area , establishing that a mhi 0.5 was correlated with better postoperative visual acuity than mih < 0.5 . ruiz - moreno et al . described the diameter hole index ( dhi ) as a ratio between minimum hole diameter and base diameter , showing the minimum diameter was the best preoperative predictive prognostic factor . different studies have shown a direct correlation between integrity of the hyperreflective line as is / os junction of photoreceptors and postoperative improvement of visual acuity . in the study of kitaya et al . , postoperative vision 0.7 was correlated with good reconstitution of the is / os junction . however , sano et al . showed that a continuous is / os line was not a reliable prognostic factor in the early postoperative period given that abnormalities of the is / os line seen on sd - oct can be gradually repaired , with achievement of a continuous is / os line at 6 months . spaide and curcio assessed the correlation of the outer retina analyzed by means of sd - oct and histopathological findings , showing that the hyperreflective line identified as is / os junction of photoreceptors corresponded to the ellipsoid portion of the photoreceptor inner segment , containing mitochondria . wakabayashi et al . using sd - oct described that reconstitution of the external limiting membrane ( elm ) was more important to predict subsequent restoration of the foveal photoreceptor layer than the ellipsoid zone restoration . restoration of elm seems to be a necessary factor for reconstitution of the ellipsoid band , with subsequent migration of photoreceptors and complete closure of the full - thickness mh . ruiz - moreno et al . have analyzed 164 eyes with mh treated by vitrectomy and ilm peeling showing that restoration of the ellipsoid portion of the photoreceptor inner segment is an important prognostic factor for visual rehabilitation after mh surgery . reopening of the hole ( figure 4 ) is one of the best known complications after initially successful mh treatment with vitreous surgery [ 67 , 91101 ] . peeling of the ilm during primary mh surgery is one of the factors that has been mostly related to the incidence of reopening , varying between 0% and 8% in eyes with ilm peeling [ 67 , 9597 ] and between 2% and 16% in eyes with no peeling [ 67 , 9395 , 97 ] . the variable percentages reported in the studies are due in part to differences in the length of follow - up , with higher rates associated with prolonged follow - up periods . paques et al . reported a 9.5% incidence with a mean follow - up of 2 years , whereas scott et al . found a 12% incidence with a mean follow - up of 7 years . kumagai et al . analyzed the results of surgery in a series of 877 cases of mh , increasing the reopening percentage to 28.1% with no ilm peeling . the incidence of recurrence was 0.39% in eyes with peeling of the ilm , increasing to 7.2% with no peeling . besides no peeling , statistically significant risk factors for reopening were myopia of more than 6 diopters and intraoperative retinal tears . retinal tears treated with laser may be one of the factors that increase the development of erm , with subsequent tangential traction and reopening of the mh . no peeling of the ilm may be associated with a higher risk of erm formation [ 92 , 93 , 97 , 100 ] . yoshida and kishi observed the presence of erm in all cases of reopening of the hole . however , kumagai et al . did not report erm in none of the cases with hole reopening assessed by sd - oct . in relation to the incidence of reopening with bilateral mh , duker et al . reported bilateral reopening in 38% of cases , christmas et al . in 59% , scott et al . in 38% , and kumagai et al . in 14.9% . cataract surgery in the postoperative period of mh surgery has been involved in the reopening of mh . paques et al . observed that 73% of cases of hole reopening occurred after a secondary cataract surgery . bhatnagar et al . reported that in the presence of cystic macular edema after secondary cataract surgery , there was a sevenfold increase in the risk of reopened holes , and garca - arum et al and sheidow and gonder reported cystoid edema in combined surgical procedures and that the incidence of hole reopening did not increase in secondary cataracts . with regard to treatment of persisting mh , ilm peeling and erm removal should be performed in those cases in which these procedures were not performed at the initial macular surgery , together with long - acting gas tamponade ( c3f8 ) and strict face - down positioning during the first postoperative days . in these patients , when ilm peeling and removal of the erm have been performed in the first surgical procedure , the success of reoperation decreases . in a series of 30 patients reported by d'souza et al . with initial ilm peel who underwent repeat surgery involving vitrectomy , enlargement of ilm rhexis , and gas tamponade with c3f8 , more extensive ilm peeling causing tangential traction due to fibrosis of dissection margin may contribute to the anatomic closure . the use of growth factors , such as platelet - derived growth factors as a stimulus of glial progenitor cells , may be useful if the ilm has been adequately peeled ( figure 5 ) , as well as the use of heavy silicone oil in patients with positioning difficulties . based on the aforementioned data and as shown in the schematic representation in figure 6 , patients with vma can be observed without the need of any intervention . in cases of vmt if the patient is asymptomatic , a follow - up control at 3 months may be sufficient . during this interval , the patient should be advised to perform periodic self - examinations with the amsler grid or monocular reading tests . in case of symptoms , intensity and disability should be assessed . there is no consensus criterion regarding the degree of vision loss that should be considered significant and amenable to treatment . however , in the tg - mv-006 y tg - mv-007 clinical trials , patients with visual acuity equal or lower than 20/25 were eligible , so that this level of visual impairment can be already considered to be susceptible of treatment . also , other causes that may justify decreased visual acuity should be excluded . metamorphopsia clinically significant for the patient and visual loss progression are also key factors at the time of adopting a more interventional therapeutic attitude . despite these considerations , a period of observation may be an option for these patients , because spontaneous resolution is still possible . in case of deciding an active treatment , the presence of other associated macular diseases , such as erm , should be excluded . when traction is 1500 m , enzymatic vitreolysis with ocriplasmin is the treatment of choice . in the presence of > 1500 m traction or erm , surgical treatment with vitrectomy 400 m in size with mvt and in the absence of erm , enzymatic vitreolysis with ocriplasmin is again the most recommendable option . in cases of holes > 400 m , or in the absence of evident vmt , or in the presence of erm , vitrectomy is the first option . patients undergoing enzymatic vitreolysis with intravitreal injection of ocriplasmin should be evaluated at 7 and 30 days . most cases of vmt or mh resolve within the first week of treatment and also at this time the occurrence of potential treatment - related complications should be excluded . if resolution of vmt and/or hole closure had not occurred after a month of treatment , the likelihood of success is highly improbable and vitrectomy can be performed . patients with lamellar mh or pseudomacular holes in which traction is usually absent are also candidates for enzymatic vitreolysis . in cases of vmt associated with other retinal diseases , such as age - related macular degeneration , diabetic macular edema , or vitreomacular interface pathology in the myope , it is still too early to make a recommendation on the impact of enzymatic vitreolysis with ocriplasmin in the treatment of these conditions , and we should await for results of ongoing clinical trials on this topic . finally , in all cases , the final decision regarding treatment with enzymatic vitreolysis with ocriplasmin or vitrectomy should be consensuated with the patient . all cases in which the use of ocriplasmin is considered a first treatment option can be successfully treated by means of vitrectomy . also , it may be possible that patients who initially are not ideal candidates for enzymatic vitreolysis may have their pathologic condition solved by treatment with ocriplasmin . favourable prognostic factors for the choice of vitreolysis have been identified including young age and phakic status , but difficulties to maintain postoperative face - down posture or the waiting lists for vitrectomy are variables that should also be considered . enzymatic vitreolysis based on the intravitreal injection of ocriplasmin is a treatment option with proven efficacy and adequate safety profile in selected patients with vmt and mh . in cases of vmt , treatment with ocriplasmin is indicated when traction is 1500 m and in the absence of concurrent macular diseases ( erm ) . in the case of mh , the hole diameter should be 400 , traction has to be present , and erm should be absent . when resolution of the process after one month of the procedure is not achieved , vitrectomy with ilm peeling would be the surgical treatment of choice .

the paper presents a review of the sequence of events of posterior vitreous detachment ( pvd ) , vitreomacular adhesion ( vma ) , vitreomacular traction ( vmt ) , and macular hole ( mh ) from their pathophysiological aspects , clinical features , diagnostic implications , and current management strategies . a treatment algorithm to be used in clinical practice in patients with vma , vmt , and mh based on the presence of symptoms , visual acuity , associated epiretinal membrane , and width of the vitreous attachment is presented . observation , pharmacologic vitreolysis with ocriplasmin , and surgical treatment are positioned as treatment options in the different steps of the therapeutic algorithm , with clear indications of the paths to be followed according to the initial presenting manifestations and the patient 's clinical course .

1. Introduction 2. Diagnosis, Definition, and Classification of VMT and MH 3. Treatment Options 4. Practical Considerations: Therapeutic Algorithm 5. Conclusions

hydrocephalus is an entity which embraces a variety of diseases whose final result is the enlarged size of cerebral ventricular system , partially or completely . among usual classifications , the most important are those who differ the communicating from non - communicating and congenital from acquired . its prevalence is near 11.5% among general population and is progressively rising with populational growth , thus representing an impressive healthy concern . congenital hydrocephalus due to a myriad of causes has a rate of 12/1000 births , being a common finding among pediatric age ( rekate , 2009 ) . the physiopathology of hydrocephalus lies in the dynamics of circulation of cerebrospinal fluid ( csf ) . there should be a disturbance either in production , circulation , or in reabsorption , resulting in positive balances and dilation of ventricular system , producing abnormal high pressure on the ventricles walls . elevated pressure reflects blocked blood flow out of the lateral ventricle . the consequent csf stasis in hydrocephalus interferes with cerebral and ventricular system development ( penn and linninger , 2009 ) . responses to elevated csf pressure can be marked oxidative changes in hydrocephalus that are reflected in the way that injured neurons metabolize neurotransmitters and myelin . contrary to the previously held belief that gliosis in the hydrocephalic brain is restricted only to the periventricular white - matter , gliosis extends through all of the cortex and the peri - aqueductal area ( penn and linninger , 2009 ) . the neighboring neuropil exhibits notable enlargement of extracellular space , synaptic plasticity and degeneration , damage of myelinated axons , and myelination delay . the role of ischemia , oxidative stress , increased calcium concentration , activation of nmda receptors , and disturbance of ion homeostasis are discussed in relationship with the fine structural alterations of hydrocephalic brain parenchyma ( castejn , 2010 ) . clinical manifestations depend especially on the time of appearance and form of onset , if acute / subacute or chronic . as a general rule , acute hydrocephalus produce pronounced symptoms as headache , vomitus , papilledema , and impaired consciousness , leading patient to coma and death ( drake , 2008 ) . chronic hydrocephalus , on other hand , produces skull enlargement , spasticity , progressive neurological deficits in children and dementia , urinary incontinence , and gait changes in elderly ( bergsneider et al . , 2008 ; the treatment , usually represented by some variation of a diversion procedure , consists in deviating csf flux and acts by reducing intracranial pressure , restoring periventricular , and global perfusion ( bergsneider et al . , 2008 ; drake , 2008 ) . in children , it is generally performed to restore csf dynamics and prevent worsening of symptoms . in chronic cases , it controls symptoms of intracranial pressure and interfere in cognitive and motor functions ( ishikawa et al . , 2008 ; thus , the form of onset is also the great determinant of cerebral tissue response , leading to physical adaptations , changing elastance and complacency , determining chemical and biological changes , including neuronal plasticity ( penn and linninger , 2009 ) . in this context , we will try to settle a link between the notable modifications to neurophysiology secondary to hydrocephalus and the ability of neuronal tissue to reassume and reorganize its functions toward adaptation . computational models such as the small - world and scale - free network might explain clinical resilience in various situations ( friston and price , 2003 ; noppeney et al . , 2004 ; achard and bullmore , 2007 ; van den heuvel et al . , 2008 ) . small - world networks predicts that neuronal cells are engaged in clustered connectivity with fewer long - range connections ( friston and price , 2003 ; achard et al . , 2006 ) . thus , there would be a shorter path length between any pair of neurons or brain regions , resulting in higher dynamical complexity , lower wiring costs , and resilience to tissue insults . a scale - free network is characterized by the existence of a small number of nodes having more connections than the other nodes . the nodes that have such a high connectivity degree are referred to as hub - nodes and are suggested to play an important role in the overall network organization ( friston and price , 2003 ) . brain resilience may be also the final result of processes such as redundancy , degeneracy , and pluripotentiality of neural systems ( friston and price , 2003 ; noppeney et al . , 2004 ) . another possible mechanism would be the local neurogenesis already reported in structures such as the basal ganglia , with preferential distribution in sub - regions of the ventral striatum ( stopczynski et al . , 2008 ) . neuronal plasticity is a continuous process where the central nervous system learn skills and remember information , structure neuronal networks in response to environment , and recover from brain and spinal cord injuries , being a fundamental tool in brain resilience to lesions ( johnston , 2009 ) . basic mechanisms that are involved in plasticity include neurogenesis , programmed cell death , and activity - dependent synaptic plasticity ( wojtowicz , 2011 ) . clinical examples of adaptive neuronal plasticity include reorganization of cortical maps of the fingers in response to practice playing a stringed instrument and constraint - induced movement therapy to improve hemiparesis caused by stroke or cerebral palsy ( ewing - cobbs et al . , 2003 ; hydrocephalus , congenital or acquired , represents a model of brain resilience too , once transient or permanent perfusional deficits generate structural and/or functional injuries , being partially or completely compensated by remaining cortical areas ( ewing - cobbs et al . , 2003 ) . much evidence shows that the brain has an astounding ability to modulate cognitive and motor skills after acute insults , during insidious neurodegenerative processes , psychological stress , or even along the aging course ( price and friston , 2002 ; meunier et al . permanent and transient lesions caused by strokes , tumors , head trauma , and hydrocephalus are good models to understand how the compensation process works , following focal or even broader damage ( price and friston , 2002 ; oliveira et al . , 2011 ) . john lorber ( 19151996 ) , a british pediatrician recognized by his work with spina bifida and ethic issues in sheffield university , had the opportunity of attending two young children with hydrocephalus presenting with normal mental development for their age . in both children , there was no evidence of a cerebral cortex , which was filled by csf . one of the children died at age 3 months , the second at 12 months . later , a young man with macrocephaly was referred to lorber ( lewin , 1980 ) . although the boy had an iq of 126 and had a first class honors degree in mathematics , he had virtually no brain . thus , he thought , there should be a tremendous amount of redundancy or spare capacity in the brain . these ideas were shared with scientifical community in a pediatric conference in 1980 . later in the same year additionally , norman geschwind ( 19261984 ) , an american neurologist at boston s beth israel hospital known for his works with behavioral neurology , also stated a certainty of capacity for reassigning functions following trauma and injuries in the brain , what should represent a high level of organization of cerebral tissue in order to promote adaptation ( berker et al . , 1992 ) . other reports even generate a scientifical query in the past , where the main question was the seemingly normal brain function with remarkable images of hydrocephalus and congenital malformations ( lewin , 1980 ) . for example , scans of a 44-year - old man s brain , showed fluid - filled ventricles , leaving little more than a thin sheet of actual brain tissue . he was married and father of two children , and worked as a civil servant . the man went to a hospital after he had mild weakness in his left leg . he used to have a shunt inserted into his head to drain away hydrocephalus as an infant and was removed when he was 14 . intelligence tests showed the man had an iq of 75 , below the average score of 100 but not considered mentally retarded or disabled , either ( feuillet et al . , we try to illustrate this scene by presenting the brain parenchyma of a normal subject followed by the brain of a normal subject with impressive hydrocephalus ( oliveira et al . , unpublished data ) and then an equally impressive hydrocephalus of a patient with profound symptoms ( oliveira et al . , unpublished data ) . the contrast among a normal brain in a normal adult ( left ) , the brain of a normal man with impressive hydrocephalus ( oliveira et al . , unpublished data ; middle ) , and an equally impressive hydrocephalus in a 54-year - old man with deep cognitive and motor impairment since childhood ( right ; oliveira et al . , unpublished data ) . the surprising question is that patients with very similar neuroradiological aspects may present with different and complex neurological impairments , from motor to cognitive . some important discussions about symptoms in hydrocephalic and non - hydrocephalic patients were already reported . previous studies of 10 sets of twins discordant for hydrocephalus in early life displayed differences in quality and quantity of development of verbal versus non - verbal cognitive functions , birth order , and hand and eye preference ( berker et al . , 1992 ) . the differences between those discordant twins seems to indicate systematic changes in pre- , peri- , and/or early postnatal organization and development of hemispheric function ( berker et al . , 1992 ) . other study considering the development of five language domains ( word finding , fluency and automaticity , immediate sentence memory , understanding of grammar , and metalinguistic awareness ) was held in children and adolescents , 75 with hydrocephalus in the first year of life , and 50 normal controls ( dennis et al . , 1987 ) . the results revealed a limited resilience of language to the effects of early hydrocephalus ( dennis et al . , 1987 ) . in adult hydrocephalus , especially idiopathic normal pressure hydrocephalus ( inph ) , it is observed recover after shunting procedures , which represents proof of brain resilience ( bergsneider et al . , 2008 ; ishikawa et al . , 2008 ; ladika and gurevitz , 2011 ) . a study displayed significant improvements at follow - up demonstrated on tests of verbal memory as well as in one test of psychomotor speed . eight of 10 patients showed improvement by more than 1 sd on at least one memory test . six of 10 patients improved significantly on more than 50% of the tests administered ( mcgirt et al . , 2008 ; simon et al . , 2009 patients elected for surgery and with good response after execution of tap test in the pre operatory period , usually present a remarkable recovery in cognitive and especially motor functions gradually ( oliveira et al . , unpublished data ) . in a report , there was a significant reversal in neuropsychological test scores with increased brain volume and increased regional cerebral glucose utilization in several brain regions after shunting of inph ( simon et al . rat models of chronic hydrocephalus suggested that disturbance in the postsynaptic integration processes , rather than axonal conduction or synaptic transmission , are more important for the production of the neurological deficits seen in chronic hydrocephalus ( kaye et al . , it was found impaired hippocampal plasticity ( tsubokawa et al . , 1988 ) . recent evidences also hypothesize the role played by dopamine d2 receptors in normal pressure hydrocephalus . in nph , d2 receptor down regulation was attenuated at 1 month after shunt surgery ( nakayama et al . , 2007 ) . a pet study showed significant increases of glucose metabolism in the cerebral cortical areas after surgery and a micro dialysis study showed a postoperative reduction in the glutamate content of the cerebral cortex , pointing that shunting and consecutively better regional perfusion reestablish the citoarquitecture and synthesis of dopamine d2 receptors , attenuating motor dysfunctions ( nakayama et al . , 2007 ) . therefore , several examples can be elicited to assign neural plasticity and resilience applied to hydrocephalic models , reassuming concepts of basic neurophysiology and discussing neural networks and integration , regeneration of neuronal tissue , and resilience to injuries . degeneracy and resilience are probably continuous and simultaneous events taking part in this complex process . we should not forget that , as long as there are large hydrocephalic , tumoral , traumatic , and ischemic samples of brain resilience and recovery , there are also cases of specific and punctiform lesions , sometimes only seem in high definition image studies , causing aggressive impairment of neurological function , even compatible with death . clinical experience and experimental models have already shown the resistance of the brain tissue to injuries , acute or chronic . until now , what we have summarized are pieces of individual reports and atypical manifestations of neurological diseases . doubtlessly , further multicenter investigations will be needed to clarify the infinite questions asked about neuronal tissue physiology . 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 .

hydrocephalus is an entity which embraces a variety of diseases whose final result is the enlarged size of cerebral ventricular system , partially or completely . the physiopathology of hydrocephalus lies in the dynamics of circulation of cerebrospinal fluid ( csf ) . the consequent csf stasis in hydrocephalus interferes with cerebral and ventricular system development . children and adults who sustain congenital or acquired brain injury typically experience a diffuse insult that impacts many areas of the brain . development and recovery after such injuries reflects both restoration and reorganization of cognitive functions . classic examples were already reported in literature . this suggests the presence of biological mechanisms associated with resilient adaptation of brain networks . we will settle a link between the notable modifications to neurophysiology secondary to hydrocephalus and the ability of neuronal tissue to reassume and reorganize its functions .

Presentation Hypothesis Scientifical Background Discussion Outcome Conclusion Conflict of Interest Statement

oct is an emerging medical imaging technology which enables cross - sectional imaging of tissue microstructure in situ and in real - time . oct can achieve 110 m resolutions and 12 mm penetration depths , approaching those of standard excisional biopsy and histopathology , but without the need to remove and process tissue specimens . oct is analogous to ultrasound imaging , except that imaging is performed by measuring the echo time delay and intensity of backscattered light rather than sound . oct imaging can be performed fiber - optically using delivery devices such as hand - held probes , endoscopes , catheters , laparoscopes , and needles which enable non - invasive or minimally - invasive internal body imaging . measurements are performed using a michelson interferometer with a low coherence length ( broadband ) light source . one arm of the interferometer illuminates the light on the tissue and collects the backscattered light ( typically referred to as sample arm ) . another arm of the interferometer has a reference path delay which is scanned as a function of time ( typically referred to as reference arm ) . optical interference between the light from the sample and reference arms occurs only when the optical delays match to within the coherence length of the light source . schematics of ( a ) time - domain , ( b ) spectral / fourier - domain , and ( c ) swept - source / fourier - domain oct systems . ( e ) a clinical vascular oct system and the fiber optic probe from lightlab imaging ( now st . jude medical , inc . ) . alternatively , oct interference signals can be detected in frequency or fourier domain . in fourier - domain oct , the reference mirror position is fixed , and echoes of light are obtained by fourier transforming the interference spectrum . these techniques are somewhat analogous to fourier transform spectroscopy and have a significant sensitivity and speed advantage compared with time - domain oct because they measure the optical echo signals from different depths along the entire axial scan simultaneously rather than sequentially . fourier - domain detection enables 10100 folds improvement in detection sensitivity and speed over the time - domain configuration . these advances greatly improve the performance of oct , enabling three - dimensional oct ( 3d - oct ) imaging in vivo . fourier - domain oct can be performed using two complementary techniques , known as spectral / fourier - domain oct and swept - source / fourier - domain oct ( ss - oct , also known as optical frequency domain imaging , ofdi ) . spectral / fourier - domain detection uses a spectrometer and a high speed line scan camera to measure the interference spectrum in parallel ( see fig . in contrast , swept - source / fourier - domain oct uses a frequency - swept laser light source and a photodetector to measure the interference spectrum ( see fig . three - dimensional imaging of biological tissue in vivo enabled by fourier - domain oct promises to have a powerful impact in disease diagnosis and therapy monitoring . up to date , many clinical applications using oct have demonstrated in a diverse set of medical and surgical specialties . several commercially - available devices have received us food and drug administration ( fda ) clearance to be sold in the market , such as imalux corporation ( fig . 1d ) whose oct system is based on time - domain mechanism for endoscopic imaging , and lightlab imaging ( now part of st . 1e ) that adapts frequency - domain mechanism for their oct system in cardiovascular imaging . to image internal organs , miniaturized catheter / endoscope imaging devices have been developed for intraluminal and intravascular imaging . other imaging devices such as laparoscopes and needle imaging device have been developed to enable solid organ imaging . development of such devices facilitates the translation of oct to clinical applications and allows clinicians to use the enhanced imaging capabilities of this technique to benefit the patients . figure 2a shows the schematic of a representative oct catheter / endoscope device consisting of a hollow cable carrying a single - mode ( sm ) optical fiber . the beam from the distal end of the fiber is focused by a gradient - index ( grin ) microlens and is directed perpendicular to the catheter axis by a microprism or micromirror . the beam can be scanned either circumferentially ( by rotating the cable ) or linearly ( by translating the cable ) to form a cross - sectional oct image . the outer diameter of the catheter / endoscope can be made small enough to image inside a human coronary artery ( see figure 2b ) . jude medical , inc . ) combined with a modified vacuum - pumped biopsy needle . this modified core - needle biopsy device includes the addition of a transparent front window for real - time oct guidance , the addition of a long steel / plastic tube through which the oct catheter is inserted , and a y - valve to allow both linear access for the oct catheter and the vacuum / pressure tube connection . figure 2d depicts a custom laparoscopic oct device imaging the ovaries in patients undergoing oophorectomy . ( a ) schematic of the distal end of an oct probe . ( b ) photograph of an intravascular imaging catheter ( 0.4 mm in diameter ) . ( c ) schematic of a modified core - needle biopsy device with a catheter - based oct probe ( figures are adapted from reference with permission ) and photographs of modified tip . ( d ) the photograph of a custom laparoscopic oct probe for imaging human ovary . figures are adapted from reference 21 with permission . since its invention in 1991 , oct has rapidly developed as a non - invasive biomedical imaging modality that enables cross - sectional visualization of tissue microstructures in vivo . the resolution of oct is one to two orders of magnitude higher than conventional ultrasound , approaching that of histopathology , thereby allowing architectural morphology to be visualized in situ and in real - time . oct enables imaging of structures in which biopsy would be hazardous or impossible , and promise to reduce the sampling errors associated with excisional biopsy . oct has been translated from bench to various clinical applications including ophthalmology , cardiology , gastroenterology , dermatology , dentistry , urology , gynecology , among others . the most developed clinical oct applications are those focusing on ophthalmic , cardiovascular , and oncologic imaging . for the application in oncology , many cancers arise from the epithelial layers , and demonstrate disruption of normal architectural morphology of tissues . the resolution and imaging field - of - view of oct is approaching those of standard biopsy and histopathology , therefore oct represents a potential method for optical biopsy of the tissue in situ , which can guide the excision biopsy to improve the sampling accuracy . oct has shown promises in detecting structural alterations associated with malignancies including those arising in the breast , bladder , brain , gastrointestinal , respiratory and reproductive tracts , skin , larynx , and oral cavity . clinical applications of oct in ophthalmology , cardiology , and gastroenterology have been reviewed extensively elsewhere . in this review , we focus on clinical oct applications in urology , particularly in bladder , ureter , and kidney . bladder cancer originates in the urothelium and is curable if diagnosed and treated early , but has a high mortality rate in advanced stages . the other problem is its high recurrence rate resulting in lifelong follow - up and possible repeated treatments , which make bladder cancer one of the most expensive cancers to manage . currently , white light cystoscopy ( wlc ) is the standard for initial bladder cancer diagnosis with several shortcomings such as flat carcinoma in situ ( cis ) is difficult to visualize . oct and several other optical imaging techniques ( such as fluorescence imaging ) have been developed to better identify and characterize bladder lesions beyond what is possible with standard wlc . over the last decade , both ex vivo and in vivo studies have been conducted on the ability of oct to detect bladder cancer by resolving the changes of bladder wall layers in urothelium , lamina propria , and muscularis propria and/or the corresponding backscattering . a 32 patient study showed that oct has high detection accuracy for real - time imaging and staging of bladder cancer adjunct to wlc ( 90% sensitivity and 89% specificity for tumor confined to the mucosa , and 100% sensitivity and 90% specificity for muscle - invasive tumors ) . another clinical study based on oct imaging with 24 patients reported an overall sensitivity of 100% , specificity of 89% , and diagnostic accuracy of 92% for superficial bladder transitional - cell carcinoma ( tcc ) . a 56 patient study showed that the overall specificity of cystoscopic oct ( 81% ) was comparable to voided cytology ( 88.9% , p = 0.49 ) , but significantly higher than wlc ( 62.5% , p = 0.02 ) in tcc diagnosis . figure 3 illustrates in vivo wlc , oct , and h&e images of normal human bladder ( fig . tcc exhibited enhanced urothelial heterogeneity as indicated by the arrows shown in figure 3e . furthermore , the same work also demonstrated better tumor margin detection using oct to guide transurethral resection ( tur ) , which is commonly used for non - muscle - invasive bladder cancer such as tcc that attributes to approximately 75% of all bladder cancer , and to enhance re - tur cases where the scar or necrosis induced by previous tur may make it difficult to identify residual or recurrent tumors by wlc . figure 4 shows in vivo wlc , oct , and h&e images of tcc post - tur ( fig . it demonstrated that oct image can differentiate recurrent tcc from scar or necrosis ( fig . cis has low diagnostic sensitivity and specificity ( e.g. , 3060% ) under routine wlc and remains a critical clinical problem . its oct image showed characteristics including no obvious urothelial thickening , slightly decreased backscattering in urothelium , and drastically diminished backscattering in lamina propria layer ( fig . finally , zagaynova et al . evaluated 28 cases with oct during tur to discriminate between muscle - invasive and non - muscle - invasive tumors with a sensitivity of 100% and specificity of 77% . figure 3 . in vivo surface ( a and d ) , cross - sectional oct ( b and e ) , and h&e - stained histologic images ( c and f ) of normal human bladder ( a c ) vs a papillary tcc ( d f ) . the morphologic details of normal bladder ( u , urothelium ; lp , lamina propria ; m , upper muscularis ) were clearly delineated by oct , but those of papillary tcc diminished . solid arrows : subsurface blood vessels ; dashed arrows : papillary features ; dashed circle : tcc , identified by oct based on increased urothelial heterogeneity ; dashed line : boundary with adjacent normal bladder . figures and captions are adapted from reference 107 with permission . in vivo surface ( a and d ) , cross - sectional oct ( b and e ) , and h&e - stained histologic images ( c and f ) of a recurrent tcc post - tur bladder tumor ( a c ) and a cis ( d f ) . oct differentiation of tcc ( left circle ) vs scar was based on low - scattering and papillary features in tcc vs ultrahigh superficial scattering with abruptly diminished underlying architecture in scar or necrotic lesion , which was nonspecific under surface image ( a ) . arrows in ( e and f ) : blood vessels . the morphology ( e.g. , lamina propria [ lp ] and muscularis [ m ] ) under cis ( u * ) diminished . cis was low backscattering and identified by oct based on increased urothelial heterogeneity and less distinguishable u - lp interface . ppv , positive predictive value ; npv , negative predictive value . the number of fresh human bladder tissue samples . computer - aided recognition of bladder cancer using oct and texture analysis is under investigation to improve the clinical utility of oct . higher oct axial resolution demonstrated the ability to differentiate healthy urothelial tissue , cis , and tcc from 142 fresh human bladder tissue samples . the reported sensitivity and specificity to detect malignant bladder are 83.8% and 78.1% , respectively . recently , real - time 3d - oct imaging was demonstrated in 3 clinical cases with bladder / ureter carcinoma to show the contrast of muscle - invasive carcinoma area , the scar tissue area from normal bladder wall , and ureter with three distinguishable layers , including the urothelium , lamina propria , and muscularis layer . one is false - positives that may be induced by scarring or inflammation of the mucosa . the other limitation is the limited field - of - view ( fov ) in both lateral and depth directions . oct was compared with high - resolution ultrasound ( i.e. , 40 mhz high frequency ultrasound , hfus ) in a rat bladder cancer model . results showed that oct could differentiate inflammatory lesions and tcc based on characterization of urothelial thickening and enhanced backscattering or heterogeneity , which hfus failed due to insufficient image resolution and contrast . on the other hand , hfus was able to stage large t2 tumors that oct failed due to limited imaging depth . multimodality cystoscopy combining oct and hfus , or the combination of oct with larger lateral fov technique such as wlc , narrow band imaging , and photodynamic diagnosis may help improve diagnosis and staging . few oct studies have been conducted in ureter , which has somewhat similar mucosal morphology as bladder that the tissue surface is covered with urothelial cells . early detection of ureteral cancer , as well as accurate tumor staging and grading , is also critical to reduce the mortality of the disease and help making the optimal treatment decisions . the staging and grading of urothelial carcinoma in ureter is challenging because the narrow caliber makes biopsy difficult and unreliable . endoscopic oct ( eoct ) is necessary to access the layer structures of the ureteral wall with sufficient resolution to stage early ureteral cancer . several ex - vivo studies in porcine ureter have demonstrated to clearly distinguish anatomical layers particularly the urothelium and lamina propria layers with better differentiation ability than endoluminal ultrasound . reported the intraluminal oct identification of anatomical layers of the healthy human ureter in vivo and the results for grading and staging upper urinary track ( uut ) urothelial carcinoma using oct . they identified several unique features by oct although this study does not have enough patients to provide information on oct s sensitivity and specificity of uut diagnosis . their study demonstrated that oct can : ( 1 ) distinguish healthy tissues from tumors ; ( 2 ) differentiate invasive and non - invasive tumors ; ( 3 ) differentiate grade 2 and 3 lesions by quantifying oct backscattering attenuation and , thus , has the potential to provide intraoperative real - time histological information on stage and grade during minimally - invasive procedures . figure 5 shows representative oct images of healthy ureter with identified urothelium , lamina propria , and muscularis layers . figure 6 shows representative oct images of invasive tumor ( namely stage t3g3 urothelial carcinoma ) where distinction among anatomical layers was not possible . inset , higher magnification reveals normal ureter urothelium ( pond sign ) , lamina propria ( asterisk ) , and muscularis ( dollar sign ) . ( b ) 520-frame volumetric data set across 52 mm trajectory along probe in approximately 5.2 s , resulting in 52 mm long by 10 mm diameter total scanned cylindrical volume . figures and captions ( a and b ) cross - sectional oct images of proximal ureter show interruption ( white asterisk ) of thin dark line ( white pound sign ) suggesting invasive tumor . distinction among anatomical layers was not possible . corresponding histology reveled t3g3 urothelial carcinoma ( black arrow ) . ( c ) 3d pullback of oct built from 520 individual cross - sectional images over 5.2 cm length . oct studies in clinical kidney diseases include applications in kidney cancer and non - destructive evaluation of transplant kidney viability or acute tubular necrosis ( atn ) . barwari et al . conducted both an ex vivo study with 14 patients and an in vivo study with 16 cases . they demonstrated the capability of oct to distinguish normal renal parenchyma from malignant renal tumors based on the backscattering properties . both studies measured higher backscattering property in malignant tumors ( measured from the surface or measured directly in the internal tumors ) than normal parenchyma . the averaged backscattering value of three benign tumors reported in the in vivo study is between the value from normal and malignant tumor but it did not show significant difference from that of normal renal parenchyma and tumors . linehan et al . imaged fresh surgical resected tissues of normal renal parenchyma and neoplasm using a laboratory oct system with lateral resolution of 10 m and axial resolution of 4 m . however , higher resolution oct is necessary to distinguish clear - cell tumors and other renal carcinoma subtypes from normal parenchyma and between carcinoma subtype themselves , which had a heterogeneous appearance on oct . figure 7 shows oct image and corresponding light microscopy of renal carcinoma , chromophobe subtype ( top panel ) and papillary subtype , grade 4 ( bottom panel ) . some defining features such as collections of large polygonal cells arranged in trabeculae in chromophobe renal carcinoma and elements of cuboidal cells surrounding a fibrovascular stalk in papillary renal carcinoma were not clearly evident on corresponding oct images . figure 7 . oct image and corresponding light microscopy of renal carcinoma , chromophobe subtype ( top panel ) and papillary subtype , grade 4 ( bottom panel ) . in the chromophobe subtype ( top panel ) , collections of large polygonal cells arranged in trabeculae are seen as areas of intermediate brightness with intervening dark spaces on oct . in the papillary subtype ( bottom panel ) , elements of cuboidal cells surrounding a fibrovascular stalk were seen on light microscopy but not visible on the oct image . bars are 500 m . figures and captions are adapted from reference 122 with permission . acute tubular necrosis ( atn ) is the most common insult to donor kidneys destined for transplantation . atn is caused by a lack of oxygen to the kidney ( ischemia of the kidneys ) , and is one of the most common causes of kidney failure . both ex vivo , and in vivo studies demonstrated the capability of oct to visualize kidney parenchyma morphology and function ( i.e. , tubular morphology , blood flow from vessels and glomeruli ) that provide information to kidney ischemic damage . figure 8 shows the hand - held oct imaging device used in the operating room ( fig . c ) . figure 8d depicts representative in vivo kidney oct images after kidney transplant showing cross - sectional profiles of superficial proximal tubules below the renal capsules . the openness of tubule lumens labeled in figure 8d reflects a functioning post - transplanted kidney . figure 8e shows the combination of morphological imaging with oct and functional imaging with doct for one patient that displayed good tubular morphology and blood flow . fairly densely packed uriniferous tubules are observed with several cortical blood vessels indicating re - perfusion . finally , video s1 shows combined oct and doct real - time images of the living kidney following its transplant as would be seen while imaging the kidney in the operation room . ( a ) transplant surgeons used the sterilized hand - held oct probe shown in ( b ) to image a transplanted human donor kidney in the operating room . ( c ) the oct imaging probe covered with transparent tegaderm ( small arrow ) . the cords leading to the probe are covered with a sterile camera sleeve ( large arrow ) . ( d ) in vivo oct imaging of human kidneys following transplantation showing open uriniferous tubules below the renal capsule . tubules appear to be fairly open and round with some degree of homogeneity throughout the images . open tubules appear round and relatively uniform across all images . also , a larger blood vessel is seen . oct is a powerful medical imaging technology that can reveal microstructure and blood flow in biological tissues in a non - invasive fashion and in real - time . current technology improvements enable 3d - oct imaging in real - time , thereby dramatically reducing the motion artifacts during image acquisition when accurate quantification of oct / doct image is essential for disease diagnosis and decision making . in addition , higher resolution might also help to enhance the classification of imaging parameters for disease diagnosis . with continued technology development and clinical translation , oct promises to enhance current clinical practice in urology .

since optical coherence tomography ( oct ) was first demonstrated in 1991 , it has advanced significantly in technical aspects such as imaging speed and resolution , and has been clinically demonstrated in a diverse set of medical and surgical applications , including ophthalmology , cardiology , gastroenterology , dermatology , oncology , among others . this work reviews current clinical applications in urology , particularly in bladder , urether , and kidney . clinical applications in bladder and urether mainly focus on cancer detection and staging based on tissue morphology , image contrast , and oct backscattering . the application in kidney includes kidney cancer detection based on oct backscattering attenuation and non - destructive evaluation of transplant kidney viability or acute tubular necrosis based on both tissue morphology from oct images and function from doppler oct ( doct ) images . oct holds the promise to positively impact the future clinical practices in urology .

Principle and Instrumentation of OCT Clinical Applications of OCT Bladder Ureter Kidney Summary Supplementary Material Disclosure of Potential Conflicts of Interest

seventy patients of ages 7 months to 11 years were included , of whom 59 ( 84% ) were < 5 years of age . during the study , of these , one died within 15 min of admission , and two died within the first 24 h after admission . one patient of age 2.5 years had severe neurologic sequelae 28 days after discharge , comprising spastic hemiparesis , blindness , and hearing impairment . at admission this child had a count of 880,205 parasites/l and plasma plasmodium falciparum histidine - rich protein-2 concentration of 1,875 ng / ml , both indicating an extremely heavy parasite burden . at the 3-month follow - up , demographic , clinical , and laboratory characteristics of the study population are described in table 1 . severe prostration , severe acidosis , convulsions , and severe anemia were the most common severity criteria . eleven patients ( 16% ) presented with decompensated or compensated shock . in addition , three patients ( 4.3% ) had blood culture proven septicemia ( unspeciated gram - negative rods , klebsiella pneumoniae , or staphylococcus aureus ) ; none of these presented with shock . pretreatment with an oral antimalarial was reported with respect to 31 patients ( 6 with quinine , 3 with amodiaquine , 13 with sulfadoxine - pyrimethamine , 8 with artemether - lumefantrine , and 1 with amodiaquine followed by artemether - lumefantrine ) . quinine within the 24 h preceding admission , the median ( range ) total dose being 16.1 mg / kg ( 10.153.7 mg / kg ) . hypoglycemia was corrected with a 10% dextrose bolus at the time of admission and in those who developed hypoglycemia after admission ( eight patients ) . peripheral blood asexual parasite counts after 24 h were negative in 12/66 ( 18% ) patients ( 24-h slide was not available for four patients , three of them as a result of death ) . the geometric mean ( 95% confidence interval ( ci ) ) parasite count after 24 h was 1,128 ( 5372,368 ) parasites/l in the rest of the study population ( n = 54 ) . the overall geometric mean ( 95% ci ) fractional reduction in parasite counts at 24 h was 96% ( 9498% ) . a total of 274 ars and dha postdose samples , randomly distributed over the first 12 h of the study , were included in the model . a one - compartment disposition model for both ars and dha was adequate to describe the observed plasma concentration time data ( table 2 ) . all combinations of two - compartment disposition models displayed significant model misspecification despite significantly lower objective function values . zero - order distribution / absorption from the injection site(s ) to the central compartment provided the best description of the data , but too few samples were collected during the absorption phase and the absorption rate was therefore fixed for an accurate estimation . interindividual variability could be estimated reliably for ars and dha clearance and ars volume of distribution , showing correlation between ars clearance and volume . when body weight was used as a fixed allometric function on all elimination clearance ( power of 0.75 ) and apparent volume of distribution ( power of 1 ) parameters , a significantly better model fit was observed ( objective function value = 14.6 ) . dha clearance increased 10.2% per unit ( g / dl ) of decrease of hemoglobin . interindividual variability in clearance decreased from 63.6% coefficient of variation to 55.3% coefficient of variation when this covariate was included , suggesting that it accounts for a limited but significant part of the observed variability . the final model described the observed data well , with adequate goodness - of - fit diagnostics ( figure 1 ) ; eta - shrinkage : elimination clearance for ars 10.6% , central volume of distribution for ars 12.2% , elimination clearance for dha 6.81 ; epsilon - shrinkage : 49.4 and 22.5% for ars and dha , respectively ) . a prediction - corrected visual predictive check of the final model resulted in no model misspecification and good simulation properties ( figure 2 ) . the numerical predictive check ( n = 2,000 ) for ars resulted in 4.35% ( 95% ci 1.459.42 ) and 5.07% ( 95% ci 1.459.42 ) of observations above and below the 90% prediction interval , respectively ; for dha , these values were 2.97% ( 95% ci 1.799.52 ) and 5.95% ( 95% ci 1.798.92 ) , respectively . ars and dha exposures after the standard 2.4 mg / kg dose were simulated at each body weight level ( 1,000 simulations each at 1-kg intervals from 6 to 25 kg ) using a uniform distribution of hemoglobin concentrations within the observed range ( 2.7213.6 g / dl ) to account for the observed covariate relationship ( figure 3a , b ) . children with body weights between 6 and 10 kg showed a mean reduction of 20.4% ( p < 0.0001 ) in dha exposure as compared with children with body weights between 21 and 25 kg ( median ( 25th to 75th percentile ) exposure : 3,380 ( 2,1305,470 ) ng h / ml in the 610 kg patients , 3,780 ( 2,4306,060 ) ng h / ml in the 1115 kg patients , 4,100 ( 2,5706,590 ) ng h / ml in the 1620 kg patients , and 4,240 ( 2,7006,840 ) ng h / ml in the 2125 kg patients ) . this suggests that smaller children need higher doses of ars to attain ars exposures similar to those in children with higher body weights . using the final model , we evaluated various dosing regimens based on body weight bands . the proposed regimen ( table 3 ) resulted in similar exposures in all weight bands after the first dose of i.m . ars ( figure 3c , d ) . the same simulations were performed assuming a uniform distribution of body weights at different levels of hemoglobin ; these resulted in lower dha exposures with decreasing hemoglobin levels ( figure 4 ) . there was no statistical difference between survivors and nonsurvivors with respect to total exposure ( ars p = 0.8060 , dha p = 0.4828 ) or maximum concentration ( ars p = 0.7655 , dha p = 0.6865 ) after the first dose . similarly , an exposure response relationship could not be established using nonlinear mixed - effects modeling ( nonmem ) in a time - to - event approach . however , this might be a consequence of the relatively low number of deaths and the high proportion of pretreatments with different antimalarial drugs , doses , and administration routes . seventy patients of ages 7 months to 11 years were included , of whom 59 ( 84% ) were < 5 years of age . during the study , of these , one died within 15 min of admission , and two died within the first 24 h after admission . one patient of age 2.5 years had severe neurologic sequelae 28 days after discharge , comprising spastic hemiparesis , blindness , and hearing impairment . at admission this child had a count of 880,205 parasites/l and plasma plasmodium falciparum histidine - rich protein-2 concentration of 1,875 ng / ml , both indicating an extremely heavy parasite burden . at the 3-month follow - up , demographic , clinical , and laboratory characteristics of the study population are described in table 1 . severe prostration , severe acidosis , convulsions , and severe anemia were the most common severity criteria . eleven patients ( 16% ) presented with decompensated or compensated shock . in addition , three patients ( 4.3% ) had blood culture proven septicemia ( unspeciated gram - negative rods , klebsiella pneumoniae , or staphylococcus aureus ) ; none of these presented with shock . pretreatment with an oral antimalarial was reported with respect to 31 patients ( 6 with quinine , 3 with amodiaquine , 13 with sulfadoxine - pyrimethamine , 8 with artemether - lumefantrine , and 1 with amodiaquine followed by artemether - lumefantrine ) . quinine within the 24 h preceding admission , the median ( range ) total dose being 16.1 mg / kg ( 10.153.7 mg / kg ) . hypoglycemia was corrected with a 10% dextrose bolus at the time of admission and in those who developed hypoglycemia after admission ( eight patients ) . peripheral blood asexual parasite counts after 24 h were negative in 12/66 ( 18% ) patients ( 24-h slide was not available for four patients , three of them as a result of death ) . the geometric mean ( 95% confidence interval ( ci ) ) parasite count after 24 h was 1,128 ( 5372,368 ) parasites/l in the rest of the study population ( n = 54 ) . the overall geometric mean ( 95% ci ) fractional reduction in parasite counts at 24 h was 96% ( 9498% ) . a total of 274 ars and dha postdose samples , randomly distributed over the first 12 h of the study , were included in the model . a one - compartment disposition model for both ars and dha was adequate to describe the observed plasma concentration time data ( table 2 ) . all combinations of two - compartment disposition models displayed significant model misspecification despite significantly lower objective function values . zero - order distribution / absorption from the injection site(s ) to the central compartment provided the best description of the data , but too few samples were collected during the absorption phase and the absorption rate was therefore fixed for an accurate estimation . interindividual variability could be estimated reliably for ars and dha clearance and ars volume of distribution , showing correlation between ars clearance and volume . when body weight was used as a fixed allometric function on all elimination clearance ( power of 0.75 ) and apparent volume of distribution ( power of 1 ) parameters , a significantly better model fit was observed ( objective function value = 14.6 ) . dha clearance increased 10.2% per unit ( g / dl ) of decrease of hemoglobin . interindividual variability in clearance decreased from 63.6% coefficient of variation to 55.3% coefficient of variation when this covariate was included , suggesting that it accounts for a limited but significant part of the observed variability . the final model described the observed data well , with adequate goodness - of - fit diagnostics ( figure 1 ) ; eta - shrinkage : elimination clearance for ars 10.6% , central volume of distribution for ars 12.2% , elimination clearance for dha 6.81 ; epsilon - shrinkage : 49.4 and 22.5% for ars and dha , respectively ) . a prediction - corrected visual predictive check of the final model resulted in no model misspecification and good simulation properties ( figure 2 ) . the numerical predictive check ( n = 2,000 ) for ars resulted in 4.35% ( 95% ci 1.459.42 ) and 5.07% ( 95% ci 1.459.42 ) of observations above and below the 90% prediction interval , respectively ; for dha , these values were 2.97% ( 95% ci 1.799.52 ) and 5.95% ( 95% ci 1.798.92 ) , respectively . ars and dha exposures after the standard 2.4 mg / kg dose were simulated at each body weight level ( 1,000 simulations each at 1-kg intervals from 6 to 25 kg ) using a uniform distribution of hemoglobin concentrations within the observed range ( 2.7213.6 g / dl ) to account for the observed covariate relationship ( figure 3a , b ) . children with body weights between 6 and 10 kg showed a mean reduction of 20.4% ( p < 0.0001 ) in dha exposure as compared with children with body weights between 21 and 25 kg ( median ( 25th to 75th percentile ) exposure : 3,380 ( 2,1305,470 ) ng h / ml in the 610 kg patients , 3,780 ( 2,4306,060 ) ng h / ml in the 1115 kg patients , 4,100 ( 2,5706,590 ) ng h / ml in the 1620 kg patients , and 4,240 ( 2,7006,840 ) ng h / ml in the 2125 kg patients ) . this suggests that smaller children need higher doses of ars to attain ars exposures similar to those in children with higher body weights . using the final model , we evaluated various dosing regimens based on body weight bands . the proposed regimen ( table 3 ) resulted in similar exposures in all weight bands after the first dose of i.m . ars ( figure 3c , d ) . the same simulations were performed assuming a uniform distribution of body weights at different levels of hemoglobin ; these resulted in lower dha exposures with decreasing hemoglobin levels ( figure 4 ) . there was no statistical difference between survivors and nonsurvivors with respect to total exposure ( ars p = 0.8060 , dha p = 0.4828 ) or maximum concentration ( ars p = 0.7655 , dha p = 0.6865 ) after the first dose . similarly , an exposure response relationship could not be established using nonlinear mixed - effects modeling ( nonmem ) in a time - to - event approach . however , this might be a consequence of the relatively low number of deaths and the high proportion of pretreatments with different antimalarial drugs , doses , and administration routes . optimal treatment strategies depend on detailed knowledge of the pharmacokinetic properties of drugs in the target population in which the drug is used . age , disease status , and severity are all factors that may affect drug absorption , distribution , metabolism , and elimination . the importance of pharmacokinetics in determining the therapeutic response is illustrated by the study of artemether , the first parenteral artemisinin derivative that was compared with quinine for effectiveness in the treatment of severe malaria in large clinical trials . in a meta - analysis of randomized trials in severe malaria , artemether significantly reduced mortality in southeast asian adults but did not do so in african children . subsequent pharmacokinetic studies showed that the oil - based artemether was released slowly and erratically from the i.m . injection site , and that this likely counterbalanced its pharmacodynamic advantages relative to quinine . despite having pharmacodynamic properties similar to those of artemether , ars is superior to artemether as a treatment because of its more favorable pharmacokinetic properties . dosing regimens for children are often derived from studies in adults , and this practice has led to substantial underdosing of several antimalarials in children . a pharmacokinetic study of sulfadoxine - pyrimethamine in african children with uncomplicated falciparum malaria showed that , with the usual dose of 25/1.25 mg / kg , the area under the concentration time curve in children 25 years of age was half that in adults . this may have caused failure of antimalarial treatment in small children , thereby contributing to the spread of resistance . it has also been shown that piperaquine exposure levels are lower in small children if a standard body weight - based dose regimen is followed . only limited data are available on the pharmacokinetics of parenteral , rectal , and oral ars in children . ars was converted rapidly into dha , the ars elimination half - life being ~26 min . this is in agreement with ars half - life values reported in other pharmacokinetic studies of i.m . this is because the elimination rate of ars is limited by the rate of absorption from the i.m . injection site ( i.e. , flip - flop pharmacokinetics ) . the volume of distribution values , maximum concentration values , and area under the concentration time curves of ars and dha are also comparable with the findings from a previous small and densely sampled pharmacokinetic study of i.m . time curve of dha in this study was considerably lower than that reported in adult patients and healthy volunteers after i.v . administration , this is still far greater than the in vitro defined dha concentration value of 2.28 ng / ml that is required for 99% inhibition ( ic ) . injection ( ~90% ) , fast absorption , and comparable estimates for ars and dha exposure support the use of i.m . our study had only a relatively small number of patients , thereby limiting its power to detect covariate relationships . a parsimonious approach ( p < 0.001 for a covariate to be retained in the model ) was applied in order to avoid artifactual covariate relationships . this has also been reported in other population pharmacokinetic studies of oral and rectal ars in pediatric and mixed adult pediatric populations . in general , physiological processes do not scale linearly with body weight ; consequently children with lower body weights will have higher body weight normalized elimination clearance values . dosing simulations using an exact dose of 2.4 mg / kg resulted in lower ars and dha exposures in small children as compared with those with body weights of 25 kg , suggesting the need for higher dosing in small children . in addition , hemoglobin concentration was also a significant covariate , resulting in lower dha exposure in more anemic children . severe anemia is a common presenting feature of severe malaria in young children . in severe malarial anemia there is usually substantial intravascular hemolysis , and this is also the cause of the degradation of ars in hemolyzed plasma samples ex vivo . hemoglobin concentration was found to be associated with reduced exposure levels of ars and dha , independent of body weight . this supports the idea that young children with severe malaria , who are generally more anemic than older children , should receive adjusted higher doses . underdosing in young children with severe malaria may have immediate adverse consequences with respect to outcomes . a recent study carried out in cambodia has shown that oral ars at a dose of 6 mg / kg / day for 7 days resulted in a reduction in neutrophil counts ( to < 1.0 10/l ) and short - term neutropenia in 19% of the patients . because oral ars has a bioavailability of ~80% , this corresponds to a total parenteral dose of 33.6 mg / kg , which would be reached only if parenteral treatment were continued for 14 doses . in the aquamat trial , the median ( interquartile range ) number of doses of parenteral treatment in surviving children was 3 ( 24 ) doses . ars is currently the first - choice treatment for severe malaria , and a product produced according to good manufacturing practices , which is who prequalified and available . to facilitate implementation of an optimized dosing regimen in the treatment of severe malaria in african children , we defined a simplified weight - band based dosing regimen based on the current population pharmacokinetics model , taking into account accuracy and practicality issues . we considered 0.5 ml to be the minimum volume of prepared ars solution that can be measured accurately and administered with commonly available types of syringes . because the weight bands are smaller for children with body weights < 14 kg , we propose an incremental ars dose increase of 5 instead of 10 mg and a dilution of 10 mg / ml for i.m . administration in this group , similar to the dosage regimen in the current study . in children with body weights of < 14 kg , doses should be split and administered in both thighs if injection volumes exceed 2 ml . in children of body weights 14 kg , larger injection volumes can be avoided by using a dilution of 20 mg / ml . binning of weight bands was also chosen in accordance with the currently available vial , which contains a dose of 60 mg , demarcating the upper limits of weight bands at 25 kg . the dosing recommendations we propose do not extend beyond the weight ranges of the children included in this study . no children with body weight < 6.5 kg were included ; therefore more studies of ars population pharmacokinetics are needed to evaluate dosing in these very small children and to support the current dosing recommendations . more extensive sampling in the first 15 min and during the first 12 h after the dose could give more information than the current study provided . in conclusion , ars and dha exposures were lower in small children after i.m . administration of ars in severe malaria , warranting dose adaptation in this group . this pharmacokinetic assessment of ars was part of the aquamat trial ( registration number isrctn 50258054 ) , a large multinational trial for which the results have been published elsewhere . this substudy was conducted at teule hospital in muheza , tanzania , from may 2009 to july 2010 . except for the additional blood sampling , ethical approval was obtained from the tanzania medical research coordinating committee and the oxford tropical research ethics committee . children 14 years with a clinical diagnosis of severe malaria confirmed by plasmodium lactate dehydrogenase ( pldh)-based rapid diagnostic test ( optimal , diamed , cressier , switzerland ) were recruited , and written informed consent was obtained from the respective parents or caregivers . severe malaria was defined as the presence of at least one of the following : coma ( glasgow coma score 10 or blantyre coma score 2 in preverbal children ) , convulsions ( duration > 30 min or 2 episodes in the 24 h preceding admission ) , respiratory distress ( nasal alar flaring , costal indrawing / recession or use of accessory muscles , severe tachypnea ) or acidotic breathing ( deep breathing ) , shock ( capillary refill time 3 sec and/or temperature gradient and/or systolic blood pressure < 70 mm hg ) , severe symptomatic anemia ( < 5 g / dl with respiratory distress ) , hypoglycemia ( <3 mmol / l ) , hemoglobinuria , severe jaundice , or , in older children , a convincing history of anuria or oliguria . patients who had received full treatment with parenteral quinine or a parenteral artemisinin derivative > 24 h before admission were excluded . physical examination was carried out at admission , and a venous blood sample was taken for peripheral blood parasite count , quantitative assessment of plasma plasmodium falciparum histidine - rich protein-2 ( a measure of total body parasite burden ) , hiv serology ( sd bio - line hiv 1/2 3.0 ; standard diagnostics , kyonggi - do , korea / determine hiv-1/2 , abbott laboratories , il ) , blood culture , liver function tests ( aspartate aminotransferase , alanine transaminase , -glutamyltransferase , total bilirubin , creatinine , and urea , by reflotron , roche diagnostics , basel , switzerland ) , hematocrit , biochemistry , and acid base parameters ( ec8 + cartridge for i - stat handheld blood analyzer , abbott laboratories , abbott park , il ) . hematocrit was reported from the i - stat reading or , when not available , measured by hemocue ( hemocue ab , ngelholm , sweden ) ( n = 5 ) . a neurologic examination was conducted at discharge , and repeated at day 28 in children who had not made a full neurologic recovery at discharge . ars ( guilin pharmaceutical factory , guangxi , china ) was administered as an i.m . injection ( 2.4 mg / kg ) shortly after admission , again at 12 h and 24 h , and daily thereafter . the contents of each 60 mg vial of ars were dissolved in 1 ml 5% sodium bicarbonate ( provided with the drug ) and further diluted with 5 ml 5% dextrose ( final concentration of 10 mg / ml ) before administration as a deep i.m . injection into the anterolateral thigh . dosing was based on the measured body weight of the patient , and injection volumes of > 23 ml were split and divided into two injections , one in each thigh . when the patient was well enough to take oral medication , but after a minimum of 24 h ( two doses of i.m . ars ) , a full 3-day course of oral artemether - lumefantrine ( co - artem ; novartis , basel , switzerland ) was given to complete the treatment . vital signs and glucose were monitored at least every 6 h and also at any sign of deterioration in clinical condition . a majority of the patients ( i.e. , other than those who were able to be orally fed ) received an infusion with dextrose 5% . blood transfusion ( 20 ml / kg ) was given to children with hemoglobin concentrations of < 5 g / dl . blood samples ( 1.5 ml ) were drawn from an indwelling catheter into prechilled fluoride oxalate tubes for ars and dha quantification before the first dose ( at baseline ) . four subsequent samples were taken from each patient at preset random time points within the following time windows : 01 , 14 , 412 , and 1224 h after the first dose . randomization of sampling times was done by computer - generated randomization ( stata version 12 ( stata , college station , tx ) . immediately after blood collection , the blood samples for drug measurements were centrifuged at 4 c at 2,000 g for 7 min . plasma samples ( 0.5 ml ) were stored at 80 c and shipped on dry ice to the moru department of clinical pharmacology , bangkok , thailand , for drug quantification . ars drug content and quality were checked in vials taken randomly from the purchase lots ( see supplementary data online ) . drug analysis . ars and dha plasma concentrations were measured using liquid chromatography tandem mass - spectrometry . quality control samples at low , middle , and high concentrations were analyzed in triplicate within each analytical batch to ensure accuracy and precision during the analysis . the lower limit of quantification was set at 1.2 ng / ml for ars and 2.0 ng / ml for dha . venous plasma concentrations were transformed into molar units and modeled as natural logarithms , using nonmem v.7 ( icon development solutions , ellicott city , md ) . ars and dha were modeled simultaneously , using a drug metabolite model with complete in vivo conversion of ars into dha ( for details , see supplementary data online ) . the first - order conditional estimation method with interaction model selection was based on the objective function values computed by nonmem , goodness - of - fit graphical analysis , and physiological plausibility . a p value of 0.05 was used in the forward step and a p value of 0.001 in the backward step to compensate for the relatively small population studied . simulation - based diagnostics ( visual and numerical predictive checks ) and bootstrap diagnostics were used to evaluate the performance of the final model . monte carlo simulations using the final model with the observed variability were performed for different body weights to obtain representative population estimates of the exposure levels during the first day of dosing ( area under the concentration time curve from time point 0 to 12 h ) after prospective dose regimens . no drug exposure target is defined for parenteral ars ; therefore , for the purpose of arriving at a practical parenteral dosing regimen , different body weight bins were evaluated to ensure similar target exposures in all weight bands in agreement with the exposure in children of body weight 25 kg . the same simulations were used to evaluate the effect of other significant covariates on drug exposure . peripheral blood smears were taken at admission and after 24 h. reduction in parasite load over 24 h , survival , and severe neurologic sequelae were evaluated as part of the pharmacodynamic analysis . the effects of ars and dha exposures on outcomes were investigated using a time - to - event analysis in nonmem , with predicted drug concentrations being used to modulate the hazard function in a traditional maximum effect ( emax ) relationship .

parenteral artesunate ( ars ) is the drug of choice for the treatment of severe malaria . pharmacokinetics data on intramuscular ars are limited with respect to the main treatment group that carries the highest mortality , namely , critically ill children with severe malaria . a population pharmacokinetic study of ars and dihydroartemisinin ( dha ) was conducted from sparse sampling in 70 tanzanian children of ages 6 months to 11 years . all the children had been admitted with severe falciparum malaria and were treated with intramuscular ars ( 2.4 mg / kg at 0 , 12 , and 24 h ) . venous plasma concentration time profiles were characterized using nonlinear mixed - effects modeling ( nonmem ) . a one - compartment disposition model accurately described first - dose population pharmacokinetics of ars and dha . body weight significantly affected clearance and apparent volume of distribution ( p < 0.001 ) , resulting in lower ars and dha exposure levels in smaller children . an adapted dosing regimen including a practical dosing table per weight band is proposed for young children based on the pharmacokinetic model .

Results Clinical details Population pharmacokinetic-pharmacodynamic analysis Discussion Methods Author Contributions Study Highlights Supplementary Material

laparoscopic pyelolithotomy was successfully performed in a pelvic kidney with an operative time of 310 minutes . the use of intraoperative fluoroscopy and a semi - automatic suturing device greatly facilitated the procedure . the patient 's operative pain was managed with 3 doses of ketorolac ; she resumed a regular diet the day after surgery , and was discharged on the first postoperative day . for patients with a large stone in the renal pelvis of an ectopic kidney , laparoscopic pyelolithotomy provides an effective approach . pelvic kidneys are typically incidental findings , but may pre - sent due to underlying obstructive or calculous disease . the anatomical characteristics of the ectopic kidney can pose a significant challenge to the treatment of calculous disease . treatment of larger renal calculi in these kidneys had been uniformly by open surgery until laparoscopically guided percutaneous nephrostolithotomy ( pcnl ) was described , initially , by esghi , et al . , and later by toth , et al . recently , 2 cases of laparoscopic pyelolithotomy have been reported in a pelvic kidney . in one , due to urine leakage , an indwelling stent and urethral catheter were maintained for 8 days . hospitalization lasted 6 days . in the second case report , intraoperative fluoroscopy and laparoscopic suturing were used to perform the procedure , but the pyelotomy closure was not tested for watertightness and the patient experienced peritoneal leakage of urine after the foley catheter was removed . this necessitated replacement of the foley catheter for an unspecified length of time and the length of hospitalization was not reported . herein we report the third case of a laparoscopic pyelolithotomy in a pelvic kidney ; in this case , use of the carterthomason needle - point suture passer ( inlet medical inc . , eden prairie , mn ) and laparoscopic suturing of the pyelotomy with an endostitch device ( auto suture , norwalk , ct ) resulted in a waterproof closure . a 63-year old woman was referred for endourological treatment of a symptomatic 2.5-cm - x-1.5 cm renal pelvis stone in her left pelvic kidney . intravenous urography ( ivu ) and abdominal / pelvic computed tomography defined the pelvic kidney 's location relative to other structures ; there was mild pyelocaliectasis noted ( figures 1 & 2 ) . ivu demonstrating ectopic right kidney and left pelvic kidney with laminated 2.5 cm stone at the ureteropelvic junction . the patient was turned to a 30-degree lateral , left side up position and a 15 mm hg co2 pneumoperitoneum was initiated with the veress needle using a lateral inflation technique . three 12 mm laparoscopic ports were placed : left lower quadrant in the midclavicular line , umbilicus , and right lower quadrant in the pararectus area . holding sutures were placed inferiorly and superiorly on the anterior surface of the renal pelvis using intracorporeal suturing ; suture delivery through the skin was accomplished via a carter - thomason needle - point suture passer ( inlet medical inc . , eden prairie , mn ) . a 3 cm long pyelotomy was made and a 10 mm biopsy forceps was used to grasp the stone , which was removed intact after the 12 mm cannula was withdrawn from the port site . a running closure of the renal pelvis was accomplished using the endostitch device with a 2 - 0 polysorb suture ( auto suture , norwalk , ct ) . the occlusion balloon catheter was changed to an indwelling 7 f double pigtail stent and a bladder catheter was left in place overnight . the total operative time was 310 minutes ( including pre- and postoperative stent placement ) . abdominal/ pelvic ct scan demonstrating left pelvic kidney with mild pyelocaliectasis and the stone at the ureteropelvic junction ( arrow at stone ) . after initiating general anesthesia , a 7 f/ 11.5 mm balloon occlusion catheter was then passed over the guidewire into the renal pelvis of the left pelvic kidney . the balloon was inflated with 1 cc of contrast material and was then snugged down at the ureteropelvic junction . postoperatively , the patient 's discomfort was managed by a total of 45 mg of intravenous ketorolac ( 3 doses ) followed by 2 doses of an oral narcotic . a follow - up ivu , one week later , revealed the left pelvic kidney to be functional , stone - free , and without extravasation . our experience ( table 1 ) is similar to the prior reports of successful laparoscopic pyelolithotomy for a large renal calculus in a pelvic kidney . placement of a retrograde ureteral catheter was instrumental in allowing expeditious localization of the renal pelvis at laparoscopy and for testing the closure of the pyelotomy . closure of the renal pelvis was facilitated by the use of holding sutures and the endostitch device . comparison of laparoscopic pyelolithotomy and pcnl for renal pelvis stones > 1.5 - 2 cm . mac= monitored anesthesia care , pcnu= percutaneous nephroureteral stent the patient experienced minimal pain and a brief hospitalization , findings consistent with previous reports of laparoscopic pyelolithotomy for a normally positioned kidney . in these reports , the operative time has averaged 2 - 5 hours using a 3- or 4-port approach . the renal pelvis was sutured closed in only one case and all patients had a surgical drain placed prior to fascial closure . while a stone - free rate of 100% was recorded among 9 patients , it is of note that 3 of those 9 patients required conversion to an open procedure . while these reports concluded that laparoscopic pyelolithotomy in a eutopic kidney was of value in a situation where shock wave lithotripsy ( swl ) or pcnl had failed or could not be done , we believe this represents a rare situation . indeed , despite a vast experience at washington university with laparoscopic renal surgery and a large surgical stone population ( e.g. an approximate annual urolithiasis case load of 500 to 600 swl , 100 to 150 ureteroscopies , and 50 to 100 pcnl ) this is the singular case of a laparoscopic pyelolithotomy at our institution . with regard to stone treatment in an ectopic or pelvic kidney , we believe the treatment strategy should be similar to that used for stones in a eutopic kidney . hence , swl or ureteroscopy remain first line therapy for smaller calculi ( less than 2 cm ) , while laparoscopic - assisted percutaneous , pure laparoscopic , or open procedures are reserved for larger calculi . from this standpoint , it is of note that 2 cases of laparoscopically guided pcnl have been reported in pelvic kidneys . the operative time was not reported for these cases and the length of hospital stay was 6 days in one report . we believe that given advances in laparoscopic equipment it may be just as simple to handle the stone removal entirely laparoscopically . the benefits of this approach would potentially include a shorter hospital stay and less postoperative morbidity as occurred in the present case . for urologists interested in laparoscopic pyelolithotomy in this situation , the use of intraoperative fluoroscopy to identify the renal pelvis , the placement of holding sutures , the use of the carter - thomason needle - point suture passer , and intracorporeal suturing with the endostitch device are recommended .

the anatomical characteristics of an ectopic kidney can pose significant challenges to the treatment of calculous disease . this report examines the feasibility of a laparoscopic approach for management of a large renal calculous in a pelvic kidney and reviews current literature regarding this subject .

Background and Objectives: Methods and Results: Conclusions: INTRODUCTION CASE REPORT DISCUSSION

bspp : bisulfite padlock probe ; charm : comprehensive high - throughput arrays for relative methylation ; cimp : cpg island methylator phenotype ; esc : embryonic stem cell ; fda : food and drug administration ; help : hpaii tiny fragment enrichment by ligation - mediated pcr ; mca : methylated cpg island amplification ; mcam : methylated cpg island amplification microarray ; medip : methylated dna immunoprecipitation ; mira : methylated - cpg island recovery assay ; mscc : methyl - sensitive cut counting ; pcr : polymerase chain reaction ; rrbs : reduced representation bisulfite sequencing . the authors are supported by the leukemia specialized program of research excellence grant p50 ca100632 .

dna methylation of promoter cpg islands is strongly associated with gene silencing and is known as a frequent cause of loss of expression of tumor suppressor genes , as well as other genes involved in tumor formation . dna methylation of driver genes is very likely outnumbered by the number of methylated passenger genes , though these can be useful as tumor markers . much of what is known about the importance of dna methylation in cancer was gained through small- and moderate - scale analysis of gene promoters and tumor samples . a much better understanding of the role of dna methylation in cancer , either as a marker of disease or as an active driver of tumorigenesis , will likely be gained from genome - wide studies of this modification in normal and malignant cells . this goal has become more attainable with the recent introduction of large - scale genome analysis methodologies and these have been modified to allow for investigation of dna methylation . several research groups have been formed to coordinate efforts and apply these methodologies to decipher the methylome of healthy and diseased tissues . in this article we review technological advances in genome - wide methylation profiling .

Abbreviations Competing interests Authors' contributions Acknowledgements

exercise is one of the most powerful non - pharmacological method of affecting cells and organs in the body1 . regular aerobic and resistance exercise training has a positive long - term impact on the cardiovascular system , which is a biologically complex adaptive system that is characterized by a variety of complex reactions to different training loads2,3,4 . while performing exercise , it is important that the body is supplied with oxygen and energy substances . this transporting function is performed by the cardiovascular system5 . the power to transport these substances to cells it has been known for long that blood flow intensification or even more accurate blood flow redistribution during exercise can increase blood supply to working muscles and consequently increase working capacity6 . nowadays , athletes and coaches are looking for the most efficient training method so as to achieve maximum results and to maintain these results for the longest possible period of time in order to minimize the probability of injury . recently , an unconventional training method has been developed that uses blood flow restriction in the musculoskeletal system , otherwise known as the kaatsu methodology . an example of this is a walking workout using blood flow restriction , which has been proven to be a useful method of improving muscle function , including muscle hypertrophy , strength , and endurance . one of the potential ways to achieve this could be training with blood flow restriction7 , 8 . training with less weight reduces stress on joints and therefore minimizes the possibility of getting injured . recently , blood flow restriction combined with low - intensity resistance exercise has been suggested as a useful exercise protocol to gain muscular strength and mass without an increase in blood pressure9 . some authors have suggested using as little as 20% of the maximum repetition weight during the training with blood restriction . as it is almost impossible to evaluate large arterial function directly , recording of an electrocardiogram ( ecg ) is one of the most practical noninvasive methods . in application of the ecg in sports physiology , st - segment depression is the key index that refers to the degree of the occurrence of myocardial ischemia . this is of special significance in adolescent or young athletes , as it can be used to recognize unphysiological states during workouts . together hereditary and congenital abnormalities of the heart are the most common causes of nontraumatic death in sport in young athletes . in middle - aged recreational athletes , more than 90% of sudden cardiac deaths occur in males , and more than 90% are caused by atherosclerotic coronary artery disease10 . such findings encourage a greater interest in noninvasive ecg methods of research and in questioning the meaning of st - segment depression at different workout and rest stages . . basically , corrected qt or jt interval ( qtc , jtc ) indices increases sudden death and other risks11 . however , some ecg parameters observed in a minority of athletes present diagnostic conundrums . the jt interval ( measured from the j - point up to the end of the t - wave ) describes the duration of ventricular repolarization , and shortening of the jt interval during physical load correlates with the increase in metabolic rate in the myocardium12 . our hypothesis states that the training gain achieved by training with the circulatory restriction applied during low - intensity exercising lies within the physiological limits of the cardiovascular system . the aim of this study was to the effect of a single occlusion training session on the cardiovascular response to bouts of exercise . the subjects in the study were amateur athletes in track and field with 46 years of training experience , and they participated in two groups : a control group without blood flow restriction and experimental group with blood flow restriction ( n=24 , mean age 22.5 1.5 years ; body mass index 24.7 0.5 kg / m ) . the weight and body mass index ( bmi ) ( tbf-300 body composition scale ; tanita , uk ltd . , west drayton , uk ) of the subjects were estimated while they were seminude ( shorts and t - shirts ) . none of the subjects exercised for at least 12 hours and before the test or ate for at least 2 hours before the test . the physical load test was carried out during the competitive period of the sports season . the study was designed to determine the effects of a single occlusion training session on the cardiovascular system . the participants underwent circulatory restriction with a 40-mm - wide cuff applied the groin13 . the participants were seated on a calf muscles trainer , and the cuff air pressure was set at 120 mmhg ( the approximate resting systolic blood pressure for each participant14 . in this study , the calf muscles ( m. gastrocnemius and m. soleus ) and the sole flexion muscle ( m. flexor digitorum brevis ) were impacted by the occlusion . as part of a coherent muscular system , the knee of the working leg was fixed at an angle of 90 , and the ankle was fixed at an angle of 70. the dynamometer was adjusted according to the foot size of the participant . maximum voluntary contraction ( mvc ) , measured in newton s ( n ) , was performed three times , and the highest value was recorded . mvc was measured only before training in order to choose the individuals training workload . arterial blood pressure ( abp ) , an important cardiovascular functional parameter , was measured using the cuff method and by listening to the korotkoff tones . a computerized analysis system kaunas - load was employed for continuous 12-lead ecg registration and analysis . changes in rr interval or heart rate ( hr ) , jt interval , st - segment depression ( sum of negative values for 12 leads ) , and in ratio of the jt / rr intervals were analysed . as shown in other studies , low - intensity resistance exercise training with occlusion ( 2050% mvc ) increases both muscle cross - sectional area and strength15 . therefore , we used a single session of training with an exercise intensity of 40% mvc . foot flexor muscle conditioning training was conducted as follows : three exercises comprising of the three sets of eight repetitions per set for each leg . a 2.5-min rest period was provided between exercises , and a 30-sec rest period was provided between sets . the participants were asked to lift the weight in time with a metronome ( 30 movement cycles per minute)16 . occlusion was applied before the exercise and was removed after each set of three exercises ( 3 sets , 3 exercises per set , and 8 repetitions per exercise ) . the arithmetic mean ( x ) , standard deviation ( s ) , and the arithmetic mean of the error ( sx ) were calculated . a two - way independent samples student s t - test was used to determine how reliable the mean difference was for performance indicator results . a significant difference between compared values this study was approved by the regional biomedical research ethics committee ( lithuanian university of health sciences , kaunas , lithuania ) ( no : be-210 , 2603 - 2015 ) . the results of the research showed that before the exercise , the average hr was 69.1 4.0 beats / min . hr showed a significant increase during exercise , increasing to 96.3 4.9 beats / min in the experimental group and to 94.1 3.8 beats / min in the control group . during recovery from the first set , hr was 84.8 5.4 beats / min in the subjects with the circulatory restriction , while the value for the other group was 82.0 3.1 beats / min . 1.changes in heart rate during exercise and at rest , the changes in the hr during the first set were the same in both groups ; however , continuation of the exercise tasks revealed that the values after each set in the control were higher than those in the experimental group . changes in heart rate during exercise and at rest before the study , there was no significant statistical difference between the groups when comparing both systolic and diastolic abp ( p<0.05 ) . in addition , we did not find any difference between groups in terms of changes in diastolic abp during exercise , and we observed a low tendency for systolic abp to increase in response to repeated sets of exercise ( table 1table 1.average increase in systolic and diastolic abp during exercise setssystolic ( mmhg)diastolic ( mmhg)set 1set 2set 3set 1set 2set 3experimental10.3 3.510.6 2.911.6 3.24.6 3.53.0 3.83.7 3.3control7.6 4.19.0 3.412.6 4.02.0 3.11.9 3.41.8 2.7 ) . at the onset of exercising , the applied occlusion had no influence on st - segment depression during exercise or at rest . jt / rr values registered before exercise during the rest and while at first set of exercises were not significantly different ; however , a tendency for a lower increase in jt / rr during the repeated exercise sets performed with partial circulatory restriction was observed ( fig . the cardiovascular system , is a vital body system and the model of human body response to exercising12 outlines it as the most important part of the supplying systems . results obtained during this study revealed that the increase in hr during the exercise was more notable in the control group compared with the experimental group . it was also determined that the application of the partial circulation restriction caused lower jt / rr ratio values in the ecg . in assessing these findings as it was shown by other authors17 , the jt / rr ratio allows depiction of the mobilization of the cardiovascular system during exercising . if we were to multiply the abovementioned ratio by 60 , it would allow us to depict the situation over time , that is , how many seconds per minute the heart ( myocardium ) was contracted and consequently how long the cardiac musculature was at rest . the data from this study indicate that cardiac muscle strain was slightly lower when the low - intensity exercise was carried out with a partial circulatory restriction . the present study found that systolic and diastolic abp were higher throughout the entire study . this corresponds to the findings of other research showing that application of occlusion increases vascular wall elasticity19 . it is important to note that vascular wall elasticity is one of the main factors that affect diastolic arterial blood pressure . the fact that certain blood vessels were clamped during the research could have also influenced the increase of diastolic abp . performing the exercised with partial circulatory restriction is not just healthier for joints ( as smaller weights are used ) ; a previous study also showed that no manifestations of thrombosis where observed when using occlusion20 . the heart reserve possibility of largely depends on whether the demand for oxygen is satisfied and on as how quickly and sufficiently oxygen is delivered to the heart during the exercise . when the myocardium is insufficiently supplied with blood there is a lack of oxygen in the myocytes . when coronary heart blood vessels are insufficiently supplied with blood , during the exercise , changes occur in the balance of metabolic processes and subsequently in electric potentials of myocytes . as a result of these processes , st - segment depression is visible in ecg records . thus assessment of the degree of ischemic episodes during exercise is also essential and indicates the functional state of the heart . partial blood flow restriction had an ambiguous effect on blood pressure changes during the exercise . systolic abp values continued to be higher during the exercise , while changes in diastolic abp were not registered . repeating the exercise increased fatigue , and the body was forced to mobilize more and more resources to perform a given exercise task . therefore , in the analysis of the research results , we assessed manifestations of variations in functional indices of the cardiovascular system , i.e. , changes of mobilization and recovery . we concluded that when a partial blood flow restriction was applied during repeated exercise , the heart mobilization and recovery features were better reflected in the jt / rr ratio than in hr . in assessment of manifestations of hr fluctuations , it was found that the amplitude of hr change in response to exercise repetitions remained the same , while the increase in mechanical work duration of the myocardium , as indicated by the jt / rr ratio , was lower , which can be interpreted as indicating that the heart could perform its pumping function with more ease . this was confirmed by the changes in jt in the ecg data , which demonstrated changes in exactly the same direction . the physiological sense of jt can be defined as follows : the jt interval in a recorded ecg is measured from the connection point j to the end of the t wave ( point j corresponds to the moment when the ecg curve after the s wave returns to isolines ) . the jt interval indicates ventricular repolarization processes and can be used as an indicator of the repolarization duration . also jt interval variation is related to changes in the intensity of myocardial metabolism21 , 22 . despite all the provided positive conclusions on the subject , we can not affirm that exercise should be performed only by applying the occlusion training method . firstly , it requires special equipment , the ability to use the special equipment , and knowledge of the application procedures . secondly , there are authors who claim that training with occlusion does not provided reliable results23 . on the other hand , in a study made by maior in 2015 , an interesting conclusion was drawn that is application of occlusion training is more effective when movement is carried out through one joint24 , 25 . thus , no unanimous conclusion can be made at present , but perhaps scientists will carry out more research in the future to determine the body s response to applying blood flow restriction with different pressure levels . in summary we conclude that applying partial blood flow restriction with occlusion training in the groin area resulted in a lower degree of hr increase in response to repeated exercise compared with training without occlusion . however , this partial blood flow restriction does not increase the myocardium load and has no significant effect on coronary vascular function . partial blood flow restriction affects the change in blood pressure during exercise in an ambiguous way . systolic abp values continue to be higher during the change in blood pressure , while diastolic abp changes are not expressed . in conclusion , this study confirmed the hypothesis that there is no additional strain on cardiac functioning during occlusion training carried out by exercising with partial blood flow restriction in the groin area . the results obtained during the study suggest that such kind of occlusion training could be applied for health training and rehabilitation purposes . occlusion training by using a low intensity of exercise triggers changes in muscular tissue similar to high - intensity exercise training . meanwhile , the cardiovascular system during exercise does not experience higher overload , which could be a risk factor for cardiac patients and physically inactive persons .

[ purpose ] occlusion training with low - intensity resistance exercises and blood flow restriction increases muscle cross - sectional area and strength . this form of training is used in rehabilitation ; therefore , the aim of this study was to examine the effect of one occlusion training session on the cardiovascular response to bouts of exercise . [ subjects and methods ] two groups took part : a control group without blood flow restriction and an experimental group with blood flow restriction . a single training session was used with the exercise intensity set at 40% of the one repetition maximum . maximum voluntary contraction , arterial blood pressure , and electrocardiogram measurements were performed . [ results ] heart rate was slightly higher in the control group . the performed training had no effect on diastolic blood pressure in either group , however , a tendency for a small systolic blood pressure increase was observed during the session in the experimental group . jt interval changes did not reveal significant differences between groups . there were no significant changes in st - segment depression during the exercise or at rest . a lower tendency for jt / rr increases was observed during the repeated exercise tasks with partial blood flow restriction . [ conclusion ] low intensity exercises carried out with a partial blood flow restriction do not result in significant overload of cardiac function .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

body language reading is of immense importance for adaptive social behavior and non - verbal communication . healthy perceivers are able to infer emotions and dispositions of others represented by point - light body movements that minimize availability of other cues ( pollick et al . , 2001 ; atkinson et al . , 2004 ; heberlein et al . , 2004 ; clarke et al . , 2005 perceivers can reliably judge emotional content of dance represented by a few moving dots placed on the dancer 's body ( dittrich et al . , 1996 ) . visual sensitivity to camouflaged point - light human locomotion is modulated by the emotional content of gait with the highest sensitivity to angry walking ( chouchourelou et al . , 2006 ) . observers can discriminate between deceptive and true intentions conveyed by body motion , and true information is precisely detected despite misleading endeavors ( runeson and frykholm , 1983 ; grzes et al . , 2004a , b ) . but how do we know whom to trust or who is attracted to us ? such judgments are vital to social interaction , and men and women appear to show profound differences in cues attended to . yet research on sex differences in visual social cognition has been mainly limited to static face images , in particular , still photographs . in accordance with widespread beliefs , females exhibit higher sensitivity to non - verbal cues : they better discriminate friendliness from sexual interest ( farris et al . , 2008 ) and are more proficient in recognition of facial emotions ( montagne et al . , 2005 ) . females without and with asperger syndrome are better at recognizing emotions from dynamic faces than males ( golan et al . , 2006 ) . moreover , females tend to better recognize emotions from faces than from voices , whereas males exhibit the opposite tendency . as a rule , however , facial expressions and static body postures can only signal emotional states and affect , but do not provide information about how to deal with it . dynamic body expressions , gestures , and actions of others are a richer and more ecologically valid source of information for social interaction ( de gelder , 2006 , 2009 ; pavlova , 2009 ) . the other important advantage of bodily expressions is that whereas face expressions ( similarly to a verbal information flow ) are believed to be easily kept under control , body movements reveal our true feelings . when emotions expressed by faces and bodies are incongruent , recognition of facial expressions is affected by emotions revealed by body ( meeren et al . , 2005 ) brain imaging indicates that emotions expressed by dynamic bodies as compared to faces elicit greater activation in a number of brain areas including the superior temporal sulcus ( sts ) , a cornerstone of the social brain ( kret et al . , 2010 ) . experimental evidence obtained primarily in patients with lesions and cortical blindness favors the assumption that emotional body language can be processed automatically , without visual awareness and attention ( for review , see tamietto and de gelder , 2010 ) . to a great surprise , however , gender impact on body language reading is largely unknown . a few studies conducted at the beginning of the 80s based on the profile of non - verbal sensitivity ( pons ) test , which includes body motion ( neck to knees ) video clips , point to the superiority of females in body language reading ( e.g. , blank et al . , 1981 ) . however , this test has some serious methodological limitations ; for example , it is based on body motion video clips of only one female actor . although sex differences represent a rather delicate topic , underestimation , or exaggeration of possible effects can retard progress in the field . the present work intends to make an initial step in filling the gap , and to clarify whether , and , if so , how perceiver 's gender affects recognition of emotional expressions conveyed by actions of others . more specifically , we ask ( i ) whether gender affects recognition of emotions represented by body motion , or , in other words , whether females excel in recognition of emotional actions ; and ( ii ) whether gender effects depend on emotional content of actions . to this end , healthy young females and males were presented with point - light displays portraying knocking at a door with different emotional expressions ( happy , neutral , and angry ) . we took advantage of a point - light technique that helps to isolate information revealed by motion from other cues ( shape , color , etc . ) . perceivers saw only a few bright dots placed on the main joints of an otherwise invisible arm ( figure 1 ) so that all other clues except for motion characteristics were abandoned . three static frames taken from the dynamic sequence representing knocking motion by a set of dots placed on the arm joints , shoulder , and head of an otherwise invisible actor . actors were seen facing right , in a sagittal view , and struck the surface directly in front of them . thirty four healthy adults , students of the university of tbingen medical school ( aged 2036 ) , were enrolled in the study . mean age of females ( 20 participants ) was 23.8 3.7 years , and mean age of males ( 14 participants ) was 22.9 2.0 years . there was no age difference between female and male participants ( t32 = 0.95 , p = 0.35 , ns ) . none had a history of neurological or psychiatric disorders including autistic spectrum disorders ( asd ) , schizophrenia , head injures , or medication for anxiety or depression . informed written consent was obtained in accordance with the requirements of the local ethical committee at the university of tbingen medical school . point - light displays were recorded during performance of knocking with different emotional content ( happy , neutral , and angry ) . we chose to use animations with happy and angry motions , because happiness and anger are reported to be quite similar on the activation dimension , and these animations tended to have fast and jerky movements ( pollick et al . , display creation is described in detail elsewhere ( pollick et al . , 2001 ) . in brief , recording was performed using a 3d position measurement system at a rate of 60 hz ( optotrak , northern digital inc . , each display consisted of six point - light dots placed on the head , shoulder , elbow , wrist , and the first and forth metacarpal joints of an otherwise invisible right hand ( figure 1 ) . point - light actors were seen facing right , in a sagittal view , and struck the surface directly in front of them . the size of all point - light knocking stimuli was standardized in such a way that in the first frame , the distance from the head to the first metacarpal joint was identical for all actors . for each emotion , six different displays with equal number of knocking performed by female and male actors were created . by using the presentation software ( neurobehavioral systems inc . , albany , ca , usa ) , each video was displayed five times per experimental session resulting in 30 trials per emotion . the whole experimental session consisted of a set of 90 displays representing three emotions in a random order , and took about 1520 min per participant . each display was shown for 1 s. we used a three alternative - forced choice paradigm . on each trial , participants indicated ( by pressing with their dominant hand one of three respective keys on a computer keyboard ) whether a display portrayed happy , neutral , or angry knocking . thirty four healthy adults , students of the university of tbingen medical school ( aged 2036 ) , were enrolled in the study . mean age of females ( 20 participants ) was 23.8 3.7 years , and mean age of males ( 14 participants ) was 22.9 2.0 years . there was no age difference between female and male participants ( t32 = 0.95 , p = 0.35 , ns ) . none had a history of neurological or psychiatric disorders including autistic spectrum disorders ( asd ) , schizophrenia , head injures , or medication for anxiety or depression . informed written consent was obtained in accordance with the requirements of the local ethical committee at the university of tbingen medical school . we used point - light displays portraying knocking arm motion ( pollick et al . , 2001 , 2002 ) . point - light displays were recorded during performance of knocking with different emotional content ( happy , neutral , and angry ) . we chose to use animations with happy and angry motions , because happiness and anger are reported to be quite similar on the activation dimension , and these animations tended to have fast and jerky movements ( pollick et al . , display creation is described in detail elsewhere ( pollick et al . , 2001 ) . in brief , recording was performed using a 3d position measurement system at a rate of 60 hz ( optotrak , northern digital inc . , waterloo , on , canada ) . each display consisted of six point - light dots placed on the head , shoulder , elbow , wrist , and the first and forth metacarpal joints of an otherwise invisible right hand ( figure 1 ) . point - light actors were seen facing right , in a sagittal view , and struck the surface directly in front of them . the size of all point - light knocking stimuli was standardized in such a way that in the first frame , the distance from the head to the first metacarpal joint was identical for all actors . for each emotion , six different displays with equal number of knocking performed by female and male actors were created . by using the presentation software ( neurobehavioral systems inc . , albany , ca , usa ) , each video was displayed five times per experimental session resulting in 30 trials per emotion . the whole experimental session consisted of a set of 90 displays representing three emotions in a random order , and took about 1520 min per participant . each display was shown for 1 s. we used a three alternative - forced choice paradigm . on each trial , participants indicated ( by pressing with their dominant hand one of three respective keys on a computer keyboard ) whether a display portrayed happy , neutral , or angry knocking . percentage correct in recognition of emotions conveyed by knocking is represented in figure 2a . in both females and males , recognition of all emotional expressions was above chance level ( p < 0.001 ) . however , recognition of happy knocking was less accurate than of neutral and angry actions . this is consistent with the outcome of previous studies on emotion recognition through point - light human locomotion ( chouchourelou et al . , 2006 ; ikeda and watanabe , 2009 ) and dance ( dittrich et al . , 1996 ) that show better recognition of angry over happy motion . recognition of happy , neutral , and angry point - light knocking by females and males . ( a ) percentage correct : males outperformed in recognition of happy knocking ( p < 0.015 ) , whereas females excelled in recognition of neutral knocking ( p < 0.016 ) and tended to over - perform in recognition of angry knocking ( p < 0.07 ) . significant differences are indicated by an asterisk ; ( b ) error rate : the lack of gender differences in error rate demonstrates that gender differences in recognition accuracy of emotional content of knocking were not caused by gender - related bias for mistaking one emotion for another . each bar represents an average ratio of the number of errors of particular type to the overall number of errors made for a display type ( e.g. , leftmost bar represents an average ratio of number of trials when happy knocking was mistaken for neutral knocking to the number of trials when happy knocking was mistaken for both neutral and angry knocking ) ; ( c ) response time to happy , neutral , and angry point - light knocking by females and males . individual number of correct responses was submitted to a 2 3 repeated - measures anova ( as assessed by the shapiro wilk test , the data were normally distributed ) with factors gender ( female / male ) and emotional expression of knocking ( happy / neutral / angry ) . this analysis revealed the lack of a main effect of gender ( f(1,32 ) = 0.21 , p = 0.648 , ns ) . however , a main effect of emotional expression ( f(2,32 ) = 82.94 , p < 0.0001 ) and interaction between the factors gender emotional expression ( f(2,32 ) = 6.23 , p < 0.003 ) were highly significant . planned pair - wise comparisons indicated that males outperformed in recognition of happy knocking ( t32 = 2.58 , p < 0.015 , one - tailed , here and below bonferroni corrected for multiple comparisons ; d = 0.84 ) , whereas females tended to over - perform in recognition of angry knocking ( t32 = 1.87 , p < 0.07 , one - tailed ) and excelled in recognition of neutral knocking ( t32 = 2.54 , p < 0.016 , one - tailed , d = 0.88 ) . the data , therefore , reveals the lack of advantage of females in recognition accuracy . instead , the findings indicate that sex effects in recognition accuracy are modulated by emotional contents of actions . error analysis ( figure 2b ) indicated that by both females and males , happy knocking was mistaken for neutral knocking in more than 80% of wrong responses ( error rate 0.84 and 0.86 for females and males , respectively ; gender difference : t32 = 0.42 , p = 0.68 , two - tailed , ns . error rate was calculated as an average ratio of the number of errors of particular type to the overall number of errors made for a display type ) . in turn , with a lack of gender differences , neutral knocking was misperceived as happy actions in about 70% of error responses ( error rate 0.68 and 0.67 for females and males , respectively ; gender differences : t32 = 0.02 , p = 0.99 , two - tailed , ns ) . in about 80% of error trials in response to angry knocking , both females and males mistook angry knocking for neutral knocking ( error rate 0.79 and 0.8 for females and males , respectively ; gender difference : t32 = 0.14 , p = 0.88 , two - tailed , ns ) . the lack of gender differences in error rate suggests that gender effects in recognition accuracy of emotional content of knocking observed in the present study are not caused by gender - related bias for mistaking one emotion for another . for response time analyses , a 2 3 repeated - measures anova was performed on individual values ( as assessed by the shapiro wilk test , the data were normally distributed ) with factors gender ( female / male ) and emotional expression ( happy / neutral / angry ) . this analysis did not reveal any effect of gender ( f(1,32 ) = 1.56 , p = 0.22 , ns ) as well as any interaction of factors gender emotional expression on response time ( f(2,32 ) = 1.42 , p = 0.25 , ns ; figure 2c ) . however , a main effect of emotional expression was significant ( f(2,32 ) = 35.16 , p < 0.0001 ) , with the fastest response to angry knocking , and the slowest response to neutral knocking ( figure 2b ) . this shows that recognition of neutral knocking was more difficult than that of angry and happy knocking . post hoc pair - wise comparisons showed no gender difference in response time to happy ( t32 = 0.09 , p = 0.93 , two - tailed , ns , average 2.00 0.39 and 1.99 0.28 s from the stimulus onset , for females and males , respectively ) , neutral ( t32 = 1.21 , p = 0.24 , two - tailed , ns ; average 2.15 0.33 and 2.28 0.3 s , for females and males , respectively ) , and angry knocking ( t32 = 0.14 , p = 0.89 , two - tailed , ns ; average 1.84 0.32 and 1.85 0.28 , for females and males , respectively ) . taken together , the findings suggest that gender does not affect speed of body language reading . for both females and males , however , the swiftness of response to body language depends on the emotional content of actions . since it is difficult to interpret negative findings within a relatively small sample size that might be considered a limitation of the study , the lack of sex differences in error rate and response time has to be further explored . the outcome of the study indicates that gender affects accuracy rather than speed of body language reading . to the best of our knowledge , the gender effect , however , is modulated by the emotional content of actions . females tend to excel in recognition accuracy of angry knocking , whereas males over - perform in recognition of happy actions . the lack of gender differences in error rate suggests that gender effects in recognition accuracy are not caused by gender - related bias . based on popular wisdom , one can expect that while women possess soft skills in social perception including high sensitivity to positive emotional signals and subtle details , men might outperform in recognition of negative menacing expressions . this assumption is based on the different evolutionary and socio - cultural roles of both genders ( e.g. , biele and grabowska , 2006 ; proverbio et al . , 2008 ) . high sensitivity of women to positive emotions has been related to their role as primary offspring care providers . social cognition in men is presumably connected with active interactions and immediate reactions , and , therefore , emotion perception is likely associated with motor programs . anger detection is usually associated with a need to act , for example , escape from a person or prepare to confront a person . these data agree with findings showing that men appear to exhibit stronger brain activation in response to positive pictures ( depicting landscapes , sport activities , families , and erotic scenes ) than women ( wrase et al . moreover , males are equally responsive to happiness conveyed through static and dynamic happy faces ( males rate the intensity of dynamic and static expressions of happiness equally high ) , whereas females are less responsive to happiness in static faces ( biele and grabowska , 2006 ) . presumably , this indicates that males are better tuned to subtle expressions of happiness in faces and actions . this might hold true , at least , for a population of young men with a high social status and educational level as those participated in the present study . the prominent outcome of the study is that females had a clear advantage in recognition of neutral knocking . this suggests that women are better tuned to the lack of emotional content in body actions . future research should clarify whether gender effects in body language reading occur with other repertoires of actions , and with other arrays of emotions . one possibility is that gender differences have neurobiological sources ( cahill , 2006 ; jazin and cahill , 2010 ) , and brain mechanisms underpinning body language reading are sex - specific . the social cognition network , commonly referred to as the social brain , primarily involves the parieto - temporal junction , temporal cortices including the fusiform face area and the sts , orbitofrontal cortices , the amygdala ( adolphs , 2003 ) , and the left lateral cerebellum ( sokolov et al . , 2010 ) . the right sts is a cornerstone for processing of meaningful body motion ( grossman and blake , 2002 ; pavlova et al . , studies of sex effects on the social brain have been limited to investigation of face expressions or body actions represented in still photographs . brain activation in females is reported to be more bilaterally distributed , presumably providing greater contribution of both hemispheres to identification of facial affect ( bourne , 2005 ; proverbio et al . , 2010 ) . females show stronger event - related potential ( erp ) response to emotional faces ( orozco and ehlers , 1998 ) . however , the findings are controversial . sex effects are found in the blood oxygen level dependent ( bold ) response of the amygdala to happy , but not to fearful faces ( killgore and yurgelun - todd , 2001 ) . on the other hand , a significant correlation between functional magnetic resonance imaging ( fmri ) activity of the amygdala and behavioral response to fearful faces both behavioral and amygdala responses to threat - related face expressions are correlated with testosterone level ( derntl et al . in accordance with widespread belief , it is reported that the female brain is more responsive to social stimuli represented in still images ( proverbio et al . , 2009 ) . recent erp findings indicate that in females , processing of actions goals occurs earlier ( proverbio et al . , 2010 ) . neuroimaging reveals that gender effects are not evident in the neural circuitry underpinning visual processing of social interaction , but rather in the regions engaged in perceptual decision making : the neuromagnetic gamma response over the left prefrontal cortex peaks earlier in females ( pavlova et al . , 2010a ) . gender effects at behavioral level do not necessarily imply that there is sex - related difference in brain activation subserving body language reading . moreover , gender differences in performance on social cognition tasks can be impacted by socio - cultural stereotypes ( pavlova et al . , 2010b ) . several types of interrelations between behavioral measures and brain mechanisms engaged in social perception should be taken into account : ( i ) sex differences both in behavioral and brain responses ; ( ii ) sex differences detectable either at behavioral level or only in brain activation ; and ( iii ) absence of sex differences both at behavioral and brain levels ( pavlova , 2009 ) . noteworthy , gender - related dimorphism in the brain may not only elicit but also prevent behavioral differences if they are maladaptive ( de vries , 2004 ) . future research should be directed at uncovering sex differences in brain activity during body language reading . such investigation would also shed light on sex differences in neuropsychiatric conditions characterized by impairments in social cognition such as asd , depression , and schizophrenia . it is known that males are more commonly affected by asd than females , with a ratio of about 4:1 ( newschaffer et al . , 2007 ) . females , however , are affected much more severely , and , therefore , in high functioning autistic individuals this ratio is even much higher . although there is some behavioral evidence that individuals with asd have difficulties in revealing information about emotions from point - light body movements ( moore et al . , 1997 ; hubert et al . , 2007 ; parron et al . , 2008 ) , it is unclear whether females and males with asd differ in body language reading . the lack of studies in females with asd calls for a thorough investigation of their profile . the other important issue for future research is sex differences in visual social cognition in survivors of premature birth . males are at a 1420% higher risk of premature birth ( melamed et al . , 2010 ) and of its complications in the brain and cognition . adolescents who were born prematurely are likely to exhibit difficulties in visual social cognition ( pavlova et al . , 2008 ) , but gender effects are largely unknown . clarification of gender impact on body language reading and underlying brain networks would provide novel insights into understanding of gender vulnerability to neuropsychiatric and neurodevelopmental impairments in visual social cognition . 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 .

body motion is a rich source of information for social cognition . however , gender effects in body language reading are largely unknown . here we investigated whether , and , if so , how recognition of emotional expressions revealed by body motion is gender dependent . to this end , females and males were presented with point - light displays portraying knocking at a door performed with different emotional expressions . the findings show that gender affects accuracy rather than speed of body language reading . this effect , however , is modulated by emotional content of actions : males surpass in recognition accuracy of happy actions , whereas females tend to excel in recognition of hostile angry knocking . advantage of women in recognition accuracy of neutral actions suggests that females are better tuned to the lack of emotional content in body actions . the study provides novel insights into understanding of gender effects in body language reading , and helps to shed light on gender vulnerability to neuropsychiatric and neurodevelopmental impairments in visual social cognition .

Introduction Materials and Methods Participants Stimuli and procedure Results Discussion Conflict of Interest Statement

aripiprazole is the third generation atypical antipsychotic and a dopamine serotonin system stabilizer ( dss ) that is effective against positive and negative symptoms of schizophrenia . it has a low propensity for extrapyramidal side effects , causes minimal weight gain or sedation , produces no elevation in serum prolactin levels and does not cause prolongation of qtc interval . it is partial agonist at d2 and 5ht1a and blocks 5ht2a receptors.[13 ] the most common adverse reactions in adult patients reported in clinical trials with dosage of 1015 mg per day were fatigue , insomnia , headache , nausea , vomiting , tremors , rigidity , akathisia , and constipation . overall incidence of acute dystonic reactions with antipsychotic medications is 2.55% , majority of which are due to typical and high potency antipsychotics . the following case report is of aripiprazole - induced oculogyric crisis ( acute dystonia ) . a 28-year - old single female was brought by her relatives to the psychiatry outpatient clinic . for last 89 months , she was interacting less with family . as per history , she was suspicious towards family members that they would poison her and also feared that few strangers would come and kill her . in addition , she was talking to herself , was occasionally irritable and had sleep disturbance . after psychiatric evaluation , her mental status examination revealed delusion of persecution which was primary , well systematized , complete , and intermittent and associated with intense fear of being harmed palpitations and irritability . she also had auditory hallucinations of an unknown male , commenting on her day - to - day work and occasionally criticizing her ; associated with the patient replying to the voice . her weight was 70 kg , and she had a family history ( mother ) of diabetes mellitus . her baseline investigations of hemogram , liver function and renal function tests , chest x - ray , and electrocardiogram were within the normal range . she was prescribed aripiprazole 10 mg orally in two divided dosages per day , eszopiclone 2 mg orally at nighttime dosage and was advised follow - up after 2 weeks . during her first follow - up visit , she showed improvement in sleep . for improvement in psychotic symptoms , aripiprazole was increased to 20 mg orally in two divided dosages and was advised follow - up after 2 weeks . after 5 days of dose increment , she was brought with complaints of upward rolling of eye balls suddenly , unexpected , occurring five to seven times a day , which were difficult to bring back to original position by the patient . after evaluation for conversion disorder , tardive dystonia , epileptic encephalopathy , anti epileptic drug intake , she was diagnosed of having oculogyric crisis ( acute dystonia ) . the patient was admitted in psychiatry ward . aripiprazole was withdrawn and immediately injection promethazine 50 mg intramuscularly was given , which was repeated after half an hour . her symptoms improved in next 1 hour and the patient was prescribed 25 mg promethazine orally in night time dosage . within 3 days of admission , she was put on aripiprazole 10 mg orally in two divided dosages for her psychotic symptoms , while continuing eszopiclone with promethazine 25 mg nocte . the patient was advised to follow up after a week . the repeat challenge with aripiprazole 20 mg orally in two divided dosages resulted into oculogyric crisis within 4 days . the patient was advised cap ziprasidone 20 mg orally in two divided doses per day ( i.e. , 40 mg per day ) with food and was asked to follow up 2 weeks later with electrocardiogram done . later uptitration of cap ziprasidone to 20 mg orally in three divided doses per day ( i.e. , 60 mg per day ) with food was done . she improved in her psychotic symptoms over 2 months with no extrapyramidal symptoms , normal electrocardiogram , and other baseline investigations . various case reports of aripiprazole - induced acute dystonia report symptoms of neck extension , torticollis , rigidity , and tongue movements.[810 ] in addition , various studies describe medications causing acute dystonic reactions and their management which are described in the table 1 . case reports of acute dystonic reactions in patients on medications such as antimalarials ( chloroquinone , hydroxychloroquine , and amodiaquine , antivertigo agents ( cinnarizine , and flunarizine ) , cocaine , buspirone , diazepam , sumatriptan , phenylpropanolamine , and ecstasy ( 3,4-methylenedioxymethamphetamine ) have also been reported . drugs causing acute dystonia and its management in this case , oculogyric crisis is the symptom of attention . this case , a female patient on third generation antipsychotic medicine ( aripiprazole ) has least propensity for dystonia.[13 ] the risk factor for developing acute dystonia in this case is history of acute dystonia . acute dystonia occurred after 5 days on increasing aripiprazole from 10 mg to 20 mg per day orally in two divided dosage . it regressed completely with stopping of aripiprazole and administration of promethazine . on repeat challenge it reappeared with the same severity . hence , on causality assessment scale by naranjo et al . , the score is 7 . numerous studies report mechanisms of acute dystonia being dopamine hypofunction resulting in a relative overactivity of cholinergic mechanisms . since aripiprazole lacks protective anticholinergic action , it can have a potential to precipitate dystonia . this was supported by the fact that , in this case , dystonia resolved with promethazine ( an antihistaminic with anticholinergic property ) . in addition , aripiprazole 's action on the d3 receptor and antagonism of 5-ht6 and 5-ht7 receptors is still unknown and hence it may play a role in oculogyric crisis . preclinical studies found that inhibitory action of aripiprazole on the serotonin transporter , which may have a potential to alter the dopamine balance in the basal ganglia region . studies mention paradoxical dopaminergic hyperfunction by either preferentially blocking presynaptic receptors or exposing the postsynaptic receptors to the natural release of dopamine from the presynaptic terminals as the dba levels drop which may result in dystonia . this justifies the judicious use of medications for treatment and prophylaxis of acute dystonia due to antipsychotics . this case outlines a significant side effect of aripiprazole , which a practitioner should be vigilant about before initiating the medication .

aripiprazole is the third generation atypical antipsychotic and a dopamine serotonin system stabilizer ( dss ) effective against positive and negative symptoms of schizophrenia . it has a low propensity for extrapyramidal side effects , causes minimal weight gain or sedation , produces no elevation in serum prolactin levels , and does not cause prolongation of qtc interval . this case report is of a patient suffering from schizophrenia ( paranoid ) . the patient developed oculogyric crisis ( acute dystonia ) with aripiprazole dose uptitration . dystonic reaction resolved with promethazine administration . naranjo 's causality assessment reveals probable association of aripiprazole with oculogyric crisis . a thorough workup and vigilance is required prior to initiation of aripiprazole in the case of schizophrenia .

INTRODUCTION CASE REPORT DISCUSSION

venous thromboembolism ( vte ) occurs to be one of the most serious complications after undergoing total joint arthroplasty.1 ) generally , pulmonary thromboembolism is generated as the secondary by - product from deep vein thrombosis ( dvt ) while its emergence may possibly trigger the chronic thromboembolic pulmonary hypertension as well as post - thrombotic syndrome.2 ) patent foramen ovale ( pfo ) , a residue of fetal circulation , is found approximately about 25 - 30% among adults ; and , it is also known that thrombus from the venous circulation rarely causes arterial thromboembolism through right - to - left shunt.3 ) in this case , the authors experienced a patient with pfo and several thrombotic disease such as pulmonary thromboembolism , dvt , and right atrial thrombus along with cryptogenic ischemic stroke after total knee arthroplasty ( tka ) . a 64-year - old female presented to the emergency room for the shortness of breath . she had been taking antiplatelet drugs ( aspirin 100 mg / day , clopidogrel 75 mg / day ) for the treatment of the right posterior temporal lobe infarction detected in brain magnetic resonance imaging for the disorientation on the third day after the operation ( fig . 1 ) . carotid and brain computed tomography ( ct ) angiography showed no evidence of atherosclerosis . afterwards , she experienced dyspnea in the second week after the operation and was performed the transthoracic echocardiography ( tte ) , which showed right atrial thrombi and was treated with the low molecular weight heparin ( lmwh ) . the symptoms , nonetheless , remained so that she had been transferred to our hospital . in the medical history her blood pressure was 127/79 mm hg while her heart rate was 89 bpm . on physical , pitting edema in her left lower leg was noted and neurologic examination was unremarkable . the chest x - rays did not show any signs of cardiomegaly as well as pulmonary edema . both d - dimer and n - terminal pro - brain natriuretic peptide were increased respectively at 2686 ng / ml , and 3310 pg / ml and on the other hand , cardiac enzymes , such as creatinine kinase - mb and troponin - i were within a normal range . tte demonstrated normal left ventricular systolic function ( ejection fraction of 63% ) , but in the right atrium two highly mobile masses ( 1.67 2.18 cm , 0.69 0.80 cm ) suggesting possible thrombi were observed . the echocardiogram also revealed right atrium and right ventricle enlargement , severe pulmonary hypertension ( pulmonary artery systolic pressure 102 mm hg ) , and d - shaped left ventricle ( fig . her chest ct showed that pulmonary thromboembolism was found in both main pulmonary arteries while dvt was observed in left femoral vein ( fig . holter ecg did not reveal arrhythmia except rare atrial premature complex and ventricle premature complex . the patient was administered to warfarin ( target inr 2 - 3 ) after using lmwh . in follow - up tte 3 weeks later , the two previous thrombi in right atrium ( ra ) disappeared . along with the improvement in ra , there was also a positive change on the extent of pulmonary hypertension . the follow up ct taken 4 weeks after the hospitalization did not reveal pulmonary thromboembolism and dvt ( fig . 3 ) . since the cause of the cerebral infarction was unclear , additional evaluation were needed . in order to examine the intracardiac shunt , the results confirmed the transference of microbubble from right atrium to left atrium without showing atrial septal defect ; and therefore , we diagnosed the patient with pfo ( fig . currently , the patient is taking warfarin continuously without any signs of abnormalities in outpatient clinic . we recommended further treatment for pfo including device closure but patient refused because of poor general condition . as vte is one of the fatal complications after undergoing tka , dvt , and pulmonary embolism ( pe ) are known to occur approximately around 13% and 3% , respectively.4 ) the occurrence period of vte after tka is reported to be 3 to 9 days after the operatio5 ) and , there are mostly cases that accompany dvt and pe and occasionally thrombi observed on several organs . particularly , it was reported that the patients with pfo even might develop arterial thromboembolism with cryptogenic stroke.3)6 ) in the present case , the authors suspected that cryptogenic stroke 3 days after the operation might possibly have been caused by the vte changed into arterial thromboembolism through pfo . pfo , one of the congenital heart diseases that can be observed even in adulthood , typically have no symptom . nonetheless , it can affect as the risk factor for several conditions including ischemic stroke , platypnea - orthodeoxia syndrome , and decompression sickness.7 ) pfo as the cause of cryptogenic stroke is not uncommon ; hence , the occurrence of ischemic stroke with either pe or dvt can be considered as the possible example of paradoxical embolism through right - to - left shunt.8 ) among the treatments on the prevention of frequent strokes within a patient who has pfo , there are medical options and invasive methods.9 ) the medical options include the use of antiplatelet agents as well as the prescription of warfarin in the patients with hypercoagulable state or venous thrombosis . in terms of invasive methods , it was used to have an operation in the past while the application of device closure is being used recently.10 ) in this case , cryptogenic stroke took place within 3 days after tka while right atrial thrombus , dvt , and pe were present in a way that the passage through right to left shunt was speculated and the result of subsequent echocardiography led to the diagnosis of a positive reaction from the agitated saline test for pfo . the patient was treated with lmwh and then administered warfarin ; and the patient discharged with the recovered conditions . in conclusion , the occurrence of pulmonary thromboembolism or dvt in a patient with pfo can trigger the development of cryptogenic stroke through right to left shunt . therefore , further evaluation of cause are essential in patients with the occurrence of cryptogenic stroke who are associated with a high risk of vte after total joint arthroplasty . also , anticoagulation treatment is necessary if a patient has a right to left shunt , such as pfo .

patients undergoing total joint arthroplasty frequently develop post - operative complication , such as deep vein thrombosis and pulmonary thromboembolism . however , it is not common coexisting deep vein thrombosis , pulmonary thromboembolisms , right atrial thrombus and acute cerebral infarction raised by thrombus through patent foramen ovale . we reported the patient who had multiple thrombi which were accompanied with a cryptogenic ischemic stroke and associated with patent foramen ovale after operation .

Introduction Case Discussion

non - alcoholic fatty liver disease ( nafld ) , the hepatic manifestation of metabolic syndrome , includes a spectrum of diseases ranging from simple steatosis , through steatohepatitis ( nash ) , to fibrosis and ultimately cirrhosis ( 1 , 2 ) . the disease definition and modalities used for diagnosis and epidemiology studies are not standardized ( 3 ) . the prevalence of nafld increased rapidly and it affects about 20% to 30% of the population in western countries ( 4 ) and 15% in china ( 5 ) . as a lipid metabolism disorder , nafld has a strong genetic component . a recent gwas from the genetics of obesity - related liver disease consortium identified lyplal1 rs12137855 for nafld in 7177 adults of european ancestry ( 6 ) . lyplal1 encodes lysophospholipase - like protein 1 , a 26 kda cytosol protein , which belongs to a subclass of lysophospholipase family ( 7 ) . recently , several single nucleotide polymorphisms ( snps ) , near human lyplal1 gene were revealed to be significantly associated with fat distribution in a relatively sex - specific pattern ( 8) , such as 3 snps including rs4846567 ( 9 ) , rs2605100 ( 10 ) and rs2820443 ( 11 ) near lyplal1 gene , which are associated with increased waist - hip ratio ( whr ) adjusted for bmi only in women not men . snp rs11118316 at lyplal1 is associated with visceral adipose tissue / subcutaneous adipose tissue ratio in both men and women ( 12 ) , and snp at rs12137855 near lyplal1 gene is strongly associated with nafld ( 13 ) . subsequent studies showed that lyplal1 plays an important role in fat distribution and lipid metabolism . lyplal1 rs12137855 , as the susceptibility gene of nafld , was widely studied , but the results were inconsistent . no research has been performed on the association between polymorphism of lyplal1 and nafld in chinese han population . the aim of our study was to investigate the association between nafld and lyplal1 in chinese han population and assess the effect of this gene on serum lipid profiles . the study was performed in accordance with the principles of declaration of helsinki and its appendices ( 14 ) . this study was approved by the ethical committee of qingdao municipal hospital ( qingdao , china ) and a written informed consent form was obtained from all patients before participation in the study . from may 2010 to may 2014 , we selected a total of 298 unrelated adult subjects , including 184 unrelated chinese patients of both genders and different ages ( 85 males , 97 females , mean age 43.18 11.53 years ) diagnosed with nafld and 114 healthy controls matched for sex and age ( 57 males , 57 females , mean age 40.77 11.47 years ) by b - type ultrasonography ( 15 ) . the subjects were collected from the department of gastroenterology and the medical center of qingdao municipal hospital . the diagnosis of nafld was performed under standard clinical evaluation conditions according to the aasid criteria . other causes of liver disease were excluded , including increased alcohol intake ( > 210/140 g / wk for males / females ) , as confirmed by at least one family member or friend and carboxydesialylated transferrin determination , viral and autoimmune hepatitis , hereditary hemochromatosis , and alphal - antitrypsin deficiency ( 16 ) . we excluded other related disease , such as subjects with type 1 diabetes mellitus and coronary atherosclerotic disease ( cad ) . the controls were confirmed as healthy by medical history , general examinations and laboratory examinations at the same hospital . blood samples of each subject for biochemical analyses were collected into ethylene diamine tetraacetic acid - containing tubes after an 12-hour overnight fast and the following information for each subject was gathered ; height , body mass , waist , hip circumference , calculating body mass index ( bmi ) equals to mass ( kg)/height ( m ) . environmental factors , such as diet and physical activity , were not recorded in this study . the blood sample tested for total cholesterol ( tc ) , triglycerides ( tg ) , high - density lipoprotein ( hdl ) and low - density lipoprotein ( ldl ) using routine enzymatic methods . serum alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) and -glutamyltransferase ( ggt ) concentrations were measured as previously described ( 17 ) . the genomic dna purification kit ( bioteke , biotechnology , beijing , china ) was used for extracting dna from peripheral blood following the manufacturer 's instructions and stored at 20c until use . genotyping for lyplal1 ( rs12137855 ) was performed by polymerase chain reaction ( pcr ) analysis using the following primers for lyplal1 polymorphism : 5'-tcctaagttcctattgtcccttca-3 ' and 5'-tgctgtggggtgagtca-3 ' . pcr amplification ( labnet , united states ) was performed as follows ; initial step of 95c for 10 minutes , followed by 35 cycles ; denaturation at 94c for 1 minute , annealing at 60c for 1 minute and elongation at 70 c for 1 minute . all pcr products were resolved using 2% agarose gel electrophoresis at 110 v for 30 minutes with a 237-base pair product in size . the lyplal1 genotypes were detected by direct dna sequencing using the abi prism sequence detection system abi3730 ( foster city , ca , usa ) . the genotyping call rate was more than 95% and the completion rate was > 99% . statistical analyses were performed using spss statistical software , version 17.0 for window ( spss inc . hardy - weinberg equilibrium between expected and observed genotype distributions was assessed using the test . genotype and alleles were estimated by chi - square test and dna distributions between nafld patients and controls were analyzed by pearson s test or fisher s exact test where appropriate . differences in characteristics between different groups were examined using student s t test , paired samples t - test or test . the strength of the association between the polymorphism and nafld was evaluated by logistic regression analysis adjusted for confounders ( age , sex , smoking and hypertension , which were considered as continuous variables ) . the study was performed in accordance with the principles of declaration of helsinki and its appendices ( 14 ) . this study was approved by the ethical committee of qingdao municipal hospital ( qingdao , china ) and a written informed consent form was obtained from all patients before participation in the study . from may 2010 to may 2014 , we selected a total of 298 unrelated adult subjects , including 184 unrelated chinese patients of both genders and different ages ( 85 males , 97 females , mean age 43.18 11.53 years ) diagnosed with nafld and 114 healthy controls matched for sex and age ( 57 males , 57 females , mean age 40.77 11.47 years ) by b - type ultrasonography ( 15 ) . the subjects were collected from the department of gastroenterology and the medical center of qingdao municipal hospital . the diagnosis of nafld was performed under standard clinical evaluation conditions according to the aasid criteria . other causes of liver disease were excluded , including increased alcohol intake ( > 210/140 g / wk for males / females ) , as confirmed by at least one family member or friend and carboxydesialylated transferrin determination , viral and autoimmune hepatitis , hereditary hemochromatosis , and alphal - antitrypsin deficiency ( 16 ) . we excluded other related disease , such as subjects with type 1 diabetes mellitus and coronary atherosclerotic disease ( cad ) . the controls were confirmed as healthy by medical history , general examinations and laboratory examinations at the same hospital . blood samples of each subject for biochemical analyses were collected into ethylene diamine tetraacetic acid - containing tubes after an 12-hour overnight fast and the following information for each subject was gathered ; height , body mass , waist , hip circumference , calculating body mass index ( bmi ) equals to mass ( kg)/height ( m ) . environmental factors , such as diet and physical activity , were not recorded in this study . the blood sample tested for total cholesterol ( tc ) , triglycerides ( tg ) , high - density lipoprotein ( hdl ) and low - density lipoprotein ( ldl ) using routine enzymatic methods . serum alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) and -glutamyltransferase ( ggt ) concentrations were measured as previously described ( 17 ) . the genomic dna purification kit ( bioteke , biotechnology , beijing , china ) was used for extracting dna from peripheral blood following the manufacturer 's instructions and stored at 20c until use . genotyping for lyplal1 ( rs12137855 ) was performed by polymerase chain reaction ( pcr ) analysis using the following primers for lyplal1 polymorphism : 5'-tcctaagttcctattgtcccttca-3 ' and 5'-tgctgtggggtgagtca-3 ' . pcr amplification ( labnet , united states ) was performed as follows ; initial step of 95c for 10 minutes , followed by 35 cycles ; denaturation at 94c for 1 minute , annealing at 60c for 1 minute and elongation at 70 c for 1 minute . all pcr products were resolved using 2% agarose gel electrophoresis at 110 v for 30 minutes with a 237-base pair product in size . the lyplal1 genotypes were detected by direct dna sequencing using the abi prism sequence detection system abi3730 ( foster city , ca , usa ) . the genotyping call rate was more than 95% and the completion rate was > 99% . statistical analyses were performed using spss statistical software , version 17.0 for window ( spss inc . chicago , il , usa ) . hardy - weinberg equilibrium between expected and observed genotype distributions was assessed using the test . genotype and alleles were estimated by chi - square test and dna distributions between nafld patients and controls were analyzed by pearson s test or fisher s exact test where appropriate . differences in characteristics between different groups were examined using student s t test , paired samples t - test or test . the strength of the association between the polymorphism and nafld was evaluated by logistic regression analysis adjusted for confounders ( age , sex , smoking and hypertension , which were considered as continuous variables ) . abbreviations : alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bmi , body mass index ; ggt , gamma - glutamyl transpeptidase ; glu , glucose ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; nafld , non - alcoholic fatty liver disease patients ; tc , total cholesterol ; tg , triglyceride . data are presented as mean sd . the genotypes distribution of lyplal1 was in accordance with the hardy - weinberg equilibrium in nafld and control groups ( pnafld = 0.323 ; pcontrol = 0.230 , respectively ) . to ensure the accuracy of our genotyping , we randomly repeated dna sequencing in 100 subjects for reverse sequencing . the genotype and allele distribution are shown in table 2 , which indicates no significant difference between the two groups ( p > 0.05 ) . the gene lyplal1 did not increase the risk of developing nafld ( or = 0.622 , 95% ci : 0.334 - 1.159 ) . p : nafld patients vs. control . to explore whether gene polymorphism affect the laboratory parameters , we compared non - carriers and carriers of variant allele ( rs12137855 ) in all subjects , nafld patients and healthy controls , respectively ( table 3 ) ; the results showed that there was a significant difference in weight , bmi and ldl . abbreviations : alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bmi , body mass index ; ggt , gamma - glutamyl transpeptidase ; glu , glucose ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; nafld , non - alcoholic fatty liver disease patients ; tc , total cholesterol ; tg , triglyceride . abbreviations : alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bmi , body mass index ; ggt , gamma - glutamyl transpeptidase ; glu , glucose ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; nafld , non - alcoholic fatty liver disease patients ; tc , total cholesterol ; tg , triglyceride . the genotypes distribution of lyplal1 was in accordance with the hardy - weinberg equilibrium in nafld and control groups ( pnafld = 0.323 ; pcontrol = 0.230 , respectively ) . to ensure the accuracy of our genotyping , we randomly repeated dna sequencing in 100 subjects for reverse sequencing . the genotype and allele distribution are shown in table 2 , which indicates no significant difference between the two groups ( p > 0.05 ) . the gene lyplal1 did not increase the risk of developing nafld ( or = 0.622 , 95% ci : 0.334 - 1.159 ) . to explore whether gene polymorphism affect the laboratory parameters , we compared non - carriers and carriers of variant allele ( rs12137855 ) in all subjects , nafld patients and healthy controls , respectively ( table 3 ) ; the results showed that there was a significant difference in weight , bmi and ldl . abbreviations : alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bmi , body mass index ; ggt , gamma - glutamyl transpeptidase ; glu , glucose ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; nafld , non - alcoholic fatty liver disease patients ; tc , total cholesterol ; tg , triglyceride . in the recent years , this gene has been widely studied , but the results were inconsistent ( 6 , 18 ) . speliotes et al . observed significant associations with both histologic nafld and ct nafld at variants or near lyplal1 ( 6 ) . our study for the first time investigated the association between lyplal1 rs12137855 and nafld in chinese han population ; we selected 184 nafld patients and 114 controls to observe the association between lyplal1 rs12137855 and nafld ; however , we did not find significant association between gene and nafld , which is in accordance with some previous findings ( 18 - 23 ) . multiple factors are involved in development and progression of nafld such as insulin resistance , obesity and oxidative stress ( 24 ) . in our study , we diagnosed nafld using routine blood testing and liver ultrasonography . lack of direct measurement of hepatic fat content by gold standard liver biopsy reduced the accuracy of diagnosis , but we observed that metabolism indicators change obviously . alt and ast are used as markers of liver fat accumulation ( 25 - 27 ) and commonly used in clinical practice ( 28 ) . we can observe significant differences in plasma concentrations of these transaminases between nafld and healthy controls . on the contrary , independent of genetic variation in lyplal1 , these results were not in accordance with paola leon - mimila and speliotes study ; they confirmed that lyplal1 rs12137855 was associated with increased tg content ( 6 , 29 ) . interestingly lyplal1-related proteins have been predicted to play a role in consecutive steps in triglyceride breakdown ( 30 , 31 ) . pnpla3 has been confirmed to increase hepatic steatosis through preventing the breakdown of triglyceride ( 32 ) . whether lyplal1 has the same function and knowing the mechanism of triglyceride breakdown need more investigations . for chinese subjects , bmi of 28 kg / m or more is an index of obesity ( 16 ) . in this study , kg / m ) than controls ( 23.04 3.47kg / m ) ( p < 0.05 ) , also higher in the variant carrier ( 25.29 3.51 independent of this gene in nafld group and control group the difference did not reach statistical significant . our study suggested that lyplal1 can influence bmi , which reflects the association with obesity indirectly . our findings for the first time found a significant difference between variant carriers and non - carriers regarding ldl ; the mechanism is not clear and needs further research . as far the studies on asian population were negative ( 18 , 19 ) , and we have reasons to doubt the correlation of the gene with nafld in asian population . to obtain more precise results , larger studies on multiple ethnic groups , such as asian indian or korean should be performed . our results may also be due to small sample size , ethnic differences in linkage disequilibrium ( ld ) patterns , ethnic - specific association and gene / environment interactions . this study provided preliminary evidence that there is no association between lyplal1 rs12137855 polymorphism and development of nafld in chinese han origin for the first time . further studies with large study samples and different ethnicity are needed to investigate the influence of this gene on nafld .

background : recent genome - wide association studies ( gwas ) identified that gene lysophospholipase - like 1 ( lyplal1 ) rs12137855 associated with non - alcoholic fatty liver disease ( nafld ) . no research has been performed regarding the association between lyplal1 and nafld in china.objectives:the aim of the present study was to investigate the association between the gene lyplal1 rs12137855 and nafld , and the effect on serum lipid profiles in a chinese han population.patients and methods : lyplal1 rs12137855 gene was genotyped in 184 patients with nafld and 114 healthy controls using sequencing and polymerase chain reaction analysis ( pcr ) . we tested serum lipid profiles using biochemical methods.results:no significant differences in genotype and allele frequencies of lyplal1 rs12137855 was found between the nafld group and the controls group ( p > 0.05 ) . subjects with the variant lyplal1 rs12137855 cc genotype had a higher mean weight , body mass index ( bmi ) and low density lipoprotein ( ldl).conclusions : our results showed for the first time that lyplal1 gene is not associated with a risk of nafld development in the chinese han population . the variant carriers of overall subjects significantly increased weight , bmi and ldl .

1. Background 2. Objectives 3. Patients and Methods 3.1. Subjects 3.2. Biochemical Analyses 3.3. Genetic DNA Extraction and Genotyping 3.4. Statistical Analysis 4. Results 4.1. Characteristics of the Study Population 4.2. LYPLAL1 rs12137855 Genotypes and Allele Distribution 4.3. LYPLAL1 rs12137855 Association with Clinical Parameters in Non-Alcoholic Fatty Liver Disease Patients 5. Discussion

a 2-year - old boy was transferred to the emergency department approximately 4 hours after having severe head trauma with loss of consciousness . on physical examination , he had a right temporoparietal scalp laceration and subgaleal hematoma ; the child had a status of decerebrate rigidity . brain ct showed intraventricular hemorrhage and subarachnoid hemorrhage ( figure 1a ) . a , head computed tomography ( ct ) illustrating the intraventricular hemorrhage . b , postoperative intraventricular external drainage resulted in a significant decrease in the intraventricular hemorrhage . c , enlargement of both the lateral ventricles with surrounding white matter edema is consistent with communicating hydrocephalus . f - i , axial and sagittal ct images showing severe , bilateral thickening of the inner table of the frontal bone with cortex compression . i , the outer table of the frontal bone was intact . an external ventricular drain was performed . one week after surgery , cranial ct revealed that the ventricle hemorrhage had disappeared ( figure 1b ) , and the drain tube was removed . one month later , repeat head ct showed dilation of the entire ventricular system associated with communicating hydrocephalus ( figure 1c ) . his consciousness was gradually recovered , and he was awakened 2 months later . the patient was discharged . at 2-year follow - up , he showed hypophrenia and dysphasia , which was diagnosed according to the diagnosis criterion of mental retardation established by the world health organization in 1985 and the s - s checkup list of dysphasia . the neuroimaging findings revealed that the lateral ventricles were smaller than before ( figure 1e ) . five years after surgery , the child was readmitted to our department due to headache , vomiting , imbalance , inappropriate behavior , and loss of interest . the patient had not developed a frontal extra - axial hemorrhage in the past 5 years . there was bilateral optic disc swelling , with hemorrhages of the optic nerve head and surrounding retina ( figure 2a ) . serum cortisol , prolactin , progesterone , estradiol , t3 , t4 , thyroid - stimulating hormone , and glucose tolerance test were within the normal limits . brain ct showed severe , bilateral thickening of the inner table of the frontal bone with cortex compression ( figure 1f - i ) . the inner table was honeycombed and red ( figure 2b and c ) , and the intracranial pressure was elevated . to achieve the decompression , a complete resection of the mass was performed , and the dura was opened in a star fashion . three - dimensional image formation was used to shape the titanium mesh for the skull neoplasty . the mass was immersed in formalin for study of the histological appearance of the mass with mineralized lamellar bone . the forehead had a good contour , and postoperative radiographs demonstrated that the bone grafts remained intact and no evidence of recurrence of the mass . baseline radiography of the skeletal system was performed and showed no additional foci of hyperostosis ( figure 3a - c ) . a - c , postoperative brain computed tomography ( ct ) demonstrating that the frontal bone was removed and replaced with titanium mesh to relieve the brain compression . hyperostosis frontalis interna is an overgrowth of bony tissue in the inner plate of the frontal bone and has been documented in the medical literature for over 300 years . the estimated incidence of hyperostosis frontalis interna in the general population is 5% to 12% . hyperostosis frontalis interna has not been reported in an asymptomatic patient younger than 10 years of age ; hence , the present patient is the first case in the medline database . the most acceptable hypothesis regarding hyperostosis frontalis interna etiology is hormonal influence on bone growth . for example , estrogen stimulation could play a part in the emergence of hyperostosis frontalis interna and explain its predominance among females . in our case , the hormone levels were normal , and interestingly , the hyperostosis frontalis interna was found after severe trauma . with the existing findings , it is difficult to address whether the trauma was related to hyperostosis frontalis interna . to the best of our knowledge , there have been no previous reports in the literature associating hyperostosis frontalis interna with brain trauma . the associated signs and symptoms are generally nonspecific and benign , but they can cluster together in some cases , giving rise to various syndromes . the most frequently presented complaints were morgagni syndrome ( headache , obesity , virilism , and hypertrichosis ) , stewart - morel syndrome ( obesity and neuropsychiatric symptoms ) , and troell - junet syndrome ( acromegaly , toxic goiter , and diabetes mellitus ) . our patient had imbalance which , to our knowledge , has not been mentioned previously . in some severe cases , hyperostosis frontalis interna leads to compression of soft tissue , dural irritation , and brain atrophy because of the thickening of the skull and decrease in the intracranial volume . it has been established that hyperostosis frontalis interna can cause diverse psychiatric disturbances such as aggressiveness , paranoia , or depression . our case tends to confirm that due to extensive hyperostosis frontalis interna , frontal lobe compression can lead to cognitive impairment and psychiatric disorders . the clinical symptoms and signs indicated the elevated intracranial pressure , which was not described in all the previous reports . hyperostosis frontalis interna is commonly an incidental finding in x - ray , ct , and mri studies . computed tomography scans , which provide the radiologist with enough information to distinguish hyperostosis frontalis interna from other bony growth , are better diagnostic tools for hyperostosis frontalis interna . in our case , the authors found bilateral frontal inner tables with diffuse uneven thickening . histologically , the process of hyperostosis frontalis interna is thought to be a deposition of new bone primarily on the inner table and a progressive development of diploe . hyperostosis frontalis interna is characterized by remodeling of the inner table of the frontal bone into a cancellous phenotype . hyperostosis frontalis interna is a benign process , and the majority of patients appear to be asymptomatic ; thus , conservative observation can be used in these cases . if hyperostosis frontalis interna leads to headaches , neurologic symptoms , psychiatric disorders , and cognitive impairment , the surgical excision of the thickened portion of the bone is the only method of treatment to relieve the symptoms . postoperatively , the patient had an uneventful recovery , and the cognitive impairment was improved . hyperostosis frontalis interna is currently regarded as an independent entity and starts to appear at a much younger age . the authors described a case in a 7-year - old boy , presenting imbalance , cognitive impairment , headache , vomiting , and papilledema with hemorrhages ; this is the first such case reported in the literature . the etiology , however , remains unclear , and it is difficult to prove the correlation between hyperostosis frontalis interna and brain injury . if present , the surgical decompression can be an effective treatment method . our case confirms that the clinical presentation was elicited due to compression of the involved cortex area .

hyperostosis frontalis interna is an unexplained irregular thickening of the inner table of the frontal bone . hyperostosis frontalis interna was first identified in 1719 by morgagni as a symptom of a more generalized syndrome characterized by virilism and obesity . most current studies have shown hyperostosis frontalis interna to be a sex- and age - dependent phenomenon , and females manifest a significantly higher prevalence of hyperostosis frontalis interna than males . in this article , the authors report the clinical case of hyperostosis frontalis interna in a 7-year - old child who had severe traumatic brain injury in the past ; review the related literature ; and discuss the clinical , radiological , and therapeutic features of this condition .

Case Report Discussion Conclusion

since the seminal report on lung protective strategy published by amato et al . in the new england journal of medicine in 1998 , whether or not and in what ways the different strategies of mechanical ventilation affect the clinical outcome of ards patients remains a controversial topic among physicians and researchers . this concise review article focuses on the lessons that the surviving sepsis campaign guidelines ( sscg ) 2012 report imparts regarding an optimized strategy for mechanically ventilating ards patients . lung protective strategy for ards patients that includes the following 7 recommendations : tidal volume should be targeted to 6 ml / kg of predicted body weight ( pbw ) plateau pressures in a passively inflated lung should be limited to 30 cm h2o . positive end - expiratory pressure ( peep ) should be applied to avoid alveolar collapse at the end of expiration . higher levels of peep should be strategically used for patients with moderate or severe sepsis - induced ards even though it is difficult to specify the absolute values of higher levels of peep , we think the ardsnet standard peep strategy is the reasonable choice for peep setting because the much higher peep strategy adopted in alveoli trial s higher peep group did not show significant improvement in survival they set peep as high as possible without increasing the maximum inspiratory plateau pressure > 28 30 cmh2o by keeping tidal volume of 6 ml / kg . recruitment maneuvers should be used in sepsis patients with severe refractory hypoxemia prone positioning should be used in sepsis - induced ards patients with a pao2/fio2 ratio < 100 mmhg in facilities that have experience with such practices . a short - term course ( < 48 h ) of neuromuscular blocking agents ( nmbas ) , along with sedatives , should be prescribed for early , sepsis - induced ards and pao2/fio2 < 150 mm hg . a serious question that must be answered is whether strict implementation of the 7 recommendations proposed in the sscg 2012 report will reduce the mortality rate in ards patients . despite recent advances in medical treatment and technologies , the mortality rate among ards patients still remains as high as ~40% [ 3 , 4 ] . one of the plausible explanations for this , despite the introduction of lung protective strategies , is that the patient populations included in the old [ 5 , 6 ] and new [ 3 , 4 ] studies might not be comparable , thereby introducing a misclassification bias . as older studies [ 5 , 6 ] tended to use relatively low levels of peep and fio2 , patients once classified as suffering ards might not meet current ards criteria . therefore , older studies might have included less severe cases , potentially under - estimating the true mortality rate among ards - afflicted patients . another possible explanation could be a decrease of the incidence of ards . in their population - based study li et al . reported a lower incidence of ards despite an increase in patients severity of illness and comorbidities . although the ards mortality rate has not changed over time , the mortality rate of patients at risk of ards has been reduced . fuller et al . reported in their systematic review that in mechanically ventilated patients who did not manifest ards at the time of endotracheal intubation , the use of lower tidal volume ventilation reduced their progression to ards . prophylactic use of lung protective strategies for those patients who are at risk of , but have not yet manifested , ards might help to avoid its progression . however , the mortality rate among patients who do suffer ards is still high . needham et al . revealed in a prospective cohort study that lung protective strategies were used in only 41% of all eligible cases . and they confirmed that , compared with non - adherence to lung protective strategies , the estimated absolute mortality risk reduction over two years in a patient with half - adherence to such strategies was 4.0% , while perfect adherence resulted in a 7.8% risk reduction . second , limiting the tidal volume to 6 ml / kg pbw and the plateau pressure to 30 cmh2o may not be sufficient to minimize lung injury in certain severe ards patients . . showed that in patients with a large , dependent , non - aerated compartment , tidal volume of 6 ml / kgpbw resulted in an increasing number of hyper - inflated compartments and a decreasing number of normally aerated compartments . in such cases , the tidal volume should be further reduced to as low as 4 ml / kg pbw according to the ardsnet protocol . third , as ards is a heterogeneous syndrome , optimization of ventilator settings for each individual would be required when mechanically ventilating ards patients . for example , among patients with recruitable lung , increasing peep may help to avoid the cyclic opening and closing of alveoli without increasing over - distention ( alveolar strain ) . however , among patients with no- or little - recruitable lung , increasing peep may not prevent such cyclic opening and closing but also cause over - distention . therefore , while raising peep might cause harm in some patients it may benefit others . alveolar recruitability may be assessed at bedside in the near future by computed tomography ( ct ) and electrical impedance tomography ( eit ) , thereby making it possible to individually optimize ventilator settings for ards patients . fourth , high - frequency oscillation ( hfo ) is now considered as an alternative to conventional ventilation ( cv ) . compared with cv , hfo was hypothesized to be a superior ventilatory strategy , as it could avoid cyclic collapse and hyperinflation of the alveoli . however , in two recent randomized controlled trials ( rcts ) comparing hfo and cv in ards adult patients , while hfo prevented severe hypoxemia , it did not improve hospital mortality rates [ 12 , 13 ] . in both trials , mean airway pressure was higher in the hfo group than in the cv group by more than 5 cmh2o . guervilly et al . demonstrated in a recent prospective study that using high - mean airway pressure in subjects under hfo worsened right ventricular function compared with cv in ards patients . sedative agents and nmba were more frequently used in hfo groups , which may have negatively affected patients prognosis . furthermore , many physicians have less clinical experience with hfo than with cv . as malhorta et al . commented in their editorial , it was not hfo itself but the hfo protocols and management strategies used in these clinical trials that were less effective than the established lung protective strategy using cv . in this context , there might be more room for hfo protocols to be better optimized for individual ards patients . in addition to ct and eit , pleural pressure and transpulmonary pressure must be taken into account when optimizing ventilator settings for individual ards patients , as it is transpulmonary pressure ( stress ) , but not airway pressure per se , that determines alveolar size ( lung volume ) during ventilation . transpulmonary pressure = alveolar pressure pleural pressure delta transpulmonary pressure / lung elastance it has been reported that overstretch - induced lunginjury occurs when alveoli are stretched above a specific threshold level [ 16 , 17 ] . in their animal study , protti et al . demonstrated that lung damage develops at strain , the ratio of delta volume / functional residual capacity ( frc ) > 2 . strain ( delta volume / frc ) = delta transpulmonary pressure / ( lung elastance x frc ) = delta transpulmonary pressure / specific lung elastance . specific lung elastance = delta transpulmonary pressure / ( delta lung volume / frc ) = lung elastance x frc ( cmh2o ) delta lung volume is the lung volume change from frc . specific lung elastance is a transpulmonary pressure that makes delta volume ( b in the fig . 1 ) . chiumello et al elucidated that the specific lung elastance was around 13.5 cmh2o in all the subgroups ( surgical patients , medical patients , ali patients and ards patients ) , and was not affected by the peep and tidal volume . as the results , any transpulmonary pressure greater than 13.5 x 2 may represent the upper threshold at which overstretch - induced lung injury occurs ( fig . this number of 27 cmh2o is close to the recommended upper transpulmonary pressure limit by grasso et al who showed that overinflation starts from plateau transpulmonary pressure of around 25 cmh2o . for example , if transpulmonary pressure was far less than 27 cmh2o and airway pressure was more than30 cmh2o , physicians should further increase peep to avoid cyclic alveolar collapse , and to permit proper ventilation of the lungs . grasso et al . reported that in patients with influenza a ( h1n1)-associated ards , a ventilatory strategy that raising peep ( and plateau pressure ) while maintaining transpulmonary pressure < 25 cmh2o lessened the possibility of respiratory failure such that the patients recovered without the use of extracorporeal membrane oxygenation ( ecmo ) . on the other hand , all patients with a transpulmonary pressure > 25 cmh2o subsequently deteriorated and required ecmo support . in such cases , raising peep might have resulted in overstretch - induced lung injury . one study proposed that when optimizing ventilator settings for individual ards patients , peep should be adjusted based on esophageal pressure measurements . in those patients who exhibit excessively high pleural pressures and inadequately low peep , for example , if a 10 cmh2o peep was applied to a patient with 15 cmh2o pleural pressure at the end of expiration , his / her transpulmonary pressure would be -5 cmh2o , a level incapable of sustaining the patency of the alveoli . therefore , peep must be increased to a level that would result in positive transpulmonary pressure at the end of expiration . in an observational study , talmor et al . showed that optimization of ventilator settings basedon transpulmonary pressure values significantly improve oxygenation and respiratory system compliance . one must be aware that the onset of spontaneous breathing during mechanical ventilation can be harmful to some ards patients . spontaneous breathing during lung protective mechanical ventilation in patients with ards could induce a large negative deflection in pleural pressure . this large negative pleural pressure could , in turn , increase transpulmonary pressure , potentially inducing lung injury . yoshida et al . demonstrated in their animal study that spontaneous breathing superimposed on cv could induce lung injury even when plateau pressure was kept below 30 cmh2o . large negative pleural pressure could also increase a transcapillary pressure gradient , thereby causing pulmonary edema . increased work of breathing due to a large increase in negative pleural pressure may be associated with elevated inflammatory cytokine levels . administration of nmbas may be advisable to ease a patient s inspiratory effort in this way thus reducing transpulmonary pressure . papazian et al . have demonstrated in a randomized controlled trial ( rct ) that short - term treatment with nmbas ( < 48 hours ) reduced mortality rates in those patients with early , sepsis - induced severe ards . using an animal ards model , yoshida et al . demonstrated that whereas spontaneous breathing could worsen severely injured lungs , it could alleviate mildly injured lungs , since lung recruitment was only possible in the latter . it is worth noting that two identical tidal volume settings could result in different outcomes , depending on whether or not spontaneous breathing is permitted . yoshida et al . suggested that in cases of severe lung injury , strong spontaneous breathing efforts and high transpulmonary pressure can lead to increased rate of cyclic alveolar opening and collapse in those affected regions surrounding the diaphragm . however , the distributionof pleural pressure could be very inhomogeneous and unpredictable when expansion of lungs is inhomogenous . in rabbits with positive pressure ventilation , egan showed that the larger the amount of closed areas , the higher is the regional overdistension ( the ratio of inflated volume to frc ) of the remaining open ones . this study implies that if a considerable part of the lung suffers collapse , the transpulmonary pressure of the area adjacent to the collapsed tissue may become very high , because vacuum effect may be produced inside the chest wall by collapsed tissue . therefore , we must be aware that imposing positive airway pressure to the patient with imhomogenous lung while a patient is paralyzed from nmbas can cause much larger transpulmonary pressure than the pressure predicted in healthy lung . it is technically difficult to insert a catheter into the esophagus , and then properly position and calibrate it . when critically ill patients are in a supine position , measurements of esophageal pressure might result in artifacts associated with body position and pathologic conditions , thus rendering it less accurate and reliable . in a canine ards model pelosi et al . found a vertical gradient of the pleural pressure in a supine position . the esophageal pressures closely matched the actual pleural pressures at the surface of the mid - lung when the animal was placed in a supine position . however , the pleural pressures in the nondependentand dependent lung legions were 7 cmh2o lower and 4 cmh2o higher , respectively , than the esophageal pressures . however , when airway pressure was increased , the measured pleural pressure in all regions changed in similar fashion . this suggests that one could estimate , with some precision , variations in pleural pressure using the measured variation in esophageal pressure . in summary , the authors believe that the protective ventilatory strategy recommended in the sscg 2012 report is sub - optimal for some ards patients . for such patients , individual optimizations of ventilator settings the authors would also propose to further reduce tidal volume , use nmbas if necessary , and if possible , determine peep settings based on transpulmonary pressure for those ards patients who failed to improve using the sscg 2012-recommended ventilatory protocol .

the mortality rate among patients suffering acute respiratory distress syndrome ( ards ) remains high despite implementation at clinical centers of the lung protective ventilatory strategies recommended by the international guidelines for management of severe sepsis and septic shock , 2012 . this suggests that such strategies are still sub - optimal for some ards patients . for these patients , tailored use of ventilator settings should be considered , including : further reduction of tidal volumes , administration of neuromuscular blocking agents if the patient s spontaneous breathing is incompatible with mechanical ventilation , and adjusting positive end - expiratory pressure ( peep ) settings based on transpulmonary pressure levels .

INTRODUCTION MORTALITY OF ARDS PATIENTS IN THE ERA OF LUNG PROTECTIVE STRATEGIES HOW TO REDUCE MORTALITY OF ARDS PATIENTS? PLEURAL PRESSURE AND TRANSPULMONARY PRESSURE CONFLICT OF INTEREST

head and neck cancer is the sixth most common type of cancer in the world , representing about 6% of all cancer cases . worldwide , more than half a million head and neck cancer cases and 320,000 deaths due to head and neck cancer are estimated to occur each year , and it is the eighth cause of cancer mortality in the world . head and neck cancer has a higher incidence in older people , primarily due to its relationship with chronic exposure to tobacco smoking and alcohol drinking . fifty - seven per cent of all newly diagnosed malignancies and 71% of all cancer deaths occur in those 65 years of age . more than 40% of head and neck cancers occur in patients older than 65 years . with increasing life expectancy , it is estimated that by 2030 , nearly 70% of the cancer cases would be diagnosed in adults with age 65 years or older . in india , 30,831,190 males and 33,998,613 females are in the age group of 65 years and above , accounting for 5.5% of the nation s population . one of the most challenging tasks for the practicing oncologist today is the care of the elderly cancer patient . in recent years , management of cancer in elderly population has not been adequately addressed as these patients have often been under - represented in clinical trials of new cancer treatments and most clinical cancer trials have had arbitrary upper age limits . majority of elderly cancer patients are less likely to receive definitive or adequate cancer - directed therapy . this has been seen not only for head and neck cancers , but also for other common malignancies . advancing age may be associated with progressive loss of stress tolerance , decline in functional reserve of multiple organ systems , high prevalence of co - morbid conditions , limited socioeconomic support , reduced cognition , and higher prevalence of depression . however , aging is highly individualized , and chronologic age may not reflect the functional reserve and life expectancy of an individual . compliance of elderly patients to intensive multimodality cancer therapy can be challenging due to significant treatment - related toxicities , the logistical demands involved in treatments , and unplanned treatment gaps introduced between the treatments . all these factors can decrease treatment compliance and prolong overall treatment time , which is known to be a detriment to therapeutic outcomes to cancer directed therapy . the present study evaluated compliance of elderly hnscc to cancer - directed therapy . to our knowledge this study is one of the first studies evaluating compliance and overall treatment time of elderly hnscc patients to cancer - directed therapy from a developing nation . our study included a total of forty - seven elderly hnscc patients ( age 65 years or older ) referred for radiotherapy from the various multi - disciplinary clinics at our institute in one unit of department of radiotherapy from july 2010 to june 2011 . the patients were assessed per stage and site of the disease , general condition , performance status , and any pre - existing co - morbidities . for the purpose of study , compliance was defined as all patients who were able to complete the stipulated treatment as intended at the primary clinic . the key factors evaluated for compliance and overall treatment time included date of registration at cancer centre , radiotherapy registration date , surgery date , radiation start and completion date , and treatment completion date . compliance was evaluated with regard to age , stage , general condition , performance status , presence of co - morbidity , and intention of treatment . for possible factors affecting compliance , statistical analysis was done using stata 9.1 software , chi - square / fischer s exact test were used to see the strength association between the two categorical variables . for all compliant patients , overall treatment time was calculated from the day of initiation of cancer - directed therapy to completion of treatment . further , to study the pattern of non - compliance , patients were divided into early non - compliance ( patients that were non - compliant during the investigation and staging work up period ) , mid - course non - compliance ( patients non - compliant after complete diagnostic work up and treatment decision , but before radiation delivery ) , and late non - compliance ( patients non - compliant during radiation delivery ) . in this cohort of 47 patients , treatment decision taken at the multi - disciplinary clinic was for radical treatment in 68% ( 32/47 patients ) , whereas the remaining 32% ( 15/47 patients ) were planned to receive palliative treatment . radical treatment included either radical radiation with or without chemotherapy in 55% ( 26/47 ) of the patients or surgery followed by postoperative radiotherapy in 13% ( 6/47 ) of the patients . in this retrospective analysis , patient and treatment characteristics the salient features were that majority ( 42/47 ) of the elderly hnscc presented in loco - regionally advanced stage ( iii iv ) , the most common site of malignancy was oropharynx ( 21/47 ) , followed by oral cavity ( 11/47 ) , larynx ( 9/47 ) , and hypopharynx ( 6/47 ) . with regard to age distribution , 72% ( 34/47 ) of the patients were between the age group of 6574 years , whereas ( 13/47 ) 28% were 75 or older . general condition was fair in most of the elderly patients ( 38/47 ) ; only ( 5/47 ) of the elderly patients were in good general condition , while the remaining ( 4/47 ) were in poor general condition . out of the 47 elderly patients , analysis of compliance to treatment decision revealed that 62% ( 29/47 ) of the elderly hnscc patients were compliant to cancer - directed therapy , whereas 38% ( 18/47 ) of the patients were not able to complete the stipulated treatment . for all compliant patients , overall treatment time was calculated from the day of initiation of cancer directed therapy to completion of treatment . the median overall treatment time for patients subjected to radical radiation therapy was 52 ( range 4799 ) days , and for radical surgery and adjuvant radiotherapy was 109 ( 95190 ) days . twenty - two per cent of the elderly patients had one or more associated co - morbidities . factors affecting compliance and the association between the categorical variables using statistical analysis by chi - square / fischer exact test are mentioned in table 2 . compliance to treatment decreased as the stage of disease increased ( 80% for early stage versus 62% in loco - regionally advanced stage ) . compliance was better for elderly patients with good to fair general condition ( 63% ) versus 50% patients with poor general condition . fifty - seven per cent of the patients having one or more co - morbidity were compliant to treatment , whereas compliance increased to 63% when patients had no co - morbidity ( p value ns ) . compliance was similar with regard to intention of treatment ( radical 63% vs. palliative 60% ) , advancing age ( age group 6574years 62% vs. 75 or older 62% ) . a further analysis on pattern of non - compliance revealed that early non - compliance to therapy was seen in 5% ( 1/18 ) of the patients , whereas majority ( 14/18 ) of elderly patients showed mid - course non - compliance . only 17% ( 3/18 ) of the patients were non - compliant to treatment during the course of radiation delivery . non - compliance to treatment has been reported to determent all parameters of disease control and survival . the assessment of patients receiving definitive concurrent chemoradiation for either locally advanced or medically inoperable nsclc treated in rtog studies revealed that prolongation of ott was associated with significantly poorer survival , with a 2% increase in the risk of death for each day of treatment prolongation . interruptions more than one week during post - operative irradiation of breast cancer adversely affected overall survival in these patients . for head and neck cancer analysis of treatment compliance in all radiation therapy oncology group ( rtog ) prospective randomized trials between 1978 and 1991 , reported a significantly reduced three - year loco - regional control ( 13% vs. 27% ) and three - year absolute survival ( 13% vs. 26% ) in patients with prolongation of treatment by 14 days or more . similarly , numerous studies have demonstrated non - compliance to treatment protocols impedes local control of head and neck cancers . these studies describe a loss of local control of 0.4% to 2.9% for each day the treatment course is prolonged , with an average of 1.7% per day . a recent analysis of critical impact of radiotherapy compliance in treatment of advanced head and neck cancers , the trans - tasman radiation oncology group ( trog 02.02 ) trial , revealed that patients with major deficiencies in their treatment plans had a markedly inferior outcome(i.e . , for deviations versus compliance as regards the treatment results2 years overall survival 50% vs. 70% and freedom from loco - regional failure , 78% vs. 54% ) . head and neck squamous cell carcinoma may have geographical variation with regard to site and stage at presentation , treatment compliance , and survival . amidst the pre - existing geographical variation with inferior treatment compliance and survival in the asian population , compliance of elderly hnscc patients is of utmost importance , as advancing age may be construed ( i ) with progressive loss of stress tolerance , ( ii ) decline in functional reserve of multiple organ systems , ( iii ) high prevalence of co - morbid conditions , ( iv ) limited socioeconomic support , ( v ) reduced cognition , and ( vi ) higher prevalence of depression all of which may be the reasons for poor treatment compliance . in the present study , elderly hnscc patient compliance in our study is comparable to overall treatment compliance for hnscc patients previously reported from the subcontinent . overall treatment time was prolonged for patients subjected to radical surgery and adjuvant radiotherapy ( median ott 109 days ) , whereas there were no such delays for patients receiving radical radiotherapy ( median ott 52 days ) . elderly patients with cancer may often have other preexisting medical co - morbidities especially related to chronic exposure to tobacco smoke and alcohol . the secondary aim of the study was to see the burden of co - morbidities in elderly hnscc patients . associated co - morbidities were present in 23% of the elderly hnscc patients , and this burden of co - morbidity was less when compared to those reported in literature . presence of co - morbidity in these patients was associated with decreased compliance ; however , the difference in compliance was not statistically significant . similarly , the study did not find a statistical significant association of compliance with age , stage , performance condition , general condition , and intent of treatment . the factors influencing compliance is elderly patients remain complex and need to be further elucidated . the highlights of this study can be : the pattern of elderly hnscc patient non - compliance revealed that majority of the elderly patients ( 83% ) were non - compliant to treatment before the initiation of radiation therapy . this was a bit surprising result as elderly patients were expected to be non - compliant to treatment during the course of radiation therapy because of development of increased acute radiation morbidity , worsening of performance status , and poor tolerance.to our knowledge the present study is the first study to report early to mid - course non - compliance to cancer - directed therapy in elderly hnscc patients . as per available records , the reasons for this early to mid - course non - compliance finding the probable explanation could be a long median radiation waiting of two months at our cancer center , during which some elderly patients may have preferred early cancer - directed therapy at some other cancer centers . further the possibility of upstaging and disease progression during the radiation waiting period can not be ignored ( majority of the patients were already in a loco - regionally advanced stage ) . this aspect of early to mid - course non - compliance to cancer - directed treatment that emerged as predominant factor in our study needs to be further evaluated , especially in developing countries where there is much load on the available machines and where the cancer patient population has probably outnumbered the available resources . the pattern of elderly hnscc patient non - compliance revealed that majority of the elderly patients ( 83% ) were non - compliant to treatment before the initiation of radiation therapy . this was a bit surprising result as elderly patients were expected to be non - compliant to treatment during the course of radiation therapy because of development of increased acute radiation morbidity , worsening of performance status , and poor tolerance . to our knowledge the present study is the first study to report early to mid - course non - compliance to cancer - directed therapy in elderly hnscc patients . as per available records , the reasons for this early to mid - course non - compliance finding could not be evaluated . the probable explanation could be a long median radiation waiting of two months at our cancer center , during which some elderly patients may have preferred early cancer - directed therapy at some other cancer centers . further the possibility of upstaging and disease progression during the radiation waiting period can not be ignored ( majority of the patients were already in a loco - regionally advanced stage ) . this aspect of early to mid - course non - compliance to cancer - directed treatment that emerged as predominant factor in our study needs to be further evaluated , especially in developing countries where there is much load on the available machines and where the cancer patient population has probably outnumbered the available resources . limitations of our study include : retrospective nature of the study and small number of patients.in view of the retrospective nature of the study , all factors for non - compliance could not be elicited . other factors which could influence non - compliance and were not available include ( a ) poor socio - economic status , ( b ) lack of social or family support , ( c ) distance from the treatment centre.elderly patients were not subjected to comprehensive geriatric assessment before treatment decisions.while the medical record documented non - compliance , the underlying reasons behind non - compliance could not be determined . retrospective nature of the study and small number of patients . in view of the retrospective nature of the study other factors which could influence non - compliance and were not available include ( a ) poor socio - economic status , ( b ) lack of social or family support , ( c ) distance from the treatment centre . elderly patients were not subjected to comprehensive geriatric assessment before treatment decisions . while the medical record documented non - compliance , the underlying reasons behind non - compliance could not be determined . in recent years , the improvements in treatment modalities and techniques have made the delivery of cancer - directed therapy safe and feasible in elderly hnscc patients . non - compliance to treatment in these patients could pose an obstacle to delivery of effective health care . our findings from india in a small cohort of elderly hnscc patients showing the issues of early mid - course non - compliance pattern need to be evaluated further . a larger prospective study , performing a comprehensive geriatric assessment and evaluating the role of comprehensive interventions combining cognitive , behavioural , and affective components to improve compliance in elderly patients , is warranted .

backgroundtreatment compliance of elderly patients to intensive multi - modality cancer therapy can be challenging and has not been adequately addressed in developing countries . the present study evaluated compliance of elderly head and neck carcinomas patients to cancer - directed therapy.methodsforty-seven elderly hnscc patients were evaluated in the present study . patients were assessed as per stage and site of disease , general condition , performance status , and any pre - existing co - morbidities . compliance was defined as patients who were able to complete cancer therapy as intended at primary clinic . non - compliance to therapy was stratified as early , mid- and late - course non - compliance . statistical analysis was done using stata 9.1 software , chi - square / fischer s exact test to see strength of association between two categorical variables that could possibly affect compliance in elderly patients.resultssixty-eight per cent of elderly patients were subjected to radical treatment , majority ( 42/47 ) presented in loco - regionally advanced stage ( iii iv ) , most common site of malignancy was oropharynx ( 21/47 ) . sixty - two per cent of elderly hnscc patients were compliance to cancer therapy . median overall treatment time for patients subjected to radical radiation therapy was 52 ( range 4799 ) days , and for radical surgery and adjuvant radiotherapy was 109 ( 95190 ) days . compliance to therapy for elderly hnscc patients was not significantly associated with advanced stage , poor general condition , intent of treatment or presence of co - morbidity . as regards to non - compliance , majority ( 14/18 ) of elderly patients showed mid - course treatment non-compliance.conclusionsnearly two - thirds of elderly head and neck carcinoma patients were compliant to cancer - directed therapy .

INTRODUCTION METHODS RESULTS DISCUSSION CONCLUSION

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abstractmacrocyclization can be used to constrain peptides in their bioactive conformations , thereby supporting target affinity and bioactivity . in particular , for the targeting of challenging protein protein interactions , macrocyclic peptides have proven to be very useful . available approaches focus on the stabilization of helices , which limits their general applicability . here we report for the first time on the use of ringclosing alkyne metathesis for the stabilization of an irregular peptide secondary structure . a small library of alkynecrosslinked peptides provided a number of derivatives with improved target affinity relative to the linear parent peptide . in addition , we report the crystal structure of the highestaffinity derivative in a complex with its protein target 1433. it can be expected that the alkynebased macrocyclization of irregular binding epitopes should give rise to new scaffolds suitable for targeting of currently intractable proteins .

Supporting information