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cardiovascular diseases ( cvds ) are multifactorial disorders which are characterized by multiple metabolic dysfunctions . epidemiologic studies indicate that increased serum lipids profiles , elevated plasma fibrinogen , and coagulation factors play critical roles . it has been estimated that this number will reach 24.8 million people in 2030 worldwide . cvds are considered to be the most common cause of death ( 2545% mortality ) and the fifth most common cause of disability . according to the world health organization statistics , the most important risk factors include tobacco usage , high blood pressure , alcoholism , high cholesterol , low fruit , and vegetable consumption , lack of appropriate physical activity and obesity . iran is the fifth country , throughout the world in terms of having high blood pressure related diseases . high blood pressure and risk factors in cvds are the main causes of death and mortality in iran and according to the report of iranian health profile survey in 1999 , the prevalence of blood pressure in adults over 30 years increased by 1245% . previous studies showed that 46% of the total deaths in 18 provinces of iran were caused by cvd . approximately , 6.6 million people between the ages of 2564 have high blood pressure , and it is estimated that 12 million persons between 25 and 46 years of age are at increased risk of cvd . oxidative stress has a pathological role in cvd , particularly its effects on low - density lipoprotein - cholesterol ( ldl - c ) oxidation , and eventually leads to inflammation status . epidemiologic studies noticed that diets rich in vegetables and fruits are affiliated with a lower risk of mortality , particularly cardiovascular diseases related deaths . it has also been indicated that the benefits of vegetable and fruit intake appear to be related to cvd . one of the most well - known sources of anti - cvd phytochemicals is garlic , which plays an effective role in the suppression and treatment of cvds . even though pharmacological interventions cause a significant reduction in high blood pressure and dyslipidemia , lifestyle modification and correcting the dietary regime can be a key step in the management of cvd . among foods which are effective in reducing inflammation status and ultimately cardiovascular parameters , garlic is used in various forms such as raw garlic , powdered garlic tablets , or extracted oil . lipid levels , blood pressure , reactive oxygen species , and other cardiovascular risk factors affected by garlic will decline that has been confirmed by several studies . epidemiological studies have demonstrated that increased consumption of polyphenols such as flavonoids and phenylpropanoids phytochemicals that are present in fruits and vegetables are associated with reduced risk of cvd . animal studies showed that erycosytryn and hesperidin have antioxidant properties , and they can decrease oxidative stress . several studies have been conducted concerning the relationship between garlic and lemon juice in reducing inflammatory biomarkers and lipid levels in patients with cvd , separately . however , the effects of garlic and lemon juice mixture on the indicators of cvds have not yet been analyzed . regarding the increasing prevalence of these diseases , we examined the effects of garlic and lemon juice mixture on some cvd risk factors such as total cholesterol ( tc ) , ldl - c , systolic blood pressure ( sbp ) , and diastolic blood pressure ( dbp ) and fibrinogen in people 3060 years old with moderate hyperlipidemia . in a parallel - designed randomized controlled clinical trial , hyperlipidemic patients 3060 years were recruited from isfahan cardiovascular research center ( icrc ) from november to december 2013 . considering the proposed formula for parallel - design randomized trials , given the type i error of 5% , the study power of 90% considering tc as the key variable . subjects were eligible if they had a fasting tc and ldl - c levels between 200240 mg / dl and 100160 mg / dl , respectively . participants were excluded if they were suffering from any other chronic diseases , including heart diseases , kidney failure , lung dysfunction , thyroid disorders , gastrointestinal disorders , rheumatoid arthritis , and other liver diseases such as hepatitis or fatty liver . participants that were pregnant lactating , or that smoked were also excluded . in addition , participants body weight should not have changed in the last 2 months . subjects were excluded if they were on medication which affected blood sugar , lipid profile , blood pressure , or anti - inflammatory markers . participants should not have had any food allergy or sensitivity to garlic or lemon juice consumption . individuals with a history of following any special diet ( vegetarian / vegan , etc . ) were excluded as well . from the revision of the medical records available in icrc , 800 patients were chosen . these subjects were invited for screening as they met nearly all of the inclusion criteria . after the screening process , 120 eligible volunteers enrolled into the study and were randomly allocated to four groups : ( 1 ) mixed with garlic and lemon juice group , ( 2 ) garlic group , ( 3 ) lemon juice group and ( 4 ) control group . of the 120 subjects randomly assigned to treatment , two subjects in the control group in the beginning of intervention were dropped out from the study because of their busy schedule and over the duration of the study , two subjects in the mixed group because of pregnancy and three subjects because of the gastric problem were eliminated from this study . one subject in the garlic group because of pregnancy and two subjects because of travel were eliminated . finally , 112 subjects continued until the end of the study . the participant flow diagram is shown in figure 1 . all participants provided informed written consent . this trial was approved by the isfahan university of medical sciences ( 392436 ) and was registered in clinical trials center 's website address ( www.irct.ir ) ( code : irct2015020720986n1 ) . in a parallel - designed randomized controlled clinical trial , professor of biostatistics did the random allocations . in this study , group blinding was not possible because the intervention involved taking or not taking garlic or lemon juice . group 1 received 20 g of raw garlic daily plus 1 tablespoon of lemon juice ( n = 30 ) , group 2 received 20 g garlic daily ( n = 30 ) , group 3 received 1 tablespoon of lemon juice daily , 2 h after dinner ( n = 30 ) , and group 4 did not receive garlic , or lemon juice ( n = 30 ) for 8 weeks . participants were asked to use garlic and lemon juice for 8 weeks . when the participants were given packets of garlic and bottle of lemon juice , we were wanted not to change their lifestyle , physical activity , or diet during the study . participants were asked to bring back the empty packets and bottles at the final session to help assure compliance . they were instructed to avoid change in diet and physical activity , so their diet and physical activity were similar , and it was checked by researchers and their records . all participants provided 3 physical activity records ( 3 days ) and 3 dietary records ( 3 days ) during the intervention at 0 , 4 , and 8 weeks . they provided 3 days of physical activity records and 3 days of dietary records ( 1 weekend day and 2 weekdays ) to certify they preserved their usual diet and activity levels during the intervention . the average amount ( g ) of food intake data , based on 3 , 3 days of dietary records , was joined with nutritionist iv software ( n - squared computing , cincinnati , oh , usa ) to conclude nutrient intake data . the nutrient database of nutritionist iv software was based on the united states department of agriculture food composition table , reclaimed for iranian foods . physical activity was declared as metabolic equivalents per hour per day . to quantify measures of blood sampling and metabolic profiles after overnight fasting , blood samples were collected from patients between 7 and 10 am . after centrifuging at 4c at 500 g for 10 min , tubes were frozen at 80c until analysis . ) . tc , triglyceride ( tg ) , high - density lipoprotein ( hdl ) were measured by photometric enzyme assay ( pars azmoon , iran ) and ldl were measured . both intra- and inter - assay coefficients of variation were < 5% for all of the used measurement kits . biochemical parameters were measured at baseline and after 8 weeks of the intervention . when participants were in the comfortable seat position , after 10 min rest body weight and height were measured with light clothing and without shoes to the nearest 0.1 kg and 0.1 cm , respectively and body mass index ( bmi ) was calculated . except height , other physical characteristics and blood pressure were measured 2 times , in study week 0 , 8 , respectively . data were analyzed using spss ( version 18 , spss , chicago , il , usa ) , and a two - sided p < 0.05 was considered to be significant . to quantify measures of metabolic profiles , blood sampling was done at the beginning of the study to establish a baseline and after 8 weeks of intervention . to ensure the normal distribution of variables the difference between the four groups based on outcome variables such as dietary intake , physical activity , and metabolic profile were compared using ancova test controlling for a baseline of these variables and age / gender . baseline general characteristics were examined using chi - square for categorical variables and one - way anova for continuous variables . we used tukey 's post hoc comparisons to identify pairwise differences when we reached a significant finding in anova . in a parallel - designed randomized controlled clinical trial , hyperlipidemic patients 3060 years were recruited from isfahan cardiovascular research center ( icrc ) from november to december 2013 . considering the proposed formula for parallel - design randomized trials , given the type i error of 5% , the study power of 90% considering tc as the key variable . subjects were eligible if they had a fasting tc and ldl - c levels between 200240 mg / dl and 100160 mg / dl , respectively . participants were excluded if they were suffering from any other chronic diseases , including heart diseases , kidney failure , lung dysfunction , thyroid disorders , gastrointestinal disorders , rheumatoid arthritis , and other liver diseases such as hepatitis or fatty liver . participants that were pregnant lactating , or that smoked were also excluded . in addition , participants body weight should not have changed in the last 2 months . subjects were excluded if they were on medication which affected blood sugar , lipid profile , blood pressure , or anti - inflammatory markers . participants should not have had any food allergy or sensitivity to garlic or lemon juice consumption . individuals with a history of following any special diet ( vegetarian / vegan , etc . ) were excluded as well . from the revision of the medical records available in icrc , 800 patients were chosen . these subjects were invited for screening as they met nearly all of the inclusion criteria . after the screening process , 120 eligible volunteers enrolled into the study and were randomly allocated to four groups : ( 1 ) mixed with garlic and lemon juice group , ( 2 ) garlic group , ( 3 ) lemon juice group and ( 4 ) control group . of the 120 subjects randomly assigned to treatment , two subjects in the control group in the beginning of intervention were dropped out from the study because of their busy schedule and over the duration of the study , two subjects in the mixed group because of pregnancy and three subjects because of the gastric problem were eliminated from this study . one subject in the garlic group because of pregnancy and two subjects because of travel were eliminated . finally , 112 subjects continued until the end of the study . the participant flow diagram is shown in figure 1 . all participants provided informed written consent . this trial was approved by the isfahan university of medical sciences ( 392436 ) and was registered in clinical trials center 's website address ( www.irct.ir ) ( code : irct2015020720986n1 ) . in a parallel - designed randomized controlled clinical trial , professor of biostatistics did the random allocations . in this study , group blinding was not possible because the intervention involved taking or not taking garlic or lemon juice . group 1 received 20 g of raw garlic daily plus 1 tablespoon of lemon juice ( n = 30 ) , group 2 received 20 g garlic daily ( n = 30 ) , group 3 received 1 tablespoon of lemon juice daily , 2 h after dinner ( n = 30 ) , and group 4 did not receive garlic , or lemon juice ( n = 30 ) for 8 weeks . participants were asked to use garlic and lemon juice for 8 weeks . when the participants were given packets of garlic and bottle of lemon juice , we were wanted not to change their lifestyle , physical activity , or diet during the study . participants were asked to bring back the empty packets and bottles at the final session to help assure compliance . they were instructed to avoid change in diet and physical activity , so their diet and physical activity were similar , and it was checked by researchers and their records . all participants provided 3 physical activity records ( 3 days ) and 3 dietary records ( 3 days ) during the intervention at 0 , 4 , and 8 weeks . they provided 3 days of physical activity records and 3 days of dietary records ( 1 weekend day and 2 weekdays ) to certify they preserved their usual diet and activity levels during the intervention . the average amount ( g ) of food intake data , based on 3 , 3 days of dietary records , was joined with nutritionist iv software ( n - squared computing , cincinnati , oh , usa ) to conclude nutrient intake data . the nutrient database of nutritionist iv software was based on the united states department of agriculture food composition table , reclaimed for iranian foods . physical activity was declared as metabolic equivalents per hour per day . to quantify measures of blood sampling and metabolic profiles after overnight fasting , blood samples were collected from patients between 7 and 10 am . after centrifuging at 4c at 500 g for 10 min , tubes were frozen at 80c until analysis . ) . tc , triglyceride ( tg ) , high - density lipoprotein ( hdl ) were measured by photometric enzyme assay ( pars azmoon , iran ) and ldl were measured . both intra- and inter - assay coefficients of variation were < 5% for all of the used measurement kits . biochemical parameters were measured at baseline and after 8 weeks of the intervention . when participants were in the comfortable seat position , after 10 min rest body weight and height were measured with light clothing and without shoes to the nearest 0.1 kg and 0.1 cm , respectively and body mass index ( bmi ) was calculated . except height , other physical characteristics and blood pressure were measured 2 times , in study week 0 , 8 , respectively . data were analyzed using spss ( version 18 , spss , chicago , il , usa ) , and a two - sided p < 0.05 was considered to be significant . to quantify measures of metabolic profiles , blood sampling was done at the beginning of the study to establish a baseline and after 8 weeks of intervention . to ensure the normal distribution of variables the difference between the four groups based on outcome variables such as dietary intake , physical activity , and metabolic profile were compared using ancova test controlling for a baseline of these variables and age / gender . baseline general characteristics were examined using chi - square for categorical variables and one - way anova for continuous variables . we used tukey 's post hoc comparisons to identify pairwise differences when we reached a significant finding in anova . from the review of the medical records available in icrc , 800 patients were selected . these subjects were called for screening as they met almost all of the inclusion criteria . after randomization , 8 person withdrew due to travel ( n = 2 ) , gastric problem ( n = 3 ) , and pregnancy ( n = 3 ) . the distribution of participants in terms of sex , weight , bmi was not significantly different between the four intervention groups . comparison of baseline metabolic profiles such as serum tc , ldl - c , hdl - c , triglyceride and fibrinogen levels , sbp and dbp revealed no significant difference between the groups . no significant differences were found between groups regarding food groups , carbohydrates , saturated fatty acids , or the majority of micronutrients as derived from the food record . they were instructed to avoid change in their physical activity and were checked by researchers . we did not find any significant difference in the physical activity levels between the four intervention groups . energy , nutrient intake , food groups , and physical activity during the intervention period were shown in table 2 . baseline characteristics of study participants nutrient intake and physical activity of study participants throughout the study changes of all variables within mixed and garlic groups were significant before and after intervention ( p < 0.001 ) . the effects of intervention within groups on general characteristics and metabolic profiles are shown in table 3 . we found a significant decrease in tc , ldl - c and fibrinogen in the mixed group in comparison with other groups ( p < 0.001 ) . no significant changes were observed regarding serum hdl and tg concentration in the mixed group in comparison with garlic and lemon juice groups ( p = 0.08 ) but this change was significant in mixed group compared with control group ( p = 0.02 ) ( p = 0.001 ) ; respectively . a greater reduction in systolic and diastolic blood pressure was observed in the mixed group compared with the lemon juice and control groups ( p = 0.01 ) ( p = 0.02 ) ; respectively . also a great reduction in bmi was observed in the mixed group compared with the lemon juice and control groups ( p = 0.04 ) . no significant differences in bmi , systolic and diastolic blood pressure between mixed and garlic groups were observed . the effects of a mixture of garlic and lemon juice , garlic and lemon juice on general characteristics and metabolic profiles are shown in table 4 . the effect of intervention on metabolic profiles and anthropometric measurements within groups the comparison on changes of metabolic profiles and anthropometric measurements between four groups multiple useful cardiovascular effects have been discovered including enhancement of fibrinolytic activity , lowering of blood pressure , reduction in cholesterol , and triglyceride . the results showed that combination of garlic and lemon juice significantly reduced serum tc , ldl - c , and blood pressure . garlic , such as many other food additives , gained substantial interest due to its effects on lipid levels . many human and animal studies have investigated the effects of garlic on lipid levels , fasting blood sugar , bmi , fibrinogen , and sbp and dbp but no studies have yet been performed on the effects of a combination of garlic and lemon juice consumption on these biomarkers . since 1993 , 25 clinical trials have been published that have investigated the hypolipidemic effects of garlic . eleven of the studies showed that garlic reduced serum cholesterol levels , but fourteen studies showed no effect on lowering cholesterol . in addition , different extracts of garlic alone have been demonstrated to lower serum cholesterol , triglycerides , and ldl in rodents and humans . maha and khalil showed that adding 8% raw garlic along with 2% cholesterol to the diet of rats decreased plasma tc and ldl - c . a number of human studies have shown that raw garlic favorably affects important risk factors for cvd . an intake of the half to one clove of garlic per day lowers cholesterol levels approximately 10% . mechanisms that explain the observed effects of garlic include a decrease in cholesterol absorption , cholesterol , and fatty acid synthesis . however , there is some evidence that garlic powder does not lower cholesterol levels , which may reflect a loss of active compound(s ) during processing . the formation of these active compounds is impressed by crushing garlic , a period of the drying process , the temperature at which garlic is dried , and humidity . . direct measurements of enzyme activity have demonstrated that garlic and various constituents prohibit human 3-hydroxy-3-methylglutaryl - coenzyme a ( hmg - coa ) reductase and squalene monooxygenase , enzymes required in cholesterol biosynthesis . this prohibition of hmg - coa reductase by garlic has also been supported in a recent study . in addition , ldl - c reduction by garlic extract may be owing to decreases of hepatic 3-hydroxy-3-methylglutaryl - coa reductase , cholesterol 7-hydroxylase , pentose - phosphate pathway activities , cholesteryl ester transfer protein activity , microsomal triglyceride transfer protein , enhanced bile acid excretion , and prohibition of hepatic fatty acid synthesis by allicin and/or other components . it should also be emphasized that certain garlic preparations ( e.g. , garlic oils ) do not indicate the degree of cholesterol lowering seen with specific powdered formulations . in this study , we did not observe significant changes in hdl - c levels between groups . aouadi showed that adding 10% fresh crushed garlic and 2% cholesterol to diet led to significant reduction in ldl - c levels , and increased hdl - c levels in rats . the present study showed that fasting blood glucose showed no significant change due to consumption of a combination of garlic and lemon juice . however , in the lemon juice group after the intervention , we observed a significant increase in fasting blood glucose compared with other groups . mohammadi and abbas showed that fasting blood glucose levels decreased in garlic group compared with hypercholesterolemia group , but this decrease was not significant . ali et al . reported that garlic administration has no effect on blood glucose , similar to this study . in our study , there was a significant reduction in mean sbp and dbps in the garlic plus lemon juice group when compared with control and lemon juice groups . since 1993 , some studies have been published on the effects of garlic on blood pressure . in one study in contrast of this , a meta - analysis published in 2001 represented that garlic has no considerable effects on blood pressure . the moderate subside of blood pressure with garlic may be because of increased nitric oxide production and a more vasodilators state . g - glutamylcysteines are compounds discovered in garlic , and these may lower blood pressure as illustrated by their ability to prohibit angiotension - converting enzyme in vitro . in this study , we observed a significant reduction in triglycerides in the mixed group compared with control group . however , no significant changes were observed in the mixed group compared with the garlic and lemon juice groups . in our study , we observed a significant reduction in fibrinogen in the mixed group compared with other groups . in some studies contrarily , a study published in 1998 concluded that garlic has no effects on fibrinogen . one important difference between our study and these other studies was the combination of garlic and lemon juice . even though the subjects were instructed to consume only 20 g garlic and 1 tablespoon of lemon juice , some significant effects were seen for the mentioned variables . in the present study , we provided garlic and lemon juice for participants and did not recommend a specific diet . the present study has some other potential limitations ; it can not suggest appropriate doses of garlic and lemon juice for people with hyperlipidemia . to establish this , other studies examining the effects of different doses of garlic and lemon juice are required . in conclusion , a combination of garlic and lemon juice resulted in an improvement in lipid levels and blood pressure of people with hyperlipidemia . further studies to determine the appropriate doses of garlic and lemon juice for these patients are warranted . in addition , the effect of a combination of garlic and lemon juice might be different based on the degree of hyperlipidemia . therefore , studies are needed to examine the effects of garlic plus lemon juice according to the degree of hyperlipidemia . this study was provided by department of clinical nutrition , school of nutrition and food sciences , isfahan , iran , grant number : 392435 . this study was provided by department of clinical nutrition , school of nutrition and food sciences , isfahan , iran , grant number : 392435 .

background : this study was performed to effects of garlic and lemon juice mixture on lipid profile and some cardiovascular risk factors in people 3060 years old with moderate hyperlipidemia.methods:in a parallel - designed randomized controlled clinical trial , a total of 112 hyperlipidemic patients 3060 years , were recruited from isfahan cardiovascular research center . people were selected and randomly divided into four groups . control blood samples were taken and height , weight , and blood pressure were recorded . ( 1 ) received 20 g of garlic daily , plus 1 tablespoon lemon juice , ( 2 ) received 20 g garlic daily , ( 3 ) received 1 tablespoon of lemon juice daily , and ( 4 ) did not receive garlic or lemon juice . a study technician was done the random allocations using a random numbers table . all participants presented 3 days of dietary records and 3 days of physical activity records during 8 weeks . blood samples were obtained at study baseline and after 8 weeks of intervention.results:results showed a significant decrease in total cholesterol ( changes from baseline : 40.8 6.1 , p < 0.001 ) , low - density lipoprotein - cholesterol ( 29.8 2.6 , p < 0.001 ) , and fibrinogen ( 111.4 16.1 , p < 0.001 ) in the group 1 , in comparison with other groups . a greater reduction in systolic and diastolic blood pressure was observed in group 1 compared with the groups 3 and 4 ( 37 10 , p = 0.01 ) ( 24 1 , p = 0.02 ) ; respectively . furthermore , a great reduction in body mass index was observed in the mixed group compared with the lemon juice and control groups ( 1.6 0.1 , p = 0.04).conclusions : administration of garlic plus lemon juice resulted in an improvement in lipid levels , fibrinogen and blood pressure of patients with hyperlipidemia .

INTRODUCTION METHODS Participants Study design Variables assessment Statistical analysis RESULTS DISCUSSION CONCLUSIONS Financial support and sponsorship Conflicts of interest

cervical cancer is the second most common cancer among iranian women.1 78% of cervical cancer happens in developing countries.23 the incidence of cervical cancer in east azerbaijan of iran , in 2003 - 2004 was 5.11 in 100000 , while it was 11.9 in 100000 for high grade and 3.68 in 100000 for low grade pre - cancerous lesions.4 about half a million cases of invasive cervical cancer are diagnosed annually.56 the risk factors for cervical cancer are : beginning sexual intercourse at early age , multiple sexual partners , history of hpv infection and genital wart in women and her partners , smoking , immune deficiency , multiparity , repetitive sexual transmitted disease , herpes simplex virus ( hsv2 ) , exposure of uterus to diethylstilbestrol ( des ) , history of intraepithelial lesion , familial history of cervical cancer , partner with penis cancer , cervical cancer in other partners of husband , low hygiene condition , and using oral contraception for a long time.4789 cervical cancer has a long pre - invasive period , so it is preventable.10 dysplasia usually has no clinical sign and its diagnosis is often made based on cytological findings using the pap smear test.8 according to agency for healthcare research and quality ( ahrq ) , the sensitivity of conventional cytology testing in detecting precancerous lesions was 51% and for diagnosis of cervical intraepithelial neoplasia grade 1 , ( cin1 ) , 2 was 47 - 62% and its specificity was 59 - 60%.9 about 30% of new cases of cervical cancer , each year , occur in women that had pap smear but due to errors in sampling , fixation , and interpretation , it has been incorrectly reported as normal , moreover in developing countries there is often lack of necessary resources to use pap smear as a screening tool for cervical abnormalities.8 because the burden of cervical cancer is high , the alternative techniques have been sought . recently , interest in direct visual inspection with acetic acid ( dvi ) has been increased . dvi method does n't need laboratory facilities and its result is identify in the same visit , so this causes save the cost and time of patients.8 numerous studies have been conducted on its sensitivity and specificity to detect cervical lesions when compared with other techniques in which its sensitivity ranged between 66 - 96% and specificity between 64 - 98%.11 the studies suggest that dvi could be a primary screening tool , with low biopsy rate especially in low - resource settings or where cytological testing services are suboptimal.12 this study was conducted to compare the results of pap smear and dvi method in diagnosis of cervical premalignant lesions . this cross - sectional study was carried out in alzahra therapeutic educational centre , tabriz , iran in 2013 on 1000 women . sample size using lin naing software and considered p1 = 0.8 , p2 = 0.65 , = 0.05 and = 90% was determined as 827 and due to probability of participant 's drop out , sample size was increased to 1000 . women aged 20 - 50 years , married and volunteers or referred by health centres or physicians for screening of cervical cancer were chosen . the exclusion criteria were pregnancy or doubt about it , hysterectomy , previous cervical cancer or pre - cancerous lesions , cryotherapy , cautery or cone biopsy for treatment of cervical disease , previous radiotherapy , abnormal vaginal bleeding , the history of genital warts or herpes , using medication for genital disease and early menopause . after approval from the research ethics committee of tabriz university of medical science , the investigator went to alzahra therapeutic- educational centre and invited women that referred for pap smear and had inclusion criteria for study . for this purpose , researcher completed the check list of participant 's selection , then , the made questionnaire and approval form was given to participants in a private place and was asked to answer the questions . the content validity was used to acquire the scientific validity of socio- demographic and midwifery information questionnaire . for this purpose , the questionnaire was given to 7 professors of tabriz university of medical science to evaluate the content of questionnaire . after collecting their idea , needed changes was done and finally used for study . in this study , first , the form of socio - demographic and midwifery information was completed , then pap smear and dvi was done for all women . for this purpose , participants went on a gynecological bed and in lithotomic position . first , pap smear was done with a plastic spatula for exocervix and a cytobrush for endocervix and sample expanded on a lamella and fixed with fixation solution ( alcohol 95% ) . then , the cervix exposed with 5% acetic acid by cotton swab for 30 seconds and observed under adequate light with magnifying glass . presence of acetowhite region in the cervix with sharp borders , abnormal finding in cervix such as polyp , leukoplakia or invasive cancer were considered as positive dvi . the pap smear results were reported after 2 week by hospital 's pathologist and if was positive ( invasive cancer , asc - us , asc - h , lsil , hsil ) , the participants called for referring to colposcopy and biopsy . the result of biopsy was evaluated by pathologist and the results compared with each other . data were analyzed through descriptive and inferential statistical tests such as frequency and chi- square tests by using spss version 13 software . their sensitivity , specificity , positive predictive value , negative predictive value , lr+ , lr- and confidence interval were determined . data were analyzed through descriptive and inferential statistical tests such as frequency and chi- square tests by using spss version 13 software . their sensitivity , specificity , positive predictive value , negative predictive value , lr+ , lr- and confidence interval were determined . in this study , participant 's mean ( sd ) of age and bmi were 33(7 ) and 27.20(4.95 ) respectively . mean ( sd ) of menarche and first intercourse age were 19(4 ) and 26(7 ) respectively . 94.4% of women referred for routine pap smear and 5.6% of women referred for genital disorders . these problems included mense problems , abdominal pain , vaginitis , infertility , post coital bleeding and etc [ table 1 ] . the reasons of women 's refer to pap smear 974(94.7% ) cases were normal and had no findings and 26(2.6% ) participants had positive results in pap smear or dvi test . 12 women had abnormal pap smear , 14 women had positive dvi and 1 woman had abnormality in both dvi and pap test which referred to colposcopy and biopsy . among women with abnormal pap smear , 9 women had atypical squamous cells of undetermined significance ( ascus ) in pap test and 3 women had dysplasia , atypical endocervical and low grade squamous intraepithelial lesion ( lsil ) results . among 14 women with positive dvi , 4 cases had hpv and koilocyte in biopsy result . 1 women with abnormality in both method had carcinoma in biopsy that referred to oncologist . in this study the sensitivity and specificity of dvi was 71.4% and 50% while it was 14.3% and 50% for pap test [ table 2 ] . sensitivity , specificity , ppv , npv , lr+ , lr- and ci of studies methods cervical cancer is one of the important health issues in many low resource settings and it is the third common female 's cancer in the world.3 almost 450000 new cases of cervical neoplasia are recorded annually . the prevalence of cervical cancer is high in countries that have poor screening programs.13 the screening failure is a major cause of cervical cancer mortality in developing countries , while it is preventable with screening programs.1014 different methods are available for cervical cancer screening.14 one of these methods is dvi with 5% of acetic acid . due to the ability of dvi in cervical cancer identification , some studies suggest this method for screening.9 pap smear is another method.15 previous studies indicated that sensitivity of dvi in identification of pre - cancerous cervix lesions is more than pap smear.161718 for example , a study in tehran showed that , sensitivity of dvi was 96% and its specificity was 44% that were higher than sensitivity and specificity of pap smear that were 42% and 10% respectively.9 another study in 2012 , reported the sensitivity and specificity of dvi , 88.8% and 99.9% while they were 37.5% and 99.6% for pap smear.19 in saharsabuddhe , et al . study , the sensitivity and specificity of dvi was reported 80% and 82.6% that were higher than pap smear ( 60.5% and 64.6% respectively).20 sensitivity of dvi in different studies was reported between 75 to 100%13141517 and its specificity was reported between 16 to 85%.16171821 also , it is reported that sensitivity of dvi is higher than pap smear while its specificity is lower than pap test.16182223 in our study sensitivity and specificity of dvi were 71.4% and 50% compared with 14.3% and 50% respectively for pap smear . in different studies positive predicative value of dvi was reported higher than pap smear.1822 in our study positive and negative predicative value of dvi was reported 35.7% and 81.8% compared with 10% and 60% for pap smear . in this study dvi had higher sensitivity than pap smear as like as other studies , but , in our study specificity of two methods was equal while it was reported different in previous studies . pap smear is a method with lower sensitivity and its main failure reason is related to false sampling and interpretation.19 on the other hand , pap smear requires laboratory facilities , cost and time that are especially more impressive in developing countries.91924 while dvi does n't need any laboratory facilities and its result is obtained at the same visit25 so patient 's follow - up is more accessible.9 since a diagnostic value of dvi is comparable to pap smear , and it performs well in detecting a high grade lesion , we conclude that dvi could be used as a screening modality for cervical cancer in low resource settings . one of the problems of dvi method is being a lot of criteria for interpretation of positive test results . proper training of health care providers and physicians is the important criteria . in dvi method , if any acetowhite lesion is considered positive , there would be a lot of false positive results that could need to refer to oncologist and it is not feasible financially for poor people . so , proper training of health care providers is one of the strategies presented in this field.26 also in this method only exocervix is measurable,27 so , it never can be used as an alternative method for pap smear , only it can consider as a supplemental method . this study was limited to females of tabriz , iran , thus it is recommended that other studies to be performed in different places of iran . lack of access to women 's health records and trust to their statements was the second limitation of this study .

background : cervical cancer is the most second common cancer among iranian women . this study was carried out to compare the results of pap smear method and direct visual inspection ( dvi ) with 5% acetic acid in cervical cancer screening in tabriz , iran.material and methods : this cross - sectional study was carried out in alzahra therapeutic - educational centre , tabriz , iran in 2013 on 1000 women . first , pap smear was done for all women , and then the cervix exposed with 5% acetic acid by cotton swab for 30 seconds and observed under adequate light . at the end , women with abnormal results in pap smear or dvi method were referred to colposcopy and biopsy . test 's sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) , lr+ , lr- and confidence interval ( ci ) were determined ( p < 0.05).results : nine - hundred and seventy - four ( 94.7% ) cases were normal and had no abnormal findings and 26 ( 2.6% ) participants had positive results in pap smear or dvi test . twelve women had abnormal pap smear ( nine women with atypical squamous cells of undetermined significance , ascus , three women with dysplasia , atypical endocervical , and low - grade squamous intraepithelial lesion , lsil results ) and 14 women had positive dvi ( four women with human papillomavirus , hpv or koilocyte , ) and one women with abnormality in both method had carcinoma in biopsy that referred to oncologist . in this study the sensitivity , specificity , ppv and npv for dvi were 71.4% , 50% , 35.7% , and 81.8% respectively in comparison with 14.3% , 50% , 10% , and 60% for pap smear.conclusion:as the dvi method has higher sensitivity and positive predictive value than pap smear , it could be used as a useful method beside the pap smear .

INTRODUCTION MATERIALS AND METHODS Data analysis RESULTS DISCUSSION CONCLUSION

colonization is usually acquired during childhood and is the most common cause of chronic gastritis . it is also associated with duodenal ulcer , gastric ulcer , gastric adenocarcinoma , and mucosa - associated lymphoid tissue ( malt ) lymphoma . in developing countries , its prevalence is relatively high . in children , the risk factors for h. pylori infection include socioeconomic status , household crowding , ethnicity , migration from high prevalence regions , and infection status of family members . the aim of this study was to determine some epidemiological aspects of the infection among children in sanandaj , which is the center of kurdistan province in west iran with a population of about 400000 . this cross - sectional population - based study was based on samples of 4-month to 15-year - old children . the children were divided into six age groups ( 4 months , 1 , 2 , 5 , 10 , and 15 years old ) . samples were collected from children aged 3 - 5 months , 10 - 14 months , 22 - 26 months , 4.5 - 5.5 years , 9 - 11 years , and 14 - 16 years for each group respectively . the lower age groups were selected randomly from healthy children who referred to primary healthcare centers for vaccination and the older ones were from 12 schools with different socioeconomic status across the city . there was a list of children in both health care centers and also in schools . we used a computer program to generate random numbers in order to draw a selected group of cases from lists . the sample size calculation was based on at least 30% prevalence rate , 5% precision , and 95% confidence interval , computed from previous epidemiological studies[24 ] . the data were collected in two phases : an interview with the mothers or participating children during april all procedures and tests were carefully explained to the parents of patients informed consent was obtained . the data regarding age , sex , duration of breastfeeding , family size , previous antibiotic usage , and infection status were recorded for each child . . a total of 458 stool samples were collected randomly from the children in all six age groups including 4 months , 1 , 2 , 5 , 10 , and 15 years old . the stool assay was performed using the dia.pro hp sa test ( dia.pro , diagnostic bioprobes srl , milano , italy ) . microplates are coated with a cocktail of affinity purified mouse monoclonal antibodies directed to the most specific h. pylori antigens . stool samples were collected using a kit consisting of a plastic spoon used to scoop a small amount of stool from the toilet paper or bowl into an airtight container . the samples were frozen at a temperature below 20c as described in the kit until analyses were performed . in the 1 incubation , the solid phase was treated with the sample , previously extracted from the stools , and simultaneously with a mixture of monoclonal antibodies to h. pylori , conjugated with peroxidase ( hrp ) . after washing out all the other components of the sample , in the 2 incubation , the color developed in the presence of the bound enzyme , which was proportional to the amount of h. pylori antigens present in the sample . stop solution was added and the results were inspected spectrophotometrically at 450 nm within 15 min of adding the stop solution . a visual determination might also be made within 15 min but we used spectrophotometry in this study to provide reproducible results . internal quality control : the controls / calibrator was checked any time the kit was used in order to verify whether the expected optical density at 450 nm ( od450 nm ) or sample od450nm / cut - off value ( s / co ) values have been matched in the analysis . the following parameters have been met continuously as recommended in the kit package - insert ( table 1 ) . requirements for quality control od450 nm : optical density at 450 nm quantitative assay : the mean od450 nm value of the calibrators was calculated . qualitative assay : the test results were calculated by means of a cut - off value determined from the o450 nm value of the cal 0ug / ml ( cal 0 ) and the od450 nm of the cal 0.1 ug / ml ( cal 0.1 ) with the following formula : cut - off = ( cal 0 + cal 0.1 ) / 2 interpretation of results : in the quantitative assay , samples showing a concentration of h. pylori antigen higher than 0.05 ug / ml was considered positive . for the qualitative assay , test results were interpreted as a ratio of the sample od450 nm ( s ) and the cut - off value ( co ) , mathematically s / co , according to the following rules : s / co of less than 1.00 was considered negative and means that the child is not infected by h. pylori , s / co of 1.01.1 was considered as undetermined and the child retested on a second sample , and s / co of greater than 1.1 was considered positive . the statistical analyses were performed using the epi info version 3.3.2 and spss software ( version 12 ) to estimate crude odds ratios and 95% confidence intervals . chi square test was used as appropriate and p value < 0.05 was considered as statistically significant . the prevalence rates of h. pylori infection based on age and sex are shown in table 2 . prevalence of h. pylori infection in different age groups and sexes there were increasing prevalence rates of h. pylori infection with increasing age in all age groups ( =98.81 , p<0.001 ) with a transient decrease in one and 10-year - old groups ( fig . 1 ) . there were no significant differences between sexes in prevalence of h. pylori infection within each group nor between groups ( p=0.6 , =0.28 , odds ratio = 1.1 , ci [ 0.76 < or < 1.63 ] ) . prevalence of h. pylori by age group table 3 shows the prevalence of h pylori infection based on family size . we found a significant increase in infection rate as the family size grew ( p=0.005 , =10.55 ) . in order to study the epidemiological effect of breastfeeding on h. pylori infection , we investigated the rate of h. pylori infection and the duration of breastfeeding in 1- to 15-year - old children ( table 4 ) . there was no significant decrease in infection rate as the duration of breastfeeding increased ( p=0.8 ) . prevalence of h. pylori infection based on family size prevalence of h. pylori infection based on duration of breastfeeding in children aged 1 - 15 years infection with h. pylori is acquired in as early as 4 months of age and even earlier . at 4 months of age at least 43% of children were positive for h. pylori antigen . by 2 years of age 66.7% of children were positive for h. pylori antigen . infection rate is cumulative with increasing age and maximize at upper age group of 15 years old . there are significant differences in the prevalence of infection worldwide and even in various parts of any specific country , which is closely related to socioeconomic status and overcrowding . in developing countries . the prevalence increases generally with age , but decreases have been noted in narrow age ranges in childhood[4 , 1113 ] . there are many reports of the declining prevalence of h. pylori over brief intervals in childhood[1417 ] . spontaneous elimination of h. pylori infection may be the reason of the declining prevalence in 10 years age group[14 , 1820 ] . other explanations are better attention to health issues in older children or use of antibiotics for other infectious diseases [ 2125 ] , differences in types of h. pylori in adults in comparison with children , and differences in special gastric receptors . however , these can not explain second peak of prevalence in 15 years age group . there are few reports about the prevalence rates of h. pylori infection in different age groups of iranian children[23 ] . most of these earlier studies had been done on considerably wide ranges of age groups , and did not include children younger than 12 years old . the age dependent acquisition of h. pylori antigen determined in our study is consistent with previous studies from developing countries[4 , 12 , 17 ] . some reports from developing countries show that most infants have high infection rate early in their life , which can reach 50% by 2 years of age[4 , 14 , 23 ] . we found no statistical difference between the rates of prevalence in male and female populations . the effects of family size on the prevalence of helicobacter were investigated in several studies[14 , 2729 ] . many environmental factors are related to an increased prevalence of infection , but the most powerful one has been the number of children residing in the house . in our study , the difference in the prevalence rate of infection between small families in comparison with larger families ( table 2 ) was statistically significant . since the children were selected randomly , the number of children in the non - breastfed group was very low to evaluate the protective effect of breastfeeding on h. pylori infection more efficiently . the duration of breastfeeding did not have a statistically consistent effect on the rate of infection . the presence of many risk factors for h. pylori infection in the community leading to high rates of infection probably obscured the protective effects of breastfeeding , if any , on h. pylori infection . a more selective study is needed to investigate the protective effects of breastfeeding on h. pylori infection in our country as described in previous reports from other regions . many studies indicate that stool antigen test is a sensitive and specific mean to diagnose h. pylori infection[3133 ] . in 2000 , a consensus report stated that two non - invasive tests , urea breath test and stool antigen test , could be used both safely and cost effectively to screen patients ( below the age of 45 ) who were positive for h. pylori without alarming symptoms : non - invasive tests are useful for primary diagnosis , when a treatment indication already exists , or to monitor treatment success or failure . sensitivity for three commercial kits ( premier platinum hp sa , femtolab cnx , and dia . pro hp ag ) was reported to be 63.6% , 88.0% , 56.0% respectively . however , the specificity was high for all tests ( reported 97.6% for dia . recently , a systematic review and meta - analysis concluded that the monoclonal technique had a higher sensitivity than the polyclonal one . hpsa assay has been proven to be a highly reliable and simple to use test for the diagnosis of h. pylori . the prevalence of h. pylori infection in children is very high in sanandaj , beginning at early infancy ( before 4 months of age ) and is cumulative with increasing age .

objectivein developing countries , helicobacter pylori infection is mainly acquired during childhood and may be a predisposing factor for peptic ulcer or gastric cancer later in life . noninvasive diagnostic tools are particularly useful in children for screening tests and epidemiological studies . we aimed to determine the prevalence of h. pylori infection among kurdish children in sanandaj , west iran.methodswe used a helicobacter pylori stool antigen ( hpsa ) test to detect h. pylori infection . a questionnaire was used to collect data about age , sex , duration of breastfeeding , and family size . a total of 458 children aged 4 months to 15 years were enrolled in this study.findingsthe mean age of enrolled children was 5.65.4 years . stool samples were positive for h. pylori in 294 ( 64.2% ) children . the prevalence of h. pylori infection increased with age ( p<0.001 ) . we found a significant increase in the infection rate as the family size grew ( p=0.005 ) . there was no correlation between a positive h. pylori status and gender ( p=0.6 ) or the duration of breastfeeding ( p=0.8).conclusionit seems that the prevalence of h. pylori infection is very high in children in sanandaj . it begins at early infancy ( before 4th month of age ) and cumulatively increases with age .

Introduction Subjects and Methods Findings Discussion Conclusion Conflict of Interest

wet age - related macular degeneration ( wamd ) is a chronic disease and a leading cause of blindness in older patients.13 following the recent introduction of anti - vascular endothelial growth factor ( anti - vegf ) therapy , wamd treatment has changed dramatically , with a switch to improving rather than maintaining visual acuity.4 in japan , after the launch of ranibizumab ( lucentis ; novartis international ag ) in 2009 , the pro re nata ( prn ) method of administration became the standard approach for anti - vegf therapy in wamd . the prn regimen consists of as - needed injections ( depending on patient symptoms ) after an initial loading phase of three consecutive monthly injections , with monthly visits to monitor symptoms.5,6 the proactive regimen was introduced with the launch of intravitreal aflibercept ( eylea ; bayer ) in 2012 . there are two approaches to proactive therapy : fixed - dosing and treat - and - extend ( t&e ) . fixed - dosing consists of injections at set intervals ( eg , every 8 weeks ) after an initial loading phase , regardless of patient symptoms.7 by contrast , t&e does not have a defined injection interval . in general , after a loading phase , monthly injections are continued until no fluid is detected , followed by 2-week extension intervals until the next injection , which is done regardless of symptoms . when fluid is detected , the extension intervals are decreased depending on the severity . unlike prn , t&e does not require patients to make monthly visits for monitoring , but rather at intervals determined by the ophthalmologist.5,8 the effectiveness of proactive therapy has been widely reported , and improvements in visual acuity have been achieved with a fewer visits.912 long - term data on anti - vegf therapy and the various regimens are also becoming available.13,14 while the majority of research focuses on the clinical outcomes of anti - vegf therapy regimens , several assessments have addressed the logistical difficulties of using anti - vegf therapy in actual practice.1518 however , none of these previous assessments address specific differences between the prn and t&e regimens . with varying visit schedules and approaches to injection criteria , it can be assumed that differences between the two regimens would impact both ophthalmologist and patient treatment experience , ultimately influencing patient adherence to long - term treatment . the aim of our research was to qualitatively assess the current issues and benefits of the prn and t&e anti - vegf therapy regimens and to focus on any issues associated with introducing t&e . here , we report how proactive therapy is used to treat wamd in clinical practices in japan and how the different regimens impact patient , caregiver , and ophthalmologist experience . qualitative assessment was performed using semistructured interviews conducted by ims consulting group , ims japan kk . interviewees were ophthalmologists selected from a third - party panel by the ims consulting group according to the selection criteria and based on their experience in treating wamd . ophthalmologists were required to have introduced proactive therapy for wamd into their practice and to have current or previous experience with the prn approach ; this stipulation was intended to ensure that the interviewees had working experience with both types of anti - vegf therapy regimens . a total of 20 ophthalmologists were recruited from university hospitals , nonacademic hospitals , and ophthalmologist clinics ; the ophthalmologists were recruited from the greater tokyo area ( 50% ) , other regional cities ( 28% ) , and rural areas ( 22% ) . interviews were conducted during the period of november 2014 through february 2015 . written informed consent was obtained from all interviewees , and with their consent , the interviews were recorded for transcripts to be used for analysis . all procedures performed in this study were approved and in accordance with the ethical standards of the institutional review board of tokyo women s university hospital and with the 1964 declaration of helsinki and its later amendments . the interview questions consisted of three parts : 1 ) issues and benefits of prn and t&e ; 2 ) logistical and operational issues of introducing proactive therapy , especially t&e , to prn practice ; and 3 ) actions taken to handle the issues raised in 2 ) . during the interview process , it became clear that two interviewees had a different understanding of either the prn or t&e treatment regimens , and their interviews were removed from the dataset . the distribution of interviewees based on their institution types were as follows : university hospital : n=7 , 39% ; nonacademic hospital : n=7 , 39% ; and clinics : n=4 , 22% . the benefits and issues of prn and t&e were evaluated from two perspectives : the patient or caregiver perspective as understood by the treating ophthalmologist and the treating ophthalmologist s own perspective . there were three main areas where the patient or caregiver experienced benefits with t&e , according to the ophthalmologists . these included the following : able to tangibly experience treatment efficacy through the sustained period of macular dryness , decrease in emotional burden associated with receiving intraocular injections , and decrease in patient / caregiver time burden ( table 3 ) . from the treating ophthalmologists perspective , the following three areas were identified : shorter consultation time per patient , decrease in burden of developing patient specific treatment plans , and decrease in overall psychological stress were those related to the increase in intravitreal injections and concerns arising from the long - term treatment . concerns over financial burden associated with an increase in intravitreal injections were highlighted from both the patient / caregiver and treating ophthalmologist perspectives . for patients who achieved dryness of the macula and stabilization of symptoms , there appeared to be difficulty in getting them to understand the significance of continuing with proactive injections , which in effect prevent their symptoms from worsening . figure 1 summarizes the different issues ophthalmologists faced when introducing t&e to their practice , according to their institution type . these issues can be possibly identified in two stages : the patient explanation phase and the operational phase . while university hospital ophthalmologists did not consider handling concerns of financial burden from long - term treatment as an issue they faced during consultations , ophthalmologists based in clinics all raised this issue as a concern . only university hospital ophthalmologists raised increased burden of managing appointments as a concern . specific examples of the issues raised in figure 1 are shown in tables 57 , organized by institution type . university hospital ophthalmologists mainly raised four issues as difficulties of practicing t&e : explaining the necessity of proactive injections as prevention , insufficient space for injections , insufficient human resources , and increased burden of managing appointments . the following six countermeasures were provided as remedies implemented : explained the efficacy / effectiveness in a more tangible way to the patient , strengthened cooperation with other local facilities and referred patients , employed part - time ophthalmologists , delegated tasks of the injection process and defined the roles of each ophthalmologist , shortened the turn - around time of patients receiving injections , and implemented a scheduling system to manage appointments . specific issues raised by nonacademic hospital ophthalmologists were the same as those raised by university hospital ophthalmologists , except for increased burden of managing appointments . a different countermeasure provided was provided injections in outpatient treatment rooms instead of operating rooms , since operating rooms require reservations making scheduling complicated . clinic - based ophthalmologists raised two issues relating to explaining the necessity of proactive injections as prevention and handling concerns of financial burden from long - term treatment . the countermeasures implemented for these issues were explained the efficacy / effectiveness in a more tangible way to the patient and prioritized patients who were referred from other hospitals and understood t&e . during the interview process , it became clear that two interviewees had a different understanding of either the prn or t&e treatment regimens , and their interviews were removed from the dataset . the distribution of interviewees based on their institution types were as follows : university hospital : n=7 , 39% ; nonacademic hospital : n=7 , 39% ; and clinics : n=4 , 22% . the benefits and issues of prn and t&e were evaluated from two perspectives : the patient or caregiver perspective as understood by the treating ophthalmologist and the treating ophthalmologist s own perspective . there were three main areas where the patient or caregiver experienced benefits with t&e , according to the ophthalmologists . these included the following : able to tangibly experience treatment efficacy through the sustained period of macular dryness , decrease in emotional burden associated with receiving intraocular injections , and decrease in patient / caregiver time burden ( table 3 ) . from the treating ophthalmologists perspective , the following three areas were identified : shorter consultation time per patient , decrease in burden of developing patient specific treatment plans , and decrease in overall psychological stress the issues surrounding t&e were those related to the increase in intravitreal injections and concerns arising from the long - term treatment . concerns over financial burden associated with an increase in intravitreal injections were highlighted from both the patient / caregiver and treating ophthalmologist perspectives . for patients who achieved dryness of the macula and stabilization of symptoms , there appeared to be difficulty in getting them to understand the significance of continuing with proactive injections , which in effect prevent their symptoms from worsening . figure 1 summarizes the different issues ophthalmologists faced when introducing t&e to their practice , according to their institution type . these issues can be possibly identified in two stages : the patient explanation phase and the operational phase . while university hospital ophthalmologists did not consider handling concerns of financial burden from long - term treatment as an issue they faced during consultations , ophthalmologists based in clinics all raised this issue as a concern . only university hospital ophthalmologists raised increased burden of managing appointments as a concern . specific examples of the issues raised in figure 1 are shown in tables 57 , organized by institution type . university hospital ophthalmologists mainly raised four issues as difficulties of practicing t&e : explaining the necessity of proactive injections as prevention the following six countermeasures were provided as remedies implemented : explained the efficacy / effectiveness in a more tangible way to the patient , strengthened cooperation with other local facilities and referred patients , employed part - time ophthalmologists , delegated tasks of the injection process and defined the roles of each ophthalmologist , shortened the turn - around time of patients receiving injections , and implemented a scheduling system to manage appointments . specific issues raised by nonacademic hospital ophthalmologists were the same as those raised by university hospital ophthalmologists , except for increased burden of managing appointments . a different countermeasure provided was provided injections in outpatient treatment rooms instead of operating rooms , since operating rooms require reservations making scheduling complicated . clinic - based ophthalmologists raised two issues relating to explaining the necessity of proactive injections as prevention and handling concerns of financial burden from long - term treatment . the countermeasures implemented for these issues were explained the efficacy / effectiveness in a more tangible way to the patient and prioritized patients who were referred from other hospitals and understood t&e . interviews with ophthalmologists revealed that compared with prn , t&e decreased emotional burden for patients / caregivers and time burden for patients / caregivers and ophthalmologists . the main issues raised were the increased number of injections and the associated financial burden from prolonged treatment duration . issues arising from the introduction of t&e at the patient explanation phase or operational phase depended on practice type . the analysis highlighted that predetermined scheduled injections ( t&e ) provided relief by ensuring that visits did not result in bad news and unforeseen injections compared with prn ; this decreased the emotional burden for patients and caregivers . droege et al19 revealed that patients were more afraid of receiving negative examination results than the actual intravitreal injections , indicating that worsening of symptoms impacts patient experience more than frequency of injections . these interview results also revealed that t&e reduced time burden for patients and caregivers in clinical practice by decreasing the amount of necessary visits . this is important because monthly monitoring of prn has been considered burdensome for ophthalmologists , patients , and caregivers in previous studies.5,9,16 for example , gohil et al20 looked at caregiver burden of prn in the uk and on average , 70% of caregivers spent at least half a day assisting their patients with clinic visits every 46 weeks , and about 25% took time off from work . according to droege et al,19 patients indicated that travel to and from visits was a major barrier to their wamd treatment adherence . combined with our results , it can be inferred that a fewer visits for t&e may result in better adherence by patients to long - term treatment . the ophthalmologists also spoke of the reality of organizing prn injections : once the patient was indicated for injection , the facility may not always be able to provide it on the same day , causing the patient to schedule another visit . if the caregiver was unable to accompany the patient for the rescheduled visit , the patient was subjected to even further delay in receiving timely treatment . this is an important issue for treating a time - sensitive condition such as wamd . a study analyzing a hospital claims database in japan showed that an average of 11.6 days elapsed between injections and the previous outpatient monitoring visits under prn.21 likewise , takahashi et al22 analyzed two hospitals in japan and found average injection delays under prn of between 0 and 104 days . these studies further indicate that injection delays are not rare with prn approaches in real - life settings . similarly , rayess et al12 found that patients receiving consecutive maintenance injections with t&e were less susceptible to recurrence of disease activity than prn , where duration of consecutive injections tends to be prolonged . previous studies reporting visual acuity outcomes with prn dosing were conducted with patients receiving injections on the same day,6,14 which , compared with treatment pattern studies , may not reflect real - life clinical practice.21,22 as reported in our analysis , t&e is able to avoid such delays and missed injection opportunities by following a predetermined injection schedule , which leads to prolonged dryness of the macula and prevention of worsening of symptoms . while previous studies have focused on ophthalmologists experience of such clinical outcomes , our analysis reveals for the first time that patients ( based on ophthalmologist feedback ) are also able to tangibly experience the effectiveness of anti - vegf treatment with t&e , which can be linked to increased treatment satisfaction and likely treatment adherence . as mentioned earlier , issues of introducing t&e into the clinical practice can be identified in two stages : the patient explanation phase and the operational phase . there were benefits associated with decreased emotional burden from simplified consultations with patients who already understood the necessity of receiving injections at their visits . however , ophthalmologists also had difficulties in explaining the preventive effect of t&e , which was identified in all institution types . they had difficulty convincing patients who had entered the maintenance phase of their treatment with stable disease activity to continue with proactive injections before any disease activity resumes . while this difficulty was common to ophthalmologists across all institution types , the management of this issue appeared to differ . this phenomenon may be attributed to the lack of a guideline for t&e articulating how to manage injections during the maintenance phase of a patient s disease state . a literature review with consensus recommendations on t&e was published in 2015 , but there remains a lack of evidence - based resource that can support japanese practice.8 issues in the operational phase were raised mostly from university hospital ophthalmologists . they have a relatively greater number of wamd patients compared with hospitals and clinics ; t&e also results in increased injections per patient and a fewer dropouts , resulting in an increased number of total patients continuing anti - vegf treatment . rearranging the patient flow and dividing staff roles to improve efficiency were provided as effective actions . however , increased staff and space for administering injections seemed to be ultimately desired even after such measures . it should also be noted that issues with staff and spatial capacity have been reported in previous studies with prn , and this could be an ongoing issue that is not unique to the practice of t&e.15,16,18,22 strengthening local clinic and university hospital relations in order to increase referrals to clinics was also raised as a potential solution to the operational phase issues . implementing integrated care pathways is still in its infancy in japan , particularly in ophthalmology . at present , there have only been pilot trials with glaucoma and ophthalmologic care for patients with dementia.23,24 disparity in care between practices is also an issue.25 this was also observed by takahashi et al.22 this variation in care further reiterates the need for an evidence - based standardized method of practicing t&e that can be followed by ophthalmologists across practices . second , interviewees were located primarily within the greater tokyo metropolitan area or other regional cities , and their responses may not reflect the realities of other regional situations . we performed subgroup analyses by region and facility types ( hospital vs clinic ophthalmologists ) to see if there were any differences . it was revealed that there were no interesting / meaningful results by region ( data not shown ) , but we did find a difference between hospital and clinic ophthalmologists . this qualitative analysis demonstrated that the benefits of t&e compared with prn as reported by ophthalmologists included decreased burden of patient consultations , decreased patient and caregiver emotional burden , and a sustained period of macular dryness . the analysis revealed that patient treatment satisfaction can be increased via t&e , and this may increase the potential for better adherence to long - term treatment . however , increased number of injections and financial burden from prolonged treatment duration remain issues associated with t&e . the ophthalmologists also experienced difficulty explaining the significance of proactive injections for prevention to patients . countermeasures to operational issues experienced by ophthalmologists varied by practice . in order to encourage better understanding of the t&e regimen , including its smooth implementation and significance for patients , a formal t&e treatment guideline providing standard practice should be considered .

purposethe purpose of this study was to monitor anti - vascular endothelial growth factor ( anti - vegf ) treatment regimens for wet age - related macular degeneration ( wamd ) in clinical practice and to determine how they impact the physician , patient , and caregiver treatment experience.materials and methodsthis was a qualitative analysis based on semistructured interviews with 20 ophthalmologists who had practiced both pro re nata ( prn ) and treat - and - extend ( t&e ) anti - vegf regimens for wamd . interview questions were constructed to assess how the different regimens affected patient and caregiver experiences ( in the opinion of the ophthalmologist ) in addition to the ophthalmologist s own experience . the interview included questions relating to 1 ) issues and benefits of prn and t&e ; 2 ) logistical and operational issues of introducing proactive therapy , especially t&e , to prn practice ; and 3 ) actions taken to handle the issues raised in 2).resultsa total of 18 interview results were eligible for analysis . the study demonstrated that the benefits of t&e compared with prn included decreased burden of patient consultations , decreased patient and caregiver emotional burden , and a sustained period of macular dryness . the issues associated with t&e were increased number of injections and financial burden from prolonged treatment duration . the ophthalmologists also experienced difficulty explaining the significance of proactive injections to patients . countermeasures to operational issues experienced by ophthalmologists varied by practice.conclusionpatients , caregivers , and the practicing ophthalmologists experienced benefits associated with a t&e regimen . however , in order to encourage better understanding of the t&e regimen , including its smooth implementation and significance for patients , a formal t&e treatment guideline providing standard practice should be considered .

Introduction Materials and methods Results Interviewee background Benefits and issues of T&E and PRN Issues arising from the introduction of T&E and countermeasures Discussion Conclusion

many studies over past 2 decades show depression disorder is one of the most important world health problems . according to the who reports , depression will be the second cause of disability , after heart diseases , in the developed countries toward 2020 . world health survey also showed that depression is more harmful on man health than other diseases such as angina , arthritis , asthma and diabetes . unfortunately despite the high prevalence of depression , it is difficult to manage the disorder because its causes are largely unknown . the other important subject which many researches engaged in is how to find correlation between depression and cardiovascular diseases ( cvds ) . various studies have reported a noticeable range of 1450% of heart diseases comorbid with depression . heart disease along with depression have produced bad prognosis in rate of relapsing and high rate of mortality because of heart illness . therefore , it is necessary to clear up the obscure points connected with the disorder in order to control and prevent the deleterious effects of depression . the use of conventional medicine strategies in combination with traditional ones can be an important way to increase our capacity to recognize and treat disease as well as reduce side effects and costs . one of the oldest and gorgeous school of medicine in the world is the persian medicine ( pm ) which has introduced some nominated scientist such as mohammad ibn zakaria razi ( rhazes ) and ibn sina ( avicenna ) . avicenna is a famous persian philosopher , thinker , and physician whose medical book named the canon of medicine has been taught in the european universities of medical sciences such as french monepoliea and belgian louvain university for many centuries . in the canon of medicin one kinds of melane , avicenna describes various kinds of mlkhly ( melancholia ) disorders as the subtitle of brain disorders . one kinds of melancholia is called cerebral melancholia is called cerebral melancholia with symptoms very similar to depression in the today 's psychiatry [ table 1 ] . comparison of symptoms of melancholia in pm with major depression on the basis of dsm - iv - tr diagnostic criteria based on avicenna , etiology of cerebral melancholia originates from heart diseases characteristically this kind of melancholia . none of the physicians before avicenna , like rufus , hippocrates , galen , muhammad ibn zakariyya al - razi ( rhazes ) , zahravi , have not mentioned any heart involvement in the melancholia etiology , although some scientists such as rhazes have made some advice to increase heart power in the process of melancholia treatment . therefore , in medicine history , avicenna is the first one who propounded correlation between cardiac disease and melancholia . at the present , studies have shown the above coexistence , and they discuss that depression is an important independent risk factor in cvd . however , avicenna believes that in most cases , some heart diseases start a process which ultimately results in depression and if the cause of depression in some cases originates from the brain , heart diseases make the problem worse . therefore , according the avicenna 's view , heart illness plays the main etiologic role in the most cases and in few events , shows a magnifying role on the depression . in spite of many studies on the depression and there are still many questions on how the etiology and treatment of depression are specially when it coincident with heart disease . for instance , however , depression in heart patients , increase the chance offside effects , recurrence of cardiac events , and its mortality rate but unfortunately clinical trial studies have not so far shown that medical treatment of depression or psychotherapy can reduce significantly side effects or recurrency or the mortality of heart diseases . in the present study we describe avicenna 's view on the depression etiology , showing some new finding confirming his view , and discuss possible mechanisms of how some heart diseases might be the main reasons of depression on the basis of pm , hope to open new approach for researchers on the etiology and treatment of depression . the most reliable references on iranian ancient medicine were determined on the basis of the following criteria : being the most used books by ancient physiciansauthor 's credibilitybeing the most cited references in papers published in peer - reviewed national and international journals in recent years.once reliable references were determined , literature search was initiated to collect data in accordance to the following steps : mlkhly , vasvasemlkhlyiee ( melancholic obsession ) , and mlkhly were picked as keywords : being the most used books by ancient physicians being the most cited references in papers published in peer - reviewed national and international journals in recent years . all data relevant to the treatment of melancholia was collectedall nonpharmacological interventions of melancholia were extractedcommon points in various references about etiology of cerebral melancholia ( depression).furthermore , the following keywords were used to search the pubmed and scopus databases : melancholia , depression , traditional medicine , cvds , myocardial infarction ( mi ) , and cardiac arrhythmias . all data relevant to the treatment of melancholia was collected all nonpharmacological interventions of melancholia were extracted common points in various references about etiology of cerebral melancholia ( depression ) . heart is the first organ which starts motion when the fetus gets life , and it is the last part to stop motion when death occurs . before discussion on heart physiology , it is necessary to describe briefly some fundamental subjects of the pm school . humors are wet and fluid matter which are produced by the liver and transferred to the heart by inferior vena cava vein and then to the other parts of body by aorta artery in order to replace the substance used by metabolism . each of these quaternary humors have a pair of different qualities of hot , cold , wet and dry forms that is , blood is hot and wet , phlegm is cold and wet , yellow bile is hot and dry and black bile is cold and dry . temperament is resulted by interaction of two opposite qualities of humors which ultimately give rise to a homogenous quality named suppose that when sugar dissolves in water , produces a solution which is fluid ( as water ) and sweet ( as sugar ) . for example , brain has a cold and wet temperament , heart a hot and dry and liver a hot and wet temperament . the combination and interaction of all organ temperaments leads to man 's special temperament . as long as this special temperament remains intact but when his temperament homeostasis changes , a process arises which ultimately causes leads to illness , a condition may be called dystemperament . avicenna scientists believe that humors are divided to thin and thick substances based on their viscosity . thick substance plays role on the formation , growth , feeding and stability of body organs . but thin humors , or so called humors vapor , when settled down in the left ventricle of heart converts to a spirit through a complicated procedure by which all the body organs are given vital , so , this spirit is called vital spirit or cardiac spirit . the existence of vital spirit in man 's body means that he is alive , and lack of vital spirit means man death . from the point of physician 's view the spirit is not , of course , anon material object such as considered in philosophy science , but has a material identity and contains very thin compounds like air , so that it has been referred to as a vaporous spirit as well . on the basis of pm , being produced by left ventricle , vital spirit spreads out all over the organ through aorta artery , so gives them life . when vital spirit travels from heart to brain base via two carotid arteries , its temperament , in a network formed in this region by these and other artery branches ( willis net ) will change and convert to sensual spirit which transfers sense and motion to all body organs including heart through nerves . therefore vital spirit in heart correlates with sensual spirit in brain and natural spirit in liver , and all of them have cross interactive effect with one another . avicenna scientists believe that each of these triple spirits carries one faculty or talent and does its own tasks through these triple faculties , including vital faculty , sensual faculty , and natural faculty . on the basis of pm , this faculty is divided in two parts , one is active , and the other is passive power . the duty of active power is cardiovascular constriction and relaxation , and passive power export source emotional states such as happiness , sorrow , anger , fearfulness , braveness , enjoy , etc . the reason why these emotions are linked with heart , is that these emotions were shown to cause the contraction and relaxation of heart spirit , and it in turn causes heart contraction and relaxation . avicenna and other physicians have demonstrated the effects of emotion son the cardiovascular system by pulse variations which again prove the correlation of these emotions with heart [ table 2 ] . pulse changes in emotional conditions conventional medicine , although assumes that emotional behaviors are controlled by brain and limbic system , but researchers confirm that there is a correlation between negative and positive emotional states , like , happiness , despair , sorrow , fear , and health specially cardiovascular health . the last important point in this section is that heart and brain have bilateral and direct connection each other . the most important connection of heart to brain is made by vital spirit towards sensual spirit through arteries , and the most important connection of brain with heart is also through sensual to vital spirit mediated by nerves , and so these two important organs , have directly reciprocal effects on each other . melancholia has been classified as one of the mental illness in pm and is described as an illness produced by changes in the quality of brain temperament ( mental state ) and prevent man from right thinking and common sense so that in patient falls is sorrow mind , fear and suspicion without any apparent reason . it is named because it was believed that the main cause of this illness comes from abnormal increase in black bile . melancholia is divided into three classes according to which organ the black bile has been accumulated in : if black bile is concentrated in brain , it is called cerebral melancholiaif black bile is condensate in organs such as stomach , liver , bladder , and extra peritoneal , it is named peritoneal melancholia ( mlenkholia - l - marqq)if black bile spreads all over the body , it is called systemic melancholia . if black bile is concentrated in brain , it is called cerebral melancholia if black bile is condensate in organs such as stomach , liver , bladder , and extra peritoneal , it is named peritoneal melancholia ( mlenkholia - l - marqq ) if black bile spreads all over the body , it is called systemic melancholia . in table 1 , melancholia symptoms in pm are compared with major depression on the basis of diagnostic and statistical manual of mental disorders , 4 edition , text revision ( dsm - iv - tr ) diagnostic criteria . in addition to this table , in dsm - iv - tr , in the subgroup of mood disorders along with description of various depression disorders , melancholic disorder is also mentioned and its diagnostic criteria are described . thus , melancholic disorder has been classified as one of the depression disorders in this accredited classification system . avicenna in describing the role of blood in producing brain diseases like melancholia , discusses that if the blood in transferring from heart to brain is bright , dilute , clear and enlivening , in other words , its physicochemical properties are natural , not only plays a major role in brain healthy , but also makes it resistant against illness , and even if mental illness is in initial stages , most probably prevents its outbreak , but if physicochemical properties of blood in going from heart to brain change , as in cardiac black bile disease , cause brain corruption and produce diseases such as melancholia . according to avicenna 's view , blood variations followed by cardiac illnesses are in most cases initiators of melancholia , although its solidity is formed in brain . avicenna , of course , states that there may be other reasons apart from heart illness which causes melancholia , but , he insists on , they can not cause this mental illness without heart collaboration . therefore , from the point of avicenna 's view the most important and greatest reason of melancholia illness roots in black bile - affected cardiac diseases . heart is the first organ which starts motion when the fetus gets life , and it is the last part to stop motion when death occurs . before discussion on heart physiology , it is necessary to describe briefly some fundamental subjects of the pm school . humors are wet and fluid matter which are produced by the liver and transferred to the heart by inferior vena cava vein and then to the other parts of body by aorta artery in order to replace the substance used by metabolism . each of these quaternary humors have a pair of different qualities of hot , cold , wet and dry forms that is , blood is hot and wet , phlegm is cold and wet , yellow bile is hot and dry and black bile is cold and dry . temperament is resulted by interaction of two opposite qualities of humors which ultimately give rise to a homogenous quality named suppose that when sugar dissolves in water , produces a solution which is fluid ( as water ) and sweet ( as sugar ) . for example , brain has a cold and wet temperament , heart a hot and dry and liver a hot and wet temperament . the combination and interaction of all organ temperaments leads to man 's special temperament . as long as this special temperament remains intact but when his temperament homeostasis changes , a process arises which ultimately causes leads to illness , a condition may be called dystemperament . avicenna scientists believe that humors are divided to thin and thick substances based on their viscosity . thick substance plays role on the formation , growth , feeding and stability of body organs . but thin humors , or so called humors vapor , when settled down in the left ventricle of heart converts to a spirit through a complicated procedure by which all the body organs are given vital , so , this spirit is called vital spirit or cardiac spirit . the existence of vital spirit in man 's body means that he is alive , and lack of vital spirit means man death . from the point of physician 's view the spirit is not , of course , anon material object such as considered in philosophy science , but has a material identity and contains very thin compounds like air , so that it has been referred to as a vaporous spirit as well . on the basis of pm , being produced by left ventricle , vital spirit spreads out all over the organ through aorta artery , so gives them life . when vital spirit travels from heart to brain base via two carotid arteries , its temperament , in a network formed in this region by these and other artery branches ( willis net ) will change and convert to sensual spirit which transfers sense and motion to all body organs including heart through nerves . therefore vital spirit in heart correlates with sensual spirit in brain and natural spirit in liver , and all of them have cross interactive effect with one another . avicenna scientists believe that each of these triple spirits carries one faculty or talent and does its own tasks through these triple faculties , including vital faculty , sensual faculty , and natural faculty . on the basis of pm , this faculty is divided in two parts , one is active , and the other is passive power . the duty of active power is cardiovascular constriction and relaxation , and passive power export source emotional states such as happiness , sorrow , anger , fearfulness , braveness , enjoy , etc . the reason why these emotions are linked with heart , is that these emotions were shown to cause the contraction and relaxation of heart spirit , and it in turn causes heart contraction and relaxation . avicenna and other physicians have demonstrated the effects of emotion son the cardiovascular system by pulse variations which again prove the correlation of these emotions with heart [ table 2 ] . pulse changes in emotional conditions conventional medicine , although assumes that emotional behaviors are controlled by brain and limbic system , but researchers confirm that there is a correlation between negative and positive emotional states , like , happiness , despair , sorrow , fear , and health specially cardiovascular health . the last important point in this section is that heart and brain have bilateral and direct connection each other . the most important connection of heart to brain is made by vital spirit towards sensual spirit through arteries , and the most important connection of brain with heart is also through sensual to vital spirit mediated by nerves , and so these two important organs , have directly reciprocal effects on each other . melancholia has been classified as one of the mental illness in pm and is described as an illness produced by changes in the quality of brain temperament ( mental state ) and prevent man from right thinking and common sense so that in patient falls is sorrow mind , fear and suspicion without any apparent reason . it is named because it was believed that the main cause of this illness comes from abnormal increase in black bile . melancholia is divided into three classes according to which organ the black bile has been accumulated in : if black bile is concentrated in brain , it is called cerebral melancholiaif black bile is condensate in organs such as stomach , liver , bladder , and extra peritoneal , it is named peritoneal melancholia ( mlenkholia - l - marqq)if black bile spreads all over the body , it is called systemic melancholia . if black bile is concentrated in brain , it is called cerebral melancholia if black bile is condensate in organs such as stomach , liver , bladder , and extra peritoneal , it is named peritoneal melancholia ( mlenkholia - l - marqq ) if black bile spreads all over the body , it is called systemic melancholia . in table 1 , melancholia symptoms in pm are compared with major depression on the basis of diagnostic and statistical manual of mental disorders , 4 edition , text revision ( dsm - iv - tr ) diagnostic criteria . in addition to this table , in dsm - iv - tr , in the subgroup of mood disorders along with description of various depression disorders , melancholic disorder is also mentioned and its diagnostic criteria are described . thus , melancholic disorder has been classified as one of the depression disorders in this accredited classification system . avicenna in describing the role of blood in producing brain diseases like melancholia , discusses that if the blood in transferring from heart to brain is bright , dilute , clear and enlivening , in other words , its physicochemical properties are natural , not only plays a major role in brain healthy , but also makes it resistant against illness , and even if mental illness is in initial stages , most probably prevents its outbreak , but if physicochemical properties of blood in going from heart to brain change , as in cardiac black bile disease , cause brain corruption and produce diseases such as melancholia . according to avicenna 's view , blood variations followed by cardiac illnesses are in most cases initiators of melancholia , although its solidity is formed in brain . avicenna , of course , states that there may be other reasons apart from heart illness which causes melancholia , but , he insists on , they can not cause this mental illness without heart collaboration . therefore , from the point of avicenna 's view the most important and greatest reason of melancholia illness roots in black bile - affected cardiac diseases . at first , it should be mentioned that with common signs of mental melancholia as it referred to in pm , and depression in conventional medicine [ table 2 ] , these two illnesses have a common root , and so , some important points about melancholia made by pm physicians such as etiology and treatment can be applied usefully to depression . the relation between some heart diseases and depression has been revealed in the last 2 decades is the same as those mentioned clearly by avicenna in his canon book a 1000 years ago avicenna explains it clearly on the basis of pm , but today researchers describe it as a two comorbid condition and only give some probable mechanism for it . the high incidence of depression , approximately 1450% among various heart diseases shows that these two illnesses come together in the most of the time . as a result , the american association of cardiovascular and pulmonary rehabilitation strongly recommended that patients with heart disease should be screen for depression and refer for cardiac rehabilitation protocols containing food , stress management , and exercise . blumenthal et al . in their studies of standard medical intervention and longtime exercise ( smile ) found that exercise is more effective than medical treatment on the reduction of depression . in addition , considerable increase of depression disorders in cardiac patients compared to common healthy population on one hand , and recurrent depression increase following heart disease on other hand raises up a viewpoint that some heart diseases may bring conditions which ultimately result in depression . however , an important question arises which makes the relation between these two diseases more complicated . the question is why , in clinical trials studies , treatment of depression in patients with heart disease have not decreased recurrent heart disease incidence or mortality considerably and has been just effective on patient acceptance of drug treatment protocol , and his life quality , in spite of studies demonstrating that depression increases the occurrence of new heart diseases , morbidity and mortality , and mi , several times in cardiac patients ? in other words , if depression disorder is the only reason for four times increase in mi and 2.75 times increase in cardiac patients mortality , why has treatment of depression not considerably decreased mi or cardiac mortality ! ? the pessimistic results of the above studies as well as the lack of randomized placebo - controlled studies in demonstrating that the depression treatment has useful clinical effect on cvd , raise serious doubt on whether depression disorder may give rise to a bad prognosis in depressed cardiac patients . that is why some scientists make suggestion that depression itself is not probably the main cause of bad prognosis in depressed cardiac patients . it seems possible to find answer from avicenna 's view for the above question , as he clearly speaks out about the etiologic role of some heart diseases on depression outbreak , and believes that these diseases make the most important cause of depression disorder . he , then , describes the role of physicochemical changes of blood properties on producing depression . this means he does not think that depression is just brain disease , but it is believed that depression is a multi - system disease , in addition to the brain , cardiovascular system and blood circulation is impaired . he , therefore , emphasizes that for depression treatment there is no way but by treating disorders of these systems along with mental therapy . at present , it is shown that cardiac arrhythmias increase of blood pressure secondary to high levels of cortisol and decrease of heart rate variability , increase of plasma level of platelet factor iv and beta - thromboglobulin , which cause raising platelet activity , endothelial vascular damage , and thrombosis , and high levels of cytokines such as interleukin i , interleukin 6 , tumor necrosis factor ( tnf ) , which play key roles on atherosclerosis , have been documented in depressive patients . in overall , it must be said that all the above conditions are in accordance with avicenna 's views of multi system causes of depression . in addition some researchers confirm the effect of collective of various biological systems on depression disorder . among them is dr . peter kramer , who writes in his book under the title of against depression . depression is not a brain disease merely ; it is also a neurological , hematological , and cvd . thus it seems that depression is not just cerebrally originated , because with such a vision , it should not result in bad prognosis for cardiac depressive patients . the failure of depression treatment on reducing morbidity and mortality should be rooted in multiple organs of depression disorder . it is believed that in conventional medicine , limbic system , especially its area of reward and punishment centers , control of excitement , but scientists have confirmed there is a link between positive and negative emotions , such as sorrow , anger , happiness , and health specially cardiovascular health . that is , positive emotional states like optimism , hope , delightfulness , reduce cvd and increase life long time , but negative emotions like hostility , fear , sever and continuous anger cause cardiovascular problems and life time shortage . some studies by kapfhammer have shown that some depressive symptoms such as enhedonia , hopelessness , and vital exhaustion , have close connection with cvds , and so proposed that these symptoms mediate , as a specific risk factor , correlation between heart disease and depression . there is an important point in pm , that is although emotion states are correlated with vital faculty and spirit , but these states , as the scientists believe , are under control of cardiac and blood temperament . to explain more , if a person has a heart with hot temperament , and his blood is dilute and clear and its quality is hot , he is , therefore , a hopeful man with happiness tendency , because of his dilute and clear blood but he tends to be anger , because of his hot heart and blood temperaments , although his angriness rises sharply then disappears quickly because of his dilute blood . now , consider another person ; he is with hot heart and blood natural temperament , but his blood is thick and turbid ( not clear and dilute ) . here , the man is in sorrow ( because of blood turbidness ) and anger ( because of hot heart and blood temperament ) tendencies , but his angriness is stable and does not diminish quickly ( because of condensed blood ) . there is a third person who , opposite to the two above persons , is with cold heart and blood natural temperament , and his blood is thick and turbid ( like the second person ) . this man is coward and slow - witted ( because of cold heart and blood temperament ) and has a sorrow tendency ( as his blood is turbid ) and his emotional states disappear slowly ( since the blood is thick ) . today , some scientists believe that it is possible to present more precise view on the origin of depression and its correlation with heart diseases if more knowledge is collected for the following questions : why do some patients with heart diseases show the best response to the treatment but some others do not?why do some patients fall in depression quickly after a heart accident , but some others show delayed or no depression?which group of depressive patients with heart diseases will demonstrate good prognosis , and which one will give bad prognosis?we believe that if future studies focus on cognition exact of blood - cardiac temperament , it will be possible to find acceptable answers to the above questions . why do some patients with heart diseases show the best response to the treatment but some others do not ? why do some patients fall in depression quickly after a heart accident , but some others show delayed or no depression ? which group of depressive patients with heart diseases will demonstrate good prognosis , and which one will give bad prognosis ? today , the most probable mechanisms of relation between depression and cvd are proposed to be the change of bio - behavioral routes . from these routes , the changes of inflammatory and immune responses are considered to be important as this ends in bad prognosis . in addition , there is another hypothesis that structural and functional changes of left ventricle can also precede clinical symptoms appearing in depressive patients . some other studies have also shown that functional disorder of left ventricle is in connection with rising higher risk of depression after mi . the functional disorder and structural changes of left ventricle in depressive patients is in accordance with the view of ibn - e - sina and other scientists of pm in that the mechanism of how cardiac disease is related to depression is the connection of vital spirit produced in left ventricle with brain sensual spirit . at first , it should be mentioned that with common signs of mental melancholia as it referred to in pm , and depression in conventional medicine [ table 2 ] , these two illnesses have a common root , and so , some important points about melancholia made by pm physicians such as etiology and treatment can be applied usefully to depression . the relation between some heart diseases and depression has been revealed in the last 2 decades is the same as those mentioned clearly by avicenna in his canon book a 1000 years ago avicenna explains it clearly on the basis of pm , but today researchers describe it as a two comorbid condition and only give some probable mechanism for it . the high incidence of depression , approximately 1450% among various heart diseases shows that these two illnesses come together in the most of the time . as a result , the american association of cardiovascular and pulmonary rehabilitation strongly recommended that patients with heart disease should be screen for depression and refer for cardiac rehabilitation protocols containing food , stress management , and exercise . blumenthal et al . in their studies of standard medical intervention and longtime exercise ( smile ) found that exercise is more effective than medical treatment on the reduction of depression . in addition , considerable increase of depression disorders in cardiac patients compared to common healthy population on one hand , and recurrent depression increase following heart disease on other hand raises up a viewpoint that some heart diseases may bring conditions which ultimately result in depression . however , an important question arises which makes the relation between these two diseases more complicated . the question is why , in clinical trials studies , treatment of depression in patients with heart disease have not decreased recurrent heart disease incidence or mortality considerably and has been just effective on patient acceptance of drug treatment protocol , and his life quality , in spite of studies demonstrating that depression increases the occurrence of new heart diseases , morbidity and mortality , and mi , several times in cardiac patients ? in other words , if depression disorder is the only reason for four times increase in mi and 2.75 times increase in cardiac patients mortality , why has treatment of depression not considerably decreased mi or cardiac mortality ! ? the pessimistic results of the above studies as well as the lack of randomized placebo - controlled studies in demonstrating that the depression treatment has useful clinical effect on cvd , raise serious doubt on whether depression disorder may give rise to a bad prognosis in depressed cardiac patients . that is why some scientists make suggestion that depression itself is not probably the main cause of bad prognosis in depressed cardiac patients . it seems possible to find answer from avicenna 's view for the above question , as he clearly speaks out about the etiologic role of some heart diseases on depression outbreak , and believes that these diseases make the most important cause of depression disorder . he , then , describes the role of physicochemical changes of blood properties on producing depression . this means he does not think that depression is just brain disease , but it is believed that depression is a multi - system disease , in addition to the brain , cardiovascular system and blood circulation is impaired . he , therefore , emphasizes that for depression treatment there is no way but by treating disorders of these systems along with mental therapy . at present , it is shown that cardiac arrhythmias increase of blood pressure secondary to high levels of cortisol and decrease of heart rate variability , increase of plasma level of platelet factor iv and beta - thromboglobulin , which cause raising platelet activity , endothelial vascular damage , and thrombosis , and high levels of cytokines such as interleukin i , interleukin 6 , tumor necrosis factor ( tnf ) , which play key roles on atherosclerosis , have been documented in depressive patients . in overall , it must be said that all the above conditions are in accordance with avicenna 's views of multi system causes of depression . in addition some researchers confirm the effect of collective of various biological systems on depression disorder . among them is dr . peter kramer , who writes in his book under the title of against depression . depression is not a brain disease merely ; it is also a neurological , hematological , and cvd . thus it seems that depression is not just cerebrally originated , because with such a vision , it should not result in bad prognosis for cardiac depressive patients . the failure of depression treatment on reducing morbidity and mortality should be rooted in multiple organs of depression disorder . it is believed that in conventional medicine , limbic system , especially its area of reward and punishment centers , control of excitement , but scientists have confirmed there is a link between positive and negative emotions , such as sorrow , anger , happiness , and health specially cardiovascular health . that is , positive emotional states like optimism , hope , delightfulness , reduce cvd and increase life long time , but negative emotions like hostility , fear , sever and continuous anger cause cardiovascular problems and life time shortage . some studies by kapfhammer have shown that some depressive symptoms such as enhedonia , hopelessness , and vital exhaustion , have close connection with cvds , and so proposed that these symptoms mediate , as a specific risk factor , correlation between heart disease and depression . there is an important point in pm , that is although emotion states are correlated with vital faculty and spirit , but these states , as the scientists believe , are under control of cardiac and blood temperament . to explain more , if a person has a heart with hot temperament , and his blood is dilute and clear and its quality is hot , he is , therefore , a hopeful man with happiness tendency , because of his dilute and clear blood but he tends to be anger , because of his hot heart and blood temperaments , although his angriness rises sharply then disappears quickly because of his dilute blood . now , consider another person ; he is with hot heart and blood natural temperament , but his blood is thick and turbid ( not clear and dilute ) . here , the man is in sorrow ( because of blood turbidness ) and anger ( because of hot heart and blood temperament ) tendencies , but his angriness is stable and does not diminish quickly ( because of condensed blood ) . there is a third person who , opposite to the two above persons , is with cold heart and blood natural temperament , and his blood is thick and turbid ( like the second person ) . this man is coward and slow - witted ( because of cold heart and blood temperament ) and has a sorrow tendency ( as his blood is turbid ) and his emotional states disappear slowly ( since the blood is thick ) . today , some scientists believe that it is possible to present more precise view on the origin of depression and its correlation with heart diseases if more knowledge is collected for the following questions : why do some patients with heart diseases show the best response to the treatment but some others do not?why do some patients fall in depression quickly after a heart accident , but some others show delayed or no depression?which group of depressive patients with heart diseases will demonstrate good prognosis , and which one will give bad prognosis?we believe that if future studies focus on cognition exact of blood - cardiac temperament , it will be possible to find acceptable answers to the above questions . why do some patients with heart diseases show the best response to the treatment but some others do not ? why do some patients fall in depression quickly after a heart accident , but some others show delayed or no depression ? which group of depressive patients with heart diseases will demonstrate good prognosis , and which one will give bad prognosis ? today , the most probable mechanisms of relation between depression and cvd are proposed to be the change of bio - behavioral routes . from these routes , the changes of inflammatory and immune responses are considered to be important as this ends in bad prognosis . in addition , there is another hypothesis that structural and functional changes of left ventricle can also precede clinical symptoms appearing in depressive patients . some other studies have also shown that functional disorder of left ventricle is in connection with rising higher risk of depression after mi . the functional disorder and structural changes of left ventricle in depressive patients is in accordance with the view of ibn - e - sina and other scientists of pm in that the mechanism of how cardiac disease is related to depression is the connection of vital spirit produced in left ventricle with brain sensual spirit . based on avicenna , depression is not only a cerebral disease , but also is a multi - system disease ; in addition to the brain , cardiovascular system and blood circulation are involved . avicenna believes that in most cases , some heart diseases initiate a process which ultimately leads to depression and if the cause of depression in some cases originates from the brain , heart will be involved and make the problem worse . therefore , according to avicenna 's view , heart disease and blood changes , in most cases have important role in etiology of depression , play as initiator and in a little cases , behave as aggravator factors . therefore , he , emphasizes that depression do nt be treated without treatment of heart disease . considering high prevalence of depression among patients with heart disease , which is shown in recent studies , we think that avicenna opinion may be correct too relates of heart disease and depression . we suggest neuroscientists study this relation on available animal models of depression , such studies we ca nt found in our searches .

background : depression is one of the most important medical problems in today 's world ; despite its high prevalence , its causes unfortunately remain not fully known . among important issues regarding this is its relation with heart diseases . based on studies this comorbidity increase morbidity and mortality and leads to worst prognosis . however the cause of such high rate of comorbidity is unclear and instead of efforts to understand this correlation has prompted the medical world to consult other medicinal disciplines , not only to find the answer but also to increase the effectiveness of treatment and decrease its cost.methods:we first reviewed the most important ancient causes for depression mentioned by avicenna and considered those as the key words for our next step . then , we made a literature search ( pubmed and scopus ) with those key words to find out new scientific findings in modern medicine about the avicenna 's suggestions.results:avicenna does not regard depression as only a mental ailment , but as a disorder resulted by the involvement of brain , heart and blood . he believed that the main causes of depressive events are rooted in heart diseases ; in most cases brain is only affected secondary to the heart . thus he declared that for the treatment of depressive disorders , the underlying cardiovascular diseases should be considered.conclusions:it is worthwhile to consider the avicenna 's recommended causes of depression and to design future scientific studies based on his suggestions .

INTRODUCTION METHODS RESULTS Heart physiology by the persian medicine approach Melancholia (description, variety) Depression and melancholia Avicenna's view in etiology of melancholia DISCUSSION Avicenna's view and new studies CONCLUSIONS

a 19-year - old woman was admitted due to slowly progressing right hemiparesis . at the time of admission , apart from a slightly drowsy mental status , her presentation was unremarkable with stable vital signs and no past medical history . on physical examination , she had multiple tiny brown pigmented macules on her face and abdomen , and a bean - shaped brownish papule on the axilla and left upper arm . magnetic resonance imaging ( mri ) of the brain demonstrated diffuse hemorrhagic infarction in the middle cerebral artery territory . the cardiac mass was resected through an open thoracotomy , and the masses on both nipples were also excised during the operation . her multiple myxomas , skin pigmentation , and familial history of cardiac myxoma were compatible with cnc . upon evaluation for endocrinologic derangement , the laboratory data including adrenocorticotropic hormone , cortisol , estradiol , and testosterone were within the normal range . follow - up was planned for the patient with regular echocardiography and ultrasonography ( us ) on her breasts and thyroid glands . during 5 years of follow - up the number of nodules on her breasts gradually increased , and numerous nodules of various sizes were found on her breasts and axilla . her thyroid glands had several tiny cystic nodules on both lobes , but the overall number or size of the nodules remained unchanged . most recently , the patient was admitted due to a perianal mass that she had for 1 year . mri demonstrated a 6.75.84.0 cm round mass occupying the subcutaneous soft tissue , abutting the inferior border of the internal sphincter . the mass showed a low signal intensity on t1-weighted imaging ( wi ) and a bright high signal intensity on t2wi , and on fat suppression t2wi . 3 ) . with the presumptive diagnosis of a malignant mucinous neoplasm , surgical resection was performed . grossly , multiple thin fibrous septa were identified within the gray to white gelatinous surface . histologic examination of the tumor showed a multilobular growth of myxoid stroma and loosely deposited spindle cells . dna was extracted using a qiaamp dna mini kit ( qiagen , hilden , germany ) from the perianal specimen . polymerase chain reaction was performed using a thermal cycler ( geneamp pcr system 9600 , perkin - elmer , wellesley , ma , usa ) for which the primers of 10 exons of prkar1a,2 exon 16 of myosin , heavy chain 8 , skeletal muscle , perinatal ( myh8),3 and 7 exons of pde11a4 were prepared ( table 1 ) . the cycle sequencing procedure was followed by the use of the bigdye terminator v3.1 cycle sequencing kit ( applied biosystems , foster city , ca , usa ) according to the manufacturer 's recommendations , and the reaction mixture was analyzed on an abi prism 3100 dna sequencer ( applied biosystems ) . the genetic analysis found a novel deletion mutation ( c.537dela ) in the prkar1a gene in exon 6 . this resulted in a frameshift mutation and introduced a premature stop codon downstream in exon 7 ( fig . 5 ) . genetic studies of the pde11a gene showed mutations in exons 1 and 20 , but these mutations were also found in the normal control dna ; we used stored normal dna that was extracted from a normal volunteer . over 500 patients of diverse ethnicity with cnc from all continents have been registered by the national institutes of health ( nih)-mayo clinic ( usa ) and the cochin hospital ( france ) , and though this is the largest registry for cnc to date , it does not include patients from a korean heritage.5 one case of a sporadic form of cnc in a korean patient that manifested as a rare psammomatous melanotic schwannoma has been reported , in which molecular studies demonstrated a loss of heterozygosity at the 17q22 - 24 locus.6 however , to our knowledge , our case is the first case of cnc in a korean patient where extensive genetic studies have been performed . cnc is diagnosed in patients with at least two of the main criteria.7,8 in the present case the patient had the characteristics of cnc including skin pigmentation , cardiac myxoma , cutaneous myxoma , breast fibroadenoma , and blue nevus . cardiac myxomas are the most common noncutaneous lesions occuring in the context of cnc.7 the mass can occur in multiples with recurrences , and it does not have a predilection for the left atrium though it did occur in the left atrium of the present case.9 a study of 353 patients in the cnc consortium found that cnc can be further classified into 3 subgroups based on the pka r - ia ( prkar1a ) gene mutation status and the presence of primary pigmented nodular adrenocortical disease ( ppnad).10 although cnc shows vast genetic heterogeneity , the disease can be grouped based on phenotype to genotype correlation . according to this subclassification , the carriers of the prkar1a mutation were grouped as cnc1 , and this phenotype correlated with disease occurrence at a younger age with a higher frequency of myxomas , schwannomas , and thyroid and gonadal tumors than those without the mutation . based on these criteria , the present case would be classified as cnc type 1 given the prkar1a mutation . cnc type 2 shows a strong linkage to the short arm of chromosome 2 ( 2p16 ) , but is beyond the context of this paper and was not further studied . the prkar1a gene encodes the type 1a regulatory subunit of the camp - dependent protein kinase , or pka.11 to date , a total of 117 different prkar1a mutations have been identified ( online database : http://prkar1a.nichd.nih.gov ) in 387 unrelated families of various ethnic origins.12 prkar1a mutations are spread along the entire coding sequence without predilection for a particular exon , although several ' hot spots ' for sequence changes have been identified . not surprisingly , the novel mutation found in our patient occurred in exon 6 , which encompasses part of the camp functional domain . the previously mentioned cnc consortium also found that although numerous pathogenic variants have been identified , the majority of prkar1a defects were premature stop codons generated by nonsense or frameshift mutations.10 the instability introduced from the premature stop codon leads to nonsense mrna mediated decay and results in either an absence or reduction in the mutant protein level.10,13 much like the majority of the prkar1a mutations that have been identified , the mutation type of the novel mutation found in our case is a premature stop codon . interestingly , the premature stop codon did not result directly from the amino acid change in exon 6 , but rather downstream in exon 7 due to the frameshift change . however , despite these extensive genetic studies on prkar1a , its role in this complex is largely unknown . given the multiple interactions with major signaling pathways and sometimes opposing effects on cellular functions , prkar1a is not a tumor suppressor gene in its classic sense.14 thus , the exact role of prkar1a in tumorigenesis remains an area for further investigation . among the endocrinologic manifestations of cnc , the most common involves the adrenal gland , more specifically in the form of ppnad.1,15 this is a rare cause of cushing 's syndrome that is pituitary - independent but adrenal - dependent.1,15,16 the genetic mutation culprit linked to ppnad has been identified as protein - truncating mutations of pde11a.17 pde11a is expressed by zona fasciculata cells in the adrenal cortex that mainly produce cortisol . cnc patients without ppnad had pde11a sequence variants not significantly different in frequency from healthy controls.4 not surprisingly , our patient did not have ppnad , but had pde11a sequence variations that are commonly found in the control group of healthy adults . previous case series have failed to establish cnc in patients with superficial angiomyxoma , thus concluding that cnc is only weakly linked to these types of myxomas.18 superficial angiomyxomas , also called cutaneous myxomas , that have been previously described in association with cnc were often small ( less than 1 cm in diameter ) and occurred most commonly in the eyelids , ears , and nipples.19 superficial angiomyxomas could range from very small sessile papules to large pedunculated polypoid masses . the present case is noteworthy because the superficial angiomyxoma was large and occurred in the deep internal anal sphincter area , which is an unusual location . in conclusion , the presence of multiple myxomas and lentiginosis should be an indication to clinicians to further investigate for the presence of cnc . numerous advancements in genetic studies have helped identify the prkar1a mutation as the culprit . as of now , several hundreds of various genetic mutations in prkar1a have been identified including our novel deletion mutation ( c.537dela ) in exon 6 . despite the numerous genetic mutations identified in recent years understanding how the diverse mutations converge into a common syndrome of phenotypes may be critical in elucidating a cure .

a case of carney complex in a korean patient is presented . the patient had the characteristics of carney complex including skin lesions , positive family history , and multiple myxomas including a superficial angiomyxoma in the perianal area . an extensive genetic analysis revealed a novel mutation in the protein kinase a type i - a regulatory subunit ( prkar1a ) gene , but not in the phosphodiesterase type 11a ( pde11a ) gene . this is the first case wherein extensive genetic studies were performed in a patient with carney complex in korea .

CASE REPORT DISCUSSION

laparoscopic adrenalectomy ( la ) is one of the successful applications of minimally invasive surgical techniques . since its first description , la has been adopted quickly as the procedure of choice to treat benign functioning and non - functioning adrenal tumours . the common indications include aldosteronoma , cushing 's syndrome , pheochromocytoma , virilising and feminising tumours , and benign non - functioning tumours . the widespread adoption of la has been partly due to the improvement in laparoscopic instrumentation and technical expertise and partly to the several trials demonstrating the superiority of the laparoscopic approach over traditional open surgery in the form of shorter hospital stay , early return to activity , less postoperative pain and fewer complications related to blood loss and surgical scar.[36 ] as experience with the technique has increased , so have the indications , with reports of large tumours ( more than 5 cm in diameter ) being successfully removed using the laparoscopic approach . however as the size of the adrenal gland increases so does the technical difficulty and increased risk of dealing with an malignant pathology of the gland . the principal concerns surrounding the laparoscopic approach to large adrenal tumours are the risk of an inadequate resection and the potential for port - site or peritoneal metastases , which would adversely affect the clinical outcome . at our institute , we followed the policy of graded approach by tackling smaller tumours in the initial part of our series before embarking onto more vascular and larger tumours . herein we report our experience with 46 laparoscopic adrenalectomies performed for large adrenal masses ( > 5 cm ) and summarise the operative and postoperative outcomes . one hundred and six laparoscopic adrenalectomies were performed by a single surgical team between 2001 and 2010 . of these 46 cases involving adrenal lesions , depending on the suspected pathology , appropriate hormonal workup was done including serum epinephrine / nor epinephrine , serum cortisol , serum acth levels , serum aldosterone and urinary vma levels . iodine 131 meta - iodobenzylguanidine ( mibg ) scan was done in patients with pheochromocytoma to rule out multiple and ectopic sites of overproduction . contrast - enhanced computerised tomography ( cect ) or magnetic resonance imaging ( mri ) were relied upon in all cases to look for the size of the gland , relation to ivc on right side , renal vein on left side and presence or absence of lymph nodes . the thin layer of fascia covering the ivc was incised along the right lateral border and the same incision was extended along the peritoneum on the inferior aspect of the liver , laterally up to the right triangular ligament . the latter maneuver aided in additional retraction of the liver and exposure of the gland and the vein . a plane was created between the adrenal gland and the ivc at the lower aspect of the tumour to reach the retroperitoneal muscle and the dissection proceeded cephalad reaching the adrenal vein . the specimen side of the vein was clipped first and with two clips on patient side , the vein was divided . the gland was then dissected free from the surrounding structures . for the left side , the peritoneum on the lateral aspect of the descending colon was serially incised and the incision extended superiorly to incise the spleno - renal ligament till greater curvature of stomach was seen . this allowed complete retraction of the spleen pancreas complex and the colon by positional gravity exposing the adrenal tumour and the kidney enveloped in the gerota 's fascia . dissection was done at the site of the renal hilum , for identification of the renal vein . the adrenal gland was then dissected free from the surrounding structures and additional adrenal branches of inferior phrenic vessels were clipped or coagulated . the difficulties due to the overhanging nature of these large adrenal tumours were overcome by use of lateral ports for retraction . increased vascularity and desmoplastic reaction , seen in large tumours especially pheochromocytoma was countered with the use of ultrasonic dissector . to avoid breech of oncologic principles of rupture and spillage , two lateral ports were joined that enabled an intact removal of the gland in a retrieval bag . the thin layer of fascia covering the ivc was incised along the right lateral border and the same incision was extended along the peritoneum on the inferior aspect of the liver , laterally up to the right triangular ligament . the latter maneuver aided in additional retraction of the liver and exposure of the gland and the vein . a plane was created between the adrenal gland and the ivc at the lower aspect of the tumour to reach the retroperitoneal muscle and the dissection proceeded cephalad reaching the adrenal vein . the specimen side of the vein was clipped first and with two clips on patient side , the vein was divided . the gland was then dissected free from the surrounding structures . for the left side , the peritoneum on the lateral aspect of the descending colon was serially incised and the incision extended superiorly to incise the spleno - renal ligament till greater curvature of stomach was seen . this allowed complete retraction of the spleen pancreas complex and the colon by positional gravity exposing the adrenal tumour and the kidney enveloped in the gerota 's fascia . dissection was done at the site of the renal hilum , for identification of the renal vein . the adrenal gland was then dissected free from the surrounding structures and additional adrenal branches of inferior phrenic vessels were clipped or coagulated . the difficulties due to the overhanging nature of these large adrenal tumours were overcome by use of lateral ports for retraction . increased vascularity and desmoplastic reaction , seen in large tumours especially pheochromocytoma was countered with the use of ultrasonic dissector . to avoid breech of oncologic principles of rupture and spillage , two lateral ports were joined that enabled an intact removal of the gland in a retrieval bag . a total of 46 adrenalectomies were done in 42 patients for tumours greater than 5 cm . the average age was 33.6 years ( range 16 - 62 years ) with the male to female ratio of 1.6:1 . the lesions were localised on the right side in 17 patients and on the left side in 21 patients with bilateral tumours in 4 patients . the mean operating time was 144 min ( range 45 to 270 min ) with the average operating time for the right side being 134 min and the left side being 138 min . bleeding was the cause of conversion in three patients of large pheochromocytomas ( size 8 cm and above ) , of which two patients were pregnant . technical difficulty in one patient of paraganglioma situated in the aorto - caval window and local invasion in a patient with adrenocortical sarcoma were the cause of conversion in the other two patients . the mean in - hospital stay was 4 days ( 2 - 8 days ) with no major post - operative complications . results are summarised in table 1 . the final histological examinations revealed pheochromocytoma ( n=26 ) , paraganglioma ( n = 4 ) , adenomyolipoma ( n=7 ) , cushing 's disease ( n=4 ) , schwannoma ( n=1 ) , tuberculosis ( n=1 ) , carcinoma ( n=2 ) and adrenocortical sarcoma ( n=1 ) . one patient developed incisional hernia through the specimen retrieval site two years postoperatively that was corrected surgically . the history of large open adrenalectomy dates back to 1890 , when knowsley thornton reported excision of a large suprarenal tumour weighing more than 20 pounds necessitating concomitant nephrectomy . in the initial years there were many innovations and modifications for the approach to adrenal gland , only intent being better access . gagner 's description of laparoscopic approach in 1992 changed the scenario , making la a gold standard for wide range of adrenal disorders . shorter hospital stay , early return to activity , less postoperative pain and fewer complications related to blood loss and surgical scar are the proven benefits of the laparoscopic approach.[36 ] three issues are of utmost importance while dealing with large adrenal tumour . first is the intra - operative technical difficulty due to distorted anatomy and overhanging on surrounding important vascular pedicles , the second being the risk of dealing with a malignant neoplasm . the third issue is retrieval of these large tumours without intra peritoneal spillage . as the size of the adrenal tumour increases , inferior vena cava ( ivc ) , liver and kidney on the right and spleno - pancreas complex and kidney on the left . large tumours often overhang the adrenal vein and the ivc on the right side and the renal vein on the left side . pheochromocytomas also elicit an intense desmoplastic reaction which leads to numerous dilated vessels in the vicinity of the tumour . however in non - functional benign large adrenal tumours , the planes are well maintained . a swimmer is faster if he / she displaces the water rather than trying to push the body forward . the same principle was applied in our series by displacing the surrounding structures without handling the tumour . use of additional lateral port for retraction helped us create a plane between the tumour and surrounding structures . vascular large tumours need to be respected or else they start crying with blood filled tears . it is important to always first clip the adrenal vein on the specimen side before clipping on the patient side ; else the vein dilates and can lead to hemorrhage . our results show that large tumours can be safely tackled using the laparoscopic approach with reasonable operative times , blood loss and conversion rates . numerous publications in recent times have demonstrated safety of the laparoscopic approach for large adrenal tumours greater than 5 cm.[81216 ] there is no real consensus on the definition of large adrenal tumour . while most publications support the size of 5 cm as large,[1720 ] there are a few recent reports suggesting a size of 6 to 8 cm that should be treated as large . analysis of our results had started in 2008 and hence the need to stick to 5 cm as the size to call it large . the mean size of the glands tackled was 7.03 cm that was similar to that published by desai et al . malignancies of the adrenal gland may arise from the cortex or medulla or may be metastatic . the relationship of size of the adrenal gland with malignancy is a grey zone with varied opinions . the risk of adrenocortical carcinoma ( acc ) in an incidentally discovered adrenal tumour is 2% for tumours less than 4 cm , 6% for tumours between 4 and 6 cm , and greater than 25% for tumours larger than 6 cm . between 5 and 26% of pheochromocytomas are malignant . numerous predictors for malignancy other than size like family history , presence of virilising features , mixed hormonal secretion , solid areas on imaging and rapid enhancement and rapid washout on mri contrast imaging have been suggested . unfortunately only local invasion and presence of metastases are the only two reliable signs which accurately predict malignancy . in our series , only 3 of the 46 tumours turned out to be malignant ( 6% ) which suggests that most large tumours without evidence of invasion or metastasis on radiology preoperatively turn out to be benign . similar results have been reported by other series.[121416 ] thus if size is the sole criterion on which the choice of operative approach is based , many patients with benign large adrenal lesions would have an unnecessary open adrenalectomy that might increase their morbidity and deprive them of the benefits of la . in the absence of unequivocally preoperative or intra - operative local invasion , the appropriate procedure for an adrenocortical carcinoma ( acc ) is simple adrenalectomy . the laparoscopic view offers an excellent magnified view and makes the dissection of the adrenal gland possible . the laparoscopic approach offers as good if not better a chance of complete resection of the adrenal gland as the open approach . long - term results of accs treated laparoscopically compare favorably well with those achieved with the open approach and recurrence of malignant disease has more to do with the biology of disease processes than surgical approach adopted at initial resection . in our small experience of three malignant cases , we noticed uncomfortable thick adherence between other neighboring organs as a criteria for malignancy that was difficult to dissect . hand - assisted laparoscopic dissection is recommended by shen et al , wherein the handport aids in retraction of the overhanging tumour and the same incision is then utilised for specimen extraction . this approach would be useful for non - functioning tumours . in our experience , size is not a cause of conversion but the obscured anatomy and possible infiltration into surrounding structures is definitely a cause . in functioning tumours like a pheochromocytoma , handling can lead to fluctuations of blood pressure and disaster . we routinely place these tumours in retrieval bags and extract the specimen by joining the incision taken for the lateral ports . la is a procedure that is performed infrequently when compared to other advanced procedures like colectomy , splenectomy and even bariatric procedures . in open surgery , surgeon 's eyes , mind and the tip of the telescope is the eye of the surgeon that is controlled by the camera person . therefore , the camera person has to read the surgeons mind allowing the surgeon to make maneuvers of his choice . if a group of individuals work together performing a given procedure repetitively , the team automatically enhances in performance . ramirez - plaza cp et al echoes this sentiment and has proposed outpatient la in selected group of patients . therefore , the concept of team work as suggested by us assumes importance in performance of la for large adrenal tumours . our results show that mere size should not be considered as a contraindication to laparoscopic approach in large adrenal masses . preoperative assessment and planning , team work , experience and adherence to anatomical and surgical principles are the key to success .

background : laparoscopic adrenalectomy ( la ) has become the procedure of choice to treat benign functioning and non - functioning adrenal tumours . with improving experience , large adrenal tumours ( > 5 cm ) are being successfully tackled by laparoscopy . this study aims to present our single unit experience of la performed for large adrenal masses.materials and methods : forty - six laparoscopic adrenalectomies performed for large adrenal lesions more than 5 cm during the period 2001 to 2010 were reviewed.results:a total of 46 adrenalectomies were done in 42 patients . the mean tumour size was 7.03 cm ( 5 - 15 cm ) . fourteen patients had tumour size more than 8 cm . the lesions were localised on the right side in 17 patients and on the left side in 21 patients with bilateral tumours in 4 patients . functioning tumours were present in 32 of the 46 patients . the average blood loss was 112 ml ( range 20400 ml ) with the mean operating time being 144 min ( range 45 to 270 min ) . five patients required conversion to open procedure . three of the 46 patients ( 6.52% ) on final histology had malignant tumours.conclusion:la is safe and feasible for large adrenal lesions . mere size should not be considered as a contraindication to laparoscopic approach in large adrenal masses . graded approach , good preoperative assessment , team work and adherence to anatomical and surgical principles are the key to success .

INTRODUCTION MATERIALS AND METHODS Surgical Technique RESULTS DISCUSSION CONCLUSION

ulcerative colitis ( uc ) is an inflammatory bowel disease ( ibd ) whose etiology is only partially known . it is characterized by diffuse inflammation of the colonic mucosa , which starts in the rectum and extends continuously along the colon . it is presented in variable clinical course with periods of remission or activity . in general , patients with uc have increased body fat and decreased lean body mass , increased production of inflammatory cytokines and they maintain a chronic low - grade inflammation [ 24 ] . cardiovascular diseases ( cvd ) are also chronic disorders associated with ( low - grade ) inflammation . atherosclerosis is the main cause of cvd and is associated with endothelial dysfunction , increased accumulation of oxidized ldl ( oxldl ) , leukocytes , and smooth muscle cells in the intima of the artery , with release of inflammatory mediators ( tnf , il-6 , ccl-2 ) , resulting in the development and expansion of the atheroma plaque . risk factors for cvd include smoking , alcohol , sedentary , diet , hypertension , and diabetes mellitus ( dm ) and dyslipidemia , as well as emerging factors such as total leukocytes count , levels of hemoglobin , and reactive c protein , il-6 , and fibrinogen [ 58 ] . several studies have demonstrated that patients with chronic inflammatory diseases such as rheumatoid arthritis and systemic lupus erythematosus have higher incidence of cardiovascular disease ( cvd ) due to chronic and systemic inflammation [ 912 ] . several studies have suggested that patients with ibd also have higher risk of atherosclerosis and cardiovascular events compared the healthy population [ 7,1319 ] . however , there are no studies evaluating this risk in uc patients when the inflammatory process is controlled . the aim of this study was to evaluate the risk of cvd in women with uc , in clinical remission , using of aminosalicylates or azathioprine and aminosalicylates and to compare the results to those obtained in healthy matched volunteers and suggest a mechanism explaining the relationship between uc and cvd . the patients were selected at the reference center for inflammatory bowel disease of the alfa institute of gastroenterology , clinical hospital , federal university of minas gerais ( ufmg ) . inclusion criteria included women aged 1860 years that did not present hypertension , dyslipidemia , diabetes mellitus , hormonal disorders , or cardiovascular disease . those presenting symptoms and signs of disease activity or using glucocorticoids or biologic agents were excluded . from 144 patients with uc monitored in the reference center , only 21 met the inclusion criteria , showing the high prevalence of risk factors for cardiovascular disease in this population and the presence of active disease phase ( supplementary figure 1 ) . all participants were in clinical remission [ do not fit the truelove and witts criteria ] by the use of aminosalicylates ( sulfasalazine or mesalazine ) ( 12 patients ) or azathioprine combined with aminosalicylate ( 9 patients ) . because the introduction of azathioprine could modify the immunological and inflammatory status of patients , we initially divided patients in an asa group ( aminosalicylates ) and an aza+asa group ( azathioprine + aminosalicylates ) to determine the inflammatory profile and cardiovascular risk ; later , we compared both treatments for remission to observe possible differences . the study was approved by the ethics committee in human research of the federal university of minas gerais ( protocol 342/11 ) . blood pressure and anthropometric data such as weight , height , body mass index ( bmi ) , waist circumference , and body composition ( body fat and body fat - free ) were also obtained . after 12 h of fasting , 20 ml blood was collected for biochemical and inflammatory markers analysis . total cholesterol , hdl - cholesterol , glycemia , total protein , albumin , hemoglobin , fibrinogen , and high - sensitive c reactive protein ( hs crp ) were assessed using commercial kits ( labstest and bioclin , belo horizonte , brazil ) . total and differential leukocyte count was obtained using a neubauer chamber , and a differential count was performed on slides using a rapid kit panoptic kit ( laborclin , paran , brazil ) . il-6 , tnf , il-1 , il-10 , tgf- , ccl-2 , and anti - oxldl antibodies were determined by elisa ( bd opteia , united states ) . the 10- and 30-year framingham risk scores ( frs ) were determined using online calculators ( http://www.framinghamheartstudy.org/ ) , considering lipid profile or bmi . unpaired mann - whitney , wilcoxon , or student s t - tests were used for continuous variables . statistical analysis was performed using spss software ( statistical package for social sciences ) version 19.0 . body weight , bmi , waist circumference , and traditional risk factors for cvd such as glycemia , triglyceridemia , total cholesterol , and its fractions were similar between groups ( tables 1 , 2 ) , while body fat was increased and fat - free mass was reduced in asa group ( table 1 ) . asa patients also showed increased total proteins due to increase in globulin , with the concomitant decreased on albumin fraction ( figure 1 ) . diastolic and systolic blood pressures another important risk factor for cvd , and hscrp and esr were higher in asa patients ( table 1 , figure 1 ) . inflammatory markers such as il-6 and ccl2 were also increased in the asa group ( figure 2 ) . nitrite concentration ( indicating nitric oxide status ) and igg anti - oxldl ( an indicator of oxldl concentration ) were increased in women in the asa group ( figure 3 ) , suggesting an increased risk of atherosclerosis in these patients . the framingham scores showed that asa patients have a higher 10-year risk for cvd , considering lipid profile or bmi . similar results were obtained when assessing the 30-year risk of cvd , showing higher relative risk of cvd in the asa group ( figure 4 ) . together , changes in blood pressure and inflammation markers show that patients with uc in clinical remission and treated with aminosalicylates have higher risk of cvd when compared to healthy subjects . patients using of aza+asa had higher bmi ( p=0.06 ) , increased waist circumference , fat mass , and blood pressure , but there was no relation with physical activity in their routines ( table 1 ) . lipid profile , glycemia , total protein , globulin , albumin , hemoglobin , esr , and fibrinogen were similar between groups ( figure 1 , table 2 ) and the crp was higher in the aza+asa group ( figure 1 ) . levels of il-6 , tnf , ccl-2 , il-10 , tgf- ( figure 2 ) , nitrite , and igg anti - oxldl ( figure 3 ) were increased in the aza+asa group , showing higher risk of atherosclerosis and cvd in this group . simultaneously , adding azathioprine treatment promotes the increase of anti - inflammatory mediators in attempting to regulate the inflammatory process . the relative risk for cvd assessed by 10- and 30-year framingham scores was higher in the aza+asa group ( figure 4 ) , suggesting increased relative risk of cvd in these patients . after comparing the patients with their respective controls , the remission treatments ( asa and aza+asa ) were compared . women in the aza+asa group presented decreased levels of total protein , globulin , esr , and lymphocytes ( figure 1 , table 2 ) compared to the asa group , suggesting better regulation of the inflammatory process characteristic of the disease . patients in the aza+asa group had higher levels of tnf , il-6 , il-10 , and tgf- compared to the asa group . others parameters were similar between groups ( tables 1 , 2 , figure 3 ) . however , the presence of these differences between both treated uc groups and the 10 and 30-year framingham risk scores were similar between groups ( figure 4 ) . body weight , bmi , waist circumference , and traditional risk factors for cvd such as glycemia , triglyceridemia , total cholesterol , and its fractions were similar between groups ( tables 1 , 2 ) , while body fat was increased and fat - free mass was reduced in asa group ( table 1 ) . asa patients also showed increased total proteins due to increase in globulin , with the concomitant decreased on albumin fraction ( figure 1 ) . diastolic and systolic blood pressures another important risk factor for cvd , and hscrp and esr were higher in asa patients ( table 1 , figure 1 ) . inflammatory markers such as il-6 and ccl2 were also increased in the asa group ( figure 2 ) . nitrite concentration ( indicating nitric oxide status ) and igg anti - oxldl ( an indicator of oxldl concentration ) were increased in women in the asa group ( figure 3 ) , suggesting an increased risk of atherosclerosis in these patients . the framingham scores showed that asa patients have a higher 10-year risk for cvd , considering lipid profile or bmi . similar results were obtained when assessing the 30-year risk of cvd , showing higher relative risk of cvd in the asa group ( figure 4 ) . together , changes in blood pressure and inflammation markers show that patients with uc in clinical remission and treated with aminosalicylates have higher risk of cvd when compared to healthy subjects . patients using of aza+asa had higher bmi ( p=0.06 ) , increased waist circumference , fat mass , and blood pressure , but there was no relation with physical activity in their routines ( table 1 ) . lipid profile , glycemia , total protein , globulin , albumin , hemoglobin , esr , and fibrinogen were similar between groups ( figure 1 , table 2 ) and the crp was higher in the aza+asa group ( figure 1 ) . levels of il-6 , tnf , ccl-2 , il-10 , tgf- ( figure 2 ) , nitrite , and igg anti - oxldl ( figure 3 ) were increased in the aza+asa group , showing higher risk of atherosclerosis and cvd in this group . simultaneously , adding azathioprine treatment promotes the increase of anti - inflammatory mediators in attempting to regulate the inflammatory process . the relative risk for cvd assessed by 10- and 30-year framingham scores was higher in the aza+asa group ( figure 4 ) , suggesting increased relative risk of cvd in these patients . after comparing the patients with their respective controls , the remission treatments ( asa and aza+asa ) were compared . women in the aza+asa group presented decreased levels of total protein , globulin , esr , and lymphocytes ( figure 1 , table 2 ) compared to the asa group , suggesting better regulation of the inflammatory process characteristic of the disease . patients in the aza+asa group had higher levels of tnf , il-6 , il-10 , and tgf- compared to the asa group . others parameters were similar between groups ( tables 1 , 2 , figure 3 ) . however , the presence of these differences between both treated uc groups and the 10 and 30-year framingham risk scores were similar between groups ( figure 4 ) . in this paper we highlight the mechanisms by which patients with uc in clinical remission are at higher risk for cvd and how drug treatment influences this . this is the first work performed in patients with uc in clinical remission that associates markers of inflammation , risk of cvd , and drug treatment . compared with healthy women , women with uc in clinical remission showed increased body fat with reduced fat - free mass . the aza+asa group still had an increase in bmi and waist circumference and in risk markers for cvd , including diseases such as diabetes mellitus and dyslipidemia . changes in body composition have been reported in ibd patients and could be related to several factors such as poor eating habits , physical inactivity , and previous use of steroids . furthermore , patients with crohn s disease have increased number of adipocytes , with reduced volume and increased inflammatory infiltrate of mononuclear cells , favoring expansion and inflammatory capacity . adipose tissue is an endocrine organ that produces and secretes inflammatory cytokines and nitric oxide , leading to a chronic low - grade systemic inflammation , increasing the risk for cvd . the expansion of adipose tissue may contribute to increased blood pressure , another risk factor for cvd that has been observed in ibd and in this study . even in remission , patients with uc have a subclinical inflammatory response in the colon , which may also contribute to the increase of these inflammatory mediators . the reduction in fat - free mass in the uc group can indicate changes in muscle mass , bone mass , and hydration status and appears to be due to poor dietary habits , physical inactivity , previous use of corticosteroids , or even due to catabolic process in common chronic diseases . the increased levels of circulating cytokines and globulins ( igg anti - oxldl and anti - cytokine antibodies ) favors the diversion of amino acids for production of inflammatory mediators affecting protein synthesis and body composition . the increase of total protein seen in the asa group is the result of increased globulins . albumin synthesis may also be affected , leading to a reduction in its level , as seen in the asa group . previous studies suggest the increase of anti - immunoglobulins cytokines in patients with chronic inflammatory diseases in an attempt to combat inflammatory process . in the aza+asa group , there were no changes in these parameters , which might be associated with better immunological and inflammatory regulation and nutritional status , which were not observed in the asa group , suggesting the effect of azathioprine in uc remission . even so , the crp levels were higher in uc groups compared to controls , while esr was higher only in the asa group , suggesting better regulation of the inflammatory process in the aza+asa group and the effect of azathioprine in this group . inflammatory cytokines such as tnf and il-6 induce hepatic production of crp , which induces expression of adhesion molecules and oxldl uptake by macrophages , favoring the development of atherosclerosis plaque and cardiovascular events . serum nitrite concentration was higher in the uc group compared to matched controls , possibly due to higher inflammatory cytokine levels that also induce inos expression , generating no and peroxynitrite , triggering changes in vascular endothelium , and increasing expression of adhesion molecules and vascular permeability , leading to increased oxidative stress and increased blood pressure [ 3234 ] , which are risk factors for cvd . although the lipid profile was similar , changes in the vascular endothelium in the uc group triggered by the increase of no , crp , and inflammatory cytokines may favor the migration of ldl in the arterial intima and its oxidation , contributing to formation atherosclerosis plaque . the increase of circulating igg anti - oxldl is related to risk of atherosclerosis and suggests that patients with uc have higher levels of oxldl than controls and higher risk of cvd . compared to the control group , the asa group showed increased serum levels of il-6 and ccl-2 , while the aza+asa group has higher levels of tnf , il-6 , ccl-2 , il-10 , and tgf- circulating . the increases in these cytokines may be derived from adipose tissue , colonic mucosa affected by uc , or of circulating inflammatory cells as mononuclear cells [ 3638 ] . il-6 plays an important role in the maintenance of chronic inflammation , preventing apoptosis of lamina propria t cells . il-6 usually binds to its membrane receptor , interacting with the gp80 and gp130 subunits , and activating the stat3 pathway , but sometimes il-6 binds to its soluble receptor and that interact with gp130 on the surface of cells ( especially those not expressing il-6 receptors ) , leading to activation of stat3 . in turn , stat3 promotes upregulation of anti - apoptotic genes , avoiding leukocytes elimination in intestinal mucosa , thus contributing to maintenance of inflammation and release of these mediators to the circulation , even during the remission phase [ 3941 ] . these processes , in turn , may interfere in the cardiovascular system , favoring changes in vascular permeability and ldl oxidation , and leading to development of atherogenesis and its complications . in this regard , we question whether il-6 could interfere with leukocyte survival in atherosclerotic plaque , contributing to its maintenance and progression . il-6 is also capable of reducing the production of no via enos , resulting in endothelial dysfunction as well as increased expression of adhesion molecules and the scavenger receptors cd36 and sr - a1 [ 38,4245 ] . vascular permeability can be altered by these factors , favoring the migration of monocytes to the intima of arteries and the up - take of oxldl , contributing once again to the formation of the foam cells and progression of atherosclerosis . ccl-2 , in turn promotes the recruitment of monocytes , thereby facilitating the migration to different locations , including adipose tissue , colon , and intima of arteries , thereby contributing to the formation of atheroma . although the levels of circulating leukocytes , especially monocytes , were similar between groups , the increased concentration of ccl-2 could induce migration of monocytes , perpetuating inflammation , but keeping monocyte concentration in the normal range in the systemic circulation . tnf were increased only in the aza+asa group , demonstrating higher severity of the disease , which can justify the use of azathioprine in this group . in general , the addition of azathioprine occurs when the patient does not respond to corticosteroids , when the patient is dependent on corticosteroids , or when the patient does not respond to conventional treatment with aminosalicylates , requiring the addition of the drug . tnf induces the secretion of other inflammatory mediators such as il-1 , pcr , and no . in addition , it also increases adhesion molecules and causes endothelial dysfunction , increasing risk of dcv [ 41,4648 ] . only the aza+asa group showed increased production of il-10 and tgf- , which are anti - inflammatory cytokines . il-10 reduces the antigen presentation and activation of inflammatory cells and reduced the activation of nf-b as well as production of inflammatory mediators , contributing to immunology homeostasis , while tgf- participates of the modulation of the activation of endothelial cells and th1 cells , inducing the formation of regulatory t cells and promoting mucosal intestinal healing . increased levels of these cytokines in patients using azathioprine compared to controls suggest a mechanism of immunoregulation , which was not seen when the asa group was compared to its matched controls . when both uc groups were compared , we noted that the aza+asa group showed increased il-6 , ccl-2 , tnf , il-10 , and tgf- , indicating that although there is increase of inflammatory cytokines , the association of azathioprine and aminosalicylate induces increase of the anti - inflammatory cytokines ( il-10 and tgf- ) , suggesting better immunoregulation in this group , and consequently reducing the risk of atherosclerosis . azathioprine reduces colonic inflammation by blocking rac1 via , resulting in apoptosis of tcd4 + lymphocytes and regulating colonic inflammation . thus , azathioprine appears to modulate t cell repertoire , reducing tcd4 + lymphocytes and preserving the il-10 and tgf- producer regulatory t cells , and promoting immunoregulation of the inflammation related to uc and cvd in this group . although aminosalicylates are associated with reduction of inflammatory cytokines and oxidative stress , the asa group was not able to induce anti - inflammatory mediators , suggesting that this group has a more inflammatory profile that leads to a more susceptible cvd status compared to the aza+asa group . the combination of azathioprine and aminosalicylates changed the profile of immune - inflammatory response in the studied patients , regulating the inflammatory process and reducing the risk of cvd , probably because the drugs have different mechanisms of action that block different inflammatory pathways . however , it is not possible to know whether the isolated use of azathioprine induces immunoregulatory response similar to that observed or whether this effect is only seen when the drugs are combined . the relative risk for cvd , estimated by the framingham risk scores at 10 and 30 years , showed that asa and aza+asa groups had higher cvd risk compared to their controls . although most patients did not show traditional risk factor for cvd , higher risk was probably due to the increase in blood pressure in uc groups . although the framingham risk scores were similar in both uc groups , the aza+asa group showed a better inflammatory profile , which is not included in the framingham score , and which could benefit these patients , improving cvd risk and its complications . we believe that previous use of glucocorticoids associated with sedentary lifestyle and poor dietary habits favor the expansion of adipose tissue . adipose tissue expansion and the inflamed bowel mucosa are factors related to higher production of cytokines and oxidative stress . increased inflammation markers result in changes in the vascular permeability , including increased expression of adhesion molecules and endothelial dysfunction , favoring the development of complications such as increased blood pressure and migration of monocytes to the arterial intima . increased inflammatory cytokines , rcp , and no also induce ldl oxidation , which was indirectly seen by anti - oxldl antibodies . together , this profile favors the development of atherosclerotic plaque , resulting in a higher risk for cvd . in turn , therapy for maintenance of clinical remission affects the inflammatory response , suggesting that the addition of azathioprine to conventional treatment with aminosalicylates decreases the inflammatory process , induces anti - inflammatory mediators , and reduces the risk of cvd in this group ( figure 5 ) . women with uc in clinical remission maintain a more inflammatory profile compared to their healthy controls , suggesting increased risk of atherosclerosis and cvd . the addition of azathioprine to treatment with aminosalicylates reduces inflammation and increases anti - inflammatory cytokines , evidencing better regulation of inflammatory processes and reducing the risk of cvd in this group when compared to that treated with aminosalicylates alone . patients with uc treated in the reference center for inflammatory bowel disease of the alfa institute of gastroenterology . of the total of 144 patients with uc treated at the reference center , 93 were women and 51 were men . among men , 48 were excluded for drug treatment ( use of corticosteroids or biology therapy ) ; 23 ( 24.7% ) were excluded for risk factors or diagnosis of cvd or other diseases ; 8 ( 8.6% ) were excluded by age ; 10 ( 10.7% ) abandoned the treatment , were not found , or did not want to participate in the study , and only 21 ( 22.6% ) women met the study criteria and wanted to participate .

backgroundulcerative colitis ( uc ) is a chronic inflammatory bowel disease with involvement of the immune system . chronic inflammatory diseases have been associated with increased risk of cardiovascular disease ( cvd ) but few studies have assessed this risk in patients with uc and the influence of drug treatment . thus , we evaluated the risk of development of cvd in women with uc in clinical remission , considering the drug treatment.material/methodstwenty-one women with uc participated in this study : 12 used aminosalicylates ( asa group ) and 9 used azathioprine added to aminosalicylates ( aza+asa group ) . the healthy control group was matched for age . we evaluated blood pressure , body composition , and biochemical and immunological parameters.resultscompared to the respective control group , the uc groups showed expansion of body fat and less lean body mass . blood pressure , pro - inflammatory cytokines , nitric oxide , c reactive protein , erythrocyte sedimentation rate ( esr ) , and anti - oxidized ldl antibodies were higher in uc groups . only aza+asa group showed increased anti - inflammatory cytokines ( il-10 and tgf- ) . framingham scores showed higher risk of cvd in uc groups . uc groups were compared and women treated with azathioprine showed reduction of total protein , globulin , esr , and lymphocytes , with increased il-6 , tnf , il-10 , and tgf-.conclusionsour data suggest that women with uc in clinical remission have a higher risk for development of atherosclerosis and cvd when compared to the control group , while women treated with azathioprine seem more protected than those treated only with aminosalicylates , due to better regulation of the inflammatory process .

Background Material and Methods Results Comparison between patients with UC in clinical remission in use of aminosalicylates (ASA) and control healthy (CT) Comparison between patients with UC in clinical remission receiving azathioprine and aminosalicylate (AZA+ASA) and healthy controls (CT) Comparison between patients in use of aminosalicylates (ASA) and azathioprine combined with aminosalicylate (AZA+ASA) for UC remission Discussion Conclusions Supplementary Data

gastroesophageal reflux disease ( gerd ) is a highly prevalent disorder affecting approximately 20% of the western population.1 furthermore in asia , where the prevalence of gerd is traditionally low , several reports suggest a rapid increase in prevalence over the last several years.24 the primary option for the treatment of gerd is acid suppressive therapy , which has been shown to be effective in reducing symptoms and healing esophagitis.5,6 a large group of gerd patients however attend surgical and gastroenterology clinics for alternative treatment options as they have persistent troublesome gerd symptoms despite of optimal acid suppression or are unwilling to undergo lifelong medical treatment.7 laparoscopic antireflux surgery ( lars ) offers an established alternative treatment option to medical therapy , reducing both heartburn and regurgitation effectively.8 many patients are however reluctant to this procedure , since new - onset dysphagia , gas - bloating , inability to vomit and/or belch are expected complications after lars.9,10 electrical stimulation of the lower esophageal sphincter ( les ) is a relatively new technique for the treatment of gerd that may address the need of a large subgroup of gerd patients , unsatisfied with medical therapy and concerned with the potential risks of lars . the aim of electrical stimulation therapy ( est ) is to enhance the barrier function of the les in order to obtain reflux control , while preserving normal les function and esophagogastric junction ( egj ) anatomy . in this review we present available data about est for gerd , including a short overview of the development of the technique , a description of the implant procedure , and safety aspects and results of the performed open - label studies.1113 the first studies on electrical stimulation of the les for the treatment of gerd were performed in canine models . ellia et al14 reported that electric stimulation ( 20 hz , pulse width of 3 msec ) with 2 pairs of electrodes caused a prolonged contraction of the sphincter complex , which was able to prevent gastroesophageal reflux in dogs with surgically - induced egj incompetence . in another animal study which investigated pulse parameters in more detail , it was found that only low frequency stimulation ( 6 cycles per minute , pulse width of 375 msec ) caused a prolonged increase in les pressure.15 the effect of stimulation was sustained even after the stimulation had stopped , while les relaxations during swallows were unaffected . high frequency stimulation ( 20 hz , pulse width of 200 sec ) by endoscopically placed micro - stimulators into the esophageal muscle layer showed similar results , but only after exceeding a 8 ma threshold.16 rodriguez et al17 were the first to investigate the effects of high ( 20 hz , pulse width of 200 sec ) and low ( 6 cycles per minute , pulse width of 375 msec ) frequency electrical stimulation on les in gerd patients . electrical stimulation with both high and low frequency caused an increase in les pressure without affecting les relaxation and les residual pressure during swallowing . high frequency stimulation was determined to be a better option as it uses less energy and increases battery life . similar to the outcomes of the animal studies , the effect of stimulation was sustained even after the stimulation was stopped . these results were replicated by banerjee et al18 using temporary stimulation leads implanted during endoscopy . the les - stimulation system ( endostim bv , the hague , the netherlands ) consists of 3 components : an implantable pulse generator ( ipg ) , a bipolar lead with 2 stitch electrodes and an external programmer ( fig . 1 ) . electrical stimulation to the les is generated by the ipg , sending electrical pulses of 5 ma at a rate of 20 hz via the bipolar lead to the 2 stitch electrodes , implanted in the les muscle . the ipg contains a non - rechargeable battery with a lifetime of 1015 years , which is inserted in a subcutaneous pocket in the anterior abdominal wall ( fig . the external programmer is used to modify parameters of the stimulation and to read therapy statistics . temporary les - stimulation studies had shown a sustained effect of est on les pressure that persisted long after stimulation was stopped , enabling the intermittent stimulation of the les.17 implantation of the ipg and bipolar lead is performed using standard laparoscopic techniques , which were described previously in detail.11 in short , after trocar placement , dissection of fat tissue is performed to expose the muscle wall of the distal esophagus . in the presence of a small hiatal hernia , repair by caudal retraction of the esophagus and tightening of the hiatus by standard surgical procedure is indicated . the bifurcated lead is then introduced into the abdominal cavity and using a guiding needle , the electrodes at the proximal end of the lead are inserted and secured into the esophageal muscle wall ( fig . 2 ) upper gastrointestinal endoscopy is performed to identify the z - line by transillumination and to avoid perforation during electrode placement . the distal end of the bipolar lead is retracted through the abdominal wall and connected to the ipg . after a functionality test of the whole implanted system by the operator , the ipg after the anesthetics have worn off , the ipg is programmed for electrical stimulation therapy . patients usually stay over in the hospital for one night for observation and are advised to wear an elastic compression bandage over the subcutaneous pocket and the ipg for 1014 days to prevent formation of a seroma . the results of the current studies demonstrate the safety of les stimulation with an excellent side effect profile . in the 2 clinical trials one sae was procedure related : a trocar perforation was identified on post - operative day one and was successfully repaired laparoscopically . the lead and the other 2 saes ( thyroid surgery and cardiac arrhythmia - atrioventricular [ av ] nodal reentrant tachycardia successfully treated with av nodal ablation ) were not device or therapy related . of the remaining 77 non - sae , 26 events had probable or definite relation to the procedure or the device ( implant site pain , localized infection and post - operative nausea ) . mild transient dysphagia was reported in 2 patients that underwent hiatal hernia repair which resolved spontaneously without intervention . no gi side effects such as diarrhea , bloating and inability to belch were reported and no cardiac side effects with les - est were observed . two open - label clinical studies investigating the efficacy and safety of est of the les using the endostim les stimulation system were initiated and showed promising efficacy with results up to 24 months of follow - up.1113 in the first open - label , single center trial performed in chile , 25 gerd patients ( 14 men , mean age 52 years ) with chronic ( > 6 months ) gerd symptoms , such as heartburn , regurgitation or retrosternal pain were included . gerd was well documented by 24-hour ph - monitoring and upper gi endoscopy , showing pathologic acid exposure time ( ph < 4.0 during > 5.0% of time ) and/or esophagitis . main exclusion criteria were the presence of hiatal hernia > 3 cm , esophagitis > los angeles grade c , long - segment barrett s esophagus or dysplasia . gerd symptoms were evaluated with gerd - health - related quality of life ( hrql ) , a disease - specific quality of life questionnaire , at baseline ( on and off proton pump inhibitor [ ppi ] therapy ) and during follow - up of electrical stimulation therapy.19 furthermore , endoscopy , 24-hour ph - monitoring and high - resolution manometry were performed for secondary study endpoints . one patient withdrew consent 2 weeks after implantation due to anxiety related to the device and multiple follow - up tests required by the protocol so the ipg was explanted despite a favorable initial symptom response . the first results of this trial showed improvement of symptom scores soon after activation of the device and at 6 months median ( interquartile range [ iqr ] ) gerd - hrql symptom score was 2 ( 04 ) , which was significantly better than the median baseline scores both on ppi ( 9 [ 610 ] , p < 0.001 ) and off ppi therapy ( 24 [ 2126 ] ) . twenty - one of 24 ( 88% ) patients were able to completely stop ppi usage . acid exposure time during 24-hour ph - monitoring improved from median of 10.1% at baseline to a median of 5.1% after 6 months ( p < 0.001).11 this study was initially designed with a 6-month follow - up period , but was extended to 2 years in order to collect additional long - term data . one patient did not enroll into the extension phase of the trial since he was planning to undergo a roux - en - y gastric bypass procedure for uncontrolled type 2 diabetes . twenty - three patients completed one year follow - up and the results were recently published.12 median ( iqr ) gerdhrql symptom score remained low at 12 months ( 2 [ 03 ] , compared to baseline off ppi [ p < 0.001 ] , and baseline on ppi [ p = 0.002 ] ) ( table ) . median acid exposure time decreased even further to 3.3% from baseline ( p < 0.001 ) and 14 of 22 ( 64% ) patients showed normal distal esophageal acid exposure time ( ph < 4.0 during < 4% of time ) . the trial also showed normalization of proximal esophageal acid exposure in subset of patients with proximal reflux at baseline . high - resolution manometry showed an increase of end expiratory and mean les pressure , whereas esophageal body function and les relaxation were unaffected ( fig . 3 ) . new - onset dysphagia or sensation of the electrical stimulation was not reported . currently , 21 patients have completed 24-month follow - up and the favorable outcomes of est both on symptom scores and acid exposure time prevail over time . seventy - one percent of patients showed either a normalized or at least 50% reduction in acid exposure time . the second ongoing open - label multicenter study was initiated at university hospitals in the netherlands , but patients were also recruited from new zealand , india , hong kong and chile . with similar inclusion and exclusion criteria as in the first open - label trial , 24 patients ( 14 men , median age 51 ) were included . currently , 18 patients have completed 3-month follow - up and 14 patients 6-month follow - up . interim results showed a positive effect on symptoms : gerd - hrql median ( iqr ) score was 4 ( 211 ) at 3 months and 5 ( 49 ) at 6 months , respectively with p < 0.001 and p < 0.01 when compared to baseline score off ppi ( 31 [ 2537 ] ) . furthermore , acid exposure time decreased from 11.3% ( 9.015.5% ) at baseline to 3.3% ( 2.59.1% ) at 3 months and to 2.6% ( 1.85.4% ) at 6 months ( fig . at present , available data on est of the les indicate that est may be an effective and safe treatment option for gerd patients searching for an alternative to medical therapy or conventional lars . the pathophysiology of gerd is multi - factorial ; several factors can lead to the prolonged exposure of the esophageal epithelium to gastric acid and cause the perception of heartburn and/or regurgitation.20,21 the primary mechanism to prevent gastroesophageal reflux however is a competent barrier function of the egj , composed of the les and the crural diaphragm.22 in gerd patients , the barrier function of the egj is impaired , allowing reflux to occur more easily than in healthy subjects.23 acid suppressive therapy does not influence the function of the egj and symptoms due to reflux of non - acid gastric contents can persist.24,25 lars focuses on reinforcement of the egj by wrapping the gastric fundus around the distal esophagus . the procedure is effective in reducing gastroesophageal reflux , but the mechanical effects of the wrap can exceed the primary goal and lead to new onset dysphagia and inability to belch.26 additional problems such as diarrhea and gas - bloating have been reported.9,27,28 due to the risks of postoperative complications , many patients dissatisfied with ppi therapy are reluctant to undergo this procedure . several alternative options for the treatment of gerd have been introduced during the last 2 decades . less - invasive endoscopic suturing devices as the endocinch ( bard endoscopic technologies , nurray hill , nj , usa ) , plicator ( ndo surgical inc . , , silver spring , md , usa ) have tried to mimic the effects of conventional anti - reflux surgery by the creation of gastroesophageal plications.2931 although early improvements in symptoms and medication use were reported in several trials , clinical efficacy or durability to the level of conventional anti - re - flux surgery have not been met.32 the enteryx procedure aimed to increase sphincter competence by injection of sponge - like material into the les . a sham - controlled study showed a moderate effect on symptoms and ppi use , however the product was withdrawn after several reports of severe adverse events related to the injection of the substance.32,33 the stretta device ( curon medical , sunnyvale , ca , usa ) delivers radiofrequency energy to the les muscle , inducing fibrosis of the les muscle and thereby augmenting its function . although a mild subjective improvement was shown in a sham - controlled trial , it failed to improve objective reflux parameters.34 all these therapies were unable to demonstrate durable and clinically meaningful improvement in distal esophageal acid exposure . another recent alternative is the magnetic sphincter , a bracelet of magnetic beads ( linx - device ; torax medical , st . a recently published study showed encouraging results on acid exposure time and symptom improvement ; however the high frequency of dysphagia requiring dilation is similar to that of conventional anti - reflux surgery and in a fraction of patients the device was removed because of sae.35 so far , no randomized controlled trials are available to fully assess the effect of the linx - device . the current results of est are promising as the 2 open - label studies have shown that est is a safe technique with significant improvement in both subjective outcomes and objective parameters of gerd . les stimulation significantly enhanced les pressure , reduced esophageal acid exposure time and improved gerd symptom scores . interestingly , the effect of est on acid exposure time increased during follow - up in the first open - label trial and also the preliminary results of the multicenter trial show a comparable improvement on acid exposure over time ( fig . 4 ) . these findings could be due to the possibility of est to non - invasively change the settings of the ipg to optimize the anti - reflux effect . however , in the current trials adjustments to the ipg settings were limited per protocol and stimulation parameters as pulse width , pulse frequency and pulse shape were similar for all patients . future trials are needed to reveal if tailored - fit electrical stimulation therapy , customized to the patients individual needs , may even further improve the anti - reflux effect of est . the mechanisms underlying the anti - reflux effect of est have not been studied in detail . the observed increase in les pressure can contribute to the anti - reflux effect of the procedure ; however other mechanisms as reduction of the number of transient les relaxations and changes in gastric motility may also play a role and need to be studied . the implant procedure is quite simple to perform for general laparoscopic surgeons and can be easily standardized for uniform performance . in comparison to laparoscopic fundoplication , there seems to be a shorter learning curve for the implantation procedure of est . therefore , it is no surprise that results from the multicenter trial in medical centers that performed only a few implantations are similar to the outcomes of the experienced single center trial . furthermore in contrast to laparoscopic fundoplication , the egj anatomy is unaltered during the implant procedure , making est easily reversible by removal of the electrodes and ipg . unlike traditional anti - reflux surgery or procedures , les - est does not adversely affect esophageal body function or les relaxation and hence could be an important therapeutic alternative for gerd patients with esophageal dysmotility that are not adequately addressed by medical management . furthermore , post - sleeve gastrectomy reflux is a new and rising subgroup of gerd patients that are inadequately treated with medication and are not a candidate for traditional anti - reflux surgery.36 these patients could be addressed with les - est and a pilot study will be started in our center to evaluate the effect of est in patients with post - sleeve gastrectomy gerd . reduction of the battery size could improve the cosmetic effect of the procedure and limit pocket related symptoms . ultimately , endoscopic implantation of rechargeable electronic microstimulators would be an ideal anti - reflux procedure ; this is however not expected in the very near future . some points need to be taken into consideration when interpreting the data published so far . the results of the multicenter trial are preliminary and long - term follow - up data need to be awaited . furthermore , study participants represent a subgroup of gerd patients with only small hiatus hernia and no severe esophagitis or barrett s esophagus . whether gerd patients with greater anatomical defects or more severe disease can benefit from est is unknown , but the combination of hiatal hernia repair with est might offer an alternative to lars . finally , the available data are obtained during non - randomized controlled studies . in order to further establish the effect of est on gerd , the technique of electrical stimulation of the les is especially suitable for a sham - controlled study , as the device can be easily activated and turned - off . in conclusion , current results suggest that est is a safe and effective therapeutic alternative that may fill the gap between ppi therapy and conventional anti - reflux surgery . more long - term data and controlled trials are required before est can be considered as an alternative for the treatment of gerd .

electrical stimulation therapy ( est ) of the lower esophageal sphincter is a relatively new technique for the treatment of gastroesophageal reflux disease ( gerd ) that may address the need of gerd patients , unsatisfied with acid suppressive medication and concerned with the potential risks of surgical fundoplication . in this paper we review available data about est for gerd , including the development of the technique , implant procedure , safety and results from open - label trials . two short - term temporary stimulation and long - term open - label human trials each were initiated to investigate the safety and efficacy of est for the treatment of gerd and currently up to 2 years follow - up results are available . the results of est are promising as the open - label studies have shown that est is a safe technique with a significant improvement in both subjective outcomes of symptoms and objective outcomes of esophageal acid exposure in patients with gerd . however , long - term data from larger number of patients and a sham - controlled trial are required before est can be conclusively advised as a viable treatment option for gerd patients .

Introduction Development of Electrical Stimulation Therapy for Gastroesophageal Reflux Disease Technique Implant Procedure Safety Efficacy Discussion

between september 2007 and august 2010 , we performed arthroscopic surgery in 207 patients . of them , the indications for surgery were hip pain with associated mechanical symptoms and intractable pain after conservative treatment for at least 12 weeks . on physical examination , all had a painful range of motion with positive impingement test ( flexion , adduction , and internal rotation ) . preoperative imaging included plain radiographs ( pelvis anteroposterior and frog leg ) and computed tomography ( ct ) arthrogram to detect bony impingement and labral tear . we excluded those who had undergone previous hip surgery and those with avascular necrosis , rheumatologic disorder and patients with arthritis on radiography ( tonnis grade > 2 ) . there were 10 men and 11 women with a mean age of 35 years ( range , 22 to 57 years ) . preoperative diagnoses included isolated labral tear in 11 hips , cam - type impingement in six hips , and pincer - type impingement in four hips . preoperative radiographs revealed tonnis grade 0 to 1 changes in 20 hips and grade 2 changes in one hip ( table 1 ) . we performed hip arthroscopy using a standard fracture table with patient in the supine position . traction was applied with slight extension and adduction of the hip joint with enough force to open the joint approximately 1 cm . two or three portals ( anterolateral , anterior , and/or posterolateral ) were placed for arthroscopic labral repair . a suture anchor was placed on the capsular side ad secured , followed by a suture passer was used to deliver a limb suture through a small portion of the substance of the labrum . an arthroscopic sliding knot was tied and passed down the cannular to secure the repair . the operation was completed by joint lavage and injection with local anesthetic . postoperatively , all patients followed a standardized rehabilitation program . immediately after surgery , passive and active range of movement was permitted . patients were kept toe - touch weight bearing for 2 weeks with range of motion encouraged but avoiding extremes of external rotation . in general , patients typically required 2 to 3 weeks of crutch assistance . patients who underwent microfracture were kept toe - touch weight bearing for 6 to 8 weeks . clinical and radiographic follow - up evaluations were performed at the 6 weeks , 3 months , 6 months , 12 months , and every 6 months thereafter . clinical evaluations were performed using harris hip score ( hhs),10 ) visual analog scale ( vas ) , western ontario and mcmaster universities ( womac ) osteoarthritis index , university of california , los angeles ( ucla ) activity , and a satisfaction survey . radiographic analyses included anteroposterior view of pelvis , frog - leg lateral view , and translateral view of hip . all radiographs were assessed using the tonnis classification,11 ) which was used to grade radiographic degenerative changes , and the outerbridge classification system,12 ) which grades the degree of chondromalacia . the difference between preoperative and postoperative outcome measures were analyzed using wilcoxon signed rank test for continuous outcome measures and fisher exact test for categorical outcome measures . eighteen patients reported that they were satisfied with the reduction or elimination of preoperative pain . although the clinical scores improved compared to preoperative scores , three patients were not satisfied with the results of the surgery . in these patients , the vas score was reduced by 2 points , womac score was reduced by 10 points , hhs score was increased by 3 points , and the ucla score was not changed . the overall rating for the 21 patients ( 21 hips ) , using the hhs was 73 points ( range , 64 to 84 points ) preoperatively and 83 points ( range , 66 to 95 points ) postoperatively ( p < 0.001 ) . four hips ( 19% ) had an excellent score , 11 hips ( 52% ) a good score , five hips ( 24% ) a fair score , and one hip ( 5% ) had a poor score ( p < 0.001 ) . the womac score was 16 points ( range , 2 to 50 points ) at the final follow - up evaluation . ucla activity at the latest follow - up improved in 16 patients and remained the same in two patients . the mean vas score decreased from 8 to 2 at the latest follow - up ( table 2 ) . at the time of surgery , arthroscopy showed synovitis in 14 hips . four of these patients had an outerbridge grade iii articular cartilage lesion of the acetabulum at the time of surgical exploration . the tonnis grade of osteoarthritis did not change in all patients at the latest follow - up . significant complications , such as infection , heterotopic ossification , thromboembolic episodes or permanent nerve injury , did not occur at the latest follow - up . eighteen patients reported that they were satisfied with the reduction or elimination of preoperative pain . although the clinical scores improved compared to preoperative scores , three patients were not satisfied with the results of the surgery . in these patients , the vas score was reduced by 2 points , womac score was reduced by 10 points , hhs score was increased by 3 points , and the ucla score was not changed . the overall rating for the 21 patients ( 21 hips ) , using the hhs was 73 points ( range , 64 to 84 points ) preoperatively and 83 points ( range , 66 to 95 points ) postoperatively ( p < 0.001 ) . four hips ( 19% ) had an excellent score , 11 hips ( 52% ) a good score , five hips ( 24% ) a fair score , and one hip ( 5% ) had a poor score ( p < 0.001 ) . the womac score was 16 points ( range , 2 to 50 points ) at the final follow - up evaluation . ucla activity at the latest follow - up improved in 16 patients and remained the same in two patients . the mean vas score decreased from 8 to 2 at the latest follow - up ( table 2 ) . four of these patients had an outerbridge grade iii articular cartilage lesion of the acetabulum at the time of surgical exploration . the tonnis grade of osteoarthritis did not change in all patients at the latest follow - up . significant complications , such as infection , heterotopic ossification , thromboembolic episodes or permanent nerve injury , did not occur at the latest follow - up . although most studies reported encouraging short to midterm results of arthroscopic treatment of labral tear , all of these studies were conducted in western countries ( table 3 ) . to our knowledge , this is the first study regarding acetabular labral repair in an asian country . eighteen of 21 patients were satisfied with relief of pain and/or improvement of their functional outcome with no progression of arthritic change of radiographic evaluation at the minimal 2 year follow - up . bedi et al.7 ) performed a systemic review to assess outcomes after surgical treatment of labral tears and femoroacetabular impingement ( fai ) . they reported 65% to 85% of patients who underwent open surgical procedures were satisfied with the outcome at a mean of 40 months after surgery as compared to 67% to 100% of patients who underwent arthroscopic surgery who were satisfied . however , most of the published research has addressed debridement of torn acetabular labrum . with increasing knowledge about the function and importance of the labrum , surgical techniques including suture anchor repair must be encouraged to maintain the function of the hip joint and decrease the development of early arthritic change . larson et al.6 ) compared the outcomes of arthroscopic labral debridement ( 44 patients ) versus refixation ( 50 patients ) in 94 patients at a mean follow - up of 3.5 years . they concluded that labral refixation resulted in better outcomes and a greater percentage of good to excellent results compared with the results of labral debridement . schilders et al.8 ) compared arthroscopic labral repair ( 69 hips ) and resection ( 32 hips ) in 96 patients ( 101 hips ) at a mean follow - up of 2.4 years . although we could not compare debridement with repair in our series , our findings are consistent with previous studies . in this study , the hhs improved from 73 points ( range , 64 to 84 points ) preoperatively to 83 points ( range , 66 to 95 points ) postoperatively , and the ucla activity improved in 16 patients and remained similar in two patients at the latest follow - up . eighty - six percent ( 18/21 ) of patients were satisfied with their arthroscopic labral repair . beaule et al.5 ) reviewed acetabular labral tear and mentioned that acetabular labral tears rarely occur in the absence of a structural osseous abnormality , such as fai . fai is a proposed etiology of early osteoarthritis in young patients.13,14,15 ) however , the incidence of hip osteoarthritis in the general population has marked ethnic and racial differences.16 ) the rate of moderate to severe idiopathic and secondary hip osteoarthritis in caucasians is 3%-6% compared with 1% in east indians , blacks , hong kong chinese , and native americans.17,18,19 ) epidemiologic data suggest that the incidence of fai and acetabular labral tear in asian countries might be lower than in western countries.16,17,18,19 ) however , if the patients have acetabular labral tear with osseous abnormality , daily activities are more seriously inconvenienced in asian patients , as the asian life style and culture requires more flexion and abduction motion of the hip joints . seventy one percent ( 15/21 patients ) in this study reported excellent and good results at the minimum 2-year follow - up . ilizaliturri et al.20 ) performed arthroscopic surgery on 19 hips who were diagnosed with fai with labral tear and reported 84% good to excellent results at the minimum follow - up of 24 months . larson and giveans21 ) compared the outcomes of arthroscopic labral debridement ( 36 hips ) with those of labral refixation ( 39 hips ) . they reported 89.7% good and excellent results in refixation groups at the minimum follow - up of 12 months . firstly , there was no control group and the total numbers of patients were small . nevertheless , this study proves the value for athroscopic procedures on acetabular labral tear with or without osseous abnormality . secondly , acetabular labral tear is frequently associated with other osseous structural abnormalities , such as cam and/or pincer - type deformities . however , we evaluated and did confirm the 86% satisfaction rate at minimum 2-year follow - up . in conclusions , arthroscopic labral repair resulted in 86% satisfaction and 71% good to excellent results at a 2-year follow - up . therefore , the hip arthroscopic procedure might be an excellent alternative to open surgery and promises greater probability of good to excellent results .

backgroundacetabular labral tear is a main cause of hip pain and disability , often requiring surgical treatment . improvements of hip arthroscopic technique have produced positive outcomes after labral repair with arthroscopy . the purpose of this study was to determine clinical outcomes and patient satisfaction after arthroscopic repair of acetabular labral tear.methodswe interviewed 21 patients ( 10 men and 11 women ; mean age , 36 years [ range , 22 to 57 years ] ) with acetabular labral tears that had been repaired arthroscopically in terms of satisfaction of the procedure . in addition , clinical outcome was assessed using visual analog scale ( vas ) score , university of california , los angeles ( ucla ) activity , western ontario and mcmaster universities ( womac ) osteoarthritis index , and harris hip score , and radiologic outcome was assessed using serial radiography . the patients were followed for 24 - 50 months.resultsthe mean harris hip score was 73 points ( range , 64 to 84 points ) preoperatively and 83 points ( range , 66 to 95 points ) postoperatively . fifteen hips ( 71% ) were rated excellent and good . the mean womac osteoarthritis index and vas scores were improved at final follow - up . ucla activity at the latest follow - up improved in 16 patients . the tonnis grade of osteoarthritis at the latest follow - up did not change in all patients . eighteen of the patients ( 86% ) were satisfied with the procedure.conclusionshigh rate of satisfaction after arthroscopic repair of acetabular labral tears is an encouraging outcome . arthroscopic treatment of labral tears might be a useful technique in patients with hip pathologies , such as femoroacetabular impingement with labral tears .

METHODS RESULTS Patient Satisfaction Clinical Outcome Radiologic Outcome DISCUSSION

heart transplantation has been used for the last three decades as a method of treating extreme heart failure . women constitute one third of heart transplant recipients ; approximately 20% of them are in reproductive age . pregnancy was contraindicated in women after heart transplantation during the 1990s ; today , it is an increasingly common challenge for clinicians . such pregnancy is always considered high - risk , as it is burdened with the risk of several typical complications . we present the case of a 30-year - old patient , who , having undergone a heart transplant due to hypertrophic cardiomyopathy , gave birth to two healthy children . the 30-year - old patient was admitted to the clinic of perinatology of the medical university of d in the 34 week of a third pregnancy due to a risk of premature labor and weak fetal movement . hypertrophic cardiomyopathy with left ventricular outflow tract obstruction was diagnosed in the described patient as early as her first year of life . taking the medical history of her family revealed that her mother and brother also suffered from hypertrophic cardiomyopathy ; they , too , underwent heart transplantation . after many years of treatment with beta blockers and verapamil , during which she remained in nyha functional class ii , the patient 's exercise capacity deteriorated between the age of 17 and 18 , and she began to suffer from dyspnea at rest . a donor was found after a month , and a heart transplant procedure was conducted at the silesian center for heart diseases . since that time , the patient has continued to receive immunosuppressive treatment ; during the described ( and the previous ) pregnancy , it consisted in the administration of tacrolimus in monotherapy at the dose of 12 mg until the 31 week of pregnancy . the patient 's gynecological history revealed that she had been monitored for 1.5 years due to a hyperechogenic lesion in her right uterine appendages . in 2005 , the patient underwent laparotomy and excision of the monitored lesion ( histopathological diagnosis : cystis serosa ovarii dextrii ) . before the pregnancy , the patient had a contraceptive intrauterine device for a period of 1.5 years . the described pregnancy was her third . in 2008 , in the 39 week of pregnancy , she gave birth by caesarean section to a son , weighing 3150 g , who was born in good general condition ( apgar score : 10 ) . performing a caesarean section in this case had been suggested by the cardiologist after consultation with the obstetrician . in 2010 , earlier , during the 16 week , the gravida received conservative treatment at the hospital due to an obstetrical hemorrhage and a risk of miscarriage . in the 30 week examinations were performed to confirm the well - being of the fetus ( ultrasonography , cardiotocography ) ; the gravida consulted a cardiac surgeon , and the next control fetal echocardiography was scheduled ( the first examination , performed during the 22 week of pregnancy , showed no changes ) . in order to stimulate fetal lung growth , the patient received a course of steroids ( two intramuscular 12 mg doses of betamethasone in an interval of 24 h ) , a hydroxyprogesterone preparation ( administered intramuscularly ) , natural progesterone dosed at 2 x 100 mg ( administered intravaginally ) , and magnesium supplementation with an oral preparation . the patient was discharged in good general condition in the 31 week of pregnancy with the recommendation that she attend a follow - up examination at the silesian center for heart diseases in zabrze . the follow - up examination , performed by a transplant specialist , revealed anemia and a rapid reduction of tacrolimus concentration in the blood . two units of packed red blood cells were transfused , and the tacrolimus dose was increased to 20 mg per day . the patient was again admitted to our clinic during the 34 week of pregnancy . at admission , the patient was in good general condition ; circulation and respiration was normal , and there was no fever ; however , the patient complained of irregular uterine contractions and dysuric symptoms . the employed diagnostic tools revealed a urinary tract infection ( culture tests revealed s. epidermidis resistant to methicillin with a titer of 10 ) , anemia ( hb 10.0 g / dl ) , thrombocytopenia ( 68 thousand/l ) , and symptoms indicating tacrolimus overdose : hyperkalemia ( k 6.2 mmol / l ) ; further examinations demonstrated a deterioration of renal function parameters ( urea 43.8 mg / dl , creatinine 1.7 mmol / l , minimal creatinine clearance rate 53.5 ml / min ) . 11 ng / ml . the dose was lowered to 15 ng / ml , and a therapeutic concentration was achieved . after hematological consultation , the diagnostic search was broadened : recent infections with parvovirus b19 and the hcv virus were excluded as causes of the hematologic disturbances ; vitamin b12 was supplemented ( initial value : 92 pmol / l , with the norm ranging from 156 to 672 pmol / l ) . echocardiography of the transplanted heart showed atrial and ventricular function and structural parameters that were typical of a long - term , good outcome of heart transplantation . fetal ultrasound performed during the 34 week of pregnancy revealed signs of an intrauterine growth restriction ; the monitored flow in the umbilical artery was normal . in the 37 week of pregnancy , the patient went into labor and gave birth by caesarean section . her newborn son weighed 2670 g and was in good general condition , without signs of growth restriction ; he was , however , treated for congenital pneumonia . based on the wishes of the postpartum mother , who was informed of the theoretical risk for the newborn associated with his mother 's tacrolimus treatment , lactation was not inhibited . on average , 30 new female heart transplant recipients are registered every year in poland . this report describes the case of a heart transplant recipient who gave birth to a second healthy child . clinical practice with regard to pregnancy management in women after the transplantation of vital organs is mostly based on expert recommendations ; there are no guidelines constructed on the basis of rct studies due to the still small number of heart transplant recipients [ 25 ] . the considerations significant for obstetricians providing care to female heart transplant recipients include : providing reliable information concerning the influence of immunosuppressive treatment on the pregnancy , referring the patient at the appropriate time to a center performing prenatal diagnostics and fetal echocardiography , monitoring the patient with respect to pregnancy - induced hypertension , and cooperating with the attending cardiologist and transplant specialist in order to determine the optimal method of delivery [ 68 ] . during the 1990s , pregnancy was contraindicated for heart transplant recipients . the improvements in the cardiac surgical care provided to patients qualified for heart transplantation and during the postoperative period result in the improved well - being of women after the transplantation and their continued ability to procreate . currently , even though it is still difficult to unequivocally pinpoint the safest moment for conception , patients with transplanted hearts give birth to healthy children both by natural vaginal delivery and by caesarean section . a heart transplant procedure in the patient 's medical history is not in itself an indication for delivery by caesarean section . the described gravida was qualified for the first caesarean section by the obstetric - cardiac team due to low height , high expected weight of the child , and the expected need for shortening the second stage of labor . female heart transplant recipients desiring offspring must be informed of both the risks related to the influence of immunosuppressants on fetal development and the dangers associated with the high number of complications pertaining to the pregnancy and transplant [ 5 , 11 ] . such complications include arterial hypertension , pre - eclampsia , infections ( especially viral ) , and intrauterine growth restriction [ 3 , 4 ] . if , before the pregnancy , the function of the transplanted heart had been normal , the physiological hemodynamic changes related to the pregnancy are usually well tolerated . changes in the body 's immune response associated with the pregnancy may result in the development of antibodies in the hla system , whose presence may lead to the loss of the transplanted organ . immunosuppressive agents must be administered during the pregnancy , even though most of them belong to category c in terms of their safety of use in pregnant women according to the food and drug administration ( fda ) . the best known agents include glucocorticoids , azathioprine , ciclosporin , tacrolimus , and mycophenolate mofetil . treatment with azathioprine , tacrolimus , glucocorticoids , and ciclosporin is allowed in pregnant women [ 1517 ] . one should remember to monitor the concentrations of ciclosporin and tacrolimus in the blood of the gravida the need for these agents during pregnancy rises , and various clinical situations , e.g. anemia or concurrent therapy with agents inhibiting the enzymes of hepatic cytochromes , may require adjustment of the dose . during the perinatal period , we monitored the concentrations of tacrolimus as often as every 2 days . of particular note is the relationship between the hematocrit levels in blood morphology and the exposure to ciclosporin or tacrolimus , which are transported primarily by erythrocytes . in anemic conditions , achieving a therapeutic concentration of each of these agents requires large oral doses , but one should remember that transfusing red blood cells will result in a rapid elevation of agent concentration , which may cause acute kidney injury or even an onset of convulsions . in the case of tacrolimus or ciclosporin monotherapy , folic acid supplementation during the first trimester mycophenolate mofetil and sirolimus should be discontinued approximately 6 weeks before a planned pregnancy due to the risk of fatal congenital defects in the fetus . however , considering tacrolimus used in monotherapy , the concentration of the agent in milk is very low , and the newborn is exposed to approximately 0.5% of the tacrolimus concentration in the mother 's blood . unfortunately , the manufacturer 's guidelines indicate that lactation should be inhibited in women receiving the agent ; therefore , decisions concerning breastfeeding ( as was the case with our patient ) should be documented with an appropriate written consent form . the postpartum mother described in this article continues to breastfeed , and the neonate shows no signs of side effects . the successful outcome of this case the birth of a second healthy child by a heart transplant recipient can be attributed to the involvement of an interdisciplinary team and numerous specialists as well as good cooperation with the patient .

pregnancy after organ transplantation is becoming relatively common . we present the case of a heart transplant recipient who gave birth to a second child . despite the fact that the transplanted heart seems to adapt well to the changes caused by pregnancy , gestation in patients after heart transplantation may be complicated by hypertension , pre - eclampsia , or preterm labor . in this article , we consider the issues of preterm uterine contractions , anemia , thrombocytopenia , and several other complications in pregnant patients with transplanted hearts . we also present current opinions regarding the use of glucocorticoids as a form of preventing breathing disorders in neonates as well as breast - feeding by mothers receiving immunosuppressive agents . pregnancies in heart transplant recipients should be considered high - risk . a second successful delivery of a healthy child remains a challenge for such patients and their doctors .

Introduction Case study Discussion Disclosure

. differentiation of nave cd4 + t cells into th17 cells requires the coordinated activities of several microenvironmental cues . the cytokines il-6 and tgf- induce th17 cell differentiation by activating retinoid - related orphan receptor ( ror)-t , a master transcription factor required for the generation of th17 cells . accumulating evidence suggests that th17 cells are major drivers of inflammation and have been implicated in the pathogenesis of multiple autoimmune and immune - mediated inflammatory diseases , including rheumatoid arthritis , multiple sclerosis , asthma , and behet s disease [ 48 ] . given the important role of il-17 cells in regulating autoimmunity and inflammation , elaborating the factors and processes that govern the differentiation of th17 cells are necessary for developing th17-targeted therapies . almost every immune cell type has been found to express vitamin d receptor ( vdr ) and/or the enzyme cyp27b1 that is required for the generation of the bioactive form of vitamin d , 1,25(oh)2d3 ( also known as calcitriol ) . the action of 1,25(oh)2d3 is based on its binding to vdr , which is a ligand - dependent transcription factor , belonging to the steroid receptor superfamily . upon ligand binding , vdr undergoes heterodimerization with the retinoid x receptor ( rxr ) and binds to hormone response elements on dna , resulting in expression or transrepression of specific gene products . both in vitro and in vivo studies have found that 1,25(oh)2d3 treatment inhibits t cell commitment to the th17 lineage as well as th17 production of il-17a . 1,25(oh)2d3 directly inhibits th17 cell differentiation through the vdr signal in cd4 + t cells , independent of the il-2 , il-10 , and stat1 signals . in addition , 1,25(oh)2d3 suppresses cytokines in th17 cells by inducing the c / ebp homologous protein ( chop ) , a molecule involved in endoplasmic reticulum stress and translational inhibition . furthermore , 1,25(oh)2d3 inhibits production of il-17a by blocking nuclear factor for activated t cells ( nfat ) , recruitment of histone deacetylase ( hdac ) , sequestration of runt - related transcription factor 1 ( runx1 ) , and induction of forkhead box p3 ( foxp3 ) , a transcription factor associating with nfat and runx1 for transcriptional repression . although previous studies have obtained some encouraging evidence , our understanding of the mechanisms by which 1,25(oh)2d3 regulates th17 differentiation remains limited . micrornas ( mirnas ) are endogenous , small , non - coding rnas approximately 22 nucleotides ( nt ) in length that are highly conserved across various species of eukaryotes . mirnas posttranscriptionally repress gene expression by binding to complimentary sequences in the 3 untranslated region ( utr ) of target messenger rna ( mrna ) . mirnas are vital for controlling many processes within the immune system including the development and differentiation of lymphocytes , secretion of cytokines and chemokines , antibody switching , and regulation of immune tolerance . mir-155 promotes th17 cell differentiation and il-17a production by targeting suppressor of cytokine signaling 1 ( socs-1 ) , which is a molecule responsible for the negative regulation of the jak - stat pathway and suppressing the inhibitory effects of the dna - binding protein jarid2 . mir-155-deficient mice show a defect in th17 differentiation and strong resistance to experimental autoimmune encephalomyelitis ( eae ) , an experimental animal model for central nervous system demyelinating disease . mir-20b expression is down - regulated in th17 cells during mouse eae , and over - expression of mir-20b inhibits th17 differentiation and ameliorates eae by targeting ror-t and stat3 . in addition , mir-21 , mir-181a , mir-210 , and mir-301a are found to be involved in the development of th17 cells [ 2125 ] . since both mirnas and 1,25(oh)2d3 are crucial regulators of th17 differentiation , it is of great interest to investigate their interaction in regulating th17 differentiation . in the present study , we aim to evaluate the expression of several candidate mirnas during th17 differentiation and the effects of 1,25(oh)2d3 on th17 differentiation and mirna expression . human peripheral blood was obtained by venipuncture from 3 young healthy subjects ( lzb , ky , and jfj ) and was collected into k2-ethylene diamine tetraacetic acid ( k2-edta ) vacutainer tubes ( shenzhen medrey medical technology , china ) . whole blood ( 4 ml ) was mixed with 4 ml of phosphate - buffered saline and layered gently on 4 ml of lymphocyte separation medium ( dakewe biotech company , china ) in a 15-ml centrifugate tube . pbmc were separated by using density centrifugation in a 25-min centrifugation step at 2500 rpm at room temperature . the mononuclear layer was collected from the interface of the blood plasma and the separation medium . after separation , pbmc were washed and cultivated at a density of 1.010 cells / ml in rpmi 1640 ( keygen biotech , china ) containing 10% fetal bovine serum , l - glutamine , nahco3 , penicillin , and streptomycin . cells were incubated in a humidified atmosphere ( 37c and 5% co2 ) . to generate th17 cells in pbmcs , anti - cd3 mab ( 0.5 ng / l ; biolegend , usa ) , anti - cd28 mab ( 0.5 ng / l ; biolegend , usa ) , recombinant human il-1 ( rhil-1 ; 100 ng / ml ) , rhil-23 ( 100 ng / ml ) , rhil-6 ( 100 ng / ml ) , and rhtgf-1 ( 25 ng / ml ) ( all cytokines from peprotech , usa ) were added to the medium on day 0 . when 1,25(oh)2d3 ( selleck chemicals , usa ) was used to treat the cells , a final concentration of 100 nm was employed according to the results of our pre - experiments ( not shown ) . after 72 hours of culture , supernatants were collected , and il-17a production in culture supernatants was analyzed by enzyme - linked immunosorbent assay ( elisa ) ( dakewe biotech company , china ) according to the manufacturer s instructions . after 72 hours of culture , total rna containing small - size rna was isolated from pbmcs using the mircury rna isolation kit ( exiqon , denmark ) . the concentration of rna was quantified using the nanodrop 2000 spectrophotometer ( thermo , usa ) . first strand cdna synthesis of mirna was performed with the mir - x mirna first - strand synthesis kit ( clontech , japan ) . for generating cdna , 5 l of 2 mrq buffer , 1.25 l of mrq enzyme , and 3.75 l of rna were used , for a total volume of 10 l . the synthesis reaction was carried out at 37c for 60 min , followed by 85c for 5 min . cdna was diluted with 90 l of ddh2o to bring the total volume to 100 l after reaction . for mirna quantification , qpcr analysis was performed using the mir - x mirna qrt - pcr sybr kit ( clontech , japan ) and the cfx96 qpcr system ( biorad , usa ) . the delta - delta ct method was used to determine the expression of hsa - mir-20b , hsa - mir-21 , hsa - mir-155 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a normalized against that of u6 snrna . the 3 primer for qpcr was the mrq 3 primer supplied with the kit , whereas the 5 primers for mirnas were designed and synthesized by takara biotechnology ( dalian ) ( table 1 ) . reactions were incubated at 95c for 10 seconds , followed by 40 cycles at 95c for 5 seconds and 60c for 20 seconds . each sample was run in duplicate for analysis . at the end of the pcr cycles , amplicons were also analyzed by agarose gel electrophoresis and visualized with ethidium bromide under uv transillumination to validate the specific generation of the expected pcr . data are presented as median with interquartile range ( irq ) and were analyzed by nonparametric statistics with stata version 11.0 . human peripheral blood was obtained by venipuncture from 3 young healthy subjects ( lzb , ky , and jfj ) and was collected into k2-ethylene diamine tetraacetic acid ( k2-edta ) vacutainer tubes ( shenzhen medrey medical technology , china ) . whole blood ( 4 ml ) was mixed with 4 ml of phosphate - buffered saline and layered gently on 4 ml of lymphocyte separation medium ( dakewe biotech company , china ) in a 15-ml centrifugate tube . pbmc were separated by using density centrifugation in a 25-min centrifugation step at 2500 rpm at room temperature . the mononuclear layer was collected from the interface of the blood plasma and the separation medium . after separation , pbmc were washed and cultivated at a density of 1.010 cells / ml in rpmi 1640 ( keygen biotech , china ) containing 10% fetal bovine serum , l - glutamine , nahco3 , penicillin , and streptomycin . to generate th17 cells in pbmcs , anti - cd3 mab ( 0.5 ng / l ; biolegend , usa ) , anti - cd28 mab ( 0.5 ng / l ; biolegend , usa ) , recombinant human il-1 ( rhil-1 ; 100 ng / ml ) , rhil-23 ( 100 ng / ml ) , rhil-6 ( 100 ng / ml ) , and rhtgf-1 ( 25 ng / ml ) ( all cytokines from peprotech , usa ) were added to the medium on day 0 . when 1,25(oh)2d3 ( selleck chemicals , usa ) was used to treat the cells , a final concentration of 100 nm was employed according to the results of our pre - experiments ( not shown ) . after 72 hours of culture , supernatants were collected , and il-17a production in culture supernatants was analyzed by enzyme - linked immunosorbent assay ( elisa ) ( dakewe biotech company , china ) according to the manufacturer s instructions . after 72 hours of culture , total rna containing small - size rna was isolated from pbmcs using the mircury rna isolation kit ( exiqon , denmark ) . the concentration of rna was quantified using the nanodrop 2000 spectrophotometer ( thermo , usa ) . first strand cdna synthesis of mirna was performed with the mir - x mirna first - strand synthesis kit ( clontech , japan ) . for generating cdna , 5 l of 2 mrq buffer , 1.25 l of mrq enzyme , and 3.75 l of rna were used , for a total volume of 10 l . the synthesis reaction was carried out at 37c for 60 min , followed by 85c for 5 min . cdna was diluted with 90 l of ddh2o to bring the total volume to 100 l after reaction . for mirna quantification , qpcr analysis was performed using the mir - x mirna qrt - pcr sybr kit ( clontech , japan ) and the cfx96 qpcr system ( biorad , usa ) . the delta - delta ct method was used to determine the expression of hsa - mir-20b , hsa - mir-21 , hsa - mir-155 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a normalized against that of u6 snrna . the 3 primer for qpcr was the mrq 3 primer supplied with the kit , whereas the 5 primers for mirnas were designed and synthesized by takara biotechnology ( dalian ) ( table 1 ) . reactions were incubated at 95c for 10 seconds , followed by 40 cycles at 95c for 5 seconds and 60c for 20 seconds . each sample was run in duplicate for analysis . at the end of the pcr cycles , amplicons were also analyzed by agarose gel electrophoresis and visualized with ethidium bromide under uv transillumination to validate the specific generation of the expected pcr . data are presented as median with interquartile range ( irq ) and were analyzed by nonparametric statistics with stata version 11.0 . a p value of < 0.05 was considered significant . to generate th17 cells , we isolated pbmc from healthy donors and activated them in vitro with anti - cd3 and anti - cd28 antibodies in the presence of polarizing cytokines il-23 , il-1 , tgf-1 , and il-6 . after 72 hours of culture , we collected the culture medium and assessed il-17a production by elisa . as shown in figure 1 , il-17a was undetectable when cells received no stimulation , whereas a high level of il-17a was detected under th17-polarizing conditions ( median [ irq ] , 2535.4 [ 2153.3 ] pg / ml , p<0.05 ) ( figure 1 ) , indicating a substantial increase in the number of il-17a - expressing t cells . we tested 1,25(oh)2d3 for its capacity to inhibit th17 differentiation by adding 1,25(oh)2d3 to the medium at a final concentration of 100 nm . after 72 hours of culture , il-17a production was significantly decreased by 1,25(oh)2d3 under th17-polarizing conditions ( median [ irq ] , 745.7 [ 473.5 ] pg / ml vs. 2535.4 [ 2153.3 ] pg / ml , p<0.05 ) ( figure 1 ) . the expression of mirnas hsa - mir-20b , hsa - mir-21 , hsa - mir-155 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a was found to be changed under th17-polarizing conditions . of these mirnas , hsa - mir-155 was significantly up - regulated ( median fold change : 3.61 , p<0.05 ) , whereas hsa - mir-20b , hsa - mir-21 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a were significantly down - regulated ( median fold change : 0.44 , 0.37 , 0.18 , 0.15 , and 0.26 , respectively ; p<0.05 ) ( table 2 , figure 2 ) . although 1,25(oh)2d3 significantly suppressed human th17 differentiation , there was no change of mirna expression after 1,25(oh)2d3 treatment ( table 2 , figure 2 ) . melting curve analysis and agarose gel electrophoresis of qpcr products confirmed that the amplicons were specific ( not shown ) . to generate th17 cells , we isolated pbmc from healthy donors and activated them in vitro with anti - cd3 and anti - cd28 antibodies in the presence of polarizing cytokines il-23 , il-1 , tgf-1 , and il-6 . after 72 hours of culture , we collected the culture medium and assessed il-17a production by elisa . as shown in figure 1 , il-17a was undetectable when cells received no stimulation , whereas a high level of il-17a was detected under th17-polarizing conditions ( median [ irq ] , 2535.4 [ 2153.3 ] pg / ml , p<0.05 ) ( figure 1 ) , indicating a substantial increase in the number of il-17a - expressing t cells . we tested 1,25(oh)2d3 for its capacity to inhibit th17 differentiation by adding 1,25(oh)2d3 to the medium at a final concentration of 100 nm . after 72 hours of culture , il-17a production was significantly decreased by 1,25(oh)2d3 under th17-polarizing conditions ( median [ irq ] , 745.7 [ 473.5 ] pg / ml vs. 2535.4 [ 2153.3 ] pg / ml , p<0.05 ) ( figure 1 ) . the expression of mirnas hsa - mir-20b , hsa - mir-21 , hsa - mir-155 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a was found to be changed under th17-polarizing conditions . of these mirnas , hsa - mir-155 was significantly up - regulated ( median fold change : 3.61 , p<0.05 ) , whereas hsa - mir-20b , hsa - mir-21 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a were significantly down - regulated ( median fold change : 0.44 , 0.37 , 0.18 , 0.15 , and 0.26 , respectively ; p<0.05 ) ( table 2 , figure 2 ) . although 1,25(oh)2d3 significantly suppressed human th17 differentiation , there was no change of mirna expression after 1,25(oh)2d3 treatment ( table 2 , figure 2 ) . melting curve analysis and agarose gel electrophoresis of qpcr products confirmed that the amplicons were specific ( not shown ) . the main findings of the present study are as follows : ( 1 ) we observed increased expression of hsa - mir-155 and decreased expression of hsa - mir-20b , hsa - mir-21 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a during human th17 differentiation ; and ( 2 ) 1,25(oh)2d3 significantly suppressed human th17 differentiation , but did not affect mirna expression during this process . as far as we know , we provide the first evidence that 1,25(oh)2d3 suppresses human th17 differentiation without influencing mirna expression . the classical function of 1,25(oh)2d3 is to maintain calcium - phosphate homeostasis and skeletal function . the study by ikeda et al . demonstrated that 1,25(oh)2d3 significantly inhibited th17 differentiation by down - regulating the mrna expression levels of rorc and aryl hydrocarbon receptor . in addition , the inhibitory effect of 1,25(oh)2d3 on th17 differentiation was found to be via the vdr signal in cd4 + t cells , but independent of il-2 , il-10 , and stat1 signals . furthermore , it was shown that 1,25(oh)2d3 suppressed th17 differentiation by blocking nfat , recruitment of hdac , sequestration of runx1 , and inducing chop and foxp3 . recently , nanduri et al . reported that 1,25(oh)2d3 repressed th17-specific genes by the smad pathway and extracellular signal - regulated kinase activation . despite of these findings , our understanding of the mechanisms whereby 1,25(oh)2d3 regulates th17 differentiation remains limited . mirnas represent a large family of about 22 nt long , noncoding , single - stranded rna molecules . in mammals , mirnas predominantly regulate gene expression by induction of mrna degradation , and they play an important role in a variety of physiological and pathological processes . an involvement in the regulation of th17 differentiation has recently been proposed for several mirnas , including mir-20b , mir-21 , mir-155 , mir-181a , mir-210 , and mir-301a [ 2025 ] . given that both 1,25(oh)2d3 and mirnas are important regulators of th17 differentiation , it is necessary to evaluate whether 1,25(oh)2d3 affects mirna expression when exerting its inhibitory effects on th17 differentiation . in this study , we focused on six candidate mirnas , including mir-20b , mir-21 , mir-155 , mir-181a , mir-210,and mir-301a , based on existing evidence from previous studies showing their relation with th17 differentiation , rather than performing mirna microarray analysis to identify candidates , because the former method was more efficient and targeted . in addition , we did not identify candidate mirnas using in silico analysis because this method was relatively unreliable . the change of mirna expression during th17 differentiation was evaluated using qpcr . in agreement with the results from previous studies , all six mirnas were found to be significantly dysregulated in the development of th17 cells , with five down - regulated ( hsa - mir-20b , hsa - mir-21 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a ) and one up - regulated ( hsa - mir-155 ) . it was noteworthy that most evidence on expression of mirnas during th17 differentiation to date has been derived from results using animal cell cultures [ 2023,25 ] , whereas we evaluated these mirnas using human cell cultures , which could provide more valuable evidence . in addition , most previous studies concentrated on a single mirna , but we evaluated several mirnas simultaneously in this study , which would provide more detailed information on change of mirna expression during th17 cell development . 1,25(oh)2d3 significantly suppressed th17 differentiation , but did not affect mirna expression during this process , suggesting that 1,25(oh)2d3-mediated th17 cell suppression was not dependent on regulation of mirna expression . we noticed that our results were distinct from those on endothelial tissues and cancer cells , which showed that vitamin d regulated mirna expression and was involved in mirna - directed posttranscriptional mechanisms . the study by zitman - gal et al . found that mir-20b expression was up - regulated by 1,25(oh)2d3 in endothelial cells exposed to a diabetic - like environment , and this change of expression played a role in the modulation of endothelial function . demonstrated that vitamin d deficiency up - regulated mir-21 expression in the aorta , which contributed to the development of atherosclerosis . in addition , cancer studies also showed that 1,25(oh)2d3 regulated the expression of a subset of mirnas with potential regulatory functions in cancer pathways [ 3032 ] . however , we did not find mirna involvement in 1,25(oh)2d3-mediated suppression of th17 differentiation in this study . our results indicate that 1,25(oh)2d3 exerts its functions via distinct mechanisms across different cell types , not through mirnas in th17 cells . our study showed that six mirnas were dysregulated during human th17 differentiation , with one significantly up - regulated ( hsa - mir-155 ) and five significantly down - regulated ( hsa - mir-20b , hsa - mir-21 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a ) .

backgroundvitamin d is an import regulator of t helper 17 ( th17 ) differentiation , but our understanding of the underlying mechanisms remains limited . in the present study , we aimed to detect the expression levels of micrornas ( mirnas ) during human th17 differentiation and evaluate the effects of 1,25-dihydroxyvitamin d3 ( 1,25(oh)2d3 ) , the bioactive form of vitamin d , on th17 differentiation and mirna expression.material/methodswe cultured human peripheral blood mononuclear cells ( pbmc ) in vitro and activated them with anti - cd3 and anti - cd28 antibodies in the presence of th17-promoting cytokines interleukin ( il)-23 , il-1 , tgf-1 , and il-6 for 72 hours . 1,25(oh)2d3 was added to the medium at a final concentration of 100 nm on day 0 . the production of il-17a in culture medium was detected by enzyme - linked immunosorbent assay ( elisa ) . the expression levels of mirnas during th17 differentiation were determined by quantitative polymerase chain reaction ( qpcr).resultssix mirnas were found to be dysregulated during human th17 differentiation . of these mirnas , hsa - mir-155 was significantly up - regulated ( median fold change : 3.61 , p<0.05 ) , whereas hsa - mir-20b , hsa - mir-21 , hsa - mir-181a , hsa - mir-210 , and hsa - mir-301a were significantly down - regulated ( median fold change : 0.44 , 0.37 , 0.18 , 0.15 , and 0.26 , respectively , p<0.05 ) . 1,25(oh)2d3 treatment significantly decreased il-17a production ( median [ interquartile range ] , 745.7 [ 473.5 ] pg / ml vs. 2535.4 [ 2153.3 ] pg / ml , p<0.05 ) . however , expression of these mirnas was not changed after 1,25(oh)2d3 treatment.conclusions1,25(oh)2d3 suppressed human th17 differentiation without affecting mirna expression .

Background Material and Methods Peripheral blood mononuclear cells (PBMC) isolation Th17 cell generation Quantitative PCR (qPCR) Statistical analysis Results 1,25(OH) Detection of miRNA expression by qPCR Discussion Conclusions

pesticide poisoning is a commonly underdiagnosed condition among farmers , particularly in rural areas and in many developing and developed countries . generally , pesticide - related morbidity is higher in rural farming communities because these locations suffer a scarcity of health care workers needed to diagnose and treat acute pesticide poisoning ( app ) . worldwide , studies report high proportions of farmers poisoned during pesticide application in vietnam ( 61% ) , ivory coast ( 55% ) , india ( 83.6% ) , myanmar ( 40% ) , and pakistan ( 55% ) . health care professionals ( hcps ) , who are responsible for the diagnosis and management of app , often receive limited training on pesticide hazards and management of pesticide - related illnesses and are sparsely available in rural areas , which often lack adequate health care facilities . most of the hcps often have limited experience of managing cases of app due to the fact that many poisonings , in particular nonsevere occupational poisoning , are not presented to hospitals . this limited knowledge is exacerbated due to the fact that clinical toxicology is a dynamic field of medicine in which new diagnostic and treatment methods are constantly being developed , and the effectiveness of diagnostic and treatment techniques is constantly being updated . in addition , pesticides used by farmers change over time and new products require new diagnostic , first aid , and treatment approaches . in tanzania , for example lack of experience in the management of app will therefore contribute to the inability of hcps to diagnose and manage app due to pesticide products with which they are not conversant . this means that surveillance systems that rely on diagnostic information from hcps will be severely weakened if hcps are not able to identify app with any reliability or accuracy . yet , without these data , surveillance systems will be unable to capture the full extent of the problem nor effectively inform appropriate preventive policies . as a result , communities from which the data are derived are not aware of the magnitude of pesticide poisoning as a public health problem nor are they given the opportunity to take preventive actions or develop community solutions . the health care services in tanzania are delivered and regulated by the ministry of health , community development , gender , seniors and children . the structure of the health system starts at village facilities and dispensaries followed by health centres located in both rural and urban areas . the health centres refer upwards to district and private hospitals , regional hospitals , and , finally , referral hospitals at the apex of the referral chain . health care professionals responsible for delivering health services at different levels include physicians , clinical officers or medical assistants , public health officers , and nurses , including nurse practitioners . clinical officers ( also known as medical assistants ) are hcps trained to assist physicians in clinical procedures . nurse practitioners are nurses trained to care for sick and injured patients and to assist physicians and clinical officers in providing clinical care . public health officers are responsible for protecting and improving the health of a community through preventive medicine , health education , control of communicable diseases , application of sanitary measures , and monitoring of environmental hazards . on average , the rate of medical doctors per capital in tanzania in 2005 was 138 000:1 , whereas that for clinical officers was 5000:1 . this is in contrast to most developed countries where the ratio of physicians exceeds 2 per 1000 of the population . ngowi et al reported poor competence among hcps in the recognition , diagnosis , and management of pesticide poisoning cases , thought to be due to inadequate training in toxicology and occupational health . the hcps in the same study were also poorly equipped to deliver appropriate care to pesticide poisoning victims . similar findings of low awareness among hcps of the problem of pesticide poisoning have been reported in other parts of east africa , south africa , costa rica , and cte divoire . although ngowi and colleagues addressed tanzanian health care worker practices in relation to the diagnosis and management of app , no studies in tanzania have examined hcp practices in relation to app surveillance . this study therefore addresses the gap in hcp knowledge and practices related to surveillance of app and also updates the study by ngowi which was conducted more than 10 years ago , prior to the 1997 introduction of mandatory reporting in the health service under the health information management system . this study therefore aimed to characterize the knowledge and experience of hcps in selected health facilities in tanzania in the diagnosis and management of app , common first aid measures , use of reporting systems , notification practices , and ability to interpret pesticide labels for the purpose of strengthening surveillance of app among farmers and community . the population included all physicians , clinical officers , and nurse practitioners working in kilimanjaro and arusha regions and who were directly responsible for diagnosing and treating potential app cases . the kilimanjaro and arusha regions were chosen because they have agricultural activities typical of rural tanzania and are geographically located close to tropical pesticides research institute ( tpri ) that facilitated logistics for data collection . an intended sample size of 91 participants was based on a priori estimate of 17% of hcps treating cases of app ( as reported by ngowi et al ) , a confidence level of 95% , and a margin of error of 8% . participants were interviewed using a semistructured questionnaire on their management of app cases and how they record and report the cases through the health management information system . they were also asked about their knowledge and practices that contribute to surveillance of app , their knowledge on pesticide label instructions , their experience in handling app , the type of first aid measures recommended for app , and their knowledge of adverse health effects of pesticides , precautionary measures contained on pesticide labels , and the classification of pesticides by world health organization ( who ) hazard class and by chemical groups . the data were collected by the principal investigator and 2 assistants between january and december 2005 . the assistants were laboratory technicians working at tpri for more than 15 years in pesticide - related research . for the study , the technicians received refresher training on pesticide classification , first aid measures for pesticide poisoning , pesticide labels , and how to administer the questionnaire for hcps . the data collection tool was pretested in january 2005 using a small sample of hcps ( n = 10 ) in selected facilities in arusha municipality before use in the main study . univariate descriptive statistics were estimated for frequencies and percentages of all categorical or numerical variables . cross - tabulations were conducted as follows : the variable knowledge on first aid ( low vs high ) was compared by the variable ever handled a pesticide poisoning and by years of working experience to identify associations with high knowledge of first aid measures . the variable familiarity with adverse health effects ( low vs high ) was compared by respondents educational level to identify whether education was associated with high familiarity with health effects of pesticides . the variable knowledge on pesticide classification ( low vs high ) was compared by respondents education level to identify whether education was associated with high knowledge of the who pesticide classification system . the variable knowledge on routes of exposure ( low vs high ) was compared by respondents years of working experience to identify whether years of experience was associated with high knowledge of routes of exposure . the variable ever handled a pesticide poisoning case vs never handled any case was compared with respondents years of working experience to identify whether increased years of experience was associated with treating cases of app . the variable high education vs low education was compared with years of working experience to identify whether long service was associated with education level . the variable type of health care facility ( government or private ) was compared with knowledge on first aid , knowledge on routes of exposure , familiarity with health effects , knowledge on pesticides classification , level of education , years of working experience , and status of handling of app cases . testing was used to compare distributions of dichotomous variables . to measure the strength of association between categorical independent and dependent variables , prevalence risk ratios spss statistical package version 16 and stata version 10.0 were used to analyse the data . participants gave informed consent prior to participation in the study and were free to decline participation without any fine or penalty . to ensure confidentiality , names were replaced by special codes , which were used in data analysis . the participants were assured that their responses would not affect their performance assessments by their managers . the study protocol was approved by tpri ethical committee and the national institute of medical research in tanzania ( ref nimr / hq / vol xi/371 ) as well as the university of cape town health sciences faculty research ethics committee ( 328/2004 ) . the population included all physicians , clinical officers , and nurse practitioners working in kilimanjaro and arusha regions and who were directly responsible for diagnosing and treating potential app cases . the kilimanjaro and arusha regions were chosen because they have agricultural activities typical of rural tanzania and are geographically located close to tropical pesticides research institute ( tpri ) that facilitated logistics for data collection . an intended sample size of 91 participants was based on a priori estimate of 17% of hcps treating cases of app ( as reported by ngowi et al ) , a confidence level of 95% , and a margin of error of 8% . participants were interviewed using a semistructured questionnaire on their management of app cases and how they record and report the cases through the health management information system . they were also asked about their knowledge and practices that contribute to surveillance of app , their knowledge on pesticide label instructions , their experience in handling app , the type of first aid measures recommended for app , and their knowledge of adverse health effects of pesticides , precautionary measures contained on pesticide labels , and the classification of pesticides by world health organization ( who ) hazard class and by chemical groups . the data were collected by the principal investigator and 2 assistants between january and december 2005 . the assistants were laboratory technicians working at tpri for more than 15 years in pesticide - related research . for the study , the technicians received refresher training on pesticide classification , first aid measures for pesticide poisoning , pesticide labels , and how to administer the questionnaire for hcps . the data collection tool was pretested in january 2005 using a small sample of hcps ( n = 10 ) in selected facilities in arusha municipality before use in the main study . univariate descriptive statistics were estimated for frequencies and percentages of all categorical or numerical variables . for the purpose of analysis , data were categorized as per table 1 . cross - tabulations were conducted as follows : the variable knowledge on first aid ( low vs high ) was compared by the variable ever handled a pesticide poisoning and by years of working experience to identify associations with high knowledge of first aid measures . the variable familiarity with adverse health effects ( low vs high ) was compared by respondents educational level to identify whether education was associated with high familiarity with health effects of pesticides . the variable knowledge on pesticide classification ( low vs high ) was compared by respondents education level to identify whether education was associated with high knowledge of the who pesticide classification system . the variable knowledge on routes of exposure ( low vs high ) was compared by respondents years of working experience to identify whether years of experience was associated with high knowledge of routes of exposure . the variable ever handled a pesticide poisoning case vs never handled any case was compared with respondents years of working experience to identify whether increased years of experience was associated with treating cases of app . the variable high education vs low education was compared with years of working experience to identify whether long service was associated with education level . the variable type of health care facility ( government or private ) was compared with knowledge on first aid , knowledge on routes of exposure , familiarity with health effects , knowledge on pesticides classification , level of education , years of working experience , and status of handling of app cases . testing was used to compare distributions of dichotomous variables . to measure the strength of association between categorical independent and dependent variables , prevalence risk ratios spss statistical package version 16 and stata version 10.0 were used to analyse the data . participants gave informed consent prior to participation in the study and were free to decline participation without any fine or penalty . to ensure confidentiality , the participants were assured that their responses would not affect their performance assessments by their managers . the study protocol was approved by tpri ethical committee and the national institute of medical research in tanzania ( ref nimr / hq / vol xi/371 ) as well as the university of cape town health sciences faculty research ethics committee ( 328/2004 ) . of the 91 hcps approached , 25 declined to participate , leaving a sample of 66 hcps from 32 facilities who were finally interviewed , representing a response rate of approximately 73% . in most facilities , there were 1 or 2 respondents ( table 2 ) . however , in the larger facilities , the number of respondents ranged up to 6 . health care providers interviewed by facility in northern tanzania . abbreviation : kcmc , kilimanjaro christian medical centre . most of the respondents were men ( 63.7% ) and they were from both private ( 53% ) and government ( 47% ) facilities in arusha and kilimanjaro regions . the facilities included a referral hospital , 2 regional hospitals , a district hospital , and 5 other private hospitals , health centres ( n = 6 ) , and dispensaries ( n = 16 ) . most of the respondents were clinical officers ( 57.5% ) and their experience in medical services ranged from 1 to more than 24 years . although the largest category had experience of 5 years or less ( 55% ) , the range of experience was wide and there were 3 participants with experience of more than 20 years in the field ( table 3 ) . respondents most frequent responses regarding knowledge on first aid and treatment in cases of pesticide poisoning included washing the contaminated area with water ( n = 23 ) , inducing vomiting if ingested ( n = 22 ) , and giving the poisoned victims fresh milk ( n = 19 ) . nineteen respondents ( 30% ) reported they do not know any first aid or treatment strategy used for victims poisoned by pesticides ( table 3 ) . table 3 indicates that many responses listed treatment options that were either incorrect or inappropriate , reported as first aid , such as administration of atropine or intravenous fluids , use of antihistamines , use of antibiotics , gastric lavage , or first aid measures ( 17.6% ) . for example , giving milk , antibiotics , and hydrocortisone are both ineffective and potentially dangerous ; the use of personal protective equipment ( ppe ) is only useful for prevention , and isolation of the victim is plainly mistaken ( table 3 ) . exactly , 50% of the respondents reported that they had never previously handled a pesticide poisoning case , 34.8% reported handling between 1 and 5 cases , and 15% handling 6 or more cases ( table 4 ) . the proportion of respondents who have handled an app case was marginally higher among staff with long work experience ( odds ratio = 1.32 ; 95% confidence interval [ ci ] = 0.9 - 1.5 ) compared with low experience . experience and knowledge of health care professionals on the management of pesticide poisoning in northern tanzania ( n = 66 ) . abbreviation : app , acute pesticide poisoning . when asked about the availability of medical laboratory testing , 53% of respondents indicated that their facilities had laboratories available on site , but none conducted any testing for biomarkers to diagnose pesticide poisoning . all the respondents reported having no standard diagnostic procedure for app , and all reported that they documented poisoning cases in the general health statistics abstracts reference books ( mtuha ) and patient register book . reporting poisoning cases and other disease conditions in this register is mandatory according to the ministry of health , community development , gender , seniors and children . only 5 respondents ( 8% ) reported having a high familiarity with the health effects of pesticides , whereas 50% admitted to having no awareness of pesticide toxicity ( table 4 ) . most of the respondents reported knowledge of possible pesticide exposure routes as oral ( 98.5% ) and inhalational ( 93.9% ) , whereas knowledge about absorption through the skin as a route was slightly lower ( 77% ; table 4 ) . most of the respondents ( 71% ) were unaware of the classification of pesticides by chemical group , and all respondents were unaware of the who hazard classification system for pesticides . pesticide chemical groups reported correctly by the respondents included organophosphates ( 37.8% ) , organochlorines ( 12.1% ) , carbamates ( 12.1% ) , and pyrethroids ( 1.5% ) . most respondents ( 55% ) reported that they had no knowledge of pesticide label safety instructions . of the 45% reporting some knowledge , the most common label instructions reported by respondents included instructions regarding storage out of reach of children ( n = 30 ) and use of ppe ( n = 27 ) . less common instructions reported were related to washing after handling ( n = 7 ) , refraining from eating while handling pesticides ( n = 8) , keeping pesticides away from food ( n = 4 ) , and avoiding pollution of the environment or water bodies ( n = 5 ) . eleven respondents ( 17% ) reported awareness of the signal word poisonous. products reported by the hcps as commonly associated with poisoning included both specific agents ( n = 31 ) and nonspecific agents ( n = 19 ) . organophosphates comprised 35% and pyrethroids comprised 16% of specific agents named . however , most commonly , the respondents were not able to specify a specific pesticide agent ( n = 35 ; table 5 ) . agents reported to be associated with poisoning as experienced by the health care professionals in northern tanzania . abbreviations : ca , carbamates ; in , inorganic ; oc , organochlorines ; op , organophosphates ; ot , other categories ; py , pyrethroids ; who , world health organization . there were marginally significant associations between educational levels of the respondents and high familiarity with pesticide health effects ( prevalence risk ratio [ prr ] high educated / low educated = 2.44 ; 95% ci = 1.05 - 5.65 ) and with high knowledge of pesticides classification ( prr high educated / low educated = 2.8 ; 95% ci = 1.3 - 6.2 ; table 6 ) . associations of knowledge about pesticides with work experience , education , and management of app among hcps in northern tanzania . there was a significant association between the status of health care facility with high knowledge on pesticide classification ( prr private facility / government facility = 1.5 , 95% ci = 1.1 - 2.1 ) ( table 7 ) . association of status of health facility and knowledge on first aid , knowledge on routes of exposure , familiarity with health effects , knowledge on pesticide classification , level of education , years of working experience , and handling of app cases among hcps in northern tanzania . there were marginally significant associations between educational levels of the respondents and high familiarity with pesticide health effects ( prevalence risk ratio [ prr ] high educated / low educated = 2.44 ; 95% ci = 1.05 - 5.65 ) and with high knowledge of pesticides classification ( prr high educated / low educated = 2.8 ; 95% ci = 1.3 - 6.2 ; table 6 ) . associations of knowledge about pesticides with work experience , education , and management of app among hcps in northern tanzania . there was a significant association between the status of health care facility with high knowledge on pesticide classification ( prr private facility / government facility = 1.5 , 95% ci = 1.1 - 2.1 ) ( table 7 ) . association of status of health facility and knowledge on first aid , knowledge on routes of exposure , familiarity with health effects , knowledge on pesticide classification , level of education , years of working experience , and handling of app cases among hcps in northern tanzania . this study updates and expands the scope of a previous investigation in tanzania ( ngowi et al ) into health care provider s knowledge and practices regarding poisoning arising from pesticides . the profile of pesticide agents now reflects usage shaped by tanzania s ratification of the stockholm and rotterdam conventions , domesticated into national law , as a result of which there are newer active ingredients and formulations which demand a new investigation of knowledge and practices . moreover , this study includes vegetable growing areas , with smaller production units , and including smaller health care facilities . most importantly , this study generates information for the first time on reporting of app by health workers through local health information systems , a crucial element for effective public health surveillance of app . the respondents in this substudy were hcps who had working experience ranging from 1 to 24 years . the study revealed poor knowledge on pesticide poisoning management , lack of familiarity with the adverse health effects of pesticides , low knowledge about pesticide chemical groups , and who categories but better knowledge about routes of dermal exposure . one intuitive explanation is that clinicians with longer working experience likely may have handled app cases which might have made them more knowledgeable . however , this was not supported by data in table 6 where long service was not associated with any of the knowledge measures . another explanation could be the fact that the respondents interviewed in the study period who were more recent graduates did not cover details about pesticides and their toxicity in their study curriculum , and app was not given much priority due to a perception that it is rare in hospitals . this may explain why many poisoning agents are frequently reported with nonspecific names for the causative agents , such as acaricides , bed bug insecticide , flower spray , and other unknown terms . this clearly limits the extent to which hcp reports can support effective surveillance for app . half of the respondents reported that they had never handled any pesticide poisoning cases , and among those who had , most had attended 5 or fewer cases in their careers . one reason for low experience could be the fact that pesticide poisoning cases are infrequently present to hospitals in tanzania , a finding also reported in india . alternatively , if they did attend cases but the diagnosis was missed , the provider did not know that they had treated an app case . the study also revealed that a large proportion of respondents had misconceptions about appropriate first aid . for example , 19 respondents ( 29% ) considered milk a first aid option for app and about one - third of respondents ( 33% ) reported inducing vomiting as one of the options for first aid for app . in fact , providing milk may give a false sense of security and delay proper treatment and hence may increase health risks . similarly , induction of vomiting is not appropriate for all products and may be contraindicated for certain agents . for example , pyridyls are corrosive products which can damage the oesophagus and upper airway if vomiting is induced . if the victim is unconscious , inducing vomiting could also result in potentially fatal aspiration of vomitus . a previous tanzanian study involving extension officers between 1991 and 1993 also reported the use of milk and inducing vomiting as options for first aid , along with other options such as use of lamb oil , fresh cattle dung , and salted water . this suggests that perceptions about the use of milk as an antidote for poisoning is prevalent among not only hcps but also the agriculture extension officers . misconceptions about the use of milk as an antidote to a range of workplace hazards are widespread in the region . a similar study conducted in united states involving a survey of primary care physicians revealed poor knowledge of the health risks associated with agriculture , and it recommended the training of hcps working in rural areas to address these health problems . the responses regarding the availability of laboratory testing indicate that although laboratories are available , none conduct any testing specific to diagnose pesticide poisoning . this finding agrees with data from record reviews at health care facilities in tanzania in which most of the cases were diagnosed through history and clinical signs . the lack of laboratory capacity to confirm diagnosis , a widespread phenomenon worldwide , for example , in india and also in the southern african region , may contribute to underdiagnosis of app cases reported in hospitals . if few cases are formally diagnosed , few will be reported , and surveillance data will underreport the extent of the problem and policymakers may not see the importance of building capacity for laboratory diagnosis , which in turn exacerbates the problem of underreporting . there is therefore a need to advocate for better diagnostic facilities , especially in rural health facilities to make the laboratory diagnosis of app possible . also , given that there is poor knowledge among hcps , better training in clinical diagnosis is also critically important . the role of poison control centres ( pccs ) in providing guidance to health professionals has been noted as an important strategy in some countries . however , as the who points out , many parts of the world lack access to pccs , particularly africa and parts of the eastern mediterranean and western pacific regions , and there is no fully operational pcc in tanzania able to provide this kind of support to health care providers facing a case of possible app . in a continent so lacking in key human resources for health , addressing this gap through establishing a pcc is an ambitious goal which is unlikely to be realized without sizable donor support and careful attention to sustainability . most of the hcps ( 55% ) could not report any safety instructions when interpreting pesticide labels . failure to interpret the label information may result in poor diagnosis and treatment as the label carries important information for the diagnosis and management of app . even if the label is available in an app case , it would appear that the hcp will not be able to interpret the label and handle the patient appropriately . taking into account that laboratory diagnosis is unavailable , hcps should be trained on , among other things , how to interpret label information for the identification and treatment of app cases . this is particularly important , given the lack of a pcc in tanzania able to provide timely and accurate guidance on pesticide poisoning to hcps , a problem common to much of africa and parts of the eastern mediterranean and western pacific regions . it is striking that the proportion of hcps reporting previous experience in managing a case of pesticide poisoning was much lower in this study ( 50% ) than reported by ngowi et al ( 80% ) . although the study by ngowi and colleagues visited mainly dispensaries and lower level facilities ( 65.3% ) , this study included fewer health care workers from dispensaries ( 44% ) . because dispensaries are mostly located in rural areas they are more likely to attend to poisoning cases , potentially including less severe occupational injuries . health care professionals working in these facilities may therefore have more experience in handling app cases . because facilities with higher status , for example , hospitals , receive more severe cases , which are less common , this could account for the smaller proportion of hcps in this study experienced in the management of poisoning cases . the findings in this study are consistent with that of a study conducted in east africa more than a decade ago , which found that more than 40% of the hcps interviewed could not recognize pesticide poisoning cases . the problem of management of app found in this study is also a problem in developed countries . for example , in a study conducted in washington , dc , and surrounding areas , it was reported that most of the hcps interviewed frequently did not diagnose pesticide toxicity from patient history and examination . exactly 64% of practitioners and 69% of nurses felt poorly prepared to answer patients questions . in all , 40% of practitioners and 26% of nurses felt that it was important to obtain more information on pesticides . this study found that knowledge about pesticide classification was significantly higher in private than government facilities ( 39.5% vs 12.0% ; p = .02 ) . this could arise from the fact that the proportion of health care workers who have handled app was somewhat higher in private facilities than government facilities ( 53.5% vs 39.1% ) but not statistically significantly so ( p = .27 ) . by handling app cases , hcps may come across different agents and their labels or containers , and this may , over time , make them knowledgeable . it may also be the result of private facilities being able to attract hcps with greater education because knowledge about pesticide classification and adverse health effects of pesticides was higher in respondents with high education ( table 6 ) . this association is to be expected as respondents who are graduates usually receive more intensive training , and hence would be likely to have more knowledge on pesticide health effects and classification . regarding documentation of poisoning cases , it was found that all hcps reported documenting all poisoning cases in the health statistics abstracts reference books ( mtuha ) and patient register book . however , this may be an overstatement because , in theory , documentation of poisoning cases is mandatory . incomplete registers , damaged pages , as well as failure to record cases . regarding agents , the study found that most of the specifically known agents reported to be associated with poisoning ( 87% ) were of who class i or ii . these products are , by definition , either highly or moderately hazardous , and their association with poisoning by the hcps was consistent with their toxic nature . although these products are restricted in tanzania , their handling and use are not well controlled due to weaknesses in enforcement . furthermore , among the specifically known agents , 25% of the products reported as associated with app were ops . organophosphates are cholinesterase - inhibiting agents , and although they were reported in low proportion in this substudy , their involvement in poisoning can not be underestimated . the proportion of unknown agents ( 41.3% ) was high indicating that many hcps either rarely handle app cases or the limited cases reported to them lack information . the study results may be weakened by a number of possible biases : information bias . health care professionals might have claimed greater familiarity with adverse health effects of pesticides than actually was true , implying that knowledge and familiarity might , in reality , have been worse than found in this study . selection bias . selection bias might also have affected the findings in that hcps who declined to participate might have done so because they were not conversant with app or may have been reluctant to disclose their lack of experience in managing app . again , this implies that estimates for knowledge and for experience with app reported in the study were likely to be overestimated than is the case in reality ie , a problem of over - reporting . however , the omission of the hcps from facilities in far - off remote areas may countereffect this overestimation if nonparticipants were used to seeing app cases . consequently , the direction of misclassification due to this selection bias is not obvious . the health facilities involved in the study included at least 1 respondent from referral hospitals , regional hospitals , district hospitals , health centres , dispensaries , and other hospitals . although not selected in a truly random manner , the spread of facilities and practitioners suggest that the sample includes hcps who typically staff such facilities and see cases of app . nonetheless , it is possible that the sample of facilities may differ from other facilities in the country . the findings should not be generalized without further studies to confirm the patterns in a representative sample of hcps . although interviews were conducted on an individual basis , in some situations respondents had opportunity to interact with other interviewed respondents before they underwent their own interview . in such situations , their answers were potentially influenced by their colleagues resulting in some degree of homogeneity of reporting . this would cause respondents to provide unrealistic responses which could have either underestimated or overestimated the knowledge and practices of hcps in relation to app . respondents may have poor memory of some events in particular events that took place more than 3 months earlier . the findings suggest that most hcps in the selected health care facilities in northern tanzania lacked adequate skills in the diagnosis and management of app and had very poor knowledge about what to do about app . the limited ability to diagnose app cases results in failure to recognize all poisoning cases arising from pesticide exposure , and this contributes to underreporting of app cases . a strong surveillance system requires hcps who are sufficiently skilled to make the diagnosis of app and report it effectively . to fill this gap , there is a need to include training on pesticide hazards , classification , and health effects in the training programmes for all categories of hcps in tanzania . to develop practical skills , it is recommended that hcps undergo practical training at institutions with experience in the management and study of pesticides , such as the tpri , which is the sole institution dealing specifically with pesticides in tanzania and therefore best placed to support clinicians in matters related to pesticides . currently , tpri has training programmes on pesticides in place conducted twice annually . although this study was undertaken in 2 regions of northern tanzania , and can only be generalized to these areas , the services and farming areas are fairly typical of other part of tanzania . for this reason , it is believed that the findings might well reflect a similar situation in the rest of the country . however , there is a need to conduct further studies in other parts of tanzania to see whether the findings are replicated .

background : acute pesticide poisoning ( app ) is commonly underdiagnosed in tanzania . studies in developing countries suggest that a lack of diagnostic skills among health care providers ( hcps ) undermines surveillance for app . this study aimed at characterizing experience and skills of tanzanian hcps regarding app diagnosis and management.methodology:the population included hcps responsible for managing app in kilimanjaro and arusha regions ( n = 91 ) . the resulting sample included 66 respondents ( response rate : 73% ) . the data were collected in 2005 using a standardized questionnaire.results:half of all respondents ( 50% ) reported handling at least 1 app case with 15% reporting handling more than 5 cases in the past . reported experience of handling an app case was marginally higher in respondents who reported 4 years of work experience in the health sector compared with those with < 4 years of work experience ( odds ratio = 1.32 ; 95% confidence interval = 0.9 - 1.5 ) . most of the respondents had high knowledge of exposure routes , reporting awareness of oral ( 98.5% ) , inhalational ( 93.9% ) , and dermal ( 77% ) routes . the study revealed low awareness of pesticide classification by chemical groups ( 29% ) or world health organization hazard ( 0% ) and weak knowledge on pesticide label instructions ( 55% ) . organophosphates accounted for 35% of the pesticide products reported by respondents as being responsible for poisoning . some treatment options were incorrectly reported as first aid options , and some reported first aid options were wrong or inappropriate.conclusions:the study revealed that hcps in northern tanzania lack adequate skills to diagnose and manage app . for effective surveillance of app , there is a need to include training on hazards , classification , diagnosis , and health effects in the training programmes for all hcps in tanzania .

Introduction Materials and Methods Population and sample Data collection Data analysis Ethical considerations Results Associations with knowledge among HCPs Discussion Study limitations Conclusions

numerous agents , from endogenous and exogenous sources , can induce dna damage in cell genome . at times , the lesion induction mechanisms of these agents are mediated by reactive oxygen species ( ros ) production . these compounds can play a role in the initial events involved in aging , mutagenesis , and cancer [ 1 , 2 ] . stannous salts , used in different areas of human daily life , and ultraviolet radiation a ( uva or near - uv light320400 nm ) , a fraction from solar light that reaches the earth surface , are examples of exogenous agents which produce lesions in dna by ros generation . different forms of stannous salts are used in food industry [ 36 ] , but major concern lies in its inorganic form such as stannous chloride ( sncl2 ) , since this is widely used in nuclear medicine as a reducing agent in the labeling of molecules and cells with technetium-99 m [ 7 , 8 ] . in this case , sncl2 is intravenously administered to the patient . several authors have been demonstrated the cytotoxic , genotoxic , and mutagenic effects of sncl2 and uva . as to sncl2 , it was described that this salt is able to produce ros in a fenton - like reaction , inducing lethality in escherichia coli ( e. coli ) , cytotoxicity and genotoxicity in eukaryotic cells , and genotoxicity in plasmid dna [ 1113 ] . mutagenic properties were also reported to sncl2 , being 8-hydroxyguanine ( 8-oxodguo ) the predominant lesion , accountable for g : c - t : a transversions , as well as mutagenicity in yeast and bacteria . concerning uva , ros production was observed in bacteria , calf thymus dna , and hela cells . in 1986 , caldeira de araujo and favre , demonstrated , by using the sos chromotest assay , that this radiation was able to induce sos functions in e. coli , suggesting that this agent had mutagenic properties . recent works have pointed out that uva mutagenic effects are associated to 8-oxodguo production [ 17 , 19 , 20 ] . another phenomenon observed by favre and coworkers , in 1985 , was that uva sublethal doses were able to induce growth delay effect in e. coli . in addition to many papers pointing toward ros production by sncl2 and uva , some researchers have demonstrated that these agents are also able to induce a direct effect on dna . for instance , de mattos and collaborators suggested that stannous ions were able to bind to dna , inducing ros formation very close to this target ; and courdavault et al . , upon studying human fibroblasts and keratinocytes submitted to uva , described production of thymine cyclobutane dimer ( cpd ) level higher than both 8-oxodguo and single strand breaks . different authors have pointed to base excision repair ( ber ) as the most important system capable of repairing oxidative damage from broad oxidative sources [ 26 , 27 ] . thus , a number of studies have been pointed to the participation of ber enzymes in lesion repair induced by sncl2 [ 14 , 28 ] and uva [ 2932 ] . in ber pathway , the injured base excision is initiated by the action of a class of dna repair enzymes called dna glycosylases , which recognize oxidative lesions and sever these from dna . the removal of ap sites is initiated by a second class of ber enzymes called ap endonucleases . ap sites are extremely toxic to cells and can be generated in dna during normal aerobic metabolism or by exogenous agents , such as ionizing radiation , uv light , and hydrogen peroxide ( h2o2 ) . the xtha protein accounts for 90% of ap endonucleolytic activity found in e. coli , being an important member of the ber mechanism . the e. coli xtha mutants are sensitive to oxidative lesions produced by agents such as near - uv and h2o2 . most of the residual ap endonucleolytic activity present in xtha mutants is encoded by the nfo gene . the endonuclease iv ( nfo protein ) , codified by nfo gene , recognizes and cleaves dna in a similar way to exonuclease iii , albeit some authors suggest that the nfo enzyme is able to recognize lesions not recognized by exonuclease iii . moreover , endonuclease iv is the only ber protein which , besides existing constitutively , can have its expression increased by the soxrs system . it has recently been described that endonuclease iv plays a role in an alternative pathway to classic ber , called nucleotide incision repair ( nir ) , in which it cleaves dna generating terminus which constitutes the dna polymerase target . thus , this pathway has the advantage of avoiding the genotoxic intermediates generated in ber mechanism . based on the above , we decided to investigate cytotoxicity and genotoxicity induced by the pretreatment of uva followed by sncl2 incubation in e. coli ber mutants . cytotoxicity was assessed through bacterial survival assays , and genotoxicity was studied by using alkaline gel electrophoresis , described by de mattos et al . stannous chloride ( sncl22h2o ) and sodium dodecyl sulfate ( sds ) were acquired from sigma chemical co. ( usa ) . sodium chloride and ethylene diaminetetracetic acid ( edta ) sodium hydroxide ( naoh ) and tris ( hydroxymethyl ) amino - methane were obtained from nuclear ( brazil ) . normal point agarose ( npa ) , low - melting point agarose ( lmpa ) and ethidium bromide were acquired from invitrogen ( brazil ) . the strains and plasmid used and their relevant genetic characteristics for this work are listed in table 1 . a starting culture for experiments was taken from overnight samples , and the cells were grown in the same medium up to exponential phase ( 1 - 2 10 cells / ml ) ; harvested by centrifugation ( 2.940 g ; 15 min ; 4c ) , washed twice and suspended in 0.9% nacl . bacterial cultures in mid - log growth phase , suspended in 0.9% nacl , were divided in two fractions , each containing 2 ml . one of them was illuminated with uva ( phillips lamp , 125 w , emitting more than 95% in 365 nm ) for 1 hour , at 25 j / m / s , totaling 90 kj / m . during irradiation , 1 ml of each fraction ( illuminated or not ) was taken and incubated with sncl2 ( 25 g / ml ) , during 1 hour , with shaking . after this , it was obtained four samples : control ( not treated ) , treated with sncl2 , uva - irradiated , and pre - illuminated plus sncl2 incubation . aliquots from each fraction were taken every 20 minutes , appropriately diluted in 0.9% nacl solution and then spread onto lb medium . after 18 hours of incubation at 37c , the colonies were counted and the number of viable cells ( n ) , obtained for the various incubation times , was matched to that observed for time zero ( n0 ) , as a measure of survival fractions . all experiments were done at least three times and data plotted represent the mean values with their respective standard deviations . untreated controls did not show significant variation ( p > .05 ) in cell number along the time ( data not shown ) . data collected were analyzed by anova , followed by the tukey 's multiple comparison test , using graphpad prism software , version 4.0 , with a 5% significance level . before anova statistical analysis , all e. coli strains assayed with this methodology were prepared as described above , in survival experiments . being the volume of cultures increased to 6 ml and the sncl2-treatment time reduced for 40 minutes . following treatments , 1.5 ml from each fraction was centrifuged and the protocol described by de mattos et al . 2008 was done with the aim to detect dna strand breaks induced by sncl2 , uva , and pre - illumination with uva , followed by sncl2 incubation . the e. coli residual fractions ( 4.5 ml ) were centrifuged , suspended in fresh lb medium and incubated with shaking for 90 minutes , at 37c . aliquot ( 1.5 ml ) from each sample was taken every 30 minutes , in order to verify the lesions repair induced by the agents used in this work . briefly , collected aliquots were centrifuged , suspended in cold te and mixed with low melting point agarose . alkaline electrophoresis assays were carried out at least three times to confirm the obtained data and the best gels were selected . the images obtained from alkaline electrophoresis were analyzed using the software image j 1.33u version to quantify the dna strand breaks . the collected data were submitted to chi - square test ( ) , adopting 5% as significance level . survival experiments have shown , for all strains tested , that the uv dose used in this work ( 90 kj / m ) was not able to induce bacterial inactivation , being considered sublethal ( data not shown ) . experiments carried out with sncl2 ( 25 g / ml ) have shown no significant differences ( p > .05 ) among inactivation levels produced in ab1157 strain ( wild type ) , bw9091 ( xtha ) , and bw372 ( nth ) mutants . however , a significant difference ( p < .001 ) was observed among wild type , bw527 ( nfo ) and bh20 ( fpg ) strains ( figure 1 ) , being nfo mutant the most sensitive . these results indicate that endonuclease iv and fpg proteins must be important in the repair of sncl2-induced lesions . a significant difference ( p < .001 ) this result was expected , as friedberg and coworkers , 2006 reported that the fpg enzyme recognizes oxidative lesions , such as 8-oxodguo . moreover , sncl2 may be able to induce this kind of damage , as supported by cabral et al . it can also be ascertained , in the same figure , that endonuclease iv enzyme could be more important than xtha gene product . this assertion is based on the fact that , in our experimental conditions , bw9091 was less sensitive than bw527 to the treatment , which could be due to nfo gene product performance that works in the first one . regarding the uva pre - treatment , followed by sncl2 incubation , it was found that this association produced a synergistic lethal effect in all strains tested , when compared to sncl2 alone ( p < .001 ) . figure 2 presents the results obtained with wild type ( ab1157 ) strain and bh20 , bw372 , bw9091 , and bw527 mutants , pre - illuminated with uva , followed by sncl2 incubation . in this case , a significant difference ( p < .001 ) was observed among these . as it had occurred with the sncl2 treatment , bw527 ( nfo ) mutant this finding corroborates , furthermore , that the product of nfo gene plays an important role in the repair of the damage induced , not only by sncl2 , but also by the association of this agent with uva . in these treatment conditions , bh20 ( fpg ) strain was more sensitive than bw372 ( nth ) mutant , suggesting , again , that fpg enzyme is more efficient than nth to recognize and remove lesions produced by the association of both agents . in order to ascertain the actual role of endonuclease iv in the e. coli survival to sncl2 and uva + sncl2 treatments , bw527 ( nfo ) strain was transformed with pbw21 plasmid carrying the nfo gene , and submitted to bacterial inactivation experiments . the wild type strain ( ab1157 ) the results demonstrated , for sncl2 treatment , that bw527 + pbw21 presented the same inactivation level ( p > .05 ) of ab1157 + pbw21 , whereas a significant difference was obtained between these strains and bw527 ( p < similar results were obtained when the strains were pre - illuminated with uva , and then incubated with sncl2 ( figure 4 ) . so , this data demonstrated that nfo enzyme accounts for e. coli survival and reinforces the fact that the lesions induced by sncl2 and by its association with uva constitute nfo protein targets . the results found in this work are similar to those obtained by levin and demple 1996 when studying the bleomicine effect on wild type ( ab1157 ) and nfo mutant ( bw527 ) e. coli cells . these authors introduced a plasmid dna containing the nfo gene in ab1157 strain and observed that this strain was able to repair the bleomicine - induced lesions . so , it was shown that nfo overexpression , in vivo , contributes to the elimination of bleomicine - induced dna lesions . in this manner , it was clear that endonuclease iv would be more important than exonuclease iii in bleomicine - induced damage repair , since its level is the same as in the nfo mutant and in the strain overexpressing nfo protein . to better understand the damage and repair mechanisms induced by the agents used in this work , alkaline gel electrophoresis was assayed to detect strand breaks in bacterial genome . results obtained with this procedure have shown that sncl2 , uva , and its association with sncl2 , in pre - illumination condition , were able to produce dna strand breaks in all tested strains . furthermore , the uva association with sncl2 generated a higher number of dna strand breaks , when compared to sncl2 treatment ( p < .0001 ) . the gel densitometrical analysis revealed , for uva treatment , that ab1157 ( wild type ) was the strain presenting the highest efficiency in lesions - induced repair by this radiation ( figure 5 ) . this result was expected , since this strain possesses all active repair mechanisms . when comparing the simple mutants ( bh20 , bw372 , bw527 , and bw 9091 ) , it was observed that bh20 ( fpg ) strain showed the slowest dna strand break repair kinetics ( figure 6 ) . this result agrees with the literature , since the fpg gene product is able to recognize 8-oxodguo lesions [ 26 , 27 , 42 ] . as exposed previously , uva radiation can induce ros generation which can lead to 8-oxodguo formation [ 43 , 44 ] . the bh20 strain display delay in lesion restoration upon a lack of fpg . the bw372 ( figure 7 ) , bw527 ( figure 8) , and bw9091 ( figure 9 ) strains were statistically identical , regarding uva - induced lesion repair capacity . .05 ) , a fact which can be explained by fpg protein functioning . this data supports the hypothesis that fpg enzyme could be important in recognizing uva - induced damages . nevertheless , the results pointed to nth , nfo , and xtha protein participation in a given repair step , since the strains deficient in these enzymes were not so efficient , as the wild type strain ( ab1157 ) , in lesion repair . in this case , it should be remembered that nth protein can also attack different residues generated by oxidative agents . in agreement , it was shown here that fpg is the preferential enzyme in recognizing uva - induced lesions , since bh20 ( fpg ) displayed a delay in lesion repair when compared to bw372 ( nth ) . this assertion is based on the fact that , during bh20 repair kinetics , a decrease was not observed in dna strand break number between the times 30 and 60 minutes of repair ( p > .05figure 6 , lanes 6 and 9 ) . in contrast , in bw372 strain , the maximum repair level was obtained in 60 minutes ( figure 7 , lane 9 ) . the residues left at the lesion site by nth or fpg enzymes action need to be processed by ap endonucleases to complete ber repair . these residues constitute exonuclease iii protein targets , and for this fact , bw9091 strain ( xtha ) was not able to complete the repair ( figure 9 ) as efficiently as in the wild type . 1988 endonuclease iv ( nfo ) protein can also take part in the ros lesion - induced repair , since this enzyme possesses a similar substratum to that of exonuclease iii . thus , in the absence endonuclease iv ( bw527 ) , restoration was not successfully completed after 90 minutes ( figure 8) , when compared with the wild type . comparing bw9091 ( xtha ) and bw527 ( nfo ) strains , it was observed that the latter was slower in the repair process than the former , since the dna strand breaks number in bw527 remains the same between the times 30 and 60 minutes ( p > .05figure 8 , lanes 6 and 9 ) . this data could suggest that nfo enzyme is more important than xtha protein in uva - induced lesions repair . such idea was supported by gel densitometrical analysis of double mutants lacking endonuclease iv which were least efficient in the repair ( data not shown ) . as cited above , the endonuclease iv is the only inducible ber enzyme and the uva dose employed in this study is known to induce growth delay in e. coli . thus , this could explain the least efficiency of bh20 ( fpg ) in uva - induced lesion repair in that , even possessing nfo enzyme , its synthesis would be partially inhibited by the growth delay mechanism . another evidence of growth delay is based on the same behavior presented by bw527 ( nfo ) , bw9091 ( xtha ) , bw372 ( nth ) , and bw434 ( xtha - nth data not shown ) strains . the mutants which possess the nfo protein were not more efficient in repairing the damages than bw527 , suggesting that nfo gene translation could be inhibited by the growth delay . the gel densitometrical analysis revealed , for sncl2 treatment , that ab1157 ( figure 5 ) was the strain which repaired the sncl2-induced lesions with the most efficiency , as expected for a wild type strain . the simple mutant analysis showed that bw527 ( nfo ) strain presented the most delay in the sncl2-lesion - induced repair ( figure 8) , being followed by bw9091 ( xtha figure 9 ) , bh20 ( fpg figure 6 ) , and bw372 ( nth figure 7 ) strains . these results point to the important roles developed by ap endonucleases ( nfo and xtha ) in processing ap sites , generated by dna glicosylases enzymes ( fpg and nth ) . when bh20 ( figure 6 ) and bw372 ( figure 7 ) strains were compared , it was observed that bh20 ( fpg ) showed the least capacity in restoring sncl2-induced damages . as literature reports , sncl2 is able to induce 8-oxodguo lesions , which constitute fpg targets [ 26 , 42 ] . so , despite the description that endonuclease iii ( nth ) could recognize and remove oxidative damages , the data obtained in this study with bh20 and bw372 mutants suggest that sncl2 would be producing 8-oxodguo , thereby reinforcing the results of cabral et al . in addition , fpg enzyme would also be required in processing this lesion . the comparison between bw527 ( figure 8) and bw9091 ( figure 9 ) revealed that the nfo deficient strain ( bw527 ) remained with the most dna strand breaks number after repair kinetics , suggesting that nfo enzyme would play a more important role than xtha in sncl2-induced damage repair . literature data showed that , among ber enzymes , exonuclease iii was required to restore sncl2-induced lesions . however , in this study , this fact was not observed with the same intensity , even when using xtha deleted strain ( bw9109data not shown ) . thus , sncl2 could be producing damages which would be preferentially recognized by endonuclease iv , as occurring with the chemical agents tert - butyl hydroperoxide and bleomicine , which produce lesions not recognized by exonuclease iii , but only by endonuclease iv . it is important to emphasize that despite the fact that endonuclease iv represents only 10% of total ap endonuclease activity it is the sole inducible ber protein . its expression may be increased 20-fold , when treatments with oxidative agents , such as paraquat , which leads to superoxide radical production , are performed . thus , in this work , it could be suggested that sncl2 would be inducing an increase in nfo levels , since this salt is able to produce ros [ 9 , 11 ] . then , upon a lack of nfo , as occurs in bw527 strain , the cell would find it more difficult to repair sncl2-induced lesions . it is known that endonuclease iv increase is depedent on soxrs system induction , in order to ascertain whether sncl2 would be able to induce the soxrs system and , consequently , endonuclease iv synthesis , sos chromotest assays were performed . the results obtained were positive ( asad , lmbo , personal communication ) , corroborating the hypothesis presented in this study . the nfo role in sncl2-damage induced repair was reinforced by double mutant analysis ( data not shown ) , in which the strains lacking nfo protein showed the slowest repair kinetics , when compared to the simple mutants . when the bacterial strains were pre - illuminated with uva , followed by sncl2 incubation , a higher dna strand break number was observed than that secured with sncl2 treatment alone . once more , the wild type ( ab1157 ) strain presented the highest efficiency in repairing the lesions induced by uva+sncl2 association ( figure 5 ) . as occurring with sncl2 treatment , bw527 ( nfo ) strain once more showed the least efficiency in restoring damages produced by uva plus sncl2 ( figure 8) . following bw527 , was the bh20 ( fpg ) strain with the lowest ability in repairing uva + sncl2-induced lesions ( figure 6 ) than the other mutants . moreover , when bh20 was compared to bw372 ( nth ) , the latter showed the smallest dna strand break number at the repair kinetics end ( figure 7 , lane 13 ) . this data suggests that uva and sncl2 association could be inducing oxidative damages , such as 8-oxodguo , as previously described [ 14 , 43 , 44 ] and fpg enzyme would be preferential in recognizing and removing this damage . the bw527 ( figure 8) and bw9091 ( figure 9 ) gel analysis reinforced the idea that endonuclease iv would be more important than exonuclease iii in restoring uva + sncl2-induced lesions , in that , at the repair kinetics end , bw527 still presented a higher dna strand break number ( figure 8 , lane 13 ) . this evidence was corroborated by double mutant analysis ( data not shown ) in which endonuclease iv deficient strains showed the least efficiency in repairing the uva and sncl2 induced lesions . the repair efficiency profiles of the strains treated with both agents follow different patterns from those revealed by the only uva illuminated strains . thus , even if the pretreatment with this radiation may induce the growth delay phenomenon , the constitutive existence of nfo protein will still warrant bw372 , bw9091 , and bh20 strains major efficiency in repairing the lesions induced by uva and sncl2 association than that of nfo mutant strain ( bw527 ) . moreover , in addition to its action on ap sites , endonuclease iv is able to recognize and cleave dna on the 5 side of various oxidatively damaged bases via nir , generating the proper ends for dna repair synthesis . as demonstrated by ischenko and colleagues , 2006 , nir is essential for handling lethal oxidative dna lesions . therefore , our study also could suggest nfo participation , via nir , in removing damages induced by sncl2 and uva + sncl2 . taken together , our results point to endonuclease iv activity via ber and possible nir in protecting e. coli cells against damages induced by sncl2 and uva + sncl2 .

stannous chloride ( sncl2 ) and uva induce dna lesions through ros . the aim of this work was to study the toxicity induced by uva preillumination , followed by sncl2 treatment . e. coli ber mutants were used to identify genes which could play a role in dna lesion repair generated by these agents . the survival assays showed ( i ) the nfo mutant was the most sensitive to sncl2 ; ( ii ) lethal synergistic effect was observed after uva pre - illumination , plus sncl2 incubation , the nfo mutant being the most sensitive ; ( iii ) wild type and nfo mutants , transformed with pbw21 plasmid ( nfo+ ) had their survival increased following treatments . the alkaline agarose gel electrophoresis assays pointed that ( i ) uva induced dna breaks and fpg mutant was the most sensitive ; ( ii ) sncl2-induced dna strand breaks were higher than those from uva and nfo mutant had the slowest repair kinetics ; ( iii ) uva + sncl2 promoted an increase in dna breaks than sncl2 and , again , nfo mutant displayed the slowest repair kinetics . in summary , nfo protects e. coli cells against damage induced by sncl2 and uva + sncl2 .

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

vasospasms of the intracranial arteries are a common and well - known complication of subarachnoid hemorrhage . they are also encountered in other disorders such as cerebral vasculitis or reversible cerebral vasoconstriction syndrome ( rcvs ) . in contrast , vasospasms of the extracranial circulation occur less frequently and have most often been associated with mechanical manipulations or ergot poisoning . recurrent spontaneous vasospasms occurring mainly within the intracranial but also within the extracranial arteries have also been reported in patients suffering from migraine headaches . we present a patient with recurrent strokes due to transient vasospastic occlusions of the internal carotid artery ( ica ) without migrainous headaches . our case supports the notion that recurrent non - migrainous vasospasms of the extracranial carotid artery likely reflect a distinct entity which can cause ischemic strokes . in july 1999 , a previously healthy 34-year - old female patient was admitted to our hospital for the first time with an acute left - sided brachiofacial hemiparesis . she reported that similar symptoms had occurred several times in the previous 10 years but had regressed spontaneously . aside from regular nicotine consumption , she had no other cardiovascular risk factors . magnetic resonance imaging ( mri ) showed an internal middle cerebral artery ( mca ) territory watershed infarct , probably of hemodynamic origin . duplex sonography on admission revealed proximal occlusions of both icas and retrograde flow in both supratrochlear arteries ( sta ) . three days later , a digital subtraction angiography showed a recanalization of the left ica and a persistent occlusion of the right ica at the origin , which subsequently never recanalized . on axial t1-weighted fat - suppressed images , the presence of intramural hematomas and thus arterial dissections as a cause of the bilateral ica occlusions could be ruled out . the patient was treated with the calcium antagonist nimodipine ( 240 mg / day ) and was heparinized with an aim for a partial thromboplastin time of > 60 s. despite this treatment , a transient occlusion of the left ica , confirmed with repeated ultrasound and magnetic resonance angiography ( mra ) , occurred again during this first admission . the patient was discharged receiving nimodipine , and an oral anticoagulation with phenprocoumon was begun . in february 2004 , she was readmitted due to a short episode with paresis of the right hand and aphasia . while mri including diffusion - weighted imaging showed no new infarction , an initial occlusion and subsequent reopening of the left ica during the course of this admission , oral anticoagulation was discontinued and the patient was discharged with aspirin and nimodipine . in march 2010 , february 2011 and may 2011 , the patient experienced recurrent episodes with transient right - sided hemiparesis and aphasia . in all three instances , mri revealed new small infarcts in the left mca territory . extensive laboratory investigations were performed twice ( including virology , bacteriology and tests for thrombophilia and vasculitis ) , but revealed no pathological results . likewise , 2 chest x - rays and an ultrasound of the abdomen were normal . similar to the findings in 2004 , mra and ultrasound repeatedly revealed an initial occlusion and subsequent reopening of the left ica ( fig . 1 , fig left ica occlusion , a reduced pulsatility in the left mca , a collateralization via the posterior communicating arteries and a retrograde flow within the ipsilateral sta were observed with ultrasound . after recanalization , ultrasound showed an antegrade flow within the ipsilateral sta and a completely normalized mca flow . since may 2011 a coincidence with smoking was presumed during the first stays , but attacks appeared also after she quit smoking . evidence is accumulating that recurrent vasospasms of the extracranial ica are a rare but important cause of ischemic disorders mainly in young patients [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ] . while previous cases have been associated with migraine [ 5 , 10 ] , we describe a patient with recurrent non - migrainous vasospasms of the extracranial ica . only a few such cases have been reported in the literature to date [ 6 , 8 , 11 , 12 , 13 , 14 ] . it is noteworthy that the recurrent migrainous and non - migrainous vasospasms of the extracranial ica vasospasms appear to occur exclusively in the intermediate portion of the ica [ 8 , 11 , 13 , 14 ] . the reason for this finding is currently unclear , but could be associated with a distinct pattern of sympathetic vasomotor innervation in the intermediate ica segment , enhancing the susceptibility of this vessel segment to sympathetic stimuli among others . it should also be emphasized that recurrent vasospasms of the extracranial ica can only be observed during a short time period , and , therefore , they can only be detected using serial ultrasound or other vessel imaging modalities . it is thus likely that the incidence of this entity is underestimated . in young patients with an acute ischemic stroke due to an ica occlusion , the most important differential diagnosis is a dissection of the carotid artery . in light of a missing wall hematoma on repeated t1-weighted fat - suppressed images , as well as the recurrence of the episodes with spontaneous resolution of the left ica occlusion within a few days , it is very unlikely that repeated dissections were responsible for the clinical course in our patient . the rapid spontaneous resolution of the ica occlusion is also an argument against the presence of an inflammatory vessel disease . although the beaded appearance of the left ica could principally be suggestive of vasculitis , extensive laboratory evaluations were performed to rule out the presence of such a disorder . in particular , there was no evidence of wegener 's disease due to missing oropharyngeal inflammation , chest x - rays were inconspicuous and c - and p - anca were negative . reversible vessel occlusions can also be observed in the context of rcvs . in the past few years , this disorder has emerged as a distinct entity and is characterized by thunderclap headaches accompanied by multifocal cerebral artery vasoconstriction . for the diagnosis of rcvs , a complete reversibility of the latter feature within 12 weeks of onset is required . the missing headaches , as well as the time course argue against the presence of rcvs as the cause of the reversible ica occlusions in our patient . currently , the optimal treatment of patients with recurrent vasospasms of the extracranial ica remains unclear . in contrast to other case reports [ 6 , 7 , 12 ] which reported positive effects of calcium channel inhibitors and corticoids , this treatment was not effective in our patient . in addition , neither oral anticoagulation nor platelet inhibitors prevented new stroke episodes in our case . recently , moeller et al . demonstrated a sympathetic hypersensitivity in a patient with an isolated vasospasm of the extracranial ica , which partially resolved using the -1-blocker prazosin . spontaneous vasospasms of the extracranial ica should be thought of in recurrent ischemic symptoms in young stroke patients . recurrent non - migrainous vasospasms of the extracranial ica likely reflect a distinct disease entity which can cause ischemic strokes .

vasospasms of the intracranial arteries are a well - known complication of subarachnoid hemorrhage and are also frequently encountered in other disorders such as migraine , cerebral vasculitis or reversible cerebral vasoconstriction syndrome . in contrast , recurrent spontaneous vasospasms of the extracranial circulation appear to be extremely rare and have most often been associated with migraine . we present a patient with recurrent strokes due to spontaneous transient vasospastic occlusions of the internal carotid artery ( ica ) without migraine over a time period of at least 13 years . initially , the patient had presented with a bilateral ica occlusion and a cerebral infarct on the right side . while the right ica remained occluded , a reopening of the left ica could be detected 3 days after this initial event . in subsequent years , both duplex sonography and magnetic resonance angiography revealed recurrent occlusions of the left ica , which resolved spontaneously within days . this case and other rare previous reports indicate that recurrent non - migrainous vasospasms of the extracranial carotid artery likely reflect a distinct entity which can cause ischemic strokes .

Introduction Case Report Discussion Conclusion

chemical warfare agents ( cwa ) such as sulfur mustard ( sm ) , a vesicant agent , were extensively used against the iranian troops and civilians during 1983 - 1988 . other proliferating stem cells may also involve and induce long - term impairments , including respiratory and ocular complications . central retinal vein occlusion ( crvo ) is a relatively common retinal vascular disorder and a frequent cause of visual morbidity in patients older than 50 years and can be associated with systemic vascular disease and coagulopathy . although most patients with crvo are over 65 years , it can occur in younger patients commonly termed papillophlebitis . younger patients frequently have an associated inflammatory cause or coagulopathy , which deserve particular attention . up to now , as far as we are aware , no early or late retinal complication of sm exposure has been reported . here , we report a fairly young man presented with non - ischemic crvo without any systemic known predisposing factor except for a history of sm exposure . a 41-year - old man was referred to our eye hospital with a complaint of sudden visual loss in his left eye . an eye examination showed that his best corrected visual acuity ( bcva ) in the right eye ( re ) was 20/20 and in the left eye ( le ) was 20/80 . relative afferent pupillary defect ( rapd ) was slightly positive in the left eye as sluggish movement of the iris . intraocular pressure ( iop ) was 13 and 15 mmhg in re and le , respectively . tear break - up time was less than five seconds , which indicated an abnormal tear film . funduscopic examination of his right eye showed no specific abnormality . in the left eye , retinal hemorrhage in all quadrants of the retina , retinal edema , venous tortuosity , and macular edema were present ( figure 1 ) . in fluorescein angiography delayed circulation , hyperfluorescence due to the breakdown of the blood - retinal barrier , venous tortuosity , and blockage due to retinal hemorrhages were obvious ( figure 2 ) . in ocular optical coherence tomography ( oct ) , macular edema was obvious and central macular thickness was increased to 357 microns ( figure 3 ) . . shows fundus photo of the patient who was exposed to sulfur mustard showing vascular tortuosity , hemorrhages , and macular edema . fluorescein angiography showing vascular tortuosity , macular edema , hemorrhages , multiple hypo and hyperfluorescence of the fundus in a 41-year - old male patient who was exposed to sulfur mustard . optical coherent tomography ( oct ) of the macula showing macular edema in a 41-year - old male patient who was exposed to sulfur mustard . hiv ( eliza ) , antinuclear antibody ( ana ) , and rheumatoid factor ( rf ) tests were also negative . all biochemical , immunological and hematologic investigations revealed no abnormalities and the results are summarized in tables 1 and 2 . results of the biochemical and immunological investigations results of the hematologic investigations the patient had no history of systemic arterial hypertension , no obvious cardiac disease , and no connective tissue diseases . however , cardiology and rheumatology consultations were requested . echocardiographies , both trans - thoracic and trans - esophageal , were negative for structural cardiac abnormalities . although all routine coagulation laboratory tests were within normal limits , we also requested hematologic consultation to rule out any coagulopathies . after 15 months , the consultations were repeated and again all were reported as normal . again , we reviewed his possible use of any drug , medication ( either chemical or herbal ) trauma , smoking , and familial history of such diseases . however , the patient had three times exposure to sm during 1987 - 1988 iran - iraq war . he had ambulatory admission and treatment in a field hospital after the last sm exposure in 1988 and recovered within two days . following written informed consent , he received four serial intravitreal injections of an anti - vascular endothelial growth factor , bevacizumab ( 1.25 mg/0.05 milliliter ) , as an off - label treatment modality . after one year , the central macular thickness decreased to 334 microns and vision improved to 20/25 . the patient was followed up for more than 24 months during which he had stable retina and vision . the patient was informed about the publication of this case report and an oral consent was obtained . sm and similar bifunctional agents have been produced as chemical weapons since the 19 century . high dose exposure to sm may result in death within hours or weeks . however , low - dose exposure may cause injury to the eyes , skin , and respiratory tract without any mortality . very low dose of sm exposure may not induce any symptom or sign and thus it is called subclinical exposure . it seems that our patient had two subclinical exposures in 1987 and one mild symptomatic sm exposure in 1988 , after which he recovered within a couple of days . however , long term effects and delayed complications of sm poisoning are well known and several reports are available , particularly on the respiratory tract , skin and the eyes . . it could be due to ocular characteristics such as high turnover rate , intense metabolic activity of corneal epithelial cells , and the aqueous - mucous surface of the cornea and conjunctiva . as it is absorbed by the lungs and skin , it may disseminate in the body and act as a toxic agent involving the internal organs as well . to our knowledge , no retinal abnormal finding ensuing sm exposure has been reported yet . endothelial cells are proposed to be injured in the sm toxicities and may induce apoptosis and necrosis in endothelial cells all over the body . this may explain the vascular permeability in acute phase after exposure , which can result in bronchial edema . long term effect of sm on vessels may predispose them to occlusion , particularly in microvasculature system such as the retina . crvo is usually associated with systemic cardiovascular diseases and coagulopathies ; and is mostly observed in elderlies . crvo in young adults is probably a general ( nonspecific ) change that results from a number of individual causes or a combination of causes . in unusual cases , the cause is known but in the vast majority of the patients , the specific cause or even the contributing factors are unknown . this is mainly due to limited literature and the paucity of histopathologic material from patients with such condition . in our case , we performed a wide array of laboratory tests , systemic work up , and consultations according to the current findings and algorithms . as all our tests and consultations were normal , we re - evaluated the patient and found a history of exposure to sm , a highly toxic chemical warfare agent that may have caused or played a predisposing factor for his non - ischemic crvo . we thus propose that sm may cause or act as a predisposing factor for non - ischemic crvo .

a 41-year - old man was referred with a complaint of visual loss in his left eye and his best corrected visual acuity was 20/80 . slit lamp examination showed arborizing conjunctival vessels and dry eye . fundus examination and fluorescein angiography revealed a non - ischemic central retinal vein occlusion . cardiovascular , rheumatologic , and hematologic work up showed no abnormal findings . an ascertained history of exposure to sulfur mustard during the iran - iraq war was documented in his medical history . four sessions of intravitreal bevacizumab injections were done as needed . after two - year follow - up , visual acuity in his left eye improved to 20/25 and macular edema was resolved without any need for further interventions . we conclude that sulfur mustard gas exposure may be considered as a predisposing factor for central retinal vein occlusion , as was found in our patient ( an iranian war veteran ) by excluding all yet known etiologies and predisposing factors .

Introduction Case Report Discussion

accidents and trauma are one of the worlds most serious , but neglected health problems and is one of the leading causes of mortality in young adults . globally , 26% of all deaths in the age group of 1544 years in 2002 were due to injury . in india , trauma is a major problem , due to a very high incidence of vehicular accidents , other accidents , injuries , crimes , and violence . infections are one of the most important causes of mortality and morbidity in traumatized patients . despite early effective treatment , such patients are susceptible to developing sepsis and multiple organ failure , which in turn are the major causes of death in these critically ill patients . monocytes have been found to play a key role in immunological response to the bacterial infections . systemic inflammation , as well as immune suppression , are thought to play a decisive role in the development of sepsis and multiple organ dysfunction syndromes . as the paradigm of sepsis pathogenesis has evolved over time , and our understanding of the different therapeutic approaches to sepsis developed , various biomarkers have been used for diagnosis of sepsis and monitoring of treatment . the diagnosis of sepsis is still a challenge till date due to the highly variable nature of its signs , symptoms , and unreliable performance of the available biomarkers . early diagnosis and stratification of the severity of sepsis will increase the possibility of appropriate and timely treatment . pro - inflammatory cytokines such as tumor necrosis factor - alpha ( tnf- ) and interleukin-6 ( il-6 ) have been implicated as principal mediators of monocytic activity and probably major mediators involved in the pathogenesis of sepsis . most of the studies on the evaluation of the role of tnf- and il-6 have been done using plasma , which depicts extracellular pooled levels . moreover , the cellular origin can not be ascertained in these extracellular assays . given the importance of sepsis diagnosis and the lacunae thereof , the present study was designed to assess the activity of monocytes with respect to production of tnf- and il-6 by flow cytometry in critically ill septic patients admitted to the intensive care unit ( icu ) of a level-1 trauma center and to correlate it with the patients outcomes . patients enrolled in the present study were those whose blood cultures ( one or more ) had grown aerobic gram - negative pathogen / s and further fulfilled the criteria for clinical diagnosis of sepsis . the presence of sepsis was confirmed when at least two of the following four clinical signs of sepsis were present : ( a ) temperature > 38.9c or < 35.5c ; ( b ) respiratory rate < 28 breaths / min or paco2 > 32 torr ; ( c ) heart rate > 100 beats / min ; ( d ) white blood cells > 15,000 or < 3500 cells / mm . five milliliter blood samples were taken from these patients for assessment of monocytic activity by flow cytometry . five milliliter of whole blood were collected from each patient and divided equally in heparinized ( bd vacutainer lithium heparin 75 usp units , bd franklin lakes nj , usa ) and plain vials from all patients on the day when their blood culture was positive ( considered as day 0 ) , and on days 2 and 5 thereafter as follow - ups to look for the patient 's outcome and correlate it with monocytic activity . serum was separated from the plain vial and stored at 20c for procalcitonin ( pct ) assay whereas the heparinized blood was processed immediately for intracellular cytokine assay . the heparinized blood was stimulated immediately as to prevent cell death over the period of time . for this , the whole blood was diluted in the ratio of 1:1 in rpmi 1640 medium ( thermo scientific , south logan , utah , usa ) , and further divided equally into two tubes . one was challenged with 1 g / ml lipopolysaccharide ( lps ) ( escherichia coli , serotype 055:b5 , sigma - aldrich , st . louis , mo , usa ) , incubated at 37c in 5% carbon dioxide ( co2 ) with 1 g / ml monensin ( bd , san jose , ca , usa ) to inhibit protein secretion for 4 h. in parallel , the other tube , taken as basal ( unstimulated ) tube was also treated similarly , but without lps to serve as a baseline comparison . after stimulation , 100 l of blood was incubated with specific monoclonal antibodies for cd45 and cd14 antigen tagged with percp cy5.5 and pe - cy7 ( bd , san jose , ca , usa ) , respectively . after incubation , red blood cells were lysed with lysis buffer ( bd , san jose , ca , usa ) . the cells were then fixed and permeabilized by fixation and permeabilization buffer ( bd , san deigo , ca , usa ) , followed by incubation on ice in dark with intracellular specific antibodies for il-6 and tnf- tagged with phycoerythrin and allophycocyanin ( bd , san jose , ca , usa ) respectively . the cells were then washed twice with wash buffer ( bd , san deigo , ca , usa ) , and the pellet was resuspended in 1% paraformaldehyde ( fischer - scientific , mumbai , india ) . samples were then analyzed using multiparametric flow cytometer ( bd facs ariaiii , usa ) . results were analyzed using bd facs diva software(bd biosciences ) and gate was set around the monocyte population , which was strongly positive for cd14 on forward scatter on dot plots . tnf- and il-6 production from monocytes also , pct levels were assessed at all time frames from serum samples by the minividas system ( biomrieux , france ) . all the patients enrolled in the study were clinically followed - up till their final hospital outcome ( discharge / death ) and their clinical and laboratory finding were recorded daily , including the microbiological culture reports of all samples received in the laboratory . detailed follow - ups of all blood culture reports of these patients were done along with daily assessment of clinical and microbiological findings . details of surgical procedure performed , antimicrobial treatment and clinical as well as autopsy reports of fatal cases to find the cause of death were similarly noted . patients enrolled in the present study were those whose blood cultures ( one or more ) had grown aerobic gram - negative pathogen / s and further fulfilled the criteria for clinical diagnosis of sepsis . the presence of sepsis was confirmed when at least two of the following four clinical signs of sepsis were present : ( a ) temperature > 38.9c or < 35.5c ; ( b ) respiratory rate < 28 breaths / min or paco2 > 32 torr ; ( c ) heart rate > 100 beats / min ; ( d ) white blood cells > 15,000 or < 3500 cells / mm . five milliliter blood samples were taken from these patients for assessment of monocytic activity by flow cytometry . five milliliter of whole blood were collected from each patient and divided equally in heparinized ( bd vacutainer lithium heparin 75 usp units , bd franklin lakes nj , usa ) and plain vials from all patients on the day when their blood culture was positive ( considered as day 0 ) , and on days 2 and 5 thereafter as follow - ups to look for the patient 's outcome and correlate it with monocytic activity . serum was separated from the plain vial and stored at 20c for procalcitonin ( pct ) assay whereas the heparinized blood was processed immediately for intracellular cytokine assay . the heparinized blood was stimulated immediately as to prevent cell death over the period of time . for this , the whole blood was diluted in the ratio of 1:1 in rpmi 1640 medium ( thermo scientific , south logan , utah , usa ) , and further divided equally into two tubes . one was challenged with 1 g / ml lipopolysaccharide ( lps ) ( escherichia coli , serotype 055:b5 , sigma - aldrich , st . louis , mo , usa ) , incubated at 37c in 5% carbon dioxide ( co2 ) with 1 g / ml monensin ( bd , san jose , ca , usa ) to inhibit protein secretion for 4 h. in parallel , the other tube , taken as basal ( unstimulated ) tube was also treated similarly , but without lps to serve as a baseline comparison . after stimulation , 100 l of blood was incubated with specific monoclonal antibodies for cd45 and cd14 antigen tagged with percp cy5.5 and pe - cy7 ( bd , san jose , ca , usa ) , respectively . after incubation , red blood cells were lysed with lysis buffer ( bd , san jose , ca , usa ) . the cells were then fixed and permeabilized by fixation and permeabilization buffer ( bd , san deigo , ca , usa ) , followed by incubation on ice in dark with intracellular specific antibodies for il-6 and tnf- tagged with phycoerythrin and allophycocyanin ( bd , san jose , ca , usa ) respectively . the cells were then washed twice with wash buffer ( bd , san deigo , ca , usa ) , and the pellet was resuspended in 1% paraformaldehyde ( fischer - scientific , mumbai , india ) . samples were then analyzed using multiparametric flow cytometer ( bd facs ariaiii , usa ) . results were analyzed using bd facs diva software(bd biosciences ) and gate was set around the monocyte population , which was strongly positive for cd14 on forward scatter on dot plots . tnf- and il-6 production from monocytes also , pct levels were assessed at all time frames from serum samples by the minividas system ( biomrieux , france ) . all the patients enrolled in the study were clinically followed - up till their final hospital outcome ( discharge / death ) and their clinical and laboratory finding were recorded daily , including the microbiological culture reports of all samples received in the laboratory . detailed follow - ups of all blood culture reports of these patients were done along with daily assessment of clinical and microbiological findings . details of surgical procedure performed , antimicrobial treatment and clinical as well as autopsy reports of fatal cases to find the cause of death were similarly noted . a total of 16 critically ill trauma patients between the age group of 18 and 65 years , who were admitted to the trauma neurosurgery and surgery icus and whose blood cultures were positive for gram - negative bacilli in the microbiology laboratory were enrolled in the study . among the enrolled patients , male ( 13 , 81% ) were more than the female ( 3 , 19% ) patients . of the 16 patients , 4 ( 25% ) had isolated abdominal trauma , whereas 4 ( 25% ) presented with both abdominal and chest trauma , 2 ( 13% ) had isolated chest trauma , and the rest ( 6 , 38% ) suffered from multiple orthopedic injuries . four patients ( 25% ) had undergone exploratory laparotomy , whereas 4 ( 25% ) other patients had to undergo tracheostomy , 2 ( 13% ) underwent screw fixation of fractures and 1 ( 6% ) each underwent splenectomy , corpectomy , and amputation respectively . of the remaining , 3 ( 19% ) had to undergo exploratory laparotomy in combination with either tracheostomy or splenectomy or gangrenous bowel resection . of these 16 patients , 12 ( 75% ) were discharged after variable lengths of hospital stay ( 1257 days ) . during their hospital stay , all of these 12 patients had one or more hospital - acquired infections . septicemia developed in all the 12 patients with acinetobacter baumannii , ( 7 [ 58% ] ) being the most commonly pathogen isolated from blood culture , followed equally by pseudomonas aeruginosa , stenotrophomonas maltophilia and candida albicans ( 2 [ 16% ] ) each and klebsiella pneumonia 1 ( 8% ) . the remaining four patients expired and the clinical and autopsy proven cause of death was cardiac arrest with septic shock . during their hospital stay , all four patients also developed septicemia and two of them had associated ventilator associated pneumonia . clinical and microbiological findings of the fatal patients tnf- : of the 16 patients , 12 ( 75% ) patients survived the sepsis after variable lengths of hospital stay . in all the patients who survived , the tnf- levels were found to increase gradually on consecutive follow - ups , but as compared with healthy controls , the levels were slightly decreased . however , in the last follow - up , the levels were slightly decreased [ table 2]il-6 : this showed varied results in all the patients who survived , but statistically , the mean of il-6 in all the patients after stimulation showed decreased level as compared to controls in every follow - up , although no uniform pattern was observed which makes the data statistically insignificant [ table 2]pct levels : out of 12 patients who survived the sepsis , pct level in eight patients were observed in normal range ( 0.5 ng / ml ) and two each were observed to be positive ( 0.52 ng / ml ) and highly positive ( 210 ng / ml ) . tnf- : of the 16 patients , 12 ( 75% ) patients survived the sepsis after variable lengths of hospital stay . in all the patients who survived , the tnf- levels were found to increase gradually on consecutive follow - ups , but as compared with healthy controls , the levels were slightly decreased . however , in the last follow - up , the levels were slightly decreased [ table 2 ] il-6 : this showed varied results in all the patients who survived , but statistically , the mean of il-6 in all the patients after stimulation showed decreased level as compared to controls in every follow - up , although no uniform pattern was observed which makes the data statistically insignificant [ table 2 ] pct levels : out of 12 patients who survived the sepsis , pct level in eight patients were observed in normal range ( 0.5 ng / ml ) and two each were observed to be positive ( 0.52 ng / ml ) and highly positive ( 210 ng / ml ) . comparison of tnf- levels in various groups of patients tnf- level in patients with fatal outcome : of the 16 patients , four patients expired due to suspected sepsis . in three patients the levels of tnf- were found to be significantly decreased on stimulation with lps as compared to healthy controls , from the day of blood culture positivity till the last follow - up on day 5 and found to be statistically significant . in the fourth patient , we have observed a rising level of tnf- on each follow - ups , though it was significantly lower compared to the healthy controls [ figure 1 and table 2]il-6 levels in patients with fatal outcome : similarly , the levels of il-6 post stimulation were also decreased in the same three patients mentioned above as compared to healthy controls but this difference was not found to be statistically significant . in the fourth patient , il-6 levels increased with every follow - up [ table 3]pct levels in patients with fatal outcome : in the three patients with decreased levels of tnf- and il-6 , pct were found to be highly positive ( 2 - 10 ng / ml ) in these patients . however , the pct level in the fourth patient with fatal outcome , who had levels of tnf- and raised il-6 levels were found to be in normal range ( 0.5 ng / ml ) . tnf- level in patients with fatal outcome : of the 16 patients , four patients expired due to suspected sepsis . in three patients the levels of tnf- were found to be significantly decreased on stimulation with lps as compared to healthy controls , from the day of blood culture positivity till the last follow - up on day 5 and found to be statistically significant . in the fourth patient , we have observed a rising level of tnf- on each follow - ups , though it was significantly lower compared to the healthy controls [ figure 1 and table 2 ] il-6 levels in patients with fatal outcome : similarly , the levels of il-6 post stimulation were also decreased in the same three patients mentioned above as compared to healthy controls but this difference was not found to be statistically significant . in the fourth patient , il-6 levels increased with every follow - up [ table 3 ] pct levels in patients with fatal outcome : in the three patients with decreased levels of tnf- and il-6 , pct were found to be highly positive ( 2 - 10 ng / ml ) in these patients . however , the pct level in the fourth patient with fatal outcome , who had levels of tnf- and raised il-6 levels were found to be in normal range ( 0.5 ng / ml ) . the decreased level of tumor necrosis factor - alpha in fatal patients following sepsis is compared to healthy control . levels of interleukin-6 in fatal cases were increased in first two follow - ups buts were significantly decreased in the last follow - up comparison of il-6 levels in various groups of patients a comparison between the cytokine levels of all the patient groups on different days are shown in tables 2 and 3 . a total of 16 critically ill trauma patients between the age group of 18 and 65 years , who were admitted to the trauma neurosurgery and surgery icus and whose blood cultures were positive for gram - negative bacilli in the microbiology laboratory were enrolled in the study . among the enrolled patients , male ( 13 , 81% ) were more than the female ( 3 , 19% ) patients . of the 16 patients , 4 ( 25% ) had isolated abdominal trauma , whereas 4 ( 25% ) presented with both abdominal and chest trauma , 2 ( 13% ) had isolated chest trauma , and the rest ( 6 , 38% ) suffered from multiple orthopedic injuries . four patients ( 25% ) had undergone exploratory laparotomy , whereas 4 ( 25% ) other patients had to undergo tracheostomy , 2 ( 13% ) underwent screw fixation of fractures and 1 ( 6% ) each underwent splenectomy , corpectomy , and amputation respectively . of the remaining , 3 ( 19% ) had to undergo exploratory laparotomy in combination with either tracheostomy or splenectomy or gangrenous bowel resection . of these 16 patients , 12 ( 75% ) were discharged after variable lengths of hospital stay ( 1257 days ) . during their hospital stay , all of these 12 patients had one or more hospital - acquired infections . septicemia developed in all the 12 patients with acinetobacter baumannii , ( 7 [ 58% ] ) being the most commonly pathogen isolated from blood culture , followed equally by pseudomonas aeruginosa , stenotrophomonas maltophilia and candida albicans ( 2 [ 16% ] ) each and klebsiella pneumonia 1 ( 8% ) . the remaining four patients expired and the clinical and autopsy proven cause of death was cardiac arrest with septic shock . during their hospital stay , all four patients also developed septicemia and two of them had associated ventilator associated pneumonia . tnf- : of the 16 patients , 12 ( 75% ) patients survived the sepsis after variable lengths of hospital stay . in all the patients who survived , the tnf- levels were found to increase gradually on consecutive follow - ups , but as compared with healthy controls , the levels were slightly decreased . however , in the last follow - up , the levels were slightly decreased [ table 2]il-6 : this showed varied results in all the patients who survived , but statistically , the mean of il-6 in all the patients after stimulation showed decreased level as compared to controls in every follow - up , although no uniform pattern was observed which makes the data statistically insignificant [ table 2]pct levels : out of 12 patients who survived the sepsis , pct level in eight patients were observed in normal range ( 0.5 ng / ml ) and two each were observed to be positive ( 0.52 ng / ml ) and highly positive ( 210 ng / ml ) . tnf- : of the 16 patients , 12 ( 75% ) patients survived the sepsis after variable lengths of hospital stay . in all the patients who survived , the tnf- levels were found to increase gradually on consecutive follow - ups , but as compared with healthy controls , the levels were slightly decreased . however , in the last follow - up , the levels were slightly decreased [ table 2 ] il-6 : this showed varied results in all the patients who survived , but statistically , the mean of il-6 in all the patients after stimulation showed decreased level as compared to controls in every follow - up , although no uniform pattern was observed which makes the data statistically insignificant [ table 2 ] pct levels : out of 12 patients who survived the sepsis , pct level in eight patients were observed in normal range ( 0.5 ng / ml ) and two each were observed to be positive ( 0.52 ng / ml ) and highly positive ( 210 ng / ml ) . tnf- level in patients with fatal outcome : of the 16 patients , four patients expired due to suspected sepsis . in three patients the levels of tnf- were found to be significantly decreased on stimulation with lps as compared to healthy controls , from the day of blood culture positivity till the last follow - up on day 5 and found to be statistically significant . in the fourth patient , we have observed a rising level of tnf- on each follow - ups , though it was significantly lower compared to the healthy controls [ figure 1 and table 2]il-6 levels in patients with fatal outcome : similarly , the levels of il-6 post stimulation were also decreased in the same three patients mentioned above as compared to healthy controls but this difference was not found to be statistically significant . in the fourth patient , il-6 levels increased with every follow - up [ table 3]pct levels in patients with fatal outcome : in the three patients with decreased levels of tnf- and il-6 , pct were found to be highly positive ( 2 - 10 ng / ml ) in these patients . however , the pct level in the fourth patient with fatal outcome , who had levels of tnf- and raised il-6 levels were found to be in normal range ( 0.5 ng / ml ) . tnf- level in patients with fatal outcome : of the 16 patients , four patients expired due to suspected sepsis . in three patients the levels of tnf- were found to be significantly decreased on stimulation with lps as compared to healthy controls , from the day of blood culture positivity till the last follow - up on day 5 and found to be statistically significant . in the fourth patient , we have observed a rising level of tnf- on each follow - ups , though it was significantly lower compared to the healthy controls [ figure 1 and table 2 ] il-6 levels in patients with fatal outcome : similarly , the levels of il-6 post stimulation were also decreased in the same three patients mentioned above as compared to healthy controls but this difference was not found to be statistically significant . in the fourth patient , il-6 levels increased with every follow - up [ table 3 ] pct levels in patients with fatal outcome : in the three patients with decreased levels of tnf- and il-6 , pct were found to be highly positive ( 2 - 10 ng / ml ) in these patients . however , the pct level in the fourth patient with fatal outcome , who had levels of tnf- and raised il-6 levels were found to be in normal range ( 0.5 ng / ml ) . the decreased level of tumor necrosis factor - alpha in fatal patients following sepsis is compared to healthy control . levels of interleukin-6 in fatal cases were increased in first two follow - ups buts were significantly decreased in the last follow - up comparison of il-6 levels in various groups of patients a comparison between the cytokine levels of all the patient groups on different days are shown in tables 2 and 3 . it is widely believed that bacterial endotoxin on entering the bloodstream triggers various alterations in the immune system , resulting in the release of various pro - inflammatory and inflammatory markers which may lead to a plethora of complications . to study the kinetics of cytokines appearing in the blood of septic patients , detailed research is needed , which would help us find some novel methods of pharmacological interventions as well as new and early diagnostic markers during the initial inflammatory response . ours is the first study from india monitoring the monocytic immune response in septic trauma patients . most of the studies on cytokine production in septic patients have been done on plasma which in our view has two disadvantages . first , studies on plasma would reflect the total production of cytokines rather than their cellular origin . second , the short half - life of pro - inflammatory cytokines results in their rapid disappearance . this does not give the true picture regarding the cellular source of cytokines at a single cell level and the actual cytokine profile of posttrauma immune response . to overcome this biological drawback , we used intracellular cytokine analysis utilizing the flow cytometric approach . thus , we targeted the pro - inflammatory cytokines producing activity of monocytes by multiparametric flow cytometry , which represents a reliable method enabling highly selective immunomonitoring of specific cells in septic patients . in septic patients who succumbed to trauma , we observed that the levels of tnf- and il-6 on stimulation with lps were strongly blunted from day 1 of getting positive blood culture reports and continued to decline till further follow - ups ( 2 and 5 day ) . whereas , in those patients who survived the sepsis , after initial low levels of tnf- , there was a gradual increase in the levels of tnf- with each follow - up , as compared to the healthy controls . hence , our data suggests a reduced capability of monocytes in whole blood from trauma patients in sepsis to release sufficient amounts of pro - inflammatory cytokines in response to the endotoxins encountered . all the patients had been started on antimicrobials based on their antibiotic sensitivity reports ; however , those patients who showed continuously decreased levels of cytokines died and those whose cytokines levels increased ultimately survived the sepsis . this highlights the fact that effectively monocytic activity is compromised during sepsis , hence limiting the ability of the patient 's immune system to adequately respond to invading microorganisms . the levels of il-6 in the septic patients who survived gave variable results , hence as a sound conclusion can not be drawn . procalcitonin levels in all except two septic patients who survived were below the cut - off levels ( 0.5 ng / ml ) , hence making tnf- and il-6 better markers for initial evaluation of sepsis . hence , our study suggests that both tnf -and il-6 would make a good marker for sepsis , though further studies are needed to substantiate it . the strength of our study was that clinical follow - ups till the final outcome and autopsy - based confirmation of the cause of death . trauma patients are a fairly uniform cohort , where underlying illness is few and most patients are in the middle age group . first , the study was done for a short duration and we need to increase the sample size . for better comparison , freshly admitted trauma patient 's monocytic cytokine profile would have given a more holistic view of the immune response . this study was done on trauma victims , who at the time of admission were not incubating any infections hence all the infection were hospital - acquired , which increases the morbidity and mortality . thus , the findings of this study can be extrapolated to predicting early sepsis development and thus preventing the vulnerable young economically productive population of our country from severe complications after injury .

introduction : trauma is one of the leading causes of mortality worldwide with infections as important causes of death in such patients . bacterial infections cause activation of monocytes with excessive synthesis of pro - inflammatory cytokines . hence , this prospective study was conducted to assess the activity of monocytes in traumatized sepsis patients using flow cytometry and to assess if they have any prognostic potential.materials and methods : a total of 16 consecutive trauma patients with sepsis and having positive blood culture were enrolled , along with four healthy controls during the period of march 2013 to july 2013 . blood from septic patients were collected on the same day when blood culture was positive and on days 2 and 5 thereafter . surface staining for monocytes with cd14 and intracellular staining for interleukin-6 ( il-6 ) and tumor necrosis factor - alpha ( tnf- ) was done and results were analyzed by flow cytometer . procalcitonin ( pct ) assay was done using minividas . complete clinical follow - up was done for the patients.results:of the 16 patients , four died due to infections by various microorganisms . isolated abdominal trauma ( 25% ) was the most common injury among the enrolled patients of sepsis . levels of tnf- were significantly decreased when stimulated with lipopolysaccharide in the fatal patients as compared to the healthy controls . patients having sepsis who survived had an increased level of tnf- during the follow - up periods.conclusion:this study showed that activity of monocytes to produce tnf- and il-6 were reduced in severe sepsis . early identification of such immune - paralysis can help in earlier intervention to salvage this vulnerable trauma population .

INTRODUCTION MATERIALS AND METHODS Patient selection Healthy control Collection of blood and stimulation Surface and intracellular staining of cells for flow cytometric analysis Multiparameter flow cytometric analysis Statistics RESULTS Demographic profile of enrolled patients Clinical profile of enrolled patients Cytokines levels in patients who survived sepsis Cytokines levels in the fatal patients DISCUSSION CONCLUSION

several genes involved in the metabolism of carcinogens have been found to be polymorphic in human populations and are associated with increased risk of cancer at some sites . this study focuses on the polymorphic enzyme glutathione transferase mu ( gt mu ) . smokers with low lymphocyte gt mu activity are at an approximately 2-fold higher risk for lung cancer and an approximately 3-fold higher risk for stomach and colon adenocarcinomas . recent cloning and sequencing of the gst1 gene has allowed the development of convenient genotyping methods based on restriction fragment length polymorphisms ( rflp ) or the polymerase chain reaction ( pcr ) . the gst1 polymorphism has been shown to be a deletion of the gene locus . to detect the presence or absence of the gene we amplified exons 4 - 5 and/or exons 6 - 7 of the gst1 gene by pcr . pcr amplification produced bands of 215-bp or 273-bp from individuals with one or two copies of the gst1 allele and no band if the individual was homozygously deleted ( 0/0 ) . in the exon 6 - 7 pcr , we co - amplified a 268-bp portion of the beta - globin gene as an internal reference standard for quantitative analysis of product yield . this allowed homozygote individuals ( + /+ ) to be distinguished from heterozygotes ( + /0 ) . we have compared the gst1 genotype to lymphocyte gt mu activity measured on trans - stilbene oxide ( tso ) in the lymphocytes of 45 individuals . low gt mu activity ( < 67 pmole / min/10(7 ) cells ) was strongly associated ( 24/24 ) with the gst1 0/0 genotype . with the exception of one individual , activities greater than 67 pmole / min/10(7 ) were associated with the presence of the gst1 allele ( 20/21 ) . individuals with the highest gt - tso activity were found to be homozygous for gst1 . ( + /+ ) , while heterozygotes ( + /0 ) generally had lower activity , suggesting a gene dosage effect in lymphocytes.(abstract truncated at 250 words)imagesfigure 2 .

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intracerebral hematoma ( ich ) and intra - sylvian hematoma ( ish ) occur in 35 - 55% of patients with subarachnoid hemorrhage ( sah ) from the ruptured middle cerebral artery ( mca ) aneurysms and that hematomas are associated with poor outcome.8)10)15 ) however , their presence is known to have little effect on the prognosis of the patients following an early operation.1)13)14 ) in our last study , we applied the hematoma grading system to predict the prognosis of ruptured mca patients . hematoma volume , midline shifting and diffuse sah were used as variables.11 ) the result showed that the grading system had a significant relationship with the patients ' glasgow outcome scale ( gos ) . van der zande et al.15 ) used computed tomography angiography ( cta ) to categorize the hematoma into pure ich and ish depending on the presence of intra - hematomal contrast enhancing vessel ( ihcev ) . however , from the surgical point of view , there seemed to be a definite difference between ich and ish . this led us to evaluate the differences in the peri - operative glasgow coma scale ( gcs ) in the 2 hematoma groups , and we wished to clarify the effect on the patients ' prognoses . we evaluated 44 ruptured mca aneurysm patients with hematoma who were admitted at our hospital between august 2005 and december 2012 . one hundred five patients with sah from ruptured mca aneurysms were admitted from august , 2005 to december , 2012 . forty - four patients ( 41.9% ) among them had ich or ish of more than 5 cc in ct scan ( table 1 ) . their age ranged from 26 to 81 years old and the male to female ratio was 1:1.44 ( m:18 , f:26 ) . among the 44 cases , 25 patients ( 56.8% ) had ich while 19 ( 43.2% ) had ish . the presence of enhancing vessels were noted in these patients by comparison of their non - enhanced ct and cta.15 ) we suggested the hematoma grading system , 1 or 2 points were assigned depending on the volume being less or greater than 25 cc . the presence of diffuse sah was assigned 1 point in the hematoma scale and 0 point for focal sah , and 1 point was assigned to midline shifting of more than 5 mm versus 0 point for those less than 5 mm ( table 2).11 ) we investigated each patient 's hematoma grade , type in ct and cta , and we compared their age , gender , hunt - hess grade and gos . the change in gcs from admission to 7 days after surgery was also compared . multiple logistic regression analysis was used to identify the independent factors predicting the patients ' prognoses . the hunt - hess grades at admission were 2 in 7 cases ( 15.9% ) , 3 in 11 cases ( 25% ) , and 4 in 24 cases ( 54.5% ) . thirty two cases ( 72.7% ) showed diffuse sah in the initial brain ct , while 12 cases ( 27.3% ) did not . twenty six had midline shifting of average 7.5 mm ( range 1 - 18 mm ) and 16 among these patients had midline shifting of more than 5 mm . the average hematoma volume was 36.73 cc ( range 5 - 107 cc ) . by hematoma grade , 5 cases ( 11.4% ) were grade 1 , 19 cases ( 43.2% ) grade 2 , 12 case ( 27.2% ) grade 3 , and 8 cases ( 18.2% ) grade 4 . the gos were evaluated in these patients ; 5 patients were categorized as grade 1 , 4 as grade 2 , 11 as grade 3 , 10 as grade 4 , and 14 as grade 5 . most of these patients needed rehabilitation and therefore their final gos was checked at the point of discharge after rehabilitation ( about 1 - 1.5 months after admission ) . the average gos of the ich patients were 3.64 , where 2 patients were categorized as grade 1 , 3 as grade 2 , 6 as grade 3 , 5 as grade 4 , and 9 as grade 5 . the average gos of the ish patients was 3.42 , where 3 patients were categorized as grade 1 , 1 as grade 2 , 5 as grade 3 , 5 as grade 4 , and 5 as grade 5 . assuming the patients with gos 4 and above to have good prognoses and those below 4 to have poor prognoses , 24 patients had gos equal to or more than 4 and 20 had less than 4 ( table 3 ) . comparing the gos of ich and ish patients , the group of hematoma type did not hold any statistical significance ( p = 0.824 ) . however , in the change of gcs , the result was different when comparing the pre - operative gcs and the post - operative gcs at day 7 ( table 4 ) . the ich group had average pre - operative gcs of 9.48 and average post - operative gcs at day 7 of 13.4 , an average improvement of 3.92 with no patient having lower gcs after operation . the ish group had average pre - operative gcs of 10.84 and average post - operative gcs of 12.26 , an average improvement of 1.42 . subsequently , in ruptured mca aneurysm patients , those with ich improved more after surgery compared to those with ish statistically ( p = 0.0391 ) . the ich patients had a larger average hematoma volume of 43.68 cc ( range 5 - 107 cc ) compared to that of the ish patients , which was 27.57 cc ( range 8 - 103 cc ) . consequently , the 14 of 25 ( 56% ) ich patients had midline shifting of more than 5 mm , while 2 of 19 ( 10.5% ) ish patients had midline shifting of more than 5 mm . intracerebral hemorrhage is associated with an increased mortality rate in patients with ruptured aneurismal sah,2)5 ) and emergent craniotomy with evacuation of the hematoma and aneurysm clipping should be considered.3)4)6)16 ) many studies have categorized hematoma into types and analyzed the difference in prognoses . shimoda et al.12 ) subdivided hematomas into temporal ich , ish , and ich with diffuse sah , and noted that the patients with ish / ich with diffuse sah fared worse compared with those with only ish / ich . prat et al.9 ) categorized hematomas into temporal ich and ish , and observed better outcomes in patients with temporal . al.7 ) grouped hematomas into frontal ich , temporal ich , and ish , and found that temporal ich had more favorable clinical outcome when compared to the other hematoma groups . van der zande et al.15 ) stated that when categorizing the hematoma types into ich and ish depending on the existence of ihcev , admission status and outcome were similar for both groups . in our previous study , we proposed a grading system for hematoma accompanied by ruptured mca aneurysm.11 ) this grading system is currently being used in our hospital as a useful tool in evaluating the patient prognosis . we attempted to confirm van der zande 's statement on prognosis by different hematoma types . we also investigated the relationship between our hematoma grading system and the types categorized by van der zande . the average hematoma grade was 2.3 ( sd 0.89 ) in the ish group and 2.8 ( sd 1.04 ) in the ich group . the group with ich generally had a larger hematoma volume and more frequent midline shifting compared to the ish group . generally , the ich group had higher hematoma grades , and this had a direct association with their gos . in our study of the ruptured mca aneurysm patients , the group with accompanying ich had a lower average gcs at admission , but improved more after surgery compared to the group with accompanying ish . this was because in ich , the hematoma is located within the brain parenchyma and thus is easily removable . ich has a larger volume in general , and therefore greater improvement in consciousness can be expected by removing this lesion . although there is no significant difference in the outcome of the 2 hematoma groups , the degree of improvement might be higher in ich group compared to ish group . ruptured mca aneurysm with hematoma , as in our previous study , proved to have a clear relationship between the hematoma grade and prognosis and this was confirmed to be statistically significant in this study . there were no notable differences in prognoses when simply subdividing the hematoma into ich and ish . however , when comparing their peri - operative gcs , a greater improvement in consciousness level after surgery was noted in ich patients .

objectiveto clarify the prognosis of the patients with intra - sylvian hematoma ( ish ) and intracerebral hematoma ( ich ) in ruptured middle cerebral artery ( mca ) aneurysms.methodswe categorized hematoma into ish and ich by the presence of intra - hematomal contrast enhancing vessel ( ihcev ) on computed tomography angiography ( cta ) . forty - four ruptured mca aneurysm patients with ich or ish were grouped by the grading system proposed by the authors in our previous study . we investigated the relevance of the following factors : patient 's age , gender , hunt - hess grade , glasgow outcome scale ( gos ) and changes in glasgow coma scale ( gcs ) between pre - operation and 7 days after operation.resultsthere were no significant differences statistically in age , gender , hunt - hess grade , and gos between the ish and ich groups . in their peri - operative gcs change , the ich group showed greater improvement compared to the ish group ( p = 0.0391 ) . the hematoma grade had a significant relevance with the patients ' gos.conclusionalthough there were no significant statistic differences in the gos of the 2 hematoma groups , there were prominent improvements of post - operative gcs in the ich group . unlike in the ish group , effective removal of hematoma was possible in most patients of the ich group . thus although there is no difference in the prognosis of the 2 groups , early surgical evacuation of hematoma seems to be effective in improving the short - term gcs score in peri - operative period .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

insulin resistance and abnormal glucose tolerance are frequently observed in chronic liver disease ( cld ) , but the precise mechanism leading to insulin resistance still remains unclear . in an attempt to explain the mechanism of insulin resistance in these patients , several investigators have studied insulin receptor bindings in patients with cld . however , the results are varied , and there remain controversies about the insulin receptor status in cld . some studies have shown that insulin receptor bindings were reduced in cld which suggested an insulin receptor defect as a primary cause of insulin resistance . however , other studies reported that insulin receptor bindings were normal , or even increased in patients with cld , thus raising doubt about the above explanation . the present study was performed to elucidate this controversy by observing the insulin receptor status of erythrocytes in patients with cld . although it is not clear whether erythrocytes reflect the insulin receptor status in more relevant tissues such as liver and muscle , it is generally agreed that insulin bindings to erythrocytes and monocytes change in a parallel direction in many situations . the studies were carried out in eleven patients with cld and in ten healthy subjects . the patient group was composed of nine men and two women ( age distribution ; 2564 years of age , mean ; 45 ) . they did not take any medications that might influence glucose intolerance , and evidence of recent gastrointestinal bleeding was absent . control group was composed of 10 healthy men ( age distribution ; 3360 years of age , mean ; 43 ) . who showed no evidence of hepatobiliary , renal or endocrine disturbances . intravenous glucose tolerance test ( ivgtt ) was performed after 25 g of glucose was infused into an antecubital vein . patients with cld were divided into two groups by fasting plasma glucose levels ( fbs ) and the results of ivgtt . group i was composed of 6 patients with fbs less than 100 mg / dl and normal glucose disappearance rate ( k - glucose ) , and group ii was 5 patients with higher fbs ( fbs>100 mg / dl ) and lower k - glucose values ( table 1 ) . glucose disappearance rate ( k - glucose ) was calculated according to the formula of amatuzio et al . as follow ; c(t)=c(o)ektwhere c(t ) is the glucose excess in mg / dl at any time t , and c(o ) is the glucose excess above basal value in mg / dl at time zero . the reticulocyte count in peripheral blood was performed in all of the samples . those with over 1% reticulocytes were discarded as reticulocytes were known to have insulin receptors of more than tenfold compared with those of more matured red cells . insulin binding to erythrocytes was studied in duplicate according to the method of gambhir with some minor modifications as previously described . twenty milliliters of overnight fasting blood was drawn into a heparinized syringe and the erythrocyte cell layer was separated by ficoll - hypaque gradient centrifugation method . the cell suspension , trace amounts of radioactive insulin [ i - a14-monoiodinated human insulin with specific activity of about 2000 ci / mmole ( amersham co. , england ) ] , and various concentrations ( 0.02 to 10 ng / ml ) of unlabeled insulin were incubated at 15c for 3 and 1/2 hours . the bound form of insulin was separated and the radioactivity was counted in a gamma counter . the nonspecific binding was defined as the radioactivity in the presence of 10 ng / ml of unlabeled insulin . the nonspecific binding was subtracted from the total binding and the results were expressed as specific i - insulin binding to erythrocytes of a concentration of 4.010 cells / ml . the results of the binding studies were presented as ; ( 1 ) the specific binding of i - insulin in the absence of competing unlabeled insulin , % ( b / t)0 , ( 2 ) the specific cell bound fraction plotted as a function of total insulin concentrations ( competition curve ) , and ( 3 ) the ratio of the cell - bound insulin to bee insulin plotted as a function of cell - bound insulin ( scatchard plots ) . in the scatchard plots , the highest insulin concentrations used was 100 ng / ml . the binding data were analyzed on the assumption that the curvature of the scatchard plots reflects the presence of two populations of insulin binding sites ( high affinity receptors and low affinity receptors ) . , and data in figures as means.e.m .. the statistical analysis of the data was performed using the unpaired t - test . 1 shows the mean competition curves for i - insulin binding to erythrocytes in three study groups . the percent specific binding in the absence of unlabeled insulin was significantly lower in the group i patients compared with that of the normal subjects . however , i - insulin binding to erythrocytes in group ii patients was very similar to that of the normal subjects . the scatchard plots of the mean values at each concentration of the bound insulin in three groups are shown in fig . apparent receptor numbers of both high and low affinity receptors tended to be lower in the group i patients than those in normal controls , but the statistical differences of these indices between two groups ( group i and normal controls ) were not significant probably due to the small size of the samples ( table 2 ) . there seem to be at least two subgroups of patients in cld : one with normal fbs , normal k - glucose and brisk insulin response to intravenous glucose loading ( group i ) , and the other with higher fbs , lower k - glucose and blunted acute insulin response to glucose loading ( group ii)(table 1 ) . present study clearly demonstrates that erythrocyte insulin receptor binding is also different between these two groups of patients . insulin binding to erythrocytes was reduced in group i patients , but was not reduced in group ii patients . this was quite surprising since if the primary cause of insulin resistance was the defect of insulin receptors in peripheral tissues , those patients with reduced insulin receptor binding would have more abnormal glucose tolerance than those with normal insulin receptor binding . this was not true in the present study , and it was suggested that insulin resistance in cld might not be due to a primary defect of insulin receptors . reduced insulin receptor binding is frequently associated with many states characterized by insulin resistance and hyperinsulinemia , such as obesity , type ii diabetes mellitus and acromegaly . cld is another state frequently associated with glucose intolerance , insulin resistance and hyperinsulinemia , and several investigators have reported that insulin receptor bindings to peripheral tissues are reduced in the patients with cld . it has been believed that down - regulation of insulin receptor by hyperinsulinemia is the cause of insulin resistance in cld . however , contradictory results have also been reported . proietto et al , have shown that insulin receptor binding was not altered in the patients with cld , okada reported that insulin binding was rather increased in the patients with cld , and that the degree of increase was more marked in the patients who also had diabetes mellitus . the result of the present study suggests that the discrepancy might be due to the differences in the study populations . in the present study , fasting insulin levels in group i patients were significantly higher than those in normal subjects ( table 1 . ) , and down - regulation by hyperinsulinemia might be the cause of reduced insulin receptor binding in these patients ( table 2 . ) . however , down - regulation of insulin receptor was not associated with glucose intolerance ( group i ) , and erythrocyte insulin receptor binding was almost normal in the patients with glucose intolerance ( group ii ) . the biological significance of this phenomenon is not clear at present , and indeed it is not clear whether the observed changes in insulin binding to erythrocytes in cld correlate with the changes in insulin binding to insulin sensitive tissues . some investigators still believe that reduced insulin receptors in hepatic tissues may be the primary cause of insulin resistance in patients with cld . however , no such direct evidence has been demonstrated until now , and at least erythrocyte insulin receptors are concerned , down - regulation of insulin receptors seems to be an epiphenomenon of hyperinsulinemia and might not be very important in the genesis of insulin resistance in these patients .

125i - insulin binding to erythrocytes was measured to evaluate the role of insulin receptors in the mechanism of insulin resistance in partients with chronic liver disease ( cld ) . patients with cld were divided into two groups by the fasting plasma glucose level ( fbs ) and the glucose disappearance rate ( k - glucose ) during intravenous glucose tolerance tests : one with normal fbs and normal k - glucose ( group i ) , and the other with higher fbs and lower k - glucose ( group ii ) . insulin receptor bindings were reduced in group i patients compared with those of normal controls , but were not reduced in group ii patients . fasting insulin levels were significantly higher in group i patients than those in normal subjects , and down - regulation by hyperinsulinemia might be the cause of reduced insulin receptor bindings in these patients . however down - regulation of insulin receptors was not associated with glucose intolerance . so it is suggested that insulin resistance in cld is not due to a primary defect of insulin receptors in peripheral tissues , and down - regulation of insulin receptors is an epiphenomenon of hyperinsulinemia .

INTRODUCTION MATERIALS AND METHODS INSULIN BINDING STUDY STATISTICAL ANALYSIS RESULTS DISCUSSION

a 45-year - old woman presented with acute bilateral visual deterioration with duration of one day . she had been taking fluoxetine , magnesium hydroxide , and hctz for one month to reduce her weight . she had visited another local ophthalmic clinic on the day of symptom onset , and had been told that the iops of the right and left eyes were 37 and 41 mmhg , respectively . she was prescribed topical iop - lowering drugs , namely , cosopt ( dorzolamide hydrochloride - timolol maleate ) and xalatan ( latanoprost ) , and oral diamox ( acetazolamide ) . her uncorrected visual acuities ( ucvas ) were 0.15 ( 20 / 150 ) , oculus dexter ( od ) and 0.1 ( 20 / 200 ) , oculus sinister ( os ) . automated refraction revealed bilateral myopia with spherical equivalents of -3.0 diopters ( d ) , od and -3.6 d , os . after correcting measured refractive errors , she could see 1.0 ( 20 / 20 ) with either eye . her iops were 12 mmhg , od and 14 mmhg os by goldmann applanation tonometry . an anterior segment examination revealed conjunctival chemosis and injection , shallow anterior chambers , and narrow angles in both eyes ( fig . 1a and 1b ) , and a funduscopic examination revealed bilateral choroidal effusions in the 360o periphery ( fig . she was diagnosed as having drug - induced myopia and asked to discontinue drugs including hctz . ten days later , she reported that her vision had recovered and that all ocular changes had disappeared ( fig . 2 ) . at this time , her ucvas were 0.8 ( 20 / 25 ) , od and 0.6 ( 20 / 33 ) , os and she had a best - corrected visual acuity ( bcva ) of 1.0 ( 20 / 20 ) in both eyes . myopia had been reduced to the prior refractive state ( spherical equivalent of -0.75d , oculi unitas ) . a 40-year - old woman presented with bilateral visual acuity deterioration , which had started 9 hours before presentation . she had been prescribed ginexin , spironolactone , and hctz at the local clinic of internal medicine to cure edematous changes of the lower extremities . her ucvas were 0.08 ( 20 / 250 ) , od and 0.06 ( 20 / 300 ) , os . automated refraction revealed bilateral myopia with spherical equivalents of -4.5 d , od and -5.25 d , os and degree of myopia was unchanged by cycloplegic refraction ( table 1 ) . after correcting measured refractive errors , she could see 1.2 ( 20 / 16 ) , od and 1.0 ( 20 / 20 ) , os . her iops were 15 mmhg , od and 16 mmhg , os by goldmann applanation tonometry . anterior segment examinations revealed conjunctival chemosis , shallow anterior chambers , and narrow angles in both eyes . she was also diagnosed as having drug - induced myopia and recommended to discontinue all three drugs . nine days after the discontinuance of medications , her ucvas were 0.8 ( 20 / 25 ) , od and 0.6 ( 20 / 30 ) , os and myopia had been reduced to the prior refractive state ( spherical equivalent of -0.75 d , od and -1.25 d , os ) . an anterior segment examination revealed that the conjunctival chemosis had disappeared and that the anterior chambers were of normal depth . a 45-year - old woman presented with acute bilateral visual deterioration with duration of one day . she had been taking fluoxetine , magnesium hydroxide , and hctz for one month to reduce her weight . she had visited another local ophthalmic clinic on the day of symptom onset , and had been told that the iops of the right and left eyes were 37 and 41 mmhg , respectively . she was prescribed topical iop - lowering drugs , namely , cosopt ( dorzolamide hydrochloride - timolol maleate ) and xalatan ( latanoprost ) , and oral diamox ( acetazolamide ) . her uncorrected visual acuities ( ucvas ) were 0.15 ( 20 / 150 ) , oculus dexter ( od ) and 0.1 ( 20 / 200 ) , oculus sinister ( os ) . automated refraction revealed bilateral myopia with spherical equivalents of -3.0 diopters ( d ) , od and -3.6 d , os . after correcting measured refractive errors , she could see 1.0 ( 20 / 20 ) with either eye . her iops were 12 mmhg , od and 14 mmhg os by goldmann applanation tonometry . an anterior segment examination revealed conjunctival chemosis and injection , shallow anterior chambers , and narrow angles in both eyes ( fig . 1a and 1b ) , and a funduscopic examination revealed bilateral choroidal effusions in the 360o periphery ( fig . she was diagnosed as having drug - induced myopia and asked to discontinue drugs including hctz . ten days later , she reported that her vision had recovered and that all ocular changes had disappeared ( fig . 2 ) . at this time , her ucvas were 0.8 ( 20 / 25 ) , od and 0.6 ( 20 / 33 ) , os and she had a best - corrected visual acuity ( bcva ) of 1.0 ( 20 / 20 ) in both eyes . myopia had been reduced to the prior refractive state ( spherical equivalent of -0.75d , oculi unitas ) . a 40-year - old woman presented with bilateral visual acuity deterioration , which had started 9 hours before presentation . she had been prescribed ginexin , spironolactone , and hctz at the local clinic of internal medicine to cure edematous changes of the lower extremities . her ucvas were 0.08 ( 20 / 250 ) , od and 0.06 ( 20 / 300 ) , os . automated refraction revealed bilateral myopia with spherical equivalents of -4.5 d , od and -5.25 d , os and degree of myopia was unchanged by cycloplegic refraction ( table 1 ) . after correcting measured refractive errors , she could see 1.2 ( 20 / 16 ) , od and 1.0 ( 20 / 20 ) , os . her iops were 15 mmhg , od and 16 mmhg , os by goldmann applanation tonometry . anterior segment examinations revealed conjunctival chemosis , shallow anterior chambers , and narrow angles in both eyes . she was also diagnosed as having drug - induced myopia and recommended to discontinue all three drugs . nine days after the discontinuance of medications , her ucvas were 0.8 ( 20 / 25 ) , od and 0.6 ( 20 / 30 ) , os and myopia had been reduced to the prior refractive state ( spherical equivalent of -0.75 d , od and -1.25 d , os ) . an anterior segment examination revealed that the conjunctival chemosis had disappeared and that the anterior chambers were of normal depth . both of our patients showed 2 to 4 diopters of transient myopia after hctz intake ( table 1 ) . because hctz was the only drug taken by both patients and all ocular signs and symptoms disappeared after cessation of this drug many reports [ 1 - 3,6 - 10 ] have been issued on the clinical characteristics of sulfa drug - induced acute ocular changes . in this report , we show photographs of choroidal effusion , radiating retinal folds , and conjunctival chemosis , which have not been previously presented . the mechanism of acute myopic shift has been suggested to involve ciliochoroidal effusion and an anterior rotation of the ciliary body . myopic shift occurs due to the anterior migration of the iris - lens diaphragm , which increases focal length . the resulting configuration has a significantly shallower anterior chamber and appositionally closes the angle of both eyes , which precipitates a glaucomatous crisis . the pathophysiology of ciliochoroidal effusion is considered to be driven by an idiosyncratic reaction , and some researchers have suggested that prostaglandins are involved . ciliary muscle spasm occurs as a manifestation of iridocyclitis , but it may also be induced by some drugs like anticholinesterase . the diagnosis of the patient can be confirmed by cycloplegic refraction . in our case studies , refractive errors , as determined by cycloplegic refraction , were similar to those detected by manifest refraction , which allowed us to rule out a ciliary muscle spasm . furthermore , because primary angle - closure glaucoma seldom develops bilaterally , the differential diagnosis is relatively easy . other clinical manifestations , such as choroidal effusion , retinal folds , and conjunctival chemosis can also aid in establishing the differential diagnosis . treatment of drug - induced myopia requires the prompt withdrawal of the offending agent , though discontinuation of medication should be conducted in consultation with an internist to reduce the likelihood of systemic side effects . a previous report also noted that laser iridotomy is not helpful , since the event is not due to a pupillary block . whenever a case of bilateral acute myopic shift with shallow anterior chambers and iop elevation is encountered , drug - induced myopia from ciliochoroidal effusion should be suspected and treated properly .

the authors experienced two cases of hydrochlorothiazide ( hctz)-induced acute - onset bilateral myopia and shallowing of the anterior chambers . two middle - aged women taking hctz , a sulfa derivative , visited our clinic complaining of acute bilateral visual deterioration . both had good visual acuity without corrective lenses before taking hctz . a complete ophthalmologic examination revealed bilateral myopic shift , intraocular pressure elevation , shallowing of the anterior chambers , choroidal effusions , radiating retinal folds , and conjunctival chemosis . approximately one week after hctz discontinuance , all ocular changes disappeared completely . physicians should be aware of the adverse ocular effects of hctz and should manage patients accordingly .

Case Reports Case 1 Case 2 Discussion

antrochoanal polyps ( acps ) are benign lesions arising from the edematous mucosa of the maxillary sinus and extending into the choana . acps constitute 4%6% of all nasal polyps . despite nasal polyposis , acps are typically unilateral . however , most of the published studies have been unanimous about preponderance of males and also its peak in early decades of life . chronic sinusitis and allergic rhinitis seem to play a major role in establishing the acp . one of the suggested etiological theories for acp described that inflammatory - related closure of osteomeatal complex and increase of pressure in maxillary sinus force mucous retention cysts to herniate into the nasal cavity . considering this theory , anatomic variations such as septal deviation , hypertrophic turbinate , and concha bullosa also pave the way for increasing the pressure difference between the middle meatus and the maxillary sinus . in this study , we examine this theory by comparing the coincidence of anatomic variation in patients with acp with normal population . between march 2014 and august 2015 , among patients referred to radiology department , kashani and alzahra hospitals ( isfahan university of medical sciences , isfahan ) by their clinicians for standard computed tomography ( ct ) scan of paranasal sinuses patients with the imaging diagnosis of acp were enrolled to the study , consecutively . seventy - eight patients with no evidence of polyposis were randomly selected and matched by sex and age with acp cases . septal deviation , unilateral or bilateral concha bullosa , and retention cyst in contralateral maxillary sinus were evaluated in both groups . data of both groups entered to ( spss version 22 , ibm corporation , somers , ny , usa ) . of the 17 patients with acp , 10 ( 58.8% ) were males and 7 ( 41.2% ) were females . ages ranged from 18 to 67 years with a mean of 38.11 11.06 years . of the 78 patients in control group , 38 ( 48.7% ) were males and 40 ( 51.3% ) were females . ages ranged from 20 to 66 years with a mean of 35.08 10.45 years . twenty - nine patients ( 37.2% ) had septal deviation , 6 ( 7.7% ) had concha bullosa , 7 ( 8.9% ) had retention cyst , and 1 ( 1.2% ) had hypertrophic turbinate . whitney u - test showed the higher coexistence of septal deviation in patients with acp in comparison with control group , significantly ( p < 0.001 ) . however , there was no significant difference in incidence of concha bullosa between two groups . chronic sinusitis which has been introduced as an important factor in the etiology of acp , instead could be the result of an obstruction of the maxillary sinus ostium caused by acp . while some studies have found significant association between acp and allergy , others fail to detect this association . role of anatomic variations of paranasal sinuses is another state of debate . in a large series of patients with acp reported by frosini et al . , the only significant correlating factor found in the patients was the anatomical variation . in their series , rate of septal deviation ( 55% ) , inferior turbinate hypertrophy , and concha bullosa were 55% , 21% , and 7% , respectively . they found that 32.3% of their cases had a contralateral mucous retention cyst . in our series , 37.2% had septal deviation , 7.7% had concha bullosa , 8.9% had retention cyst , and 1.2% had hypertrophic turbinate . while some authors consider etiologic role for some of the anatomic variations in establishment of acp , the others claim these variations are secondary to the presence of acp . diagnosis is based on physical examination , includes anterior rhinoscopy and nasofibroscope and ct scan . ct scan is helpful not only in the diagnosis of acp but also in choosing the appropriate surgical approach as it offers valuable information about the boundaries of each polyp . magnetic resonance imaging has been also used in acp . on t1-weighted images , acp is intermediate to low signal ; and on t2-weighted images , it is heterogeneously high . the major imaging differential diagnoses for acp are retention cysts , acute sinusitis with prolapsed mucosa , inverted papilloma , maxillary sinus mucocele , sinonasal organized hematoma , nasoethmoidal encephalocoele , esthesioneuroblastoma , and juvenile nasopharyngeal angiofibroma . this study showed that septal deviation is an anatomic variation which is significantly concomitant with acp . larger studies are needed to show the role of other anatomic variations in patients with acp . all authors contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work .

background : this study was designed to determine the prevalence of concomitant anatomic variation of paranasal sinuses in patients with antrochoanal polyp ( acp).materials and methods : among patients referred by their clinicians for standard computed tomography scan of paranasal sinuses septal deviation , concha bullosa , and retention cyst were evaluated in both patients with acp as well as in a control group.results:of the 17 patients with acp , fifteen patients ( 88.2% ) had septal deviation and two patients ( 11.8% ) had concha bullosa . none of the patients with acp had retention cyst or hypertrophic turbinate . of the 78 patients in control group , twenty - nine ( 37.2% ) had septal deviation , six ( 7.7% ) had concha bullosa , seven ( 8.9% ) had retention cyst , and one ( 1.2% ) had hypertrophic turbinate . septal deviation in patients with acp has higher incidence ( p < 0.001).conclusion : this study showed that septal deviation is an anatomic variation which is significantly concomitant with acp . larger studies are needed to show the role of other anatomic variations in patients with acp .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest AUTHORS' CONTRIBUTION

human homeostasis , defined as the internal state necessary for establishing systemic equilibrium and maintaining stability , has arguably shifted in less than a century . it is well documented that individuals adhering to a phytochemical - depleted diet and relatively sedentary lifestyle are more likely to develop obesity , contract chronic conditions such as diabetes , cardiovascular disease , and cancer , and spend a significant segment of their later lives dependent upon health care and pharmaceuticals . trends in consumerism and shifts in societal preferences toward convenience and modernization continue to peak in the united states and other westernized countries , and are now apparent in developing countries around the world . an estimated 1.234 million cases of colorectal cancer occurred worldwide , with 60% of these cases arising in developed regions ( globocan colorectal cancer data , 2008 ) . between the years of 19831987 and 19982002 , colorectal cancer incidence rates for both genders increased in 27 out of 51 registries of the iarc , most notably within regions of recent urbanization and economic transition such as most of asia and numerous south american countries ( center et al . , 2009 ) . when incidence , death , and 5-year prevalence rates are examined among world cultures ranging from traditional to modern , the differences in statistics are startling . the three regions with the lowest rates ( east mediterranean , african , and indian ) had on average a 26-fold lower incidence rate , a 16-fold decrease in related death , and a substantial 41-times lower 5-year prevalence of colorectal cancer when compared to rates within the category of more developed regions it is interesting to note that cultures exhibiting the lowest rates of colorectal cancer are also more likely to follow a pastoral way of life reflecting reliance upon the natural world . the reduced risks of cancer and other chronic diseases enjoyed by these individuals are attributable in some ways to genetic disposition , but also correlate largely with environmental factors arising from their retention of preventive dietary and lifestyle practices ; the same practices that have allowed human beings to utilize their environments in order to survive them throughout the course of evolution . however , considering the pace at which urbanization , industrialization , and technology spread in the twentieth century , a historically unprecedented scenario of environmental exposures has emerged within a timeframe too rapid for an appropriate adaptive physiological response . lifestyles has promoted persistent pro - disease phenotypes within certain populations that are concerning and costly in terms of time , resources , and quality of life . age - standardized incidences for colorectal cancer , as well as most other cancers , were highest in australia , canada , western europe , japan , and the united states ; the lowest incidences were reflected for the majority of the african continent ( except for south africa ) , india , the middle east , and south american countries surrounding the amazon basin ( globocan colorectal cancer data , 2008 ) . notably , colorectal cancer rates stabilized in the us in the mid-1980s , and have steadily declined since ( seer data ) , especially in distal as compared to proximal cases , likely contributable to positive societal factors such as increased awareness , early detection through endoscopic screening , and advances in preemptive outpatient polyp removal ( umar and greenwald , 2009 ) . however , influences such as these have not prevented the rapid rise in colorectal cancer observed since 1985 in japan and the republic of korea , with rates now comparable to those of the us and canada ( globocan colorectal cancer data , 2008 ) . largely by virtue of conflict , these two countries were directly influenced by american culture , now apparent in the lives of post - war japanese and korean generations . clearly this form of cancer , in addition to other chronic diseases , is directly associated with acculturation falling under the umbrella of westernized culture . the simple clues to overcoming this caveat can be realized by examining ways in which modern lifestyles and environments have altered human physiology , and how the traditional peoples of the world still practice prevention . instead of a few minor deviations , humans have both consciously and unknowingly introduced numerous hurdles to maintaining appropriate physiological functioning . the general awareness that a failure to eat right and exercise can lead to poor health is now extensively supported by scientific data demonstrating the molecular and cellular toll of failing to safeguard one s health . the physical body can be thought of as a sophisticated machine that relies on proper fuel , regular flushing , and routine upkeep in order to function at its maximum efficiency . if organisms are largely what they eat , and humans are turning to nutrient - poor , yet toxin - rich food , it is no surprise that signs of wear and fatigue are evident in many populations . chronic inflammation is recognized or suspected to be an underlying cause of a plethora of modern pathologies , including cardiovascular disease , diabetes , autoimmune disorders , neurological dysfunctions , and cancer . imbalances on molecular and cellular levels that promote inflammation are numerous and arise predominately from intrinsic factors such as redox fluctuations that evoke free radical damage and stress signaling to resolve acute challenges . whereas short - term signals are necessary for survival , it is in the case of unresolved insult that the body begins to waste its energy and develop abnormal responses to internal and external triggers , thus gradually promoting susceptibility to chronic diseases such as cancer ( coussens and werb , 2002 ) . humans have the means for controlling inflammation through a number of innate pathways including the cholinergic pathway which strongly influences cytokine production and suppression . another inherent mechanism for counteracting inflammation is dependent upon vitamin d , a nutrient once obtained in abundance through uv exposure , but now found to be severely lacking in the serum of humans living in developed regions . when obtained at appropriate levels , vitamin d acts as a prohormone via its nuclear receptor in cooperation with the retinoid x receptor to bind dna and modulate anti - inflammatory gene expression . in addition to this and other modern - day nutrient deficiencies , humans in fast - paced and demanding societies now tend to display physical symptoms of prolonged mental and emotional stress , which can induce chronically elevated cortisol , cytokine , and chemokine levels . although modern living can be mentally rigorous at times , the requirement of physical labor for survival has been nearly eliminated in certain cultures , thereby leading to dramatic reductions in physical energy expenditures . this trend is coupled with unprecedented increases in hyperconcentrated caloric intake from unnaturally dense foods , such as high - fructose corn syrup , that lessen perceptions of satiety after a meal . in addition to being more susceptible to weight gain , a consistently static body is less likely to efficiently circulate oxygen and nutrients as well as filter lymph contents , further emphasizing how modern living has escalated homeostatic imbalance . while calorically excessive , the westernized diet lacks essential nutrients including vitamins , minerals , and mediators of oxygenation , thereby promoting acidification , hypoxiation , and dehydration of the body . given that various tissues and fluids must exist within a narrow ph range in order to function properly , ph imbalances can contribute to altered homeostasis . cancer cells and tumor microenvironments are typically within a relatively acidic range due to their preference for anaerobic , lactic acid - generating metabolism . it should be noted that chemotherapy , phototherapy , and even hyperthermia induce tumor acidification , and the spread of melanoma in a mice study was shown to be fostered by an acidic microenvironment ( rofstad et al . , 2006 ) . it has been demonstrated that bioelectrical signaling serves as a regulator of cellular proliferation and differentiation , and it is possible that altered membrane potential can influence ion flux in a way that would allow for preferential uptake or exclusion of compounds by cancer cells ( sundelacruz et al . , 2009 ) . given the amount of sodium present in processed food , it is clear why molecular activities linked to sodium channels are often dysregulated . furthermore , the estimated fourfold increase in refined sugar intake in the last century has merely provided fuel for glycolysis - addicted cancer cells . when the body is full of solutes and deficient in its intended solvent , cellular osmotic balance is threatened by dehydration . in addition to a lack of water , optimal flushing of waste products from the body has been limited by the switch from whole to processed grains , which eliminated a major source of the fibrous content once consumed by humans . gut microflora composition is also altered in modern times , as beneficial levels of probiotics intended to aid in digestion are depleted by poor diet and excessive antibiotic use . these evidences of recent alterations in human physiology support the idea that environmentally induced imbalances set the stage for cancer and other chronic diseases . modern diets contain significantly higher levels of pro - inflammatory compounds in comparison to the anti - inflammatory nutrients common in the diets of early peoples ( simopoulos , 2008 ) . considering that americans have doubled their consumption of meat and dairy in the last century , a disproportionally large intake of animal fat compared to plant fats has occurred . different lipids have different metabolic functions and the essential fatty acids ( fas ) must be obtained via the diet for biosynthesis of other key compounds , some of which have opposing effects on inflammation . the ratio of omega-6 to omega-3 unsaturated fa consumption of primitive humans is estimated to have been roughly 1:1 , with high amounts of anti - inflammatory molecules such as epa and dha . today s omega-6:3 consumption in the average american is estimated to be almost 17:1 ( simopoulos , 2002 ) . this indicates a relatively rapid and dramatic increase in the intake of pro - inflammatory molecules like arachidonic acid , the precursor of prostaglandins and thromboxanes , coupled with a decrease in the intake of vital anti - inflammatory fas such as alpha - linolenic acid ( ala ) . therefore , eicosanoid - controlled mechanisms that accelerate cardiovascular and inflammatory pathologies such as colon cancer are upregulated by the westernized diet . fatty acid - induced chronic inflammation combined with dna methylation is suspected to fuel colon cancer growth as prostaglandin e2 was shown to silence tumor suppressor genes and promote intestinal tumor progression ( xia et al . , 2012 ) . the accumulation of excessive body fat from unhealthy sources is further deleterious as visceral adipose tissue can have a direct pro - inflammatory effect on surrounding tissues ( lysaght et al . , 2011 ) , and adipose tissue - derived stem cells have been shown to participate in building the breast tumor microenvironment to facilitate growth and metastasis ( muehlberg et al . , 2009 ) . elevated consumption of low - density lipoprotein ( ldl ) has a direct inflammatory effect on ldl receptor - expressing cells , and an altered distribution of cholesterol and sphingolipids within the plasma membrane can drastically influence a cell s response to its environment via lipid signaling . levels of inflammatory enzymes such as cyclooxygenase-2 ( cox-2 ) and inducible nitric oxide synthase ( inos ) are also likely to be upregulated in individuals adhering to the western diet , as well as levels of inflammatory cytokines such as il-6 and tnf-. altered cell signaling involving the egfr - associated ras / raf and pi3k / akt pathways can be induced via inflammation , and constitutive activation of these cascades is common in gi cancers ( risio , 2011 ) . the introduction and perpetuation of these complex arrays of cellular imbalances can manifest into chronic disease when left unchecked . this effect can occur within a short span of time , as immigrants who adopt a western lifestyle soon acquire increased colorectal cancer risk . factors that interfere with homeostasis by creating physiological instability are most apparent at the cellular and subcellular level , but are likely influential on the most basic , quantum interactions that drive the atomic and molecular bonding crucial for cellular functions . chronic inflammation can lead to nucleic acid damage , lipid peroxidation , protein misfolding and accumulation , prolonged heat shock induction , excessive growth factor secretion , and unnecessary immune activation or subversion . the later effects may be mediated by perturbations in regulatory t - cell numbers and functions , which are now suspected to play an important role in the developing epidemic of allergic , autoimmune , and inflammatory conditions ( fazekas de st groth , 2012 ) . unresolved inflammation has numerous tumor - promoting effects ( mantovani et al . , 2008 ) , and is an underlying factor behind the fatigue and debilitating pain plaguing millions of otherwise healthy people . however , inflammation is a vital component of normal pathways that initiate immune responses , wound healing , and apoptosis in severely damaged cells . therefore , acute inflammatory responses , much like acute stress , are beneficial for an organism s survival by inducing a swift and potent response to cellular changes , but chronic conditions can lead to a perpetual state of perceived wound healing . inflammation is thoroughly involved in all steps of carcinogenesis including initiation via genetic and epigenetic gene expression changes , progression promoted by aberrant cell signaling , and creation of the local and distant tumor microenvironments ( coussens and werb , 2002 ) . it is now thought that these processes are occurring within a specific cell type , known as a cancer initiating cell or cancer stem cell ( csc ) . this unique cellular niche retains key stem - like properties allowing it to be responsible for driving tumorigenesis in the same way that it would otherwise drive wound healing and tissue regeneration . given that embryonic and adult stem cells appear to perform a range of roles , from therapeutic to pathological , it can be argued that local and systemic environments determine these roles by checking and balancing or dysregulating their behavior . in an attempt to repair prolonged cellular damage , proliferative fibroblasts promote local fibrosis and scarring , and angiogenesis occurs to increase delivery of wound healing mediators . all the while , circulating cytokines and chemokines may sustain a chronic inflammatory state by inducing excessive neutrophilic actions that cause further free radical damage . tissue - derived and mesenchymal stem cells are intimately involved in the repair and regeneration of inflamed tissue . homeostasis of the intestinal epithelium , the majority of which gets replaced weekly , is maintained by stem cells residing within crypt bases that respond to cues from the microenvironment such as morphogenetic signaling through wnt , notch , hedgehog , and bmp ( medema and vermeulen , 2011 ) . three stem cell compartments exist within the human colon : crypt epithelial stem cells , subepithelial stem cells which include pericryptic myofibroblasts , and immigrating mesenchymal stem cells . these specialized stem cell niches interact to orchestrate epithelial cell differentiation , renewal of the colonic mucosa , and chronic inflammatory responses underlying colorectal oncogenesis ( sipos et al . , 2012 ) . one major genetic instability often linked to the initiation of colon cancer is chromosome 1p deletion , which otherwise encodes the glutathione - s - transferases and other enzymes involved in detoxification , as well as numerous tumor suppressors , dna repair , and checkpoint genes vital for preventing malignancy ( payne et al . , 2011 ) . studies in human gi cancers and rodent models indicate that loss of heterozygosity of the adenomatous polyposis coli ( apc ) gene is associated with early tumorigenesis . it was found that silencing of this tumor suppressor could occur in the absence of mutation or a gain or loss of genetic material , providing evidence that epigenetic regulation is involved ( amos - landgraf et al . , 2012 ) . as we will discuss , cellular alterations arising from complex epigenetic changes are an important level of gene expression regulation beyond dna mutation and are highly pertinent to cancer causation and its prevention . silencing of critical tumor suppressor functions along with gains in oncogenic expression can be transformative in a long - lived stem cell that has already acquired various mutagenetic and epigenetic changes , or could lead to plasticity within a terminally differentiated cell causing it to reacquire stem - like properties and transform ( rapp et al . , 2008 ) . extrinsic or innately driven accumulation of chromosomal instabilities within a stem cell can allow for unchecked clonal expansion of this cell and its progenitors into cscs . these transformed stem - like cells then build and recruit a protective niche of heterogeneous tumor tissue and stroma where they can remain highly functional , relatively quiescent , treatment - resistant , and poised to migrate if surgically disturbed . cscs can also facilitate the epithelial - to - mesenchymal transition ( emt ) and migrate along the sdf-1/cxcr4 chemotaxis pathway , which is implicated in angiogenesis and metastasis ( liekens et al . it is clear that correlations exist between modernization , acculturation , and increased risk for chronic diseases such as colorectal cancer , which is now the third leading cause of all deaths in industrialized countries ( barone et al . , 2011 ) . according to globocan worldwide data , the three cultures with the lowest cancer rates ( east mediterranean , african , and indian ) interestingly consume the most potently anti - inflammatory , plant - based diets in the world , while maintaining adherence to their traditional ways of life . currently , one in three americans contracts cancer and one in four american deaths is cancer related ( national cancer institute , 2005 ) . the steady rise in life expectancy previously observed within the us population is now forecasted to begin declining ( olshansky et al . , 2005 ) , suggesting that for the first time in centuries , american children will not live as long as their parents . therefore , for the sake of posterity , it is time to acknowledge and address risk factors arising from our daily habits and living environments in order to reverse rising global trends in chronic disease . explicit links between environmental exposures and cancer have already been made in the case of ionizing radiation and asbestos , and other parallels remain to be found within the ranks of the 80,000 chemicals currently registered with the us epa , the vast majority of which remain untested . given that health challenges such as infection , injury , and childbirth are medically manageable in modern times , the average life expectancy has increased greatly . since aging cells are more prone to oncogenesis , it could be argued that the observed trends in cancer rates are due to the simple fact that humans are living long enough to contract it ( dunn , 2012 ) . age is a considerable risk factor , but it does not account for incidences in young and otherwise healthy individuals such as those contributing to the rising rates of acute lymphoblastic leukemia ( all ) and brain cancer in children in recent decades . considering that certain early cultures did enjoy a longer lifespan , age would have been a risk factor then as well , in addition to mutation through inheritance or opportunistic infection . cancer in antiquity did exist , as bone malignancies have now been identified in mummified remains , although at a 0% rate in samples from children and a 1.2% rate in adult specimens ( zink et al . , 1999 ) . it must be noted that this analysis did not account for cancer in non - osseous locations , so overall cancer rates in ancient egyptians were surely higher , but still only a fraction of the 33% cancer incidence rate for present - day americans . in spite of limited historical records and pathological evidence , it is generally thought that the occurrence of malignancy was remarkably low in ancient times compared to modern times , suggesting a role for present - day environmental exposures ( david and zimmerman , 2010 ) . also rising are incidences of cancer in uncommon locations , such as the esophageal - gastric junction and the salivary gland , with trends currently unaccounted for ( zheng et al . , 1997 ) . it should be acknowledged that the number of cancer related deaths in the us is on the decline due to significant advances in oncology . however , in spite of our medically progressive society , cancers induced by poor diet and lack of exercise are still causing one third of all preventable deaths , with another proportion attributable to tobacco and alcohol use ( american cancer society , 2012 ) . a big - picture analysis reveals that homeostasis has largely been redefined by the abrupt changes in daily habits such as low phytochemical intake , electromagnetic field ( emf ) exposure , and toxin accumulation largely absent from human existence until recent times . these factors now constitute the unintended health and environmental legacy of westernization , with health impacts arising on a global scale that may have serious implications for adaptation in the modern world . according to a recent report from the president s cancer panel , the vast majority of cancers arise from environmental factors , directing attention to the fact that toxins play a significant , yet unappreciated role in carcinogenesis and the lives of americans ( national cancer institute : president s cancer panel , 2009 ) . there are many types of common environmental and physiological contaminants including heavy metals , radioactive isotopes , industrial solvents , agricultural chemicals , petroleum byproducts , and plastic components , some of which are classified as endocrine disrupting compounds ( edcs ) . the endocrine system is comprised of a network of vital chemical messengers ( hormones ) and cellular targets that together orchestrate homeostasis in liaison with the nervous system . many environmental edcs such as bpa , diethyl hexyl phthalate ( dehp ) , and the parabens act as xenoestrogens . thus , these compounds are capable of exerting potent hormonal disruption via the estrogen receptors ( ers ) , which are fairly promiscuous in ligand binding , and mediate non - genomic and genomic signaling via presence in multiple cellular locations . the mechanisms allowing edcs to influence estrogenic signaling arise from direct er interactions , as well as indirectly through metabolic enzymes involved in estrogen synthesis , such as aromatase ( aro ) , and transcription factors like the aryl hydrocarbon receptor ( ahr ; shanle and xu , 2011 ) . endocrine disrupting compounds may also exert their effects by acting as anti - androgens or interfering with the hypothalamic - pituitary - adrenal axis and/or thyroid gland functioning , the later shown to be the case for triclosan , the active ingredient in most antibacterial soaps ( dann and hontela , 2011 ) . xenohormones can induce changes in estrogen- and androgen - responsive tissues including the developing breasts , ovaries , prostate , and testis ; it is noteworthy that prostate and breast cancer are predicted to be the most common non - skin cancers diagnosed in 2012 according to the nci , having now advanced past lung cancer . the increase in breast cancer incidence is not restricted to women , as cases in men are becoming more frequent . a recent study found the proportion of girls with breast development at age eight to be higher than reports from 10- to 30-years ago ; 18% of white , 43% of black , and 31% of hispanic girls evaluated had undergone the onset of puberty by 8 years old ( biro et al . , 2010 ) . therefore , when present in utero and during early reproductive development , low levels of edcs may contribute to or counteract endogenous hormone production and xenoestrogen exposure is likely capable of promoting feminine phenotypes . bpa is found in polycarbonate plastics , metal can linings , dental sealants , epoxy resins , and register receipts . this chemical appears to be strongly linked to premature puberty in females , the induction of male infertility , and spontaneous miscarriage due to its hormonal , mutagenic , and genotoxic activities ( tiwari et al . , 2012 ) . dehp is one of the many plasticizers used to soften polyvinyl chloride ( pvc ) during manufacturing and may constitute a large percentage of a final product s weight , from which it actively leaches out upon hydrolysis , heat , and/or pressure . pvc plastic and phthalates are used in a wide array of products making them nearly ubiquitous in our lives in the form of plumbing pipes , electrical cords , food packaging , vinyl floors , faux leather handbags , cosmetics , infant chew rings , as well as medical catheters , blood bags , and tubes . given the prevalence of plastic in healthcare , indirect accumulation of edcs within the body can be a side - effect of hospitalization , and detoxification of medical settings should become a priority of administrators , as medical care should not contribute to the causes of disease ( ruzickova et al . , 2004 ) . accountability within product manufacturing is highly urged , as xenoestrogens like the parabens should not be included in infant shampoo formulations , and pvc , which may also contain stabilizers such as lead and cadmium , should not be present in children s toys . the common household and food service industry practices of freezing , microwaving , and storing food in plastic can serve to accelerate leaching of harmful components . furthermore , burning or disposing of plastic waste in landfills directly releases toxins into the air , soil , and groundwater . plastics are petroleum byproduct - based , thus contaminants linked to petroleum acquisition and separation also find their way into cheap consumer products , the demand of which further stimulates the toxic effects of the oil industry . given that expression of ers occurs in numerous cell types , with an increase in estrogen - responsive genes often found in colorectal cancers , the effects of these compounds in the gi tract are under investigation . dehp was shown to stimulate expression of the multi - drug resistance transporter ( mdr1 ) gene in colon cancer cells by binding to the steroid and xenobiotic receptor ( sxr ; takeshita et al . , 2006 ) . animal studies exploring the effects of bpa found it to be present in the small intestine almost immediately following oral ingestion , with evidence that it was reabsorbed into the colon ( sakamoto et al . , 2002 ) . perinatal exposure of bpa at human reference doses was shown to alter intestinal barrier dynamics , influence gi pain reflexes , and induce a potent inflammatory response in adult female offspring ( braniste et al . , 2010 ) . the effects of bpa and other xenoestrogens have been more extensively investigated in breast tissue , in which cellular activity is tightly regulated by the presence of ers , so the relevancy of these studies may extend to er expression in colon cancer . whereas the growth - promoting er isoform tends to be upregulated in hormonally responsive breast cancers , the er isoform , which likely serves a tumor suppressor role , is downregulated via a variety of mechanisms ( fox et al . , 2008 ) . it is possible that xenoestrogen exposure early in development primes breast tissue for chemically induced cancer not only in females , but also in males , and may set determinants in utero for other chronic inflammatory diseases . pre - pubertal exposure of bpa in rodent mammary tissue was shown to upregulate expression of steroid receptor coactivators ( srcs ) , er , egfr , and phospho - igf-1r , leading to activation of downstream kinases including erk and akt ( lamartiniere et al . , 2011 ) . xenoestrogenic compounds at humanly bioactive doses have been shown to exert pro - tumorigenic epigenetic changes in breast epithelial cells through repression of mirna-9 - 3 ( hsu et al . , 2009 ) , increased expression of the histone methyltransferase ezh2 upon in utero exposure ( doherty et al . , 2010 ) and silencing of the lamp3 gene ( weng et al . , 2010 ) . the proliferation of many cell types , including adipocytes , is accelerated by certain xenoestrogens which have been described as obesogenic because they promote obesity via numerous mechanisms including the reduction of basal metabolic levels and increased appetite stimulation ( grun , 2010 ) . bpa and other edcs have been linked to aberrant genetic programming during fetal development that likely increases susceptibility for adult obesity , arguing that modern trends in unusually large infant and adolescent body sizes may also be partly attributable to early edc exposure ( vom saal et al . , 2012 ) . attention should be given to the fact that bpa levels are detectable in over 90% of individuals tested ( lamartiniere et al . , 2011 ) , along with a slew of other edcs such as the parabens , which were found to be present in 99% of breast tissue samples in a study of mastectomy patients ( barr et al . , 2012 ) . aside from a direct role in carcinogenesis , exposure to these compounds carries possible repercussions for sex differences in the developing brain through abnormal hormonal influences such as altered estrogen to androgen ratios and thyroid levels . in one human study , reduced masculine play was observed in a group of school - aged boys with higher levels of certain phthalates present in their systems in comparison to boys with low levels of these compounds ( swan et al . , 2010 ) . animal studies have revealed that gestational exposure to bpa can alter social behavior in juvenile and adult mice , with higher mrna levels of the glutamate transporter slc1a1 in the brains of females . to support the role of bpa in epigenetic regulation , this same study found altered expression of the dna methyltransferase ( dnmt ) genes dnmt1 and dnmt3a in the brains of mice exposed in utero ( wolstenholme et al . cholinergic signaling in the brain was found to be altered in mice after a single dose of bpa at 10 days old , which manifested in changes in spontaneous behavior , lowered adaptation to new environments , hyperactivity , and reduced nicotine sensitivity in adulthood ( viberg et al . , 2011 ) . in addition , bpa demonstrated the ability to enhance spinogenesis in adult hippocampal neurons , with rapid effects observed for nanomolar doses , indicating large responses from small amounts ( tanabe et al . , 2012 ) . considering the rapid rise in childhood and adult adhd , coupled with the alarming 26% climb in autism - spectrum disorders within just a 2-year span ( 20062008 ; cdc , 2012 ) , it is clear that modern environmental factors are affecting cognition in a significant manner . endocrine disrupting compounds - induced hormonal imbalances can also have drastic effects on reproductive organs . studies by one group in male rats exposed neonatally to bpa revealed reduced spermatogenesis through downregulation of cell junctional proteins ( salian et al . , 2009b ) , in addition to reduced fertility in subsequent generations , demonstrating vertical transmission of exposure - related effects ( salian et al . , 2009a ) . male sexual differentiation disorders , such as hypospadias , have increased in recent decades and it is likely that the accompanying increases in xenoestrogen and/or anti - androgen exposures are a factor ( sultan et al . , 2001 ) . altered expression of er and er in the testis of adult rats subjected in utero to bpa was shown to correlate with dna hypermethylation of both promoters , accompanied by upregulation of dnmt3a and dnmt3b expression ( doshi et al . , 2011 ) . this suggests that edcs can produce epigenetic feedback loops to auto - regulate their target cellular receptors . it is sobering to consider the mixtures of these epigenetically active chemicals that likely circulate now within the majority of people in developed regions . a recent analysis of umbilical cord blood content from ten minority newborn babies indicated detectable levels of 232 environmental contaminants , including bpa ( 90% of samples tested ) , dehp , fire retardants , polycyclic musks , rocket fuel , and banned pesticides ; all present at the time of birth . to date , a total of 414 chemicals have been detected in 186 people during the course of testing and constitute what the environmental working group ( ewg ) has termed , the human toxome . the cdc has conducted its own nationwide biomonitoring program over the last decade and was reported to have detected 203 chemicals distributed among the thousands of individuals tested ( environmental working group , 2009 , pollution in people report ) . other persistent organic pollutants ( pops ) , such as dioxins , polychlorinated biphenyls ( pcbs ) , organochlorine pesticides like ddt ( banned in the us in 1972 ) , and polybrominated diphenyl ethers ( pbdes ) are exhibiting a boomerang it is possible for a compound to travel thousands of miles and transfer from smaller to larger predators , until finding its way onto a plate in the form of meat , dairy , or seafood like tuna and mackerel . upon ingestion , these long - lived , lipophilic molecules integrate into adipose tissue . we absorb the majority of toxins through food , with common culprits including processed meats containing carcinogenic preservatives , items stored in bpa - lined cans , and soft drinks and deserts containing high levels of artificial ingredients . even fresh produce can carry large toxin loads from conventional chemical - based farming practices , with tests showing apples carried combinations of 57 chemicals and a single celery sample contained 14 pesticide residues ( environmental working group , 2011 , pesticides in produce report from 2000 to 2009 usda and fda data ) . common herbicides and pesticides can induce potent cellular changes , and atrazine was shown to stimulate growth in normal colonic epithelial cells , as well as colon cancer cells , with the highest proliferative effects notably observed for lower doses ( greenman et al . atrazine is the most commonly used herbicide in the us with 75% of all cornfields treated each year , making it a prevalent contaminant of ground and drinking water . epidemiological studies revealed that atrazine exposure in women living in farming communities was linked to increased menstrual cycle irregularities and altered reproductive hormone levels ; notably , this effect was observed at levels below the us epa s maximum contaminant level ( cragin et al . , 2011 ) . in addition , gender influences have been observed in wildlife exposed to atrazine , apparent by partial or complete feminization of male fish , amphibians , and reptiles ; the effects of which are statistically significant and consistent across vertebrate classes ( hayes et al . , 2011 ) . atrazine has been shown to exert hormonal changes at low , ecologically relevant doses in adult male african clawed frogs through demasculinization , leading to the ability to produce viable eggs . therefore , not only do edcs have implications for human health , they also threaten lower order species , likely contributing to global declines in amphibian populations ( hayes et al . , 2010 ) . the modern methods by which food is prepared and served have also introduced toxic chemicals into the body . perfluorinated compounds ( pfcs ) , including perfluorooctanoate ( pfoa ) and perfluorooctane sulfonate ( pfos ) , are components of non - stick cooking surfaces as well as stain and water - repellant food packaging and fabric coatings . pfcs are known peroxisome proliferators and hepatocarcinogens , and have been shown to alter membrane potential and intracellular ph in colon cancer cells , based on the hydrophobicity of the compound ( kleszczynski and skladanowski , 2009 ) . in a study of pfoa effects in the f344 rat liver , this chemical was shown to inhibit gap - junctional intercellular communication which serves as an epigenetic marker of tumorigenesis , in an erk and pc - plc - dependent manner ( upham et al . , 2009 ) . in human liver cells , pfoa upregulated transcription of the de novo dna methylator , dnmt3a , leading to hypermethylation of the glutathione - s - transferase pi ( gstp ) promoter , which could serve to limit the tumor - suppressive role of gst during detoxification ( tian et al . , 2012 ) . alteration of gstp methylation was also observed in rat livers following prenatal exposure to pfos ( wan et al . , 2010 ) , further emphasizing the epigenetic capabilities of common chemicals . it has also been suggested that pfcs suppress cytokine secretion by immune cells via alteration of nf-b activity , and pfoa was shown to be the least active of all pfcs assessed ( corsini et al . , 2012 ) . a prospective study of a birth cohort from a faroe island hospital revealed that high concentrations of pfcs in maternal pregnancy serum and child serum were associated with severely reduced levels of diphtheria and tetanus antibodies in vaccinated children at the ages of five and seven . these results could have implications for the development of humoral immunity and effectiveness of childhood vaccines ( grandjean et al . , 2012 ) . bpa exposure is also implicated in aberrant immunity , as exposed b1 cells were shown to upregulate autoantibody production in vitro and in vivo , with upregulated er expression in b1 cells from aging mice that had developed lupus ( yurino et al . , 2004 ) . like bpa , neonatal exposure to pfos and pfoa has been shown to induce neurobehavioral issues in mice via cholinergic signaling , with symptoms worsening with age ( johansson et al . , 2008 ) , and this effect may have implications for this pathway in inflammation and cancer . cholinergic pathways are also involved in the neurotoxic mechanism of the commonly used insect repellant , deet , which has also been shown inhibit cholinesterases , leading to acetylcholine excess within synapses ( corbel et al . , compounds consisting of highly electronegative atoms , such as fluorine , are adept at stealing electrons from cellular structures and may participate in forming reactive species such as the hypochlorous radical in the case of chlorine . while they do afford convenience and functionality , these products have long half - lives within the environment and body , making them damaging not only the consumer , but to the workers manufacturing them and residents of the surrounding towns . in fact , one epidemiological study with large sample sizes found that people living near a pfc - producing plant in west virginia had a sevenfold increase in pfoa blood levels as compared to the us average . children living in this community were exposed to the chemical likely in utero or via drinking water and breast milk , and had 44 and 42% more pfoa and pfos , respectively , in their systems than their mothers . these results demonstrate the enhanced vulnerability of the young to toxin accumulation , and suggest that markers of early exposure events can persist for decades ( mondal et al . , 2012 ) . the protective blood - brain barrier is not present in utero and the placenta is not a superb shield against a mother s circulating metabolome , therefore the rapidly developing fetus is highly vulnerable to chemical exposure , of which can lead to irreversible neurological and organ insult ( grandjean and heindel , 2008 ) . given that the body has not evolved to degrade and clear this type of molecule , exposure to chemicals such as pfcs is suspected to lead to dysfunction of the kidneys and liver , which are vital for maintaining homeostasis . these findings argue that humans in certain societies undergo a lifelong bioaccumulation of toxins from conception , through major developmental stages and into adulthood . there is a growing body of evidence for fetal origins of adult disease , and the data presented here implies that epigenetic programming within stem cells during key prenatal and neonatal stages is influenced greatly by environmental toxin exposure . a growing child experiences a crucial phase of developmental plasticity in which epigenetic marks are made in response to dynamic environmental changes that will ultimately manifest during transition phases later in life ( hochberg et al . , 2011 ) . animal studies have emphasized the influence of epigenetics on phenotype ; for example , perinatal exposure of yellow agouti mice to bpa at humanly relevant doses was found to induce global gene methylation changes capable of altering coat color distribution in the offspring ( anderson et al . , 2012 ) . direct correlations have been made between urinary levels of bpa in humans and the development of avoidable medical disorders and morbidity ( lang et al . , 2008 ) . an a large study of a representative sample of american adolescents and adults , a significant relationship was found between urinary phthalate and bpa metabolite concentrations and altered thyroid levels , demonstrating that adverse effects due to edcs are already measurable in the us population ( meeker and ferguson , 2011 ) . therefore , women with child - bearing abilities exert dramatic influences on the health , wellbeing , and reproductive capabilities of future generations on levels previously unrecognized . by crossing the placenta during pregnancy and into breast milk during lactation , these compounds are contaminating what should be the purest of physiological environments . not meant to be taken as hyperbole , isolated and moderate contact with these compounds is unlikely to be a cause for concern , but daily low - dose exposures may carry subtle , yet severe implications in the form of hermetic responses . given that a range of exposures can occur for one individual , including heavy metals like lead and mercury , common carcinogens such as formaldehyde and benzene , electromagnetic radiation from wireless communications , and pharmaceutical drugs ( which can be present in drinking water ) , the human body is facing an unprecedented level of adverse and potentially synergistic effects . if the environment we live in is admittedly polluted with changes already apparent in marine life and lower order mammals , what natural defenses exist within our cells to ensure optimum functioning in this time of modern genetic pressure ? prevention through primitive plant and human adaptations may be the intended route for counteracting and reversing the imbalances we have postulated to lead to chronic disease . presented in table 1 is an overview of common endogenous compounds of both synthetic and natural origin theorized to play a role in human homeostasis , or the disruption thereof . examples of exogenously obtained compounds , including common environmental toxins and phytochemicals , theorized to play a role in the acceleration or prevention of chronic disease by influencing homeostasis . + , promoting effect ; , attenuating effect ; aa , anti - angiogenic ; ach , acetylcholine ; ahr , aryl hydrocarbon receptor ; am , antimicrobial ; aox , antioxidant ; ar , androgen receptor ; aro , aromatase ; cvd , cardiovascular disease ; dm , diabetes mellitus ; ed , endocrine disruption ; er , estrogen receptor ; i , immunological effects ( a : autoimmune , q : acquired ) ; m , mutagen ; mr , microbial resistance ; nb , neurological / behavioral effects ; ob , obesity ; pop , persistent organic pollutant ; r , reproductive effects ; src , steroid receptor coactivator ; sxr , steroid xenobiotic receptor;tg , transgenerational ; ths , thyroid hormones ; top , topoisomerase . the designation of cancer was first used by hippocrates in reference to the crab - like appearance of certain tumors . known as the father of western medicine , he also coined the phrase , let food be thy medicine , and medicine be thy food , demonstrating his respect for the relationship of diet to human health . as opposed to the chemical cocktails that co - emerged with modernization and now exist within humans , it is equally relevant to consider the compounds now physiologically absent that were once components of human diets . plant - eating organisms have acquired sophisticated adaptations to use phytochemicals , some of which are shown in figure 1 , as defense against a wide variety of threats , ranging from microbial infection to cancer . if plant compounds are protective and cancer is common in phytochemical - deprived societies , then in one sense , cancer can be approached as a deficiency disease , reminiscent of what g. edward griffin proposed in his book world without cancer ( griffin , 1974 ) . it is acknowledged that the body does not operate properly without recognized nutrients such as vitamin c , b12 , and b6 , much less the complex array of obscure phytonutrients once obtained by humans living in tropical and temperate regions . epidemiological data collected from underdeveloped regions has drawn links between primitive diets and comparatively low risk for common cancers , and may explain the elevated longevity enjoyed in certain geographic locations . it is also instructive to review published studies on the composition of paleolithic diets versus that of modern diets ; the depletion of plant - based compounds is striking . structural examples of phytochemicals with known or suspected medicinal properties such as antioxidant , antimicrobial , anti - inflammatory , and anti - cancer activity , likely stemming from protective and defensive roles in their plant sources . many contain compounds that act as direct or indirect antioxidants , participate in detoxification and elimination of waste , activate regulatory t - cell responses , and modulate cell signaling to induce apoptosis in damaged cells while promoting differentiation in others . ingestion of certain fruits and vegetables can promote balanced ph levels by alkalinizing the blood and tissues via their high mineral and chlorophyll content . weak acids from citrus juices and plant vinegars can also buffer ph through conversion to strong bases during metabolism , as in the case of citric acid and acetic acid . it should be noted that many fruits contain high sugar content , which could feed cancer cell metabolism . however , given that cancer cells will preferentially overexpress certain glycolytic enzymes , it is possible to exploit the warburg effect to identify new metabolic targets in malignancy ( resendis - antonio et al . , 2010 ) . invasive tumors from human breast and colon tissues have been reported to upregulate an array of enzymes , including glycosidases , in comparison to normal cells ( bosmann and hall , 1974 ) . it is interesting to note that plants produce glycosidic compounds only broken down in the presence of certain enzymes , suggesting that they may act on trojan horse principles . by increasing its uptake of sugar - containing compounds , a cancer cell can inadvertently bring in attached cytotoxic groups such as cyanide ( cn ) . cn - containing glycosides and nitrilosides exist in thousands of plants including strawberries , alfalfa sprouts , spinach , pecans , and in particularly high concentration in apricot kernels and other seeds , thus responsible for their characteristically bitter taste . it should be noted that bitterness is an important sensory indicator of potently anti - inflammatory , yet potentially poisonous compounds ( in excessive amounts ) within a plant . in medicinal chemistry , there is little distinction between a drug and poison , and the specific poisoning of cancer cells is also the basis of chemotherapy . in one study , six bioactive compounds extracted from peach seeds , including amygdalin , showed marked antitumor activity , two of which were as effective as the green tea ( gt ) polyphenol egcg . more importantly , the most potent compounds contained a carboxyl group or hydrogen instead of cn , thereby demonstrating that glycosidic compounds can exist without the threat of presumed toxicity ( fukuda et al . , 2003 ) . the reported historical use of apricot kernel extract for pain reduction and inflammation was investigated in lipopolysaccharide - exposed mouse microglial cells , and results revealed an anti - inflammatory effect likely mediated through suppression of cox-2 and inos expression ( chang et al . , 2005 ) . amygdalin also induced apoptosis in du145 and lncap prostate cancer cells through caspase-3 activation , involving the downregulation of bcl-2 and upregulation of bax ( chang et al . another study revealed that amygdalin treatment lead to decreased expression of exonuclease - i and topoisomerase - i in snu - c4 colon cancer cells , demonstrating that this compound can induce cell cycle arrest in malignant cells ( park et al . amygdalin and other glycosidic compounds were abundant in the diets of early humans , and are commonly eaten by animals in the wild ; the dietary lack thereof may contribute to increased cancer rates observed not only in humans , but also domesticated animals . another recent discovery has suggested that plant micrornas ( mirnas ) can play a protective cellular role by participating in a unique form of interspecies signal transduction . secreted vesicles containing exogenous mirnas can accumulate to measurable levels in the plasma of humans upon oral ingestion of rice . it has been shown that these intact rnas are absorbed through the gi tract into the blood stream where they specifically bind to mammalian receptors , such as the ldl receptor adaptor protein 1 ( ldlrap1 ; zhang et al . , 2012b ) . there is also evidence that peptides from terrestrial and marine plant are antineoplastic , antifungal , antibacterial , antiplatelet , and antimalarial , along with the benefits of displaying little antigenicity , low - molecular weight , and easy absorption ( tan and zhou , 2006 ) . rare cyclic peptide conformations exist within plants that offer greatly enhanced stability and bioavailability over the linear peptides found in mammals . cyclopentapeptides obtained from the root of the aster tataricus plant were shown to have in vivo antitumor effects , and synthesized cyclic astins were able to induce caspase - mediated apoptosis in a human papillary thyroid carcinoma cell line ( cozzolino et al . , 2005 ) . how this and other anti - cancer mechanisms serve a defensive role in plants is up for debate , and whether they originated in an extremely early common ancestor or were the result of extensive human and plant adaptation is also not clear . the plant likely produces these secondary metabolites in place of an adaptive immune response to promote its own defense against dna damage and unwanted foreign growth . this may be why extracts of certain plants such as neem ( azadirachta indica ) , tea tree ( melaleuca alternifolia ) , and rosemary ( rosmarinus officinalis ) are able to exhibit such broad spectrum inhibition of microorganisms and high degrees of predator deterrence via adverse sensory and metabolic reactions . volatile plant compounds may also defend against predation through interference with insect and mammalian reproductive efficiency . neem extract was shown to be larvicidal against mosquitoes ( dua et al . , 2009 ) , found to inhibit early stages of rodent embryogenesis and implantation ( mukherjee et al . , 1999 ) , and has been historically used as a human contraceptive in india . these proposed repressive effects on embryogenesis could also influence the mechanisms allowing for trophoblastic - like growth of invading tumors and cscs . aside from these implications , neem extract has been demonstrated to be gastroprotective by limiting hyperacidity and ulcer development in vivo ( maity et al . , 2009 ) , shown to be potently anti - mutagenic in chemically exposed fish ( farah et al . , 2006 ) , and acted as an oral chemopreventive agent by stimulating phase ii detoxifying enzymes in a model of hamster buccal pouch carcinogenesis ( subapriya et al . , 2005 ) . these preliminary studies demonstrate the diverse medicinal mechanisms remaining to be characterized for the plant known in ancient times as sarva roga nivarini , or the optimum acquisition of secondary metabolites from plants depends upon growth conditions , similar to the way that human phenotype is largely dependent upon environment . phytochemical levels tend to be higher when harvest occurs during the early morning hours surrounding sunrise or when an impending threat has been detected , as in the case of increased quercetin and diallyl sulfide levels following the crushing of a garlic bulb . the region of the plant from which the highest levels of nutrients can be extracted is also variable . typically , the colorful outer peels of fruits carry significant levels of protective and defensive compounds for counteracting uv light and pathogens . high concentrations of beneficial compounds are also found in seeds , kernels , and sprouts in order to ensure survival of the plant s genetic material and developing seedling . research is currently exploring the effects of exogenous compounds on plant metabolite production . in one study , an analysis of dried samples from tomatoes grown in the us during a 10-year period found that organic tomatoes contained on average 79% more quercetin and 97% more kaempferol when compared to conventionally grown samples ( mitchell et al . , 2007 ) . however , an italian study found that organically grown tomatoes contained more salicylic acid , but less lycopene and vitamin c , in addition to higher cadmium and lead when compared to the conventionally grown group ( rossi et al . , 2008 ) . even different cultivars of grapes raised in controlled agronomic environments were shown to differ in their flavonol content . when tested on caco-2 colon cancer cells , each of the three extracts were effective at limiting growth and inducing apoptosis , but evoked different responses at lower doses , demonstrating that chemopreventive properties can vary with cultivation ( dinicola et al . , 2010 ) . it would be interesting to explore whether or not modern agricultural practices have induced epigenetic changes within the plant genome beyond the genetic modifications already intentionally introduced , and how this affects medicinal properties . grape skin and seed contain resveratrol , which has been shown to have many chemopreventive properties by protecting colon cells from chemotherapy - induced cytotoxicity ( cheah et al . , 2009 ) and potentiating colon cancer cell apoptosis ( radhakrishnan et al . , 2011 ) . resveratrol demonstrated an anti - inflammatory effect in a model of experimental colitis by rebalancing redox status , downregulating adhesion molecules , and limiting immune cell invasion ( abdallah and ismael , 2011 ) . this compound was also shown to mediate anti - inflammatory actions through reduced nf-b activation upon lps treatment of human colon cancer cells ( panaro et al . , 2012 ) . dietary - feeding of grape seed extract ( gse ) in azoxymethane - treated f344 rats was able to prevent aberrant crypt foci formation likely via suppression of -catenin , nf-b , cox-2 , and inos levels ( velmurugan et al . , 2010a ) . gse was also shown to inhibit intestinal polyp formation and growth in conjunction with increased apoptosis , decreased levels of inflammatory markers , and increased expression of the cyclin - dependent kinase inhibitor cip1/p21 in apc ( min/+ ) mice ( velmurugan et al . , 2010b ) . displaying selectivity , resveratrol performs an antioxidant role for normal cells , yet acts as a pro - oxidant for malignant cells ; dna damage and apoptosis were observed to be enhanced in the low ph environment established by glycolytically active cancer cells ( muqbil et al . , 2012 ) . the selective induction of oxidative stress within cancer cells may underlie the proapoptotic actions of gse through erk1/2 phosphorylation and arrest of the cell cycle via p21 induction ( kaur et al . , 2011 ) . the flavonoid procyanidins found in gse have been shown to regulate additional cell signaling and mitogenic pathways important for cancer . in prostate cancer cells , gse greatly limited autocrine feedback of egf / egfr / erk signaling and induced jnk - related apoptosis in the du145 androgen - independent line ( tyagi et al . , 2003 ) . proanthocyanidins from gse also induced apoptosis in the caco-2 colon cancer cell line , partially mediated through attenuation of pi3k signaling ( engelbrecht et al . , 2007 ) . our laboratory has investigated the effects of gse on cell viability of the colon cancer lines hct116 , ht29 , hct115 , and caco-2 , and our results revealed cytotoxic effects in all four lines , which vary widely in their genetic profiles ( sokolosky and wargovich , unpublished data ) . roots , leaves , and tree barks can also contain powerful substances recognized for their medicinal values in ayurvedic , chinese , and traditional south american medicine . gt , the most widely consumed beverage worldwide after water , has received recent popularity due to its broad spectrum ability to fight inflammation and decrease the risks for many types of malignancy . polyphenols from gt have been shown to possess extremely high antioxidant capacities given their numerous hydroxyl groups , as well as the ability to enhance b- and t - cell mediated immunity ( notably within the tumor microenvironment ) . the most abundant polyphenol from gt , egcg , has been shown to bind the metastasis - associated 67-kda laminin receptor at nanomolar concentrations , expression of which can sensitize cancer cells to this compound s anti - proliferative effects ( tachibana et al . , 2004 ) . egcg exhibited selective apoptotic and growth limiting properties in cancer cells through caspase cleavage , inhibition of nf-b , and modification of cell cycle protein expression ( butt and sultan , 2009 ) . in addition to inducing cancer cell apoptosis , egcg also induced death in bone - resorbing osteoclasts in an fe and h2o2 dependent manner , with two of the egcg hydroxyl groups being crucial for this biological activity ( nakagawa et al . , 2007 ) . inhibition of osteoclast function can serve to remodel the hematopoietic stem cell ( hsc ) niche , thereby inducing differentiation and reducing hsc numbers in vivo ( lymperi et al . , 2011 ) . therefore , compounds such as egcg may be able to induce differentiation of cscs , similar to how vitamin d3 can induce differentiation in mesenchymal stem cells ( piek et al . , 2010 ) as well as many forms of cancer ( gocek and studzinski , 2009 ) . furthermore , certain gt catechins , including egcg , exhibit not only cellular , but species specificity as reflected by their ability to inhibit mammalian dna polymerases in vitro , but not plant or prokaryotic polymerases ( mizushina et al . , it has been shown by our laboratory that egcg can inhibit cox-2 expression during colon carcinogenesis ( peng et al . , 2006a ) , and exert anti - proliferative effects during ras - induced transformation of intestinal epithelial cells ( peng et al . upstream events at the plasma membrane which lead to aberrant activation of ras and other kinases can be mediated by lipid rafts that act as signaling hubs . these lipid regions reflect variable compositions between transformed and benign cells as well as apoptotic and viable cells , which may mediate the targeted effects observed with egcg ( patra et al . , 2008 ) . by reorganizing lipid rafts of colon cancer cells , egcg caused sequestration of egfrs away from the cell surface via endosomal vesicles ; the process of which can be chemopreventive by limiting egf - induced pathway activation ( adachi et al . , 2008 ) . egcg was also shown to downregulate expression of enzymes involved in gluconeogenesis in murine intestinal cells and caco-2 colon cancer cells , demonstrating that gt may be able to limit glycolytic activity in premalignant and malignant tissues ( yasui et al . , 2011 ) . in a large , randomized clinical trial investigating the effects of gt extract on the recurrence of metachronous colorectal adenoma , the patient group that consumed the extract ( dosage equivalent to about 10 cups of gt per day ) experienced a 50% reduction in incidence along with smaller sizes of relapsed tumors ; no negative side effects were observed in the extract - treated patients ( shimizu et al . , 2008 ) . egcg was also shown to suppress colony formation ability of mcf-7 breast cancer cells while delaying onset and reducing the size of tumors in a xenograft model . these effects correlated with potent inhibition of hsp70 and hsp90 , thus demonstrating that heat shock responses are targets of egcg antitumor mechanisms ( tran et al . , 2010 ) . additional findings involving the role of numerous phytonutrients in the chemoprevention of colon cancer are available , as the colon is an attractive model of study given the accumulation and bioavailability of these compounds in this region after ingestion . however , it should be noted that conflicting data exists concerning the combined effects of a phytochemical - rich diet and chemotherapy or radiotherapy . one study found that egcg significantly lowered the apoptotic abilities of radiotherapy in du145 prostate cancer cells , suggesting that the potent antioxidant properties of one counteract the free radical generating mechanisms of the other , either of which would be more beneficial alone ( thomas et al . , 2011 ) . on the other hand , a study of breast cancer patients undergoing radiotherapy found that concurrent egcg intake led to lower levels of secreted factors like vegf and mmp-9 and mmp-2 , suggesting anti - angiogenic and anti - metastatic effects . when sera obtained from these patients was added to mda - mb-231 cultures , anti - proliferative and proapoptotic changes occurred , and egcg treatment was able to enhance the apoptotic efficacy of -radiation in these cells , which correlated with reduced akt and nf-b activity ( zhang et al . , 2012a ) . in another human - based study , mucosal cells from colon cancer patients revealed significant changes in diagnostically relevant dna methylation as compared to normal colonic mucosa , many of which led to altered expression of glycolytic hormones and enzymes . this suggests that certain dietary compounds will be metabolized differently between the groups of patients , which should be a consideration when selecting therapy , especially metabolically targeted therapy ( silviera et al . , 2012 ) . therefore , the dynamic interactions evoked in the presence of various pro- and anti - inflammatory dietary compounds , plant - based phytochemicals , and environmental toxins within a cancer patient could have a profound and clinically relevant influence on their response to conventional and/or alternative therapy . in light of the complex array of factors underlying chronic disease in modernized societies , it is likely that epigenetic regulation of gene expression via exogenously obtained compounds plays a critical role in the emergence of homeostatic imbalance , as shown in figure 2 . in addition to focusing on the cellular and organ level , the entire physiological system must be considered , including circulating metabolites of environmental toxins and plant compounds , to yield a more comprehensive understanding of a living organism . the human metabolome database is an updated , annotated library of metabolites found to exist within the human body , and can serve as a resource for scientists and clinicians ( wishart et al . , 2009 ) . by integrating an individual s genomic , proteomic , and metabolomic information , the identification of new biomarkers will be vital to further defining colorectal cancer risk , designing individualized therapies , and planning practical preventive interventions relevant to the population of focus ( risio , 2011 ) . panels to assess circulating inflammatory mediators and xenobiotic compounds in the blood could represent a new class of measurable markers in personalized health care . the dynamic epigenetic interplay of inflammation , environmental toxins , and phytochemicals theorized to lead to homeostatic imbalance and chronic disease in modernized societies ; the manifestation and extent of which depends upon the hypothetical timing of critical gene expression changes in stem cells during embryogenesis , wound healing , and colon carcinogenesis ( in this scenario ) , with other outcomes plausible . tumor progression is a dynamic process in which the commitment to malignancy is not firm , but fluctuates between progression , stabilization , and regression dependent upon adaptation , or the lack thereof , to selective stimuli . precancerous cells exhibit a range of evolutive speeds , representative of a complex array of genetic and non - genetic factors , and these speeds can be influenced by environmental switches within the human epigenome ( risio , 2011 ) . according to the maximum genetic diversity hypothesis , evolution from simpler to more complex organisms involves an elevation in epigenetic complexity that occurs at the expense of genetic ( mutation - driven ) diversity among individuals . this inverse relationship between two forms of gene expression regulation can expand upon classical darwinian definitions of evolution to further explain how diverse phenotypes arise within a closely related population in a relatively short span of time ( huang , 2008 ) . it is in this way that one identical twin can develop a metastatic tumor while the other twin , in spite of sharing the same germ line dna , can remain cancer - free by virtue of their unique epigenetic profiles . it is possible that sporadic and familial cancers without clear causation are the result of both inherited genes for susceptibility and inherited epigenetic footprints that run in a particular lineage . epigenetic changes passed on to daughter cells are just as relevant as mutations , especially in organs that undergo high rates of renewal while simultaneously being exposed to high toxin levels such as the lungs , breasts , and gi tract . alarmingly , common toxins have now been shown to exert inherited effects in animal models in which an exposure to an edc in a pregnant female induces epigenetic modifications that are passed on to each subsequent generation of males , as evidenced by altered dna methylation patterns in the germ line . this transgenerational ( tg ) effect occurred in the absence of direct exposure of the offspring to the causative agent ( anway et al . , 2005 ) . further investigation into the tg actions of mixed toxins in f0 gestating rats revealed effects in the f1-f3 generations such as early puberty and spermatogenic apoptosis stemming from the original f0 exposure . epigenetic markers within differential dna methylation regions of sperm cells from unexposed offspring conveyed correlations with specific ancestral exposures , with 499 genes identified to be associated with tg effects ( manikkam et al . , 2012 ) . additional elucidation of the in vivo epigenetic mechanisms of common toxins in normal , precancerous , and malignant cells is strongly encouraged . based on present data , it is important for the scientific and medical communities to increase the general public s awareness of toxins in their every lives , as well as promote practical and affordable methods of prevention . given that edc exposure is virtually unavoidable in some cases , once bioaccumulation has occurred , therapies and interventions that accelerate detoxification and elimination of persistent toxins from the body would be clinically relevant and could ameliorate current management approaches for chronic illness ( genuis , 2011 ) . by practicing prudent avoidance of toxins and obtaining consistent levels of dietary phytochemicals , we can restore and maintain physiological balance by limiting negative epigenetic changes and promoting protective ones . for example , cooking with onions and garlic can neutralize many of the carcinogens created by high - heat preparation of food , thereby limiting a preventable exposure . thus , to maintain epigenetic balance it is imperative to properly fortify the body by consuming a diverse diet rich in whole plant foods , herbs , and spices . gse was shown to exert potent epigenetic regulation in androgen - responsive lncap cells by inhibiting histone acetyltransferase ( hat ) activity up to 80% , in addition to significantly decreasing androgen receptor mediated transcription ( park et al . , 2011 ) . resveratrol has been shown to directly counteract the negative effects of toxin exposure by acting as an antagonist for the ahr , of which dioxin and benzo[a]pyrene are ligands . therefore , prophylactic resveratrol could offer a non - toxic and potent way to limit expression of dioxin - inducible genes like cytochrome p4501a1 and il-1 , upon aryl hydrocarbon exposure ( casper et al . the prevention of bpa - induced toxicity in sertoli cells was also achievable through pre - treatment with ginsenoside compounds from ginseng , suggesting that the effects of environmental exposures can be limited if phytochemicals are present ( wang et al . remarkably , egcg is able to hydrogen bond within the active site of dna methyltransferase ( dnmt ) , thereby inhibiting its cancer promoting functions and turning on previously silenced genes in prostate cancer cells ( fang et al . , 2003 ) . egcg has also been shown to induce histone deacetylation and promoter hypomethylation to achieve repression of htert , the catalytic subunit of telomerase , which is overexpressed in 90% of cancers . this effect induced apoptosis in mcf-7 breast cancer cells , and egcg also induced apoptosis in hl60 leukemia cells , although via a different mechanism ( berletch et al . , 2008 ) . htert promoter demethylation by egcg was also observed in er+ mcf-7 and er - mda - mb-231 breast cancer cells through inhibition of dnmt and hat activity ( meeran et al . , 2011 ) . another study found that egcg epigenetically repressed promoter methylation of the tumor suppressors p16 and p15 , thereby restoring their expression and inhibiting proliferation of colon cancer cells ( berner et al . , egcg can also alter levels of the universal methyl donor , s - adenosylmethionine , and the methyltransferase inhibitor , s - adenosylhomocysteine , thereby indirectly influencing epigenetic regulation ( park et al . , 2012 ) . therefore , phytochemicals offer chemopreventive mechanisms important for reestablishing tumor suppressor activity previously silenced during oncogenesis . it should be noted that exogenous hormone exposure can occur via ingestion of certain plants , but these non - steroidal natural compounds appear to behave differently than synthetic edcs . genistein and resveratrol , which are classified as phytoestrogens , were shown to exert epigenetic regulation of the estrogen receptor 1 ( esr1 ) gene in colon cancer cells , demonstrating that a wide array of mechanisms can be modulated by bioactive food compounds ( berner et al . , 2010 ) . it was also shown that genistein can inhibit proliferation by downregulating wnt5a promoter methylation in colon cancer cells ( wang and chen , 2010 ) . it is interesting to note that genistein and other isoflavones mediate their effects in humans through selective binding to er as opposed to er ( barone et al . , 2011 ) . whereas the growth - promoting er isoform tends to be upregulated in hormonally responsive cancers , signaling through the er isoform , which may serve a tumor suppressor role , is often downregulated via a variety of mechanisms . it was found that genistein promoted anti - proliferative and proapoptotic effects through er in the large and small intestine ( schleipen et al . , 2011 ) , circumvention of such may be why cancers specifically upregulate expression of the growth - promoting er isoform . in respect to normal adipocytes , genistein was shown to inhibit leptin secretion by limiting glucose metabolism ( szkudelski et al . , 2005 ) . genistein , resveratrol , and quercetin were shown to exhibit enhanced inhibition of adipogenesis and promotion of apoptosis when used in combination to treat human and murine adipocytes , effects of which correlated with a decrease in glycerol 3-phosphate dehydrogenase activity ( park et al . , 2008 ) . these results suggest that dietary intake of various phytoestrogens may carry implications for appetite control , sugar metabolism , and aberrant glycolytic activity within cancer cells , with effects being opposite of those observed for edcs . however , the full gamut of phytoestrogen activity remains to be characterized as studies in breast cancers have revealed different effects depending upon er , as well as her2 expression . the relative expression levels of er and er within a normal or cancerous cell can greatly influence proliferation , endocrine signaling , and response to various er ligands ( chang et al . , 2006a ) , both plant - derived and manmade . as for synthetic edcs , bpa , and others bind to er and stimulate signaling , but upon binding to er , fail to trigger the anti - proliferative activities of this receptor subtype ( bolli et al . , bpa also may bind to the estrogen - related receptor gamma ( err ) as indicated in neuronal studies ( tanabe et al . , it should be noted that in certain situations , er signaling may be capable of preserving the epithelial phenotype and limiting emt , the effect of which has been observed in breast cancer cells ( guttilla et al . , 2012 ) , although the loss of hormone - responsiveness would overcome this possible anti - metastatic effect of excessive er stimulation via edcs . the tissue - selective agonist and antagonistic effects of xenoestrogens occur based in the relative expression of er isoforms , much in the way that hormonal contraceptives and the selective er modulator , tamoxifen , can exhibit tissue - specific effects in the ovaries and breasts . in general , xenohormones are a risk factor in colon cancer through imbalanced nuances of er and er expression ; a tip in either direction could have cancer promoting consequences . therefore , phytoestrogens may be the natural answer for balancing the effects of artificial xenoestrogens , as these two types of compounds appear to serve as agonists and/or antagonists for similar cellular targets , as detailed in table 1 and figure 3 . in fact , the estrogenic potency of phytoestrogens may be greater than environmental estrogens , as genistein was shown to stimulate respective er transcriptional activity at much lower nanomolar concentrations than those required for bpa ( kuiper et al . maternal dietary supplementation with folic acid ( methyl donor ) or genistein was able to limit the cpg hypomethylation otherwise induced by bpa exposure , demonstrating that epigenetic balance can be restored through appropriate nutrient intake ( dolinoy et al . , 2007 ) . the full epigenetic regulatory spectrum of natural compounds is currently being defined and additional mechanisms involving the gene expression modifying enzymes , dnmts , hats , and histone deacetylases ( hdacs ) are likely to be elucidated . structures of common xenoestrogens capable of influencing estrogenic signaling through agonistic and/or antagonistic mechanisms . unparalleled selectivity is reflected in natural products , as well as extremely low or absent toxicity when compared to other chemopreventive agents such as the non - steroidal anti - inflammatory drugs , which can induce bleeding and ulceration . whereas inhibitors of cox-1 and cox-2 have been shown to counteract chronic inflammation and lower cancer risk , they carry substantial cardiovascular concerns and liver toxicity with prolonged use . the apparent influence of fat intake on inflammation can be exerted beneficially by establishing an ideal fa ratio through consumption of omega-3 rich foods and restriction of omega-6 sources . in a model of experimentally induced colitis , ala , an omega-3 fa , was able to reduce oxidative stress and counteract inflammation via suppression of nf-b and cox-2 activity ( hassan et al . , 2010 ) . in another colitis study , krill oil , rich in omega-3 fas , acted in an anti - inflammatory manner to decrease oxidative stress and cytokine secretion in dextran sulfate sodium - treated mice ( grimstad et al . , 2012 ) . in addition to ideal fa ratios , an approach to addressing inflammation could be based on intake of flavorful , sometimes pungent , and often bitter plant compounds in their native configurations from whole - food sources like turmeric , ginger , cinnamon , broccoli , chili peppers , coffee , tea , dark chocolate , and fruit seeds . a phytochemically diverse diet creates beneficial synergistic effects , and certain compounds can facilitate proper transport and absorption of other nutrients , as in the case of piperine from black pepper and vitamin c intake with plant - based meals . in addition to nutrient intake , consumption of probiotics can establish and maintain beneficial gut microflora that stimulate immunity and assist in the elimination of toxins . for example , it was shown that when fed a diet containing a bifidobacterium or lactobacillus strains , bpa - exposed rats had a reduced bpa blood concentration over time and 2.4-fold increase in bpa fecal excretion ( oishi et al . , 2008 ) . therefore , dietary alterations can be powerful tools for counteracting modern environmental exposures by restoring inflammation to a proper range in which chronic states are suppressed but vital acute responses still efficiently occur . we can acknowledge the finely tuned adaptations of mammalian cells for plant compounds and draw on the order of sophistication and simplicity only attained in nature . in the lab , we can explore the development of nutraceuticals with slight chemical modifications that increase their absorption , bioavailability , and stability in vivo while maintaining natural potency . a novel prodrug form of egcg containing peracetate - protected hydroxyl groups was shown have increased stability and enhanced uptake within breast cancer cells , where it was able to selectively repress telomerase expression in an epigenetic manner , as compared to non - transformed control cells ( meeran et al . , 2011 ) . in another study , acylated catechin derivatives had enhanced anti - cancer and anti - angiogenic properties compared to fa - conjugated epicatechins in hct116 cells , with stearic acid - modified catechin demonstrating the highest level of efficacy ( mizushina et al . , 2011 ) . by elucidating the structure - activity relationships and computationally visualizing the molecular interactions of phytochemicals , chemists can seek to improve their pharmacokinetic / dynamic profiles . however , a chemically modified and synthetically derived compound may not exhibit the same subtle nuances that it would if produced in its natural state , so plant - based culture systems for experimental and large - scale phytochemical production may be preferable . it is important to recognize that drug drug interactions between bioactive food compounds and pharmaceutical agents are a concern , as cytochrome - mediated drug metabolism and/or bioavailability may be altered by phytochemicals in some scenarios . in the presence of egcg , studies have found increased bioavailability of tamoxifen ( shin and choi , 2009 ) , but decreased bioavailability for the rtk inhibitor sunitinib ( ge et al . , 2011 ) . decreased efficacy of the boronic acid - based proteosome inhibitor bortezomib has also been linked to egcg , with direct binding of the two compounds shown to mediate the interaction ( golden et al . , 2009 ) high egcg intake during pregnancy is also contraindicated as recent findings link this topoisomerase - inhibiting flavonoid to mll gene cleavages and translocations , implicated in infant leukemia ( strick et al . , 2000 ) . another limitation of oral phytochemical usage can be absolute bioavailability , mostly due to oxidation , rapid absorption , and/or microbial enzymatic cleavage in the gi tract . however , consistent dietary intake is likely sufficient for establishing blood and tissue concentrations within the low range required for chemopreventive effects . egcg accumulation within prostatectomy tissue was achievable with daily gt consumption by prostate cancer patients , although it should be noted that 50% of the egcg present had been methylated to 4-meegcg , which appears to reduce its antitumor effects ( wang et al . , 2010 ) . it is interesting to note that when combined with quercetin , which inhibits catechol - o - methyltransferase and drug efflux pumps , egcg methylation was decreased by threefold and uptake increased by fourfold in lung cancer cells ( wang et al . , 2012b ) . once again , these findings support the idea that chemopreventive properties are maximized when a variety of plant - based foods are consumed . as an alternative to maintaining systemic bioavailability from oral dosage , therapeutic levels may be achieved locally with bioengineered delivery systems such as implantable scaffolds and hydrogels constructed from natural polymers . non - oral routes of phytochemical dosage such as inhalation , intravenous , or epidermal application would also be worth investigating . modern drug discovery , design , and delivery have relied heavily on cues from nature , and although not a panacea , natural products have revealed numerous medicinal mechanisms that could positively impact human health , with many remaining to be investigated . even without physiological administration , plants can prevent disease by eliminating airborne toxins such as benzene and vinyl chloride , the monomer of pvc , by virtue of photosynthetic and metabolic processes ( doty et al . , 2007 ) . by virtue of large or small - scale production leading to broad availability at grocery chains and markets , plant - based nutrition is accessible , relatively affordable , and easily promoted to society as an important preventive measure against chronic disease . although , translating this knowledge to common practice is growing increasingly difficult in westernized cultures whose populations opt for nutrient and phytochemical - depleted fast foods . researchers can perform discovery science to expose modern roots of disease and further illuminate our knowledge of prevention through the generation of additional hypotheses . we know little about the mechanisms of cellular uptake , transport , and metabolism of many of the compounds mentioned . in an effort to mimic human prevention and disease scenarios , experimental animal models should be employed that reflect realistic dietary conditions ( high in fat and low in nutrients and fiber ) , such as the new western - style diet ( nwd1 ) , which is known to induce inflammatory responses and increase colon cancer occurrence in rodents ( bastie et al . , 2012 ) . it should be noted that any exogenous compound , including a natural compound at a high enough concentration , can act as a stressor in cell - based models that ultimately do not reflect physiological conditions . this may explain cases of conflicting data obtained between in vitro and in vivo phytochemical studies , and experiments must be comprehensively designed to address this caveat . in the clinic , practitioners can incorporate holistic - based medicine within their repertoire to treat a patient s tumor as a symptom while working with them to adjust the underlying physiological imbalances that set the stage for cancers to thrive . although phytochemical - based approaches can not reverse extensive pre - existing damage or address aggressive , late - stage disease , they can become a mainstay of prevention for healthy patients and an important intervention for patients with manageable conditions . defining new frontiers in oncology involves reevaluating old paradigms in the context of modern needs , thereby revamping the historically practical wisdom that established the foundation of medicine . the nineteenth century french physiologist claude bernard defined the concept of milieu intrieur , or homeostasis , when he proposed to his colleagues that , the terrain is everything , the germ is nothing . according to legend , he proceeded to drink a glass of cholera - tainted water to prove his point ; if only we could be so bold in our endeavors to dethrone , as siddhartha mukherjee phrased it , the emperor of all maladies . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

the advent of modern medicine has allowed for significant advances within the fields of emergency care , surgery , and infectious disease control . health threats that were historically responsible for immeasurable tolls on human life are now all but eradicated within certain populations , specifically those that enjoy higher degrees of socio - economic status and access to healthcare . however , modernization and its resulting lifestyle trends have ushered in a new era of chronic illness ; one in which an unprecedented number of people are estimated to contract cancer and other inflammatory diseases . here , we explore the idea that homeostasis has been redefined within just a few generations , and that diseases such as colorectal cancer are the result of fluctuating physiological and molecular imbalances . phytochemical - deprived , pro - inflammatory diets combined with low - dose exposures to environmental toxins , including bisphenol - a ( bpa ) and other endocrine disruptors , are now linked to increasing incidences of cancer in westernized societies and developing countries . there is recent evidence that disease determinants are likely set in utero and further perpetuated into adulthood dependent upon the innate and environmentally - acquired phenotype unique to each individual . in order to address a disease as multi - factorial , case - specific , and remarkably adaptive as cancer , research must focus on its root causes in order to elucidate the molecular mechanisms by which they can be prevented or counteracted via plant - derived compounds such as epigallocatechin-3-gallate ( egcg ) and resveratrol . the significant role of epigenetics in the regulation of these complex processes is emphasized here to form a comprehensive view of the dynamic interactions that influence modern - day carcinogenesis , and how sensibly restoring homeostatic balance may be the key to the cancer riddle .

Modernization, Acculturation, and Risk for Colorectal Cancer Inflammation, Homeostatic Disruption, and Cancer Low-Level Environmental Exposures Phytochemical Depletion Rebalancing the Tipped Epigenetic Scale Conflict of Interest Statement

clinical presentation of gas under the diaphragm and acute abdomen can be due to rupture of viscus , perforated peptic ulcer disease and rarely spontaneous . a 38 year old woman presented in accident and emergency ward with a history of acute abdominal pain . she gave a history of dyspepsia , symptoms of gastritis in the past and was treated with omeperazole . she was seen by surgeons who advised for an erect chest x - ray which showed gas under the diaphragm implying the diagnosis of perforated peptic ulcer disease . erect chest x - ray highlighting the presence of subphrenic gas she was seen b they gynaecology team in view of history and acute abdomen . a detailed history was elicited and patient reported having had sexual intercourse earlier on the same day and denied the pain following it . her vital signs were stable and abdominal examination revealed diffuse vague tenderness , no sign of distension or guarding and vaginal examination was difficult and no abnormalities detected . in view of the clinical symptoms and radiological findings patient was taken for emergency laparatomy . there was no perforation of viscus but there was a defect noted in the vaginal vault . gas under the diaphragm can be due to like perforation in stomach , duodenum due to peptic ulcer disease , in jejunum or illeum by inflammatory bowel disease or cancer can lead to pneumoperitoneum . this can present as an acute symptom.(1 ) 10% of cases diagnosed on pneumoperitoneum are not due to perforation of an air containing viscus but due to sexual activity , peritonitis , gas forming bacteria and some rare causes like iucd . first case of pneumoperitoneum due to post coital perforation after abdominal hysterectomy has been reported in 1980.acute abdomen may or may not be the symptom ( 2,3 ) . vaginal evisceration may or may not be a presenting symptom.(3,4 ) in our patient the acute symptoms and x - ray findings lead for an early laparatomy . this condition could have been managed conservatively if the pain has not been severe . the vault repair can be corrected laparoscopic or vaginally and a major laparatomy could be avoided embarrassment and modesty often prevent patients form talking about sexual activity . clinical examination during acute pain , particularly vaginal examination , is more painful for patients . the condition is rare but gynaecologist and surgeons should be aware of the situation and appropriate team work is essential to deal with the rare situations .

gas under the diaphragm can be due to like perforation in stomach , duodenum due to peptic ulcer disease , in jejunum or illeum by inflammatory bowel disease or cancer can lead to pneumoperitoneum . we present a rare cause of pneumoperitoneum following abdominal hysterectomy .

INTRODUCTION CASE REPORT DISCUSSION

the limiting factor in these flaps is the unpredictable blood supply , which , if insufficient , may produce irreversible damages to the microcirculation . this damage results in partial or complete flap necrosis and renders the wound more susceptible to infection , thereby causing further healing impairment ; thus , improving distal blood supply in the random skin flap is an important goal . the cellular damage that occurs during tissue reperfusion after ischemia is the result of a cascade of events involving free - radical oxygen and inflammatory mediators . molecular oxygen plays a central role in the reparative healing process and is one of the critical nutrients of the wound . hyperbaric oxygen ( hbo ) increases the tolerance of tissue to ischemia and enhances free - radical formation ; however , hyperoxia can increase the biochemical defence mechanisms against free radicals and improve the survival probability of ischemic tissue . the hbo has a protective effect on microcirculation possibly by interfering with the deleterious action of activated neutrophils on microvascular endothelium . one of the most important components of the intracellular antioxidant system is glutathione , a powerful active radical scavenger that is depleted in ischemia - reperfusion ( ir ) injury . n - acetylcysteine ( nac ) is a prodrug that supplies bioavailable cysteine for glutathione replenishment . in the presence of overwhelming active oxygen species ( ros ) , the antioxidant nac prevents some of these deleterious effects , indicating an involvement of oxidative stress during hbo exposure . the effects of hbo in improving the survival of a random skin flap are already known . thus , it is important to study its combination with other substances , such as enzymes , nac , and so on , to potentiate their effect to be applied to patients . thus , in this study , our aim is to investigate the role of hbo , nac , and hbo plus nac on the necrosis area of random rat 's skin flaps of a modified mcfarlane flap design . the experimental protocol ( # 1431/03 ) was approved by the ethics committee of the federal university of so paulo ( unifesp ) . all the procedures strictly followed the existing regulations about animal experimentation ( brazilian college on animal experimentation , cobea ) . thirty - two male wistar rats weighing 280 - 300 g were kept in individual cages in acoustically isolated rooms at 25c , with artificial illumination as well as chow and water ad libitum . the animals were randomly divided into four groups : g - s ( sham group , n = 8) , g - nac ( nac , n = 8) , g - hbo ( hbo , n = 8) , and g - hn ( hbo plus nac , n = 8) . after 6 h of fasting without solid diet and 4 h without liquid diet , the animals received 5 mg / kg of acepromazin i.m . subsequently , after 10 min , they received a combination of 50 mg / kg of ketamin ( ketalar , medical division of pfizer do brazil , so paulo , brazil ) and 10 mg / kg of xylazin i.m . a rectangular area ( 2 8 cm ) was longitudinally marked with ink , based on 7 cervical vertebra and running to caudal position with the spine as a central landmark . 15 ) , and a flap skin was displaced from the muscular dorsal layer and the cranial portion was preserved from incision [ figure 1 ] . a polyethylene film was placed over the muscular region , covering all the wound area and acting as a barrier between the skin and muscles [ figure 2 ] . an interrupted 3.0 nylon suture ( mononylon , ethicon , so paulo , brazil ) was employed to fix the flap into the original place . 15 ) , and a flap skin was displaced from the muscular dorsal layer and the cranial portion was preserved from incision a polyethylene film was placed over the muscular region , covering all the wound area and acting as a barrier between the skin and muscles a dose of 300 mg / kg of nac ( fluimucil acetylcysteine 300 mg/3 ml , zambon laboratrio farmacutico ltd . , so paulo , brazil ) was intraperitoneally injected into the g - nac or g - hn groups after the elevation of skin flap , and consecutively for every 24 h for 7 days . on the other hand , distilled water of 1 ml ( distilled water , isofarma , so paulo , brazil ) was intraperitoneally injected into the g - s and g - hbo groups after the elevation of skin flap , and consecutively for every 24 h for 7 days . hbo procedure was carried out in a hyperbaric chamber for experimental animals of university regional do alto uruguai campus erechim ( uri ) . before pressurization , 100% medical oxygen was flushed through the chamber for 5 min to displace the room air . the oxygen pressure was then increased at a constant rate to reach a pressure of 2.4 atmosphere . all the animals of g - hbo and g - hn groups were exposed to 100% oxygen at 2.4 ata for 2 h ( once a day ) , 15 min after flap fixation , and every 24 h for the consecutive 7 days . the sequence of procedures in each group is summarized in figure 3 . the animals in g - s ( n = 8) received distilled water intraperitoneally 15 min after flap elevation for 7 consecutive days ; those in g - nac ( n = 8) received 300 mg / kg of nac intraperitoneally after flap elevation for 7 consecutive days ; those in g - hbo ( n = 8) were exposed to 100% oxygen at 2.4 ata for 15 min following the delay procedure and for 2 h a day each for 7 consecutive days in a hyperbaric animal experimental chamber flushed with 100% oxygen , and distilled water was intraperitoneally injected ; and those in g - hn ( n = 8) received the combination of nac and hbo for 7 consecutive days . g - nh : flap proceedings and eight daily injection of nac , followed by hbo every day ( twice a day ) , all the animals were examined and the occurrence of fever , incision infection , and liquid stools or refuse of the chow or drinking water were recorded . once any sign of severe suffering was identified , the veterinarian interrupted the research and the animals were euthanized . on the eighth day the dorsal area was photographed from a standard distance by a 7.2-mega pixel digital camera ( sony p-200 , sony , japan ) , and the images were saved in jpeg format . under anaesthesia and after the collection of samples , the animals were put in a chamber and flushed with co2 until cardiorespiratory arrest . on the eighth postoperative day , the flap area was photographed and compared with that recorded on the first day of the experiment . the photographs were captured by the computer software image pro plus 4.5. mean flap necrosis area was then assessed for all groups . the areas of flap necrosis were expressed as mean and standard deviation ( spss 11.0 version ) . significance of differences in necrosis was determined by one - way analysis of variance ( anova ) , applying post - hoc test of bonferroni . a p value of 5% ( p the experimental protocol ( # 1431/03 ) was approved by the ethics committee of the federal university of so paulo ( unifesp ) . all the procedures strictly followed the existing regulations about animal experimentation ( brazilian college on animal experimentation , cobea ) . thirty - two male wistar rats weighing 280 - 300 g were kept in individual cages in acoustically isolated rooms at 25c , with artificial illumination as well as chow and water ad libitum . the animals were randomly divided into four groups : g - s ( sham group , n = 8) , g - nac ( nac , n = 8) , g - hbo ( hbo , n = 8) , and g - hn ( hbo plus nac , n = 8) . after 6 h of fasting without solid diet and 4 h without liquid diet , the animals received 5 mg / kg of acepromazin i.m . subsequently , after 10 min , they received a combination of 50 mg / kg of ketamin ( ketalar , medical division of pfizer do brazil , so paulo , brazil ) and 10 mg / kg of xylazin i.m . under general anaesthesia , the dorsal regions were shaved and the animals were fixed in the prone position . a rectangular area ( 2 8 cm ) was longitudinally marked with ink , based on 7 cervical vertebra and running to caudal position with the spine as a central landmark . 15 ) , and a flap skin was displaced from the muscular dorsal layer and the cranial portion was preserved from incision [ figure 1 ] . a polyethylene film was placed over the muscular region , covering all the wound area and acting as a barrier between the skin and muscles [ figure 2 ] . an interrupted 3.0 nylon suture ( mononylon , ethicon , so paulo , brazil ) was employed to fix the flap into the original place . 15 ) , and a flap skin was displaced from the muscular dorsal layer and the cranial portion was preserved from incision a polyethylene film was placed over the muscular region , covering all the wound area and acting as a barrier between the skin and muscles a dose of 300 mg / kg of nac ( fluimucil acetylcysteine 300 mg/3 ml , zambon laboratrio farmacutico ltd . , so paulo , brazil ) was intraperitoneally injected into the g - nac or g - hn groups after the elevation of skin flap , and consecutively for every 24 h for 7 days . on the other hand , distilled water of 1 ml ( distilled water , isofarma , so paulo , brazil ) was intraperitoneally injected into the g - s and g - hbo groups after the elevation of skin flap , and consecutively for every 24 h for 7 days . hbo procedure was carried out in a hyperbaric chamber for experimental animals of university regional do alto uruguai campus erechim ( uri ) . before pressurization , 100% medical oxygen was flushed through the chamber for 5 min to displace the room air . the oxygen pressure was then increased at a constant rate to reach a pressure of 2.4 atmosphere . all the animals of g - hbo and g - hn groups were exposed to 100% oxygen at 2.4 ata for 2 h ( once a day ) , 15 min after flap fixation , and every 24 h for the consecutive 7 days . the sequence of procedures in each group is summarized in figure 3 . the animals in g - s ( n = 8) received distilled water intraperitoneally 15 min after flap elevation for 7 consecutive days ; those in g - nac ( n = 8) received 300 mg / kg of nac intraperitoneally after flap elevation for 7 consecutive days ; those in g - hbo ( n = 8) were exposed to 100% oxygen at 2.4 ata for 15 min following the delay procedure and for 2 h a day each for 7 consecutive days in a hyperbaric animal experimental chamber flushed with 100% oxygen , and distilled water was intraperitoneally injected ; and those in g - hn ( n = 8) received the combination of nac and hbo for 7 consecutive days . g - nh : flap proceedings and eight daily injection of nac , followed by hbo every day ( twice a day ) , all the animals were examined and the occurrence of fever , incision infection , and liquid stools or refuse of the chow or drinking water were recorded . once any sign of severe suffering was identified , the veterinarian interrupted the research and the animals were euthanized . on the eighth day the dorsal area was photographed from a standard distance by a 7.2-mega pixel digital camera ( sony p-200 , sony , japan ) , and the images were saved in jpeg format . under anaesthesia and after the collection of samples , the animals were put in a chamber and flushed with co2 until cardiorespiratory arrest . on the eighth postoperative day , the flap area was photographed and compared with that recorded on the first day of the experiment . the photographs were captured by the computer software image pro plus 4.5. mean flap necrosis area was then assessed for all groups . the areas of flap necrosis were expressed as mean and standard deviation ( spss 11.0 version ) . significance of differences in necrosis was determined by one - way analysis of variance ( anova ) , applying post - hoc test of bonferroni . a p value of 5% ( p in figure 4 , the mean of necrosis ( % ) is shown by groups . the average flap necrosis area was 18.3% in g - s , 24.3% in g - nac , 12.6% in g - hbo , and 14.9% in g - hn group . means and standard deviation of the percentage of necrosis on the random skin flaps in g - s , g - nac , g - hbo , and g - hn groups . the means of necrosis in all the experimental groups were lesser than those of the g - s group . there was a significant difference between g - hbo < g - nac ( p < 0.01 ) and g - hn < g - nac ( p < 0.001 ) ( anova test ) in table 1 , the results of macroscopic evaluation ( area in mm ) for each group are presented . means , standard deviation , and range of necrosis area ( mm ) in the skin flaps of g - s , g - nac , g - hbo , and g - hn on day 8 g - hbo group showed a smaller area of flap necrosis , when compared with g - s , but was not significant ( p = 0.12 ) ; however , when compared with the g - nac group , a significant difference was noted ( p < 0.01 ) . the flap survival in g - hbo was the same as that noted in g - hn , and when compared with the g - nac , the g - hn group showed significant difference ( p < 0.01 ) . in this study , however , the combination of nac and hbo , in contrast to the expectations , had no better survival , when compared with administration of hbo alone . there has been much controversy over the angiogenic properties of hbo . in skin wounds , the angiogenic properties induced by hbo are derived from the increase in oxygen tension that may persist for several hours after hbo . comparison of g - s and g - nac groups revealed that repeated on - off exposures produce a favourable environment in random flaps . in this study , hbo administered after surgery improved the survival of random skin flaps . this finding is in contrast to the original hypothesis that suggests that extra oxygen increases the production of free radicals that would increase flap necrosis . hong jp showed that when hypoxic tissue is exposed to hbo , there is an increase in po2 in the plasma and a reduction in po2 , and the rate of diffusion of oxygen increases . nac is a precursor of glutathione , a potent antioxidant that inhibits the induction of pro - inflammatory cytokines . glutathione also induces the production of nitric oxide synthase ( inos ) as well as adhesion molecule 1 . this antioxidant also induces the production of a vascular cell adhesion molecule 1[101517 ] and stimulates the production of nitric oxide ( no ) . in this study , the flaps treated with hbo alone led to improvement in the average survival than those treated with a combination therapy with both hbo and nac , suggesting that these agents do not potentiate one another . the groups treated with distilled water and nac alone exhibited the worst results in this experiment . the doses of nac ( 300 mg / kg / day ) utilized in this study were chosen owing to the reported low toxicity of this drug and favourable results in the protection of random skin flaps in rat model . the plastic barrier interposed between the flap and donor bed hbo in association with nac may be responsible for a protective effect on the deleterious outcome of nac . when nac is used alone , the measures of necrotic areas were found to be higher in the g - nac ( 24% ) . on the other hand , with the combination of hbo and nac , the measures of necrotic areas probably , in this study , the correct concentration of nac could have inhibited angiogenesis and wound - healing response . this inhibition is considered to occur through an imbalance in the cellular redox state or an undetermined mechanism . it has been demonstrated that hbo can increase the tolerance of tissue to ischemia , diminish metabolic disturbances , improve tissue microcirculation , and reduce platelet aggregation . these characteristics , combined with the ability of plasma to carry dissolved oxygen to areas where red blood cells can not reach , have been shown to have a beneficial effect on oxygenation of many hypoxic tissues . in the present study , the combination of hbo and antioxidants therapy did not improve survival above and beyond the effect of hbo administered alone , suggesting that the potential toxic effects of hyperoxia from ros were not minimized by antioxidant therapy with nac . oxidizing species , such as free radicals and hydrogen peroxide , may serve as cellular messengers mediating complex redox - sensitive processes , such as extracellular matrix formation , cytokine action , angiogenesis , and cell motility . knight et al . demonstrated in rabbits that high dose of nac has no significant difference in the survival of flaps . furthermore , ramon et al . showed an increase in the survival of the transverse rectus abdominis myocutaneous flap treated with hbo alone . demonstrated in rats that hbo associated with vitamin e and c could improve the survival of epigastric island skin flaps , while rocha et al . reported that hbo alone showed a protective effect in the ischemic skin flap , which was associated with reduced expression of apoptosis however , despite these results , multicenter prospective clinical studies are clearly needed to compare hbo treatment with other mechanical or pharmacologic interventions , to improve wound healing for grafting or to support flap survival . with the experimental model , it can be concluded that hbo produced an improvement in the distal blood supply of the random skin flap ; however , this improvement was not significant . furthermore , the effect of combination with nac was not significantly different on the survival of flap , when compared with the hbo therapy alone .

objective : our aim is to investigate the role of hbo ( hyperbaric oxygen ) , nac ( n - acetylcysteine ) , and hbo plus nac on the necrosis area of random rat 's skin flaps of a modified mcfarlane flap design.materials and methods : thirty - two male wistar rats were randomly divided into four groups : g - s ( sham : n = 8) , g - nac ( nac : n = 8) , g - hbo ( hbo : n = 8) , and g - hn ( hbo plus nac : n = 8) . a rectangular skin flap ( 2 8 cm2 ) was dissected from the muscular dorsal layer , preserving the cranial pedicle . polyethylene film was placed over the muscular layer and an interrupted 3.0 nylon suture was employed to fix the flap into the original place . on the eighth day , full - thickness biopsy samples ( 2 1 cm2 ) were collected from the proximal , middle , and cranial areas of the skin flap , and in a site away from the flap labelled as the control area.results:the measurements of necrotic areas in the groups were 18.3% in g - s , 24.3% in g - nac , 12.6% in g - hbo , and 14.9% in g - hn . significant difference was observed between the groups g - hbo and g - hn as well as g-nac.conclusion:hbo is associated with reduced area of necrosis of skin flap . the g - nac group was associated with poor results when examined in isolation . the association between hbo and nac did not produce favourable results with respect to the use of hbo alone . these findings suggest that the diffusion of oxygen through the interstitial space was the determining factor of more favourable results of hbo .

INTRODUCTION MATERIALS AND METHODS Ethical committee Animal housing and groups Anaesthesia Surgical procedure NAC and distilled water administration procedures HBO procedure Sequential daily procedures Seven days follow-up Euthanasia Determination of flap necrosis area Statistical analysis RESULTS DISCUSSION CONCLUSION

cytomegalovirus ( cmv ) infection is a common cause of morbidity and mortality among patients receiving immunosuppressive treatment . gastrointestinal infection by cmv includes mainly involvement of the large bowel , especially in patients with underlying inflammatory bowel disease . gastritis caused by cmv infection in immunocompromised patients , although quite rare , seems to be important from both a clinical and therapeutic point of view . moreover , diffuse gastritis due to cmv infection has been described in bone marrow transplant patients , in patients after cord blood transplantation for acute myeloid leukemia and after rituximab treatment for non - hodgkin 's lymphoma . it must be stressed however that development of cmv gastritis and gastric ulcer has also been noticed in immunocompetent subjects . gastric outlet obstruction ( goo ) due to cmv infection has very rarely been described . we present a woman with long - standing rheumatoid arthritis who developed diffuse gastritis accompanied by gastric ulcer and goo due to cmv infection while receiving methotrexate and non - steroidal anti - inflammatory drugs for a long period of time . a woman aged 67 years was admitted to our department because of abdominal pain located mainly in the epigastrium and right side of the abdomen accompanied by weight loss and fatigue . the symptoms had appeared eight months before with a clinical episode compatible with gastroenteritis . personal history included rheumatoid arthritis and the patient had been on regular treatment with methotrexate for more than twelve years . during the last few months she was a current smoker . on physical examination , except for some tenderness on palpation of the epigastrium and right abdomen , no significant findings were noticed . g / l ) and hematocrit ( 31% ) , increased erythrocyte sedimentation rate ( 39 mm/1st h ) and fibrinogen ( 411 mg / dl ) , increased crp and a1-acid - glycoprotein ( 17 and 1.44 serum tumor markers , gastrin , vitamin b12 and folic acid were normal . blood and stool cultures were negative . igg and iga antibodies against yersinia enterocolitica [ yop m ( 2a ) ] ( western blot ) were negative . serum igg anti - cmv antibodies were positive while serum igm anti - cmv antibodies were negative . a computerized tomography scan ( hydro - ct ) of the abdomen revealed thickening of the gastric wall located mainly in the antrum . upper gastrointestinal endoscopy demonstrated multiple linear exudative gastric ulcers , the largest being 3 cm in diameter . the 9 mm endoscope was finally passed through the pylorus , although with great difficulty . histological examination of specimens obtained from the gastric ulcer , the surrounding area and other parts of the stomach revealed the presence of diffuse gastritis . abundance of hyperchromatic epithelial cells with nuclear viral inclusions ( owl 's eye ) was demonstrated . repeat upper gastrointestinal endoscopy performed few days later because of worsening of symptoms , as well as histology of gastric specimens , confirmed the previous results . however , this time and despite great efforts , passing the endoscope through the pylorus was impossible . consequently , treatment with intravenous ganciclovir ( 2 5 mg / kg / day ) was initiated with slow clinical improvement seen after 3-week treatment . however , after 6 weeks continuous treatment her symptoms resolved almost completely and on a subsequent endoscopy only signs of healing ulcers were seen . the intriguing element in this case is the atypical manifestation of the patient 's disease state , namely goo accompanied by severe gastric ulceration without the heralding symptoms of emesis , inability to eat , bloating , early satiety and epigastric fullness . the interest in this case is the clinical scenario in which cmv , although a common viral factor especially in immunocompromised patients , was not an asymptomatic bystander , but instead was etiologically associated with the disease . the present case obviously leads to the assumption that the appearance of goo in immunocompromised patients must guide the physician not to proceed immediately with surgical restoration of the continuity of the gastrointestinal tract , but to wait for the results of the histology . after confirmation of diagnosis of cmv infection , the physician must also wait for the results of the specific antiviral treatment before considering a surgical approach . our case also raised some diagnostic dilemmas because of the absence of pathognomonic endoscopic features for cmv infection and also created difficulties concerning both the right medical treatment and the best way of follow - up . the message for the clinical gastroenterologist is to search for the possibility of the existence of cmv infection in immunocompromised patients with a clinical picture compatible with goo by obtaining a large number of biopsies and by asking specifically for cmv staining , especially if infection by helicobacter pylori is not present . an important point regarding the diagnosis and course of cmv infection could be the number of infected cells in the biopsy specimens in relation to the severity of endoscopic lesions . in a relevant study , no correlation between cmv inclusion bodies count and severity of symptoms or length of survival could be demonstrated . on the other hand , coexistence of other potential pathogens , such as herpes simplex , mycobacteria and candida , the coexistence of other infective agents was excluded by clinical and laboratory findings ( normal chest x - ray , negative mantoux test ) as well as special staining of biopsy specimens ( ziel nielsen - pas ) . another interesting point in this case was the negative serum igm anti - cmv antibodies despite the presence of cmv in the gastric biopsies . however , it is generally accepted that in patients with active cmv infection , negative serologic test can be obtained as this test is an unreliable marker of diagnosis of current infection due to low sensitivity . described two immunocompetent patients with gastric ulcer as the only manifestation of cmv infection . both patients required antiviral treatment , and one case evolved to goo requiring surgery the latter case , as well as the case described by mohammed et al . and our own , are probably the only cases of cmv gastritis accompanied by goo described so far in the literature . some descriptions suggest that the clinical picture of cmv gastritis is dominated by epigastric pain having some special characteristics : it is decreasing in supine position of the patient and it is increasing while sitting , standing or walking . in our patient the cardinal symptom was also abdominal pain mainly located in the epigastrium and having the same characteristics as those described in the literature . diagnosis of cmv gastritis can be very difficult if one relies only on the findings of upper gastrointestinal endoscopy as the endoscopic picture is not characteristic . the protean radiologic features are not specific either and may mimic several other conditions . it has been suggested that diagnosis of cmv gastritis can be facilitated by the use of qualitative pcr . however , we suggest that clinical suspicion plays the most important role in making the correct diagnosis . the clinician should always consider cmv infection as a possible cause of gastritis , especially in immunosuppressed patients . consequently , the endoscopist must advise the histopathologist accordingly in order to search for the presence of cmv infection . the pathophysiological consequences of cmv gastritis are not well understood either , although reduced gastric emptying and dysmotility have been described . in all cases of cmv gastritis described so far , epigastric pain completely resolved after treatment with ganciclovir , as it happened in our patient . prolonged low - dose ganciclovir prophylaxis after heart transplantation reduces the incidence of cmv disease in cmv - mismatched patients and reduces the morbidity of cmv disease in cmv - positive recipients . we can not answer the question whether the aphthous ulcers seen in the terminal ileum in the first colonoscopy were actually part of a disseminated cmv infection or the result of the consumption of nsaids . however , the disappearance of the lesions in the follow - up colonoscopy after completion of anti - cmv treatment might be a clue to cmv ileitis . goo caused by cmv infection can be noticed in immunocompromised patients . the clinician must search for the presence of cmv infection in all immunocompromised patients presenting with a clinical picture of goo by obtaining enough biopsies and by asking the histopathologist to specifically stain the specimen for cmv infection , especially if infection by helicobacter pylori is not present .

a case of gastric outlet obstruction accompanied by diffuse gastritis and gastric ulcer due to cytomegalovirus infection is presented . the patient , a woman aged 67 years with a long history of rheumatoid arthritis under immunosuppressive treatment ( methotrexate ) , was admitted to our department complaining mostly of abdominal pain located in the epigastrium and the right abdomen . upper gastrointestinal endoscopy revealed the presence of gastritis accompanied by ulcer in the prepyloric area and gastric outlet obstruction due to cytomegalovirus infection which was confirmed histologically . the patient responded well although after many weeks to specific treatment with antiviral treatment ( ganciclovir ) . it is concluded that gastric outlet obstruction caused by cytomegalovirus infection can be observed in immunocompromised patients . the clinician must search for possible cytomegalovirus infection in all immunocompromised patients presenting with a clinical picture of gastric outlet obstruction by obtaining enough biopsies and by asking the histopathologist to specifically stain the specimen for the presence of cytomegalovirus , especially if infection by helicobacter pylori is not present .

Introduction Case Report Discussion Conclusion

liver diseases caused by various factors , including viruses , chemicals , drugs , alcohol , genetic factors , or a patient 's own immune system , represent a significant public health problem because liver damage can progress to liver failure . liver inflammation may be acute , flaring up and resolving within a few weeks to months , or chronic , enduring over many years . chronic hepatitis can simmer for 20 years or more before causing significant symptoms related to advanced liver damage , including cirrhosis ( scarring and liver failure ) , liver cancer , or death . hepatitis represents a worldwide health problem in humans , for which the currently available pharmacological treatments are inadequate . however , the development of new drugs requires proper animal models relevant to human hepatitis [ 2 , 3 ] . most liver diseases , such as viral hepatitis , autoimmune hepatitis , primary biliary cirrhosis , primary sclerosing cholangitis , and liver allograft rejection , are caused by activated t lymphocytes , which infiltrate and destroy the liver parenchyma , leading to liver injury . the injection of mice with the t - cell mitogenic plant lectin concanavalin a ( cona ) results in the polyclonal activation of t lymphocytes , inducing a liver - specific inflammatory response that mimics activated t - cell - mediated hepatitis . cona - induced hepatitis is an ideal animal model that simulates the pathophysiology of human viral and autoimmune hepatitis and has been used extensively to clarify various aspects of human t - cell - mediated liver diseases [ 511 ] . two mechanisms , apoptosis and necrosis , are frequently involved in acute and chronic liver injury . apoptosis is a precisely regulated and genetically determined process that can be induced by death - receptor - mediated extrinsic pathways or by intrinsic mechanisms activated by intracellular stress . apoptosis is executed by a group of intracellular cysteine proteases called caspases . with caspase activation and the subsequent cleavage of their intracellular substrates , in contrast , necrosis is associated with organelle swelling , cytoplasmic membrane breakdown , and ensuing inflammatory responses . recently , a novel type of cell death called necroptosis was identified by degterev et al . . these researchers used tumor necrosis factor- ( tnf- ) and fas ligand to induce necroptosis in different cells , including jurkat cells and mouse embryonic fibroblasts [ 16 , 17 ] . necroptosis , or programmed necrosis , is a caspase - independent mode of cell death that prevails when caspases are not activated or are inhibited . the term necroptosis refers only to regulated necrotic cell death , which is mediated by receptor - interacting protein 1 ( rip1 ) kinase activity . therefore , by definition , necroptosis is inhibited by necrostatin-1 ( nec-1 ) , 5-(1h - indol-3-ylmethyl)-3-methyl-2-sulfanylideneimidazolidin-4-one , a small - molecule inhibitor of programmed cell necrosis that has shown promise as a neuroprotectant in adult rodent models of myocardial ischemia and traumatic and ischemic brain injury [ 16 , 19 ] . the inhibition of necroptosis by nec-1 reduced the infarct volume and ameliorated the neurological deficit in a mouse model of middle cerebral artery occlusion . however , recent studies have revealed that the receptor - interacting serine - threonine kinase family member rip plays a crucial role in regulating the switch between apoptosis and necroptosis . when caspases are inhibited , stimulation with tnf- triggers necroptosis , mediated by rip1 and rip3 . the mechanism by which rip kinases orchestrate apoptotic or necroptotic cell death has been studied intensively [ 22 , 23 ] . this type of cell death is characterized by the massive degradation of the cellular contents , including essential organelles such as the mitochondria , in membrane vesicles called autophagosomes , which then fuse with lysosomes to become autolysosomes [ 24 , 25 ] . autophagy allows a cell to survive in the harsh environment with self - help behavior . to a certain extent , autophagic cells play a protective role but , beyond this point , result in tissue damage . there is increasing evidence that blocking the cell death pathways can significantly reduce the organ and tissue damage caused by inflammation [ 26 , 27 ] . therefore , reducing liver cell death is considered to be a reasonable treatment for potential acute liver injury . nec-1 protects cells by inhibiting rip1 kinase activity and possible downstream anti - inflammatory effects and perhaps by other mechanisms . currently , the mechanism by which nec - l inhibits cona - induced acute liver injury and cell death is unclear . with the establishment of animal models of experimental liver injury , it is possible to explore the mechanism of nec-1 's action , to screen drugs , to treat acute liver injury , to examine the pathophysiological changes that occur in acute liver injury , and to significantly improve the level of clinical treatment available . therefore , this study is designed to assess the role of rip1 and autophagy in the protective effect of cona - induced liver injury . we show that preconditioning of nec-1 can inhibit the rip1 kinase pathways and reduce the cona - induced injury in vivo . in addition , we also found that nec-1 treatment can degrade the level of autophagy in hepatocytes after cona - induced injury . cona , dimethyl sulfoxide ( dmso ) , and rip1 were purchased from sigma aldrich ( st . louis , mo , usa ) . antisera against microtubule - associated protein light chain 3 ( lc3 ) and beclin-1 were purchased from abcam ( abcam cambridge , ma , usa ) . anti - rip1 ( 0.2 g / ml ; h-207 , santa cruz biotechnology , santa cruz , ca , usa ) is a rabbit polyclonal antibody that does not cross react with rip2 or rip3 . anti - tnf- ( 0.1 g / ml ; millipore ; billerica , ma , usa ) is a rabbit polyclonal antibody raised against recombinant mouse tnf-. other antibodies used in the present study include goat anti - il2,6 and goat anti - caspase polyclonal antibodies ( santa cruz biotechnology ) . the rna pcr kit was purchased from takara ( takara biotechnology , dalian , china ) . dulbecco 's modified eagle 's medium and fetal bovine serum were obtained from invitrogen corporation ( carlsbad , ca , usa ) . male c57bl/6 mice ( 68 weeks old , 20 2 g ) were obtained from shanghai slac laboratory animal co. ltd ( shanghai , china ) . they were housed in plastic cages with controlled light - dark cycles and fed a standard diet with water , in a controlled - temperature ( 25 1c ) and humidity ( 50 5% ) environment . the experiments were performed according to the national institutes of health guidelines for the care and use of laboratory animals and were approved by the committee on the ethics of animal experimentation of hirosaki university . the mice were housed and maintained under specific - pathogen - free conditions in the animal resource facility of university of tongji , and all experiments were preapproved by the institutional animal care and use committee of that institution ( iacuc approval no . cona was dissolved in pyrogen - free normal saline solution ( nss ) at a concentration of 2.5 mg / ml and injected intravenously at a dose of 20 mg / kg body weight to induce hepatitis , as previously described . nec-1 was dissolved in 0.5% dmso at a concentration of 5 mg / ml and was injected intraperitoneally at a dose of 1.8 mg / kg body weight . the mice were randomly divided into four groups : the normal control group was injected via the tail vein with nss alone ; the vehicle group was injected intraperitoneally with dmso without cona ; the model cona group was injected via the tail vein with cona without nec-1 treatment ; the nec-1 pretreatment group was injected intraperitoneally with nec-1 1 h before the cona challenge . at the end of the experiments , the mice were killed at the indicated time points after cona injection , and their serum and liver tissue samples were collected . at the same time , we calculated the differences in the mortality of the mice . hepatocyte damage was assessed at the indicated time points after cona injection with the measurement of plasma alanine transaminase ( alt ) and aspartate transaminase ( ast ) activities using an automated clinical analyzer ( olympus au1000 ; olympus , tokyo , japan ) . the other groups of animals were subjected to a tail vein injection of cona ( 20 mg / kg ) , and their orbital blood was collected after 8 h , 12 h , and 20 h. the animals were killed after anesthetization with an intraperitoneal injection of sodium pentobarbital ( 62.5 mg / kg ) . paraffin sections ( 5 m thick ) were stained with hematoxylin - eosin ( h&e ) and examined by light microscopy for inflammation and tissue damage by one blinded pathologist . the percentage injury was determined by measuring the total area of portal inflammation , lobular inflammation , and necrosis and comparing it with the uninjured area in the corresponding region . the liver tissue specimens were cut into 4 m sections and dewaxed , hydrated , and pretreated with a heat - induced antigen retrieval technique . the sections were blocked and incubated overnight at 4c with antibodies directed against rip1 , tnf- , and lc3 at 1 : 100 dilutions and with a secondary antibody diluted 1 : 50 for 60 min at room temperature . each antibody was diluted in tris - buffered saline ( tbs ) , 2% bovine serum albumin ( bsa ) . the negative control antibodies consisted of species - matched immunoglobulin ( ig ) fractions , which were also immunoglobulin g- ( igg- ) subclass - matched where appropriate , used at the same dilution as the secondary antibodies . staining was documented with a digital camera ( olympus ) mounted on a microscope ( leica , wetzlar , germany ) at 200 magnification . hepatocytes with brown staining in the cytoplasm demonstrated the normal localization of rip1 , tnf- , and lc3 . ten separate high - power fields were chosen randomly in each section , and five mice from each group were examined . total rna was isolated and transcribed into cdna using the rneasy mini kit ( qiagen , hilden , germany ) and the high capacity cdna reverse transcription kit ( applied biosystems , foster city , ca , usa ) , respectively . the resulting cdna was used as the template for pcr with primers specific for tnf- , ifn- , il2 , il4 , il6 , il10 , and glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) ( see table 1 ) . frozen livers were sonicated in phosphate - buffered saline ( pbs ) containing 0.1% tween 20 and protease inhibitors . an aliquot ( 90 g ) of each liver lysate was separated on a 10% sodium dodecylsulfate - polyacrylamide gel and transferred onto a nitrocellulose membrane ( hybond ecl ; amersham biosciences , freiburg , germany ) . the membranes were incubated in blocking buffer ( 5% nonfat milk powder in tbst [ tbs with 0.1% tween 20 ] ) for 3 h and then incubated overnight at 4c with gentle shaking with specific primary antibody directed against rip1 ( diluted 1 : 1000 ) and monoclonal anti - tnf- ( 1 : 500 ) , anti - ifn- ( 1 : 500 ) , anti - il2 ( 1 : 500 ) , anti - il6 ( 1 : 500 ) , anti - lc3 ( 1 : 500 ) , or anti - caspase-3 ( 1 : 1000 ) antibody . after the membrane was washed , it was incubated at room temperature for 1 h with a horseradish - peroxidase - labeled secondary antibody ( sigma - aldrich ) . the membranes were visualized with enhanced chemiluminescence ( supersignal ; pierce , rockford , il , usa ) . fresh liver tissues collected from the mice were fixed in 4% paraformaldehyde on ice for 1 h. the fixed liver tissues were washed three times with pbs for 5 min on ice before they were dehydrated overnight in 30% sucrose ( dissolved in pbs ) at 4c . the tissues were infiltrated with oct for 2 h on day 2 and then frozen and stored at 80c . sections ( 5 m ) were cut with a freezing microtome and stored at 20c . before analysis , the prepared sections were dried at room temperature for 5 min , and the oct was dissolved in pbs for 5 min . the cell membranes were ruptured with 0.2% triton x-100 at room temperature for 20 min . nonspecific antigen - binding sites were blocked with 5% bsa , and the sections were then incubated overnight with antibody directed against tnf- , rip1 , beclin-1 , lc3 , il2 , or il6 ( 1 : 1000 ) at 4c . after the samples were incubated with anti - rabbit antibody for 30 min on day 2 , the cell nuclei were stained with dapi ( 4,6-united amidine-2-phenylindole ) ( 1 : 1000 ) . mice were treated as described above , and the liver was flushed with 1 ml nss and then perfused with 2 ml 4% glutaraldehyde in pbs and postfixed in 1% oso4 . livers were sectioned and photographed using a transmission electron microscope ( jeol , jem 1230 ) at 80 or 60 kv onto electron microscope film ( kodak , estar thick base ) and printed onto photographic paper . differences among the experimental groups were determined with the unpaired student 's t - test or analysis of variance ( anova ) followed by the bonferroni post hoc test or tukey 's test when f was significant . the blank control group mice had good statement ; their activity , behavior , and vital signs were normal and no animal died . two hours after injection with cona , the model group showed reduced activity , apathy , vertical hair , and coiled body signs . at 12 h , mouse mortality was 13% in the model group , which differed significantly from that in the blank control group ( p < 0.05 ; see table 2 ) . the vehicle ( dmso ) group and model ( cona ) group showed similar symptoms . the intervention ( nec-1 ) group mice also displayed the symptoms and signs of acute liver injury , but they were alleviated relative to those in the model group . the overall mortality rates in the vehicle group , cona group , and nec-1 group were 30% , 27% , and 7% , respectively . the difference between the nec-1 group and cona group was statistically significant ( p < 0.05 ) , but there was no significant difference between the vehicle group and the cona group ( p > 0.05 ) . this suggests that nec-1 reduced the death rate in mice with cona - induced acute liver injury ( see table 3 ) . to determine the protective effects of nec-1 against cona - induced liver injury , nec-1 first , we established a mouse model of acute liver injury using the tail vein injection of cona ( 20 mg / kg ) . serum alt and ast levels and liver tissue biopsies were examined as indicators of liver injury . at 8 , 12 , and 20 h after injection with cona , serum alt and ast levels had changed dynamically ( alt : 810.8 340.9 u / l at 20 h , with a peak of 1888.4 155.9 u / l at 12 h ; ast : 667.6 149.9 u / l at 20 h , with a peak of 902.4 150.8 u / l at 12 h ) ( see table 4 and figures 1(a ) and 1(b ) ) . serum alt and ast levels were significantly elevated in the mice after the administration of cona , whereas nec-1 pretreatment significantly attenuated the cona - induced elevation of serum alt and ast ( see table 5 and figures 1(c ) and 1(d ) ) . to examine the histological changes in the liver after cona injection in the presence or absence of nec-1 , we stained the liver tissues with h&e . at 8 , 12 , and 20 h after cona injection , the hepatic lobules showed inflammatory cells infiltrating the confining ducts , large numbers of infiltrating mononuclear lymphocytes , and a proportion of necrotic liver cells in cona - induced liver injury model groups ( figure 2(a ) vii ix ) . flaky necrosis was visible 20 h after the cona injection with a pathological examination of the liver tissues ( figure 2(a ) ix ) . histopathological studies of the mouse livers were used to determine the effects of nec-1 on cona - induced liver injury . as shown in figure 2(b ) , a light microscopy examination revealed extensive inflammatory infiltration and large areas of necrosis in the livers of the cona - treated mice . in contrast , mice pretreated with nec-1 showed minor liver damage ( figure 2(b ) vii ix ) . we conducted a detailed analysis of the mrna expression of intrahepatic cytokines ( il2 , il4 , il6 , il10 , tnf- , and ifn- ) in untreated ( blank ) , vehicle - treated , cona - treated , and nec-1-treated mice using pcr . our results show that , in the cona - treated model mouse liver tissues , tnf- , ifn- , il2 , il4 , and il6 mrna expression was significantly higher than in the blank control group . after the nec-1 intervention , the expression of these cytokines ( tnf- , ifn- , il2 , il4 , and il6 ) was significantly reduced compared with that in the model group ( p < 0.05 ) , but the expression of il10 did not differ significantly ( p > 0.05 ) from that in the model group ( figure 3(a ) ) . thus , cona - induced hepatic injury is associated with changes in the levels of inflammatory cytokines . the levels of proinflammatory cytokines such as ifn- , il2 , and il6 in the liver tissues were assayed by western blotting after the administration of cona to untreated ( blank ) , dmso - pretreated , and nec-1-pretreated mice . western blotting showed that ifn- , il2 , and il-6 protein expression in the model group showed upward trends , whereas their expression was lower in the nec-1 intervention group , and the differences were statistically significant ( p < 0.05 ; figure 3(b ) ) . as shown in figure 3(c ) , immunofluorescence showed that the expression of il2 and il6 was significantly lower in the nec-1 intervention group than in the untreated and dmso - pretreated model group . its serine / threonine kinase activity is essential for the necrotic death pathway but is not required for either nf-b activation or apoptosis , which rely on the intermediate and death domains of the protein . the small - molecule inhibitor of necroptosis , nec-1 , is a potent inhibitor of rip1 kinase activity . therefore , we investigated whether nec-1 affects hepatic rip1 expression , using western blotting and immunocytochemical staining in mice with cona - induced liver injury . the hepatic expression of rip1 protein was examined at 8 , 12 , and 20 h after the cona challenge . as shown in figure 4(a ) , rip1 protein expression was significantly upregulated at 12 h in the cona - treated group and was clearly downregulated in the nec-1-pretreated group . furthermore , as shown in figure 4(d ) , the rip1 levels in the nec-1 group were significantly reduced in the cytoplasm of the hepatocytes and in the area of necrosis 12 h after cona injection compared with those in the negative control and vehicle groups . these data suggest that nec-1 pretreatment inhibits rip1 release and expression , thus protecting the mice from cona - induced hepatic injury . it is well known that rip1 is more strongly expressed after cona - induced liver injury . therefore , we examined the changes in rip1 in the liver tissues collected from the three experimental groups after 12 h , using immunofluorescence . rip1 was clearly localized in the cytoplasm and was expressed at low levels in the nec-1 group . therefore , the levels of proinflammatory cytokines such as tnf- in the liver tissues were assayed by western blotting after the administration of cona to the model and nec-1-pretreated mice . as shown in figure 4(a ) , the increase in intrahepatic levels of tnf- in response to cona was prevented by pretreatment with nec-1 . therefore , the prevention of cona - induced liver injury by nec-1 is associated with the inhibition of the release of proinflammatory cytokines , such as tnf-. cleavage by caspase 3 was measured to establish the extent of hepatocyte apoptosis . as shown in figure 4(b ) , hepatocyte apoptosis was observed in the livers of mice treated with cona . these data suggest that nec-1 pretreatment does not affect hepatocyte apoptosis in cona - induced hepatitis but confirm that necroptosis and apoptosis are two different types of programmed cell death . liver tissues with cona - induced injury exhibited an increased autophagic flux and enhanced conversion of lc3 , a marker of the activation of autophagy in liver tissues . cona induced autophagosome formation in the hepatocytes , which was detected with tem , and this was significantly reduced by the nec-1 intervention , as was the expression of the autophagy - related proteins beclin-1 and lc3 , detected with immunofluorescence and western blotting ( figures 5(a)5(c ) ) . electron microscopic assessment of cona - induced acute liver injury at 12 h showed the ultrastructural morphological changes of liver tissue necrosis . the cona model group displayed large areas of necrotic disintegration , with uniform nuclear chromatin in the hepatocytes and a small amount of condensation , although the integrity of the nuclear membrane was less clear ; there were many mitochondria within the cytoplasm , but the organellar structure was not clear , although phagosomes were visible . the nec-1 signal in the hepatic nuclear chromatin was homogeneous , with a small amount of agglutination . the membrane integrity was unclear , there were many mitochondria within the cytoplasm , and the structures still had integrity ( figure 5(d ) ) . in this study , we found that cona - induced liver injury was ameliorated in nec-1-treated mice and that this amelioration was accompanied by a reduction in the expression of proinflammatory cytokines and rip1 . serum alt and ast levels were significantly reduced in the nec-1-treated mice after injection with cona compared with those in the model mice . liver diseases with various etiologies , including viral and autoimmune hepatitis , represent a significant health problem , and , in most cases , there are no effective medical therapies . the death receptor family plays a critical role in regulating cell numbers and eliminating harmful or virally infected cells . agonistic stimulation of the death receptors is known to lead to two alternative cell fates : either the activation of nf-b to promote cell survival or the induction of apoptosis , which leads to cell death . now , a third pathway , designated necroptosis or programmed necrosis , has been identified . interestingly , a death - domain - containing kinase , rip1 , is involved in the mediation of all three pathways , and its kinase activity is specifically involved in regulating necroptosis . the availability of nec-1 , a specific inhibitor of rip1 kinase , has made it possible to distinguish the distinct functional domains of rip1 . necrostatin,1,5-(1h - indol-3-ylmethyl)-(2-thio-3-methyl)-one , a small - molecule inhibitor of programmed cell necrosis , has shown promise as a neuroprotectant in adult rodent models of myocardial ischemia and traumatic or ischemic brain injury [ 16 , 28 , 32 ] . nec-1 protects cells by inhibiting rip1 kinase activation , with possible downstream anti - inflammatory effects , and by other mechanisms [ 19 , 28 ] . a recent study showed that treatment with nec-1 reduced necrotic cell death and increased apoptotic cell death . nec-1 also reduced hypoxia - ischemia - induced oxidative damage to proteins and attenuated markers of inflammation coincidental with reduced activation of nuclear factor-b and caspase 1 . in vitro , necrostatin has little effect on classic apoptotic cell death but , instead , strongly inhibits programmed cell necrosis . cona , a lectin with mannose specificity that can induce acute hepatic inflammation , was tested for its therapeutic effects on hepatoma . cona - induced hepatitis is a well - known murine model of fulminant hepatitis attributed to excessive immune responses , such as autoimmune hepatitis . previous studies have shown that the pathogenesis of cona - induced hepatitis is mainly mediated by the release of inflammatory cytokines . of those cytokines , tnf- is secreted by hepatic kupffer cells and acts as a major mediator of inflammation - induced hepatocyte apoptosis when it binds to its receptors . the identification of the rip1 kinase inhibitor nec-1 has allowed researchers to identify the involvement of necrotic cell death in an increasing number of pathological conditions . indeed , rip1 kinase activity is not essential under most apoptotic conditions , but it is crucial for the activation of the regulated form of necrosis recently designated necroptosis . pcr analysis revealed that the expression of il2 , il4 , il6 , il10 , tnf- , ifn- , and gapdh mrnas in the livers of untreated ( blank ) and dmso - treated mice was significantly upregulated 12 h after their injection with cona ( figure 3 ) . nec-1 is a small molecule chosen from an extensive chemical library for its ability to inhibit the cell death caused by tnf- stimulation in the context of caspase inhibition . it is an allosteric inhibitor of rip1 and is a key signaling intermediate in the cell death described as programmed necrosis . in vitro , the inhibition of rip1 kinase blocks the cell death caused by death - receptor signaling in the presence of caspase inhibition . in experimental models of acute liver injury , liver cell death mainly occurs through necrosis and apoptosis , and different models of hepatic inflammation show different apoptosis / necrosis ratios . the severity of the experimental acute inflammation in the liver correlates positively and directly with the degree of liver cell necrosis and correlates negatively with the level of apoptosis , the exact mechanism of which is not yet clear . in the present study , there was no significant change in the expression of rip1 in the first 8 h in the mouse model of acute liver injury induced with cona , but its expression was significantly upregulated in the first 12 h and 20 h after cona treatment . programmed necrosis in acute liver injury and its underlying molecular mechanism have not been clarified . zhang et al . reported that , in rip3 mice produced using gene knockout technology , cona - induced acute liver damage with liver cell necrosis was significantly reduced . our research and other studies indicate that programmed necrosis may be involved in cona - induced acute liver injury . nec-1 is a specific chemical inhibitor of programmed necrosis , and has been shown to inhibit the phosphorylation of rip1 , thereby inhibiting rip1 function and the necrotic process . nec-1 also protects against hypoxia - ischemia / reperfusion brain damage and inhibits retinal - detachment - caused photoreceptor cell necrosis and retinal ischemia / reperfusion injury . if acute liver injury is a necrosis - related disease , does nec-1 have a similar protective effect in the liver ? to address this question , we investigated the effects of nec-1 administered by intraperitoneal injection on cona - induced acute liver injury in vitro . we found that nec-1 significantly reduced the levels of serum transaminases , inhibited liver cell necrosis , reduced the severity of acute liver injury , and confirmed the presence of programmed necrosis . liver cell necrosis and inflammatory responses play an important role in the development of acute liver injury . the necrosis of liver cells is an important source of the inflammatory response and the increased production and release of inflammatory cytokines ; nec-1 does not inhibit tnf--induced nf-b activation . therefore , we speculate that the inhibition of the production of inflammatory mediators by nec-1 is caused by its reduction of necroptosis during cona - induced hepatitis . nec-1 prevents the programmed necrosis of hepatocytes by inhibiting the expression of tnf- and rip1 in vivo , thereby inhibiting the process of hepatocyte necrosis . this confirms that the nec-1 intervention can reduce cona - induced acute liver injury . recent evidence supports the view that enhancing autophagy may be a novel approach to improve hepatocyte viability and function after i / r injury , and such a hepatoprotective role of autophagy may be associated with its antiapoptotic and anti - inflammatory activity . in the present study , we found that autophagy was inhibited by nec-1 preconditioning in hepatocytes in vitro . these results suggest that autophagy is also a key mediator in the hepatoprotective effect of nec-1 intervention . in summary , our study is the first to confirm that nec-1 has a significant protective effect against acute liver injury in mice , suggesting that the programmed necrosis of liver cells may have important clinical implications for the inhibition of acute liver injury , providing new ideas and targets for the treatment of this disease . nec-1 , the role of its targets , and the molecular mechanism of programmed necrosis require further study .

objective . necrostatin-1 ( nec-1 ) inhibits receptor - interacting protein 1 ( rip1 ) kinase and programmed necrosis . this study was designed to examine the protective effects and mechanisms of nec-1 in concanavalin a- ( cona- ) induced hepatitis in mice . methods . c57bl/6 mice were exposed to cona via tail vein injection and injected intraperitoneally with nec-1 or vehicle . levels of serum liver enzymes and histopathology were determined . levels of inflammatory cytokines with cona - induced hepatitis were determined with real - time polymerase chain reaction ( real - time pcr ) . the expression of tnf- , rip1 , and lc3 was detected with immunohistochemical staining . the expression of tnf- , ifn- , il2 , il6 , caspase 3 , rip1 , beclin-1 , and lc3 protein was assessed by immunofluorescence and western blotting . autophagosomes were observed with transmission electron microscopy ( tem ) . results . amelioration in liver functions and histopathological changes and the suppression of inflammatory cytokine production were observed in nec-1-injected mice . western blotting analysis showed that the expression of tnf- , ifn- , il2 , il6 , and rip1 was significantly reduced in the nec-1-injected mice , which was confirmed by immunofluorescence and immunohistochemistry . autophagosome formation was significantly reduced by nec-1 treatment , as the expression of beclin-1 and lc3 , determined with immunofluorescence and western blotting . conclusion . these results demonstrate that nec-1 prevents cona - induced liver injury via rip1-related and autophagy - related pathways .

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

breast cancer is the most common cancer affecting women and has an enormous impact on their health . the incidence of breast cancer varies between countries with the highest incidence in the united states and northern europe . in the united states breast cancer makes up 30% of all cancers in women , while in the netherlands the lifetime risk for a woman for developing breast cancer is about 11% . the aetiology of breast cancer is varied : inherited genetic susceptibility , acquired genetic changes , and effects of endogenous and exogenous environment factors . there is limited and indirect evidence that self - examination and physical examination can help in decreasing mortality because tumours smaller than 10 mm will not be detected in the majority of the cases . most breast cancers are detected with mammography in either a screening situation or by the discovery of a palpable breast mass . randomised trials have shown that screening with mammography in the age category of 5070 years can reduce mortality by about 25% . however , there is no consensus at the moment about the value of screening younger women with mammography . one of the reasons is the lower sensitivity of mammography in women below the age of 50 years . this is because young , pre - menopausal , women have denser breasts compared to post - menopausal women , resulting in an increased chanced that a malignancy will be missed on mammography . in a diagnostic setting , magnetic resonance imaging ( mri ) is a very sensitive tool for the detection of breast cancer . especially for invasive breast cancer , the sensitivity of this imaging technique this sensitivity is not influenced in any way by the amount of glandular tissue present in the breast . the role of mri as a screening modality has not yet been outlined . in the literature , mri has only been evaluated as a screening tool for women with an increased risk for developing breast cancer [ 68 ] . in this paper the role of mri in the screening of women with an increased risk for developing breast cancer is discussed . there are two categories of women who have an increased risk for developing breast cancer . approximately 20%30% of women with breast cancer have a positive family history and about 20% of these individuals have a first degree relative with breast cancer . only about 5%10% of all cases of breast cancer are caused by inherited factors . the most common gene mutations are the brca 1 and brca 2 . in 1990 , hall and co - workers identified chromosome 17 q 21 as the location of a susceptibility gene for early onset breast cancer , now known as the brca 1 gene mutation . shortly after that narod described a linkage between the genetic marker d17 s 74 on 17 q 21 and ovarian cancer . in two different studies the suggestion has been made that about 3% of all breast cancers are caused by the brca 1 gene [ 12 , 13 ] and about 45% of all hereditary cases of breast cancer are caused by the brca 1 mutation . mutations in the brca 1 are most commonly seen in russia , followed by israel and italy . women who are carriers of the brca 1 gene mutation have a lifetime risk ( ltr ) for developing breast cancer of approximately 80% . other malignancies suggested to have an increased prevalence in these families are ovarian cancer , prostate and colonic cancer . brca 1 associated breast malignancies tend to have a high malignancy grade and are often oestrogen and progesterone receptor negative . median age of onset of breast cancer in this group is younger than 45 years . approximately 35% of all inherited breast cancers are caused by the brca 2 mutation , first identified by wooster et al . . the estimated ltr for developing breast cancer in this group is somewhat lower than in the brca 1 group . it has been suggested by ursin that in this group the use of oral contraceptives also increases the risk for developing breast cancer . non - hodgkin s lymphoma has been reported and also prostate cancer and bladder cancer [ 19 , 20 ] . the brca 2 mutation is associated with a 6% ltr of male breast cancer , which means a 100-fold increase over the general male population . at the moment little is known about the malignancy grade and receptor status in the brca 2 group . for both the brca 1 and brca 2 mutation carriers , the ltr for a contralateral breast cancer is about 65% . there are other more sporadic hereditary diseases with an increased risk for developing breast cancer . it is an autosomal dominant disease causing an increased risk for developing among others breast cancer , different types of sarcoma and leukaemia . in this group 30% of all malignancies cowden s disease or multiple hamartoma syndrome showed an increased risk for both benign breast disorders like fibroadenomas and nipple malformations and breast malignancies [ 23 , 24 ] . other hereditary diseases such as ataxia telangiectasia and the peutz jeghers syndrome ( hamartomous polyps in the small bowel ) and the muir torre syndrome ( a variant of hereditary non - polyposis colon cancer ) also give an increased risk for breast cancer . another group of women with an increased risk for breast cancer consists of women who have an individual risk factor . patients with a history of lobular carcinoma in situ ( lcis ) have a somewhat increased incidence of developing an invasive cancer . the national surgical adjuvant breast and bowel project ( nsabbp ) suggested an incidence of 13 invasive malignancies in 1000 women with an lcis . in this group ductal carcinoma in situ ( dcis ) also gives an increased risk for an invasive breast malignancy . about 30% of women not treated postoperatively with irradiation developed an invasive malignancy after a mean interval of 6 years [ 27 , 28 ] . invasive lobular carcinoma is characterized by multifocality in the ipsilateral breast and appears to be more often bilateral than other types of invasive malignancies . in the updated results of the nurses health study , post- or per - menopausal use of hormones showed an excess risk for developing breast cancer in the group of women with current or recently used hormones . women treated with irradiation of the chest for , for instance , ( non-)hodgkin s lymphoma , also have an increased risk for developing breast cancer . the excess risk in this category of patients is dependent on dose and age at irradiation . the younger the age at time of exposure to irradiation , the younger the onset of breast cancer . currently there are four possible breast screening modalities : clinical examination , mammography , ultrasound and mri . the primary goal of breast examinations during screening is the detection of breast cancer at an as early stage as possible in order to reduce mortality . clinical examinations of the breasts and self - examination as a screening procedure have been poorly evaluated . there is only limited and indirect evidence that these methods could help in decreasing mortality due to breast cancer . as mentioned , small malignant lesions ( < 10 mm ) will not be detected by palpation in the majority of cases . kriege and co - workers showed a sensitivity of only 17.8% of clinical breast examination in a screening setting . the younger the woman , the more glandular tissue there is , the denser the breasts are , and the lower the sensitivity of mammography . for women in the age group 4050 sensitivity ranges from about 50% to 80% , while in the age group over 50 the sensitivity ranges from 70% to about 90% . the sensitivity of mammography in the case of invasive lobular carcinoma ( ilc ) is lower than in women with invasive ductal carcinoma ( idc ) . in 1973 , the breast cancer detection demonstration project ( bcddp ) was started . in the subsequent 7 years more than 280000 women were screened . the screening depicted about half of all breast cancers detected in the screened area . in addition , the distribution of stage was more favourable in the screened population than in a control group in that area , so overall long - term survival was also better . in 1998 in the netherlands a mortality reduction of 13% was reached in the age category 5574 since the beginning of screening in 1990 with conventional mammography . other screening trials like the health insurance plan of new york ( hip - study ) showed a mortality reduction of 30% in the screened group compared to the control group . analysis of age specific mortality reduction indicates that screening for breast cancer has a special benefit in older women above the age of 50 and less in the younger age group . in addition , tabar et al . showed that the likelihood of dedifferentiation of a tumour is much higher in women younger than 40 years . survival is also influenced by both tumour grade and the size of the tumour [ 36 , 37 ] . there is currently no evidence that ultrasound ( us ) has a role as a screening modality for breast cancer . the two most important roles of us are differentiation between cystic and solid lesions in the breast and us guided biopsy of solid breast lesions . the most currently used investigation technique is the flash 3d technique which includes one series of images pre - contrast and five series of images after intravenous administration of contrast medium containing gadolinium . smoothly outlined round or oval lesions tend more to be benign , while speculated lesions are more suspicious of a malignancy . if a lesion shows a wash out on kinetic behaviour , this is highly suspicious for a malignancy . if there is progress in signal intensity over time this is more characteristic for a benign lesion . although the sensitivity of mri for detecting invasive breast cancer is more than 95% , the value in detecting dcis , especially dcis grade i , is lower . the sensitivity for dcis is described by orel et al . to be in the range of 75% . this is in comparison to the results of boetes et al . , who also described a sensitivity of about 75% in this group . in a series of 17 patients , they missed four cases of dics , 3 grade i and 1 grade iii . the problem with screening with mri is the relatively low specificity , which means a relatively high number of false - positive findings . almost all women with a genetic predisposition for developing breast cancer are younger than the age of 50 . so the value of screening with conventional mammography is doubtful in this group of women . especially in young women in a screening situation kuhl was the first to describe the results of mri in a screening situation for women with an increased risk for developing breast cancer . a group of 192 asymptomatic and six symptomatic women were evaluated . in the symptomatic group in the same year tilanus - linthorst described an asymptomatic group of 109 women with a 25% or more lifetime risk , in which 12 gene carriers also were included . in this group mri detected three malignancies occult on mammography . thirteen malignancies in this group showed a cancer on mri while mammography detected five . in 2003 , morris and co - workers described a group of 367 women , retrospectively . mri detected 14 more malignancies than mammography . in 2004 , the results of screening a group of 1909 women with both mri and mammography were described by kriege et al . . in the ltr group of 15%30% , the detection rate for cancer was 7.8 per 1000 women , in the ltr of 30%50% the detection rate was 5.4 per 1000 . however , in the group of carriers of brca 1/2 the incidence of malignancy was 26.5 per 1000 women . the overall sensitivity for the detection of breast cancer was 40% for mammography and 71.5% for mri ( fig . if only the invasive cancers were taken into account the sensitivity of mammography dropped to 33% and of mri increased to 79% . in the mrisk study group 43% of all invasive malignancies were smaller that 10 mm , but in two selected control groups only , 14 and 12.5% , the tumour was smaller than 10 mm . the negative node status was also better in the mrisk group than in both control groups with 21.4% compared to 52.4 and 56.4% , respectively . they evaluated 649 women with a total of 1881 screens both with mammography and mri . the combination of both imaging techniques showed a sensitivity of 94% . the difference in sensitivity was especially seen in the gene carrier group . however , as stated by liberman , any method of screening for breast cancer has the potential for both benefit and harm . the benefit of screening is especially the detection of a malignancy as early as possible . although prognosis of small breast cancers is better , the detection of a small cancer does not guarantee an improved survival rate . the real value of screening can only be proved by randomized controlled trials with death as an end point . the data published to date show that screening with mri has benefit for the group of women at high risk for developing breast cancer . however , if a centre proceeds to screen with mri it should follow technical and interpretative guidelines and there should be the possibility of performing mr guided biopsies [ 46 , 47 ] . in the screening of women with an increased risk for developing breast cancer detection needs to be done at a young age . therefore mammography is of limited value . because the sensitivity of mri is high for detecting breast cancer and because this sensitivity is not influenced by the amount of glandular tissue present , as with mammography , mri is the best modality available at this time for the screening of women with an increased risk for developing breast cancer . ( a ) , ( b ) the mammography of a 39-year - old woman obtained during annual screening because of a familial history of breast cancer . ( a ) , ( b ) the mammography of a 32-year - old woman obtained during annual screening because of a proven brca 2 gene carrier . ( c ) the mri from the same day . in the medial part of the right breast an irregular strong enhancing area was seen .

breast cancer is the most common cancer affecting women . in the screening of women for breast cancer , mammography is the most used imaging modality . women with an increased risk for getting breast cancer can develop a malignancy at a relatively young age compared to other women . the increased risk for developing breast cancer can usually be found in a positive familial history . this positive familial history is based on a gene mutation in 510% of cases . the most common gene mutations are brca1 and brca2 . this risk makes it necessary to start screening these women at a young age . mammography , however , has proven to be less reliable in younger women because its sensitivity is lowered due to the dense breast tissue often present in this group . mri has a higher sensitivity for detecting breast cancer compared to mammography . mri is not influenced by the density of the breast tissue . this makes breast mri the best modality available for the screening of women with an increased risk for developing breast cancer .

Introduction Increased risk for breast cancer Screening of the breast Conclusion Figures and Tables

worldwide , metastases are observed in 20%-30% of patients with an initial diagnosis of renal cell carcinoma ( rcc ) , even in those with localized rcc who undergo curative nephrectomy during follow - up . in the immunotherapy ( it ) era , cytokine - mediated it using interferon alpha ( ifn- ) or interleukin-2 ( il-2 ) or both were the first - line systemic treatment modalities for metastatic rcc ( mrcc ) . further attempts to augment cytokine - based it activity ( e.g. , inclusion of vinblastine and 5-fluorouracil [ 5-fu ] chemotherapy ) led to slightly improved objective response rate ( orr ) . however , the efficacy of such combinations was likely due to the cytokine effect , because the addition of vinblastine did not improve the progression - free survival ( pfs ) or overall survival ( os ) . the reported median survival time of mrcc in the it era was 12 months and the 2-year survival rate was 10%-20% , mainly due to a low orr of 10%-20% , with long - term durable responses only observed in 5%-7% of patients undergoing high - dose il-2based therapy . recent advances in understanding of the biology and genetics of rcc have led to the introduction of several novel multi - pathway targeted agents . clinical trials in treatmentnave mrcc patients conducted in western and asian countries have shown that targeted therapy ( tt ) had superior efficacy when compared with cytokine - based it . in terms of pfs and os , the clinical outcome of patients with mrcc has improved dramatically since the introduction of tt , which has now replaced it as the standard systemic treatment for mrcc . to facilitate prompt management strategies and achieve the optimal therapeutic response , physicians should be aware of the efficacies and limitations of systemic mono , combinational , and sequential therapies of diverse therapeutic agents for the treatment of mrcc . however , few large studies comparing the outcome of it and tt in patients with mrcc have been reported , although small comparative studies have been conducted . the current study assessed the outcome of systemic tt and it administered to a large number of patients with mrcc over a 10-year period at a single korean cancer institution . data from 262 patients with mrcc treated with systemic agents between 2003 and 2013 were evaluated retrospectively . patients who were administered it as a systemic therapy between 2003 and 2013 were selected from the prospectively recorded rcc database of the national cancer center hospital , and those administered tt who were treated between 2006 and 2013 were selected . of the 262 patients , 135 ( 51.5% ) underwent first - line it regimens based on mono or combinational therapy with ifn- , il-2 , 5-fu , or vinblastine . the remaining 127 patients ( 48.5% ) underwent first - line systemic tt . patients with no follow - up history and who had incomplete rcc treatment histories were excluded . additional information about the clinical outcomes of 96 patients treated with either tt or it as a sequential second - line systemic therapy was also evaluated . the choice of it or tt was at the discretion of the treating urologist ( j.c . ) and was based on the patient s histopathology and coverage by the national health insurance system . combination it comprised different combinations of subcutaneous recombinant human il-2 ( aldesleukin , proleukin , chiron corporation , emeryville , ca ) or recombinant human ifn- ( ifn - alpha-2a , roferon - a , hoffmann - la roche , nutley , nj ) , and intravenous 5-fu ( jw pharm , seoul , korea ) or vinblastine ( united pharm , seoul , korea ) . the triple or quadruple regimens were administered as follows : il-2 , 20 miu / m on days 35 of weeks 1 and 4 , and 5 miu / m il-2 on days 1 , 3 , and 5 of weeks 2 and 3 ; ifn- , 6 miu / m on day 1 of weeks 1 and 4 and on days 1 , 3 , and 5 of weeks 2 and 3 , and 9 miu / m on days 1 , 3 , and 5 of weeks 5 - 8 ; 5-fu , 750 mg / m once weekly during weeks 5 - 8 ; with ( quadruple ) or without vinblastine ( triple ) at 0.1 mg / kg once weekly during weeks 5 - 8 . the dual combination regimen of vinblastine plus ifn- was administered as ifn- at 9 miu / m on days 1 , 3 , and 5 each week , and vinblastine at 0.1 mg / kg every 3 weeks . all tt was administered orally . for sunitinib , each cycle consisted of 50 mg / day for 4 weeks followed by a 2-week hiatus . subjects were administered consecutive 10 mg of everolimus once a day per week and 5 mg of axitinib twice a day per week as the second - line tt . the treatment response was assessed every 4 - 6 weeks for sorafenib , pazopanib , everolimus , and axitinib . during follow - up , meticulous history taking , physical examination , routine blood tests , abdominal computed tomography ( ct ) , radionuclide bone scan , simple chest radiography , and/or chest ct , and electrocardiography were performed at regular intervals . in addition , mandatory thyroid function tests and echocardiography were performed in patients scheduled to receive tt . all responses were evaluated on ct imaging according to the response evaluation criteria in solid tumor ( recist ) ver . therapeutic responses were assessed at the end of each cycle ( at 8 weeks for the triple and quadruple it regimens ) or every 3 - 4 cycles ( at 9 - 12 weeks for the dual it regimen ) . for tt , response was assessed at the end of two cycles and treatment was continued until disease progression . time to progression and death were assessed using kaplan - meier analysis with the log - rank test . the orr , pfs , and os of the first- and second - line therapies were assessed according to the systemic agent and the clinical outcomes of sequential treatment with it and tt , and their prognostic stratifications , were also evaluated according to the memorial sloan kettering cancer center ( mskcc ) and heng criteria . , college station , tx ) . a p - value of < 0.5 was considered statistically significant . following approval by the institutional review board ( irb ) of the research institute and hospital national cancer center ( irb no . ncc2015 - 0212 ) , patient record / information was anonymized and de - identified prior to analysis . all study protocols were conducted according to the ethical guidelines of the world medical association declaration of helsinki - ethical principles for medical research involving human subjects . data from 262 patients with mrcc treated with systemic agents between 2003 and 2013 were evaluated retrospectively . patients who were administered it as a systemic therapy between 2003 and 2013 were selected from the prospectively recorded rcc database of the national cancer center hospital , and those administered tt who were treated between 2006 and 2013 were selected . of the 262 patients , 135 ( 51.5% ) underwent first - line it regimens based on mono or combinational therapy with ifn- , il-2 , 5-fu , or vinblastine . the remaining 127 patients ( 48.5% ) underwent first - line systemic tt . patients with no follow - up history and who had incomplete rcc treatment histories were excluded . additional information about the clinical outcomes of 96 patients treated with either tt or it as a sequential second - line systemic therapy was also evaluated . the choice of it or tt was at the discretion of the treating urologist ( j.c . ) and was based on the patient s histopathology and coverage by the national health insurance system . combination it comprised different combinations of subcutaneous recombinant human il-2 ( aldesleukin , proleukin , chiron corporation , emeryville , ca ) or recombinant human ifn- ( ifn - alpha-2a , roferon - a , hoffmann - la roche , nutley , nj ) , and intravenous 5-fu ( jw pharm , seoul , korea ) or vinblastine ( united pharm , seoul , korea ) . the triple or quadruple regimens were administered as follows : il-2 , 20 miu / m on days 35 of weeks 1 and 4 , and 5 miu / m il-2 on days 1 , 3 , and 5 of weeks 2 and 3 ; ifn- , 6 miu / m on day 1 of weeks 1 and 4 and on days 1 , 3 , and 5 of weeks 2 and 3 , and 9 miu / m on days 1 , 3 , and 5 of weeks 5 - 8 ; 5-fu , 750 mg / m once weekly during weeks 5 - 8 ; with ( quadruple ) or without vinblastine ( triple ) at 0.1 mg / kg once weekly during weeks 5 - 8 . the dual combination regimen of vinblastine plus ifn- was administered as ifn- at 9 miu / m on days 1 , 3 , and 5 each week , and vinblastine at 0.1 mg / kg every 3 weeks . all tt was administered orally . for sunitinib , each cycle consisted of 50 mg / day for 4 weeks followed by a 2-week hiatus . for sorafenib , each cycle consisted of consecutive 400 mg twice daily per week . for pazopanib , each cycle consisted of consecutive 800 mg once daily per week . subjects were administered consecutive 10 mg of everolimus once a day per week and 5 mg of axitinib twice a day per week as the second - line tt . the treatment response was assessed every 4 - 6 weeks for sorafenib , pazopanib , everolimus , and axitinib . during follow - up , meticulous history taking , physical examination , routine blood tests , abdominal computed tomography ( ct ) , radionuclide bone scan , simple chest radiography , and/or chest ct , and electrocardiography were performed at regular intervals . in addition , mandatory thyroid function tests and echocardiography were performed in patients scheduled to receive tt . all responses were evaluated on ct imaging according to the response evaluation criteria in solid tumor ( recist ) ver . therapeutic responses were assessed at the end of each cycle ( at 8 weeks for the triple and quadruple it regimens ) or every 3 - 4 cycles ( at 9 - 12 weeks for the dual it regimen ) . for tt , response was assessed at the end of two cycles and treatment was continued until disease progression . time to progression and death were assessed using kaplan - meier analysis with the log - rank test . the orr , pfs , and os of the first- and second - line therapies were assessed according to the systemic agent and the clinical outcomes of sequential treatment with it and tt , and their prognostic stratifications , were also evaluated according to the memorial sloan kettering cancer center ( mskcc ) and heng criteria . statistical analyses were performed using stata software ( release 9.2 , statacorp . , college station , tx ) . following approval by the institutional review board ( irb ) of the research institute and hospital national cancer center ( irb no . ncc2015 - 0212 ) , patient record / information was anonymized and de - identified prior to analysis . all study protocols were conducted according to the ethical guidelines of the world medical association declaration of helsinki - ethical principles for medical research involving human subjects . during the treatment period ( median duration , 4.3 months ) and the 24-month follow - up period , the first - line orr , pfs , and os were 41.9% , 8.1 months , and 16.8 months , respectively ( table 1 ) . during these same periods , the second - line orr , pfs , and os were 27.5% , 6.5 months , and 15.3 months , respectively . significantly better prognostic outcomes of second - line pfs were observed in the tt group ( 7.1 months ) compared with the it group ( 2.1 months ) ( p=0.002 ) . however , second - line os was not significantly different between the tt ( 16.6 months ) and it ( 8.6 months ) groups ( p=0.636 ) . in subgroup analyses of first - line systemic therapies , the tt , it , and sequential groups had a pfs of 9.3 , 6.4 , and 5.7 months ( p=0.819 ) , respectively , and an os of 15.8 , 16.5 , and 40.6 months ( p=0.014 ) , respectively ( table 2 , fig . 1 ) . pazopanib provided the best median pfs response , followed by sorafenib , sunitinib , and it ( p=0.053 ) ( table 2 , fig . the quadruple it regimen had superior pfs to that of it mono , dual , or triple regimens ( p=0.522 ) ( table 2 ) . among the 14 it - treated patients who achieved a complete response ( cr ) , eight ( 5.9% ) remained alive with a mean response duration of 104.542.8 months ( median , 123 months ; range , 33.1 to 156.7 months ) ; four of these ( 3.15% ) also received tt treatment . for the seven tt - treated patients who achieved a cr , four remained alive , with a median response duration of 22.8 months ( range , 13.9 to 54.8 months ) ( table 2 ) . for the risk - stratified subgroup analyses that were dependent on the mskcc and heng criteria ( table 3 ) , the clinical outcomes of pfs for each stratified prognostic group were followed subsequently in order according to their consecutive risk groups . when stratified according to the mskcc or heng criteria , treatments had differential outcomes for median os . for the favorable mskcc risk group in the first - line therapy , tt showed a superior os compared with it or sequential it plus tt ( p < 0.001 ) ( table 3 ) . in contrast , for the favorable heng risk group , it had superior os compared with tt or it plus tt ( p < 0.001 ) . other prognostic outcomes of first - line therapies and pfs and os of second - line therapies , as calculated using the mskcc and heng risk group models , are summarized in table 3 . since the introduction of tt , the therapeutic regimen paradigm has shifted from it to tt in mrcc . the prognoses have also improved dramatically in the clinical setting . in the current study , differences in prognoses in mrcc patients treated either with it , tt , or sequential therapy as the first- and second - line systemic therapies were examined at a single center during 10 years . the current study showed that the median pfs and os of it regimens was superior or equivocal to those observed in other western studies on it , and their orr of 41.9% and disease control rate of 69.5% ( 31.1% and 54.8% ; including 59 follow - up loss and death patients ) were superior to those reported in a previous korean study on it ( 18% and 51% , respectively ) ( table 1 ) . the use of 5-fu or vinblastine chemotherapy in conjunction with it increased the pfs by 0.5 - 3 months in 40.0% of the patients undergoing it treatment , and quadruple it resulted in the best pfs ( 8.8 months ) compared with all other it combinations ( 5.8 - 6.2 months ) ( table 2 ) . this suggests that the clinical benefit of an it with the addition of a chemotherapy regimen should be carefully considered in terms of whether the increased survival gains would outweigh the likely increase in adverse events . patients previously treated with high - dose il-2 achieved a 15% of orr with a median duration of 54 months , suggesting that high - dose il-2 treatment may have been curative in a small minority of patients . there are no clear guidelines regarding patient selection ; however , retrospective studies have suggested that il-2 was more efficacious in patients with a clear cell histology and favorable prognosis . ifn- , another systemic immunotherapeutic agent for mrcc , has consistently demonstrated low but reproducible responses ( 10%-20% ) with occasional durable responses with a pfs and os of 5 - 5.6 and 4.1 - 21.8 months , respectively . this is similar to the data of the patients in the current study undergoing ifn- monotherapy who had similar pfs and os ( 5.8 months ) ( table 2 ) and 19.3 months ( 19.3 months , data not shown ) . a combination of il-2 and ifn- showed a better clinical outcome in terms of orr and pfs when compared with il-2 monotherapy ( table 2 ) . in addition , sequential therapy with the combined it and tt regimen resulted in a better os ( 40.6 months ) compared to single tt ( 15.8 months ) and single it ( 16.5 months ) treatment regimens ( p=0.014 ) ( table 2 ) . however , this outstanding sequential therapy os may have resulted from our selection of patients who were capable of surviving it prior to tt and were expected to have a good performance based on favorable prognostic modeling with a fairly low tumor burden and a history of nephrectomy . in particular , patients with rapid progression might be excluded from participation before beginning tt . therefore , conditional survival may be an important methodology when evaluating the prognosis of mrcc patients treated with sub - sequential tt . in recent decades , compared with it , multiple single tts have shown greater activity in terms of pfs , specifically when compared with ifn- . however , a clinically relevant increase in cr and os has not been reported and the role of tt in increasing the curability of mrcc has not been fully elucidated . from a pharmacological viewpoint , the unsatisfactory results of tt to induce a cr may be explained in part by their mechanism of action , which appears to be more cytostatic than cytotoxic . the current study also showed that the os ( 15.8 months ) , orr ( 33.9% ) , and cr ( 5.6% ) of tt were either similar or inferior to the os ( 16.5 months ) , orr ( 31.1% ) , and cr ( 10.4% ) of it ( table 2 ) . the percentage of patients reaching cr for sunitinib , sorafenib , and pazopanib as first - line tt was reported as 1%-3% in earlier real - world clinical trials , post - marketing all - patient surveillance trials , and pivotal trials [ 16 - 18 ] . the higher cr rate ( 5.6% ) in this study would actually be much lower than observed because patients who stopped therapy after achieving cr during a substantial follow - up had a higher rate of relapse than patients who continued sequential therapy , as shown in a recent , large , retrospective study evaluating mrcc patients who were in cr during tyrosine kinase inhibitor treatment ( either alone or in combination with local treatment ) . another possible explanation for achievement of high cr would be that it is often integrated with tt with surgery , radiotherapy , or both . reported a similarly increased cr rate in comparison with the current study in patients treated with tyrosine kinase inhibitors plus surgery ( 4.5% ) when compared to those treated with medical therapy alone ( 1.8% ) . the current study also showed a significantly longer pfs ( 11.2 months ) and greater cr rates ( 5.4% ) in patients who underwent nephrectomy plus tt compared to patients who underwent tt alone ( pfs , 5.6 months ; cr rate , 1.0% ; data not shown in tables ) . previous extended - access and post - marketing all - patient surveillance trials have reported 16 - 22%/25.9%/39% of orrs and 5.2 - 9.4/6.0/13.7 months of pfss for sunitinib , sorafenib , and pazopanib in mrcc patients , respectively [ 16 - 18,21 ] . the orrs for sunitinib , sorafenib , and pazopanib reported in the current study were superior to those reported in previous studies ( 44.1% , 27.3% , and 62.5% , respectively ; data not shown ) ; except for the pfs inferior or similar to previous studies ( 8.5 , 9.0 , and 11.0 months , respectively ) [ 16 - 18,21 ] . additional evaluation of second - line systemic therapies for their orr , pfs , and os ( 27.5% , 6.5 months , and 15.3 months , respectively ) was similar or superior to that of previous trials with second - line therapy in cytokine- or tt - refractory mrcc patients ( orr , 9% to 23% ; pfs , 4 to 7.4 months ; os , 11.5 to 16.3 months ) . these differences of orr , pfs , and os are likely due to follow - up time , differences in clinical policies between clinicians surrounding various treatment modalities , differences in baseline study populations such as disease burden , nephrectomy rate , and physical body composition , as has been previously demonstrated for other malignancies . regarding the analyses of prognostic models , patients with favorable mskcc or heng risk group showed longer survival . in agreement , in the current study , those with favorable mskcc or heng risk group of first- and second - line therapy had a significantly better prognosis compared to those with poor or intermediate risk group ( table 3 ) . when stratified according to mskcc and heng criteria , the pfs for all groups in the first - line therapy was better in those treated with tt compared to those treated with it . however , those with a favorable heng risk group treated with it showed a superior os in the first - line therapy , compared to all other groups and treatments . it was a retrospective study with a small patient population , with a possible patient selection bias based on the era of when systemic therapy was initiated . the relevance of the stratified findings based on mskcc and heng criteria is not fully understood and should be developed further in future large - scale multicenter studies with a more diverse patient population . although the orr of tt and it were comparable in this study , tt demonstrated superior pfs and os compared with it in patients with mrcc . this supports the hypothesis that the clinical outcomes of patients with mrcc have shown significant improvement since the introduction of tt .

purposethe purpose of this study is to compare the outcomes of first - line systemic targeted therapy ( tt ) and immunotherapy ( it ) in patients with metastatic renal cell carcinoma ( mrcc).materials and methodsthis study was a retrospective review of the data of 262 patients treated with systemic it or tt with tyrosine kinase inhibitors between 2003 and 2013 . the objective response rate ( orr ) , progression - free survival ( pfs ) , and overall survival ( os ) were assessed using response evaluation criteria in solid tumor ver . 1.0 criteria and the kaplan - meier method with log - rank test.resultsduring the median 4.3-month treatment and the 24-month follow - up period , the orr / pfs / os of the overall first - line and second - line therapy were 41.9%/8.1 months/16.8 months and 27.5%/6.5 months/15.3 months , respectively . the first - line tt / it / sequential it had a pfs of 9.3/6.4/5.7 months and an os of 15.8/16.5/40.6 months ( all p < 0.05 ) . the second - line of tt / it had a pfs of 7.1/2.1 months ( both p < 0.05 ) and an os of 16.6/8.6 months ( p=0.636 ) , respectively . pazopanib provided the best median pfs of 11.0 months ( p < 0.001 ) and a quadruple it regimen had a superior pfs ( p=0.522 ) . for os , sequential treatment with it and tt was superior compared to treatment with either it or tt alone ( 40.6/16.5/15.8 months , p=0.014 ) . the prognosis according to the memorial sloan kettering cancer center model showed that favorable / intermediate / poor risk groups had a pfs of 8.5/10.4/2.3 months , and an os of 43.1/20.4/5.6 months , respectively . the prognosis calculated using the heng model showed that the favorable / intermediate / poor risk groups had a pfs of 9.2/3.9/2.7 months , and an os of 32.4/16.5/6.1months , respectively ( all p < 0.001).conclusionin patients with mrcc , tt provided a better pfs and os compared with it .

Introduction Materials and Methods 1. Patients 2. Treatment regimens 3. Targeted therapy 4. Follow-up 5. Statistical analyses 6. Ethical statements Results Discussion Conclusion

the in - vivo study on ophthalmic products is done on experimental animals , usually on rabbit 's eye . preparation volume 50 l is instilled topically in the center of the lower cul - de - sac . eyes of the animal are observed for signs of irritation and injury . in vitro methods diclofenac sodium solid lipid nanoparticles ( slns ) , prepared with goat fat and phospholipids evaluated using bio - engineered human cornea , produced from immortalized human corneal endothelial cells ( henc ) , stromal fibroblasts , and epithelial cells . sustained release and high permeation of diclofenac sodium through the bio - engineered cornea were achieved . the bovine corneal opacity and permeability ( bcop ) assay has been accepted as a valid in - vitro alternative method to the draize eye irritation test with the development of an improved laser light - based opacitometer ( llbo ) for the analysis of the complete corneal surface instead of dual beam op - kit opacitometer . confocal laser scanning microscopy study on the transport mechanism through the corneal epithelium of nanostructured lipid carrier was carried out in rabbits in vivo . the corneas were isolated and rinsed in physiological saline to remove the adhesive fluorescence fragment . after 30 minute administration in to eyes , the interaction with corneal epithelia to be investigated by observing the corneal samples using confocal laser scanning microscopy ( clsm ) . the epiocular tissue model is a new in - vitro test method for ocular irritation , produced by mattek corporation tissue production facilities . the epiocular tissue model shows in - vivo like morphological and growth characteristics that are uniform and highly reproducible . the tissue consists of highly organized basal cells , which progressively flatten out as the apical surface of the tissue similar to the normal in - vivo corneal epithelium . this test could be applied as an alternate to rabbit eye test in the toxicological assessment of ophthalmic preparations . the in - vivo study on ophthalmic products is done on experimental animals , usually on rabbit 's eye . preparation volume 50 l is instilled topically in the center of the lower cul - de - sac . eyes of the animal are observed for signs of irritation and injury . in vitro methods diclofenac sodium solid lipid nanoparticles ( slns ) , prepared with goat fat and phospholipids evaluated using bio - engineered human cornea , produced from immortalized human corneal endothelial cells ( henc ) , stromal fibroblasts , and epithelial cells . sustained release and high permeation of diclofenac sodium through the bio - engineered cornea were achieved . the bovine corneal opacity and permeability ( bcop ) assay has been accepted as a valid in - vitro alternative method to the draize eye irritation test with the development of an improved laser light - based opacitometer ( llbo ) for the analysis of the complete corneal surface instead of dual beam op - kit opacitometer . confocal laser scanning microscopy study on the transport mechanism through the corneal epithelium of nanostructured lipid carrier was carried out in rabbits in vivo . the corneas were isolated and rinsed in physiological saline to remove the adhesive fluorescence fragment . after 30 minute administration in to eyes , the interaction with corneal epithelia to be investigated by observing the corneal samples using confocal laser scanning microscopy ( clsm ) . the epiocular tissue model is a new in - vitro test method for ocular irritation , produced by mattek corporation tissue production facilities . the epiocular tissue model shows in - vivo like morphological and growth characteristics that are uniform and highly reproducible . the tissue consists of highly organized basal cells , which progressively flatten out as the apical surface of the tissue similar to the normal in - vivo corneal epithelium . this test could be applied as an alternate to rabbit eye test in the toxicological assessment of ophthalmic preparations . most of the formulation efforts aim at maximizing ocular drug absorption through prolongation of the drug residence time in the eye as well as to slow drug release from the delivery system and minimize pre - corneal drug loss . nanostructured platforms provide the effective ways for delivery of insoluble drugs to the target site . as these platforms for ophthalmic delivery are very recent and the research continues , formulation factors such as loading of drug , release rate , stability , and therapeutic effectiveness for these needs to be optimized .

the topically applied drugs as drops are washed off from the eye in very short period , resulting in low ocular bioavailability of drugs . number of approaches have been attempted to increase the bioavailability and the duration of action of ocular drugs . this review provides an insight into various novel approaches ; hydrophilic nanogels , solid lipid nanoparticles , and nanosponges applied very recently in the delivery of insoluble drugs , prolonging the ocular residence time , minimize pre - corneal drug loss and , therefore , bioavailability and therapeutic efficacy of the drugs . despite various scientific approaches , efficient ocular drug delivery remains a challenge for pharmaceutical scientists .

None Ophthalmic products toxicity evaluation Conclusion

in things necessary , unity ; in things doubtful , liberty . in the previous issue of critical care , nil - weise and colleagues investigate the effectiveness of the semiupright position ( and elements of the ' ventilator bundle ' ) in preventing ventilator - associated pneumonia ( vap ) . however , while physicians try to reconcile the art with the science of medicine by attempting to apply at the bedside the results of clinical trials , intensive care patients receive , on average , only about one half of the recommended core clinical interventions . this inconsistency in clinical decision - making and care provision is not due exclusively to variation in case mix and facilities but is the result of the intrinsic complexity of critical care , on one hand , and the physician 's heuristics ( that is , intuitive judgments ) on the other . errors of omission , such as failure to perform routine tasks or an intended plan , or the loss of key information at handover are particularly relevant today , when the management of critically ill patients is more complex , the working shift pattern of health - care professionals requires multiple daily handovers , and patients and physicians experience discontinuity in care . the standardization of the process of care , therefore , requires structuring the task environment and compensating for heuristics to facilitate consistent and reliable delivery of best evidence based practice . this is the aim of care bundles , defined by the institute of healthcare improvement as a ' a group of interventions related to a disease process that , when executed together , result in better outcomes than when implemented individually ' . the definition implies that its elements should function as a package and that its effectiveness comes from the excellence of the supporting evidence and its consistent comprehensive execution . although proponents of standardized , protocol - driven care see the conceptual advantages of bundles ( that is , that they simplify decisions , reduce omissions and errors in medical reasoning , promote goal - orientated care , and deal with areas of uncertainty by giving a pragmatic but consistent solution ) , adherence to bundles depends on the interplay between factors that act as barriers or enablers . commonly , lack of knowledge , unavailability of resources , high costs , nursing convenience , fear of adverse events or patient discomfort , and ( most importantly ) disagreement on the strength of the supporting evidence in conjunction with external barriers can affect a physician 's ability to execute recommendations . in this context , nil - weise and colleagues report a systematic review and the recommendations of a european expert panel on the benefits and disadvantages of the semi - upright position in the prevention of vap . the authors found nonsignificant reductions in the incidence of vap ( clinically suspected and microbiologically confirmed ) and in mortality with semi - upright position with no sufficient data to quantify harm ( for example , venous thromboembolism , hemodynamic instability , or patients ' discomfort ) . in this context , the expert panel suggested that the semi - upright position be used as the preferred option but only in the absence of clear contraindications ( for example , spinal injury ) and with necessary restrictions ( for example , nursing tasks , medical interventions , and patients ' wishes ) . expert opinions and adherence to recommendations often seem dissociated from the strength of clinical evidence . for instance , some strategies considered to be ineffective by trials have high rates of adherence , whereas others found to be effective ( for example , continuous subglottic aspiration and digestive decontamination ) have a high rate of nonadherence . in addition , regardless of ( or , occasionally , despite ) the strength of evidence , some clinicians oppose the concept of care bundles in general , arguing that bundles ( a ) are used by industries as a marketing tool , ( b ) deprive clinicians of clinical autonomy , ( c ) are inefficient ( similar benefit could be achieved with fewer elements ) or ineffective as they may divert from the implementation of a more effective set of interventions not included in the bundle , ( d ) increase the risk of over- or under - treatment ( that is , not all patients need all elements of the bundle all of the time ) , ( e ) may be inappropriately adopted as a measure of organizational performance , or ( f ) suffer from positive publication bias and lack of external validity in ' the real world ' or contain elements that are not plausibly related to the bundle 's objectives ( for example , thromboprophylaxis and vap prevention ) . in the paper by nil - weise and colleagues , the expert panel recommended upright head elevation , despite a lack of strong supporting evidence , as did the uk national institute for health and clinical excellence ( nice guidance psg002 ) . this apparent discrepancy between ' evidence - based ' and ' common - sense ' recommendations may reinforce the perception that committees ' advice is ' unscientific ' or attempts to drive consensus rather than reflect it . given these uncertainties , the question for the practicing clinician is , should we use bundles and protocols ? in the presence of clear evidence , the answer seems simple : if the components are scientifically sound , yes ; but when the evidence is conflicting , a common - sense approach is necessary . the best guess involves the consistent use of protocols for routine and common practice , as overall they are unlikely to cause significant harm and are more likely to be beneficial . however , the optimal balance between protocolized versus individualized care will change among institutions , depending on staffing and case mix , and with the availability of new research . in this context , using compliance to each element of a bundle as an indicator of performance may not reflect quality of care unless other considerations of risks and benefit of the proposed interventions are included and the reasons for deviation are reported . in the meantime , we should strive to implement current performance systems to deliver the consistent engineered care that patients and their families expect and deserve .

care bundles aim to improve standard of care and patient outcome by promoting the consistent implementation of a group of effective interventions . however , a variety of barriers prevent their full application in clinical practice . here , we discuss some of the benefits and limitations of care bundles in the delivery of safer and more effective and consistent health care .

None Abbreviations Competing interests

pneumatosis sistoides intestinalis ( psi ) a rare condition that is characterized by gas - filled cysts in the intestinal submucosa and subserosa . psi may occur as primary or secondary and often seen terminal ileum in the gastrointestinal tract . respiratory infections , tumor or collagen disease , traumas , immunosuppression and the use of steroids are associated with psi . imaging tools , endoscopy , surgery and sometimes pathological examinations are used in the diagnosis . although psi is usually benign disease , sometimes high mortality is observed . in asymptomatic patients are used hyperbaric oxygen treatment , antibiotics and rest to bowel ; in patients with signs of peritonitis are recommended to surgical treatment . we want to discuss 3 case of with psi that followed - up and treated in our clinic . a male patient aged 62 was hospitalized for emergency treatment of previously diagnosed free air under the diaphragm . at the time of arrival the patient reported supraumbilical pain , nausea and vomiting in the past two weeks . the problems of abdominal discomfort and pain connected with taking food had started 21 years earlier , with intermittent remissions and there was a history of stomach bleeded before 15 years . a few months before , the intensity of the symptoms had increased and continued with nausea , vomiting and weight loss ( 15 kg in a year ) and his body mass index ( bmi ) was 17 kg / m . he often received proton pomp inhibitors and there was not history of gastroscopy . on physical examination the abdomen was tenderness and defense at palpation in the epigastrium . in laboratory analysis hemoglobin was 10.3 contrast - enhanced abdominal ct was detected free - air in the abdomen ( fig . 2 ) . the patient was admitted and prepared for the operating theatre where the operation was started . after opening the abdominal cavity and exploration , the following finding was present : stenotic pylorus with an abnormally increased stomach and gas - filled cysts were seen in the terminal ileum ( figs . after that we decided to make the first resection of that antrectomy and were performed retrocolic , isoperistaltic gastrojejunostomy . specimens were sent to the pathology department . at first , when the specimen sections from the ileum and proximal colon ; macroscopic findings were noted that cystic lesions were filled with air and in greatest diameter 2 cm . under the microscope , additionally , the cysts were not communicated with each other and in different sizes which means that the finding at the terminal ileum was pneumatosis cystoides intestinalis ( figs . at second , operative material of stomach with increased dimensions , decreased elasticity , tall and prominent mucosal folds without macroscopic erosions and ulcerations , astenotic place on the pylorus with the lumen passable for 0.6 cm ( fig . 3 ) . microscopic finding , there was seen chronic inflammatory infiltration with lymphocytes and intestinal metaplasia into the antrum with bleeding erosions focus and an oedematic edge of the pylorus . he resumed normal activity in the follow- up visit 1 month after the second surgery . a male patient aged 70 was admitted with abdominal pain and vomiting in emergency room . he has received proton pump inhibitor therapy for long years and weight loss ( bmi = 17 kg / m ) . the abdomen was tenderness and defense at palpation in the epigastrium and upper right quadrant . x - ray showed to free - air under diaphragm . in the laboratory white blood cell was 9 10/l and amylase was 220 intraperitoneal free air and millimetric contrast agent leakage in curvature minor of the stomach has been detected with contrast - enhanced abdominal ct . were seen gas - filled cysts in the terminal ileum and pyloric stenosis that 2 1 cm diameter and permitting passage . he was discharged on postoperative day 8 and performed upper gastrointestinal endoscopy a month later . we detected pyloric stenoses that did not allow of endoscop passage in to the duodenum . pathology reports were benign and determined diffuse intestinal metaplasia and widespread involvement of helicobacter pylor . the patient resumed normal activity in the follow - up visit 20 day after the surgery . a male patient aged 81 was admitted with abdominal pain from internal medicine of clinics . the patient had been operated before 4 years and had congestive heart failure , chronic lung disease and prostate disease . was detected median incision scar and only tenderness at palpation in the epigastrium and there was no acute abdomen . the patients had gas or stool output and white blood cell was 5.6 10/l . free - air was determined under diaphragm and intraperitoneal in x - ray and abdomen ct , respectively . upper gastrointestinal endoscopy and colonoscopy were performed and detected hiatus hernia , erythematous pangastritis and grade 2 internal hemorrhoids . the 2 - 4 lt / dk oxygen and antibiotic therapy were given to patients . a male patient aged 62 was hospitalized for emergency treatment of previously diagnosed free air under the diaphragm . at the time of arrival the patient reported supraumbilical pain , nausea and vomiting in the past two weeks . the problems of abdominal discomfort and pain connected with taking food had started 21 years earlier , with intermittent remissions and there was a history of stomach bleeded before 15 years . a few months before , the intensity of the symptoms had increased and continued with nausea , vomiting and weight loss ( 15 kg in a year ) and his body mass index ( bmi ) was 17 kg / m . he often received proton pomp inhibitors and there was not history of gastroscopy . on physical examination the abdomen was tenderness and defense at palpation in the epigastrium . in laboratory analysis hemoglobin was 10.3 contrast - enhanced abdominal ct was detected free - air in the abdomen ( fig . 2 ) . the patient was admitted and prepared for the operating theatre where the operation was started . after opening the abdominal cavity and exploration , the following finding was present : stenotic pylorus with an abnormally increased stomach and gas - filled cysts were seen in the terminal ileum ( figs . after that we decided to make the first resection of that antrectomy and were performed retrocolic , isoperistaltic gastrojejunostomy . specimens were sent to the pathology department . at first , when the specimen sections from the ileum and proximal colon ; macroscopic findings were noted that cystic lesions were filled with air and in greatest diameter 2 cm . under the microscope , additionally , the cysts were not communicated with each other and in different sizes which means that the finding at the terminal ileum was pneumatosis cystoides intestinalis ( figs . at second , operative material of stomach with increased dimensions , decreased elasticity , tall and prominent mucosal folds without macroscopic erosions and ulcerations , astenotic place on the pylorus with the lumen passable for 0.6 cm ( fig . 3 ) . microscopic finding , there was seen chronic inflammatory infiltration with lymphocytes and intestinal metaplasia into the antrum with bleeding erosions focus and an oedematic edge of the pylorus . he resumed normal activity in the follow- up visit 1 month after the second surgery . a male patient aged 70 was admitted with abdominal pain and vomiting in emergency room . he has received proton pump inhibitor therapy for long years and weight loss ( bmi = 17 kg / m ) . the abdomen was tenderness and defense at palpation in the epigastrium and upper right quadrant . x - ray showed to free - air under diaphragm . in the laboratory white blood cell was 9 10/l and amylase was 220 u / l . intraperitoneal free air and millimetric contrast agent leakage in curvature minor of the stomach has been detected with contrast - enhanced abdominal ct . were seen gas - filled cysts in the terminal ileum and pyloric stenosis that 2 1 cm diameter and permitting passage . he was discharged on postoperative day 8 and performed upper gastrointestinal endoscopy a month later . we detected pyloric stenoses that did not allow of endoscop passage in to the duodenum . pathology reports were benign and determined diffuse intestinal metaplasia and widespread involvement of helicobacter pylor . the patient resumed normal activity in the follow - up visit 20 day after the surgery . a male patient aged 81 was admitted with abdominal pain from internal medicine of clinics . the patient had been operated before 4 years and had congestive heart failure , chronic lung disease and prostate disease . was detected median incision scar and only tenderness at palpation in the epigastrium and there was no acute abdomen . the patients had gas or stool output and white blood cell was 5.6 10/l . free - air was determined under diaphragm and intraperitoneal in x - ray and abdomen ct , respectively . upper gastrointestinal endoscopy and colonoscopy were performed and detected hiatus hernia , erythematous pangastritis and grade 2 internal hemorrhoids . the 2 - 4 lt / dk oxygen and antibiotic therapy were given to patients . pneumatosis sistoides intestinalis ( psi ) a rare condition that is characterized by gas - filled cysts in the intestinal submucosa and subserosa , is most frequently observed in the terminal ileum of gastrointestinal tract . psi is more frequently seen in the first days of life and 60 years of age and its estimated prevalence is 0.03% . its etiology is unclear and there are plenty of theories about the pathogenesis of pnemautosis intestinalis , including mechanical , bacterial , pulmonary , and caused by the increase in the mucosa permeability . our 3 patients were over the age of 60 and gas - filled cystic lesions were localized in the ileum . h. pylori can be considered in etiology of psi that identified in the biopsy of 3 patients ( figs . pneumatosis cyistoides intestinalis is classified as primary disease ( 15% ) of unknown cause and secondary ( 85% ) associated to gastrointestinal diseases , as intestinal obstruction , cystic fibrosis , peptic ulcer , diverticula , inflammatory bowel disease , mesenteric infarction , chronic intestinal pseudo - obstruction , respiratory infections , tumor or collagen disease , traumas , immunosuppression and the use of steroids . we believe that psi develops in our three patients which secondary to gastrointestinal obstruction or pyloric stenosis . the history of patient , symptoms and physical examination are important for diagnosis of psi . diarrhea , constipation , rectal bleeding , tenesmus , nonspecific symptoms such as weight loss and abdominal pain may occur in symptomatic patients . volvulus , intestinal obstruction , blood pressure pneumoperitoneum , rectal bleeding , intussusception and intestinal perforation can be seen by 3% . there was rectal bleeding in a patient and in three of our patients had abdominal pain and weight loss ( table 1 ) . these include plain radiographs , usg , barium series , ct , ct - colonoscopy , magnetic resonance imaging and mri - colonography , endoscopy , colonoscopy . x - ray can show intraperitoneal free - air and abdominal ct can detect pneumoperitoneum , portomesenteric venous air , bowel wall thickening , bowel dilatation . reported in their work of eighty - four patients that importance of ct in the diagnosis of psi . a definitive diagnosis is difficult with the upper gastrointestinal endoscopy and colonoscopy , because of submucosal cysts can be mixed with polyps . a laparoscopic exploration is quite useful to confirm a pci diagnosis , if the physical examination findings are suspicious , and particularly in cases that are not preoperatively diagnosed clearly using the above - mentioned radiological methods . diagnostic laparoscopy provides the convenience of converting to open surgery as well as confirming the diagnosis . treatment of psi depends on the underlying cause , so that include elemental diet , antibiotics , steroids , hyperbaric oxygen therapy and surgery . in asymptomatic patients with free - air at x - ray and abdominal ct reporting gastrointestinal perforation direction , is a great handicap for the surgeon . so we should be careful in the diagnosis of disease . because of spontaneous remission can see rate of 50% in treating the disease and may occur gas - filled cysts again after surgery . conservative approaches , including nasogastric decompression , intestinal rest , antibiotic therapy and oxygen , are recommended for patients with positive examination findings and normal biochemical parameters who are confirmed radiologically to have no intestinal ischemia or perforation . in asymptomatic patients can be given hyperbaric oxygen ( hbo ) or absolute atmospheres ( non - hbo ) therapy may be given . oxygen therapy has long been recognized as an effective therapy for pi , leading to cyst regression on imaging and symptom resolution . applying 250 mmhg po2 pressure or 70% oxygen inhalation for 5 days or 2.5 atmospheres of hyperbaric oxygen pressure for 150 min / day for 3 consecutive days can lead to resolution of gas collection within a cyst . . gave hbo therapy to seven patients and atmospheres absolute ( ata , non - hbo ) to 20 patients in asymptomatic patients with psi . hbo therapy was given mean of 4.7 days , ata treatment was given mean of 14.6 days and have found lower recurrence rates . in asymptomatic patients with psi is not necessary surgery treatment . early surgical intervention should be considered in any patient with signs of perforation , peritonitis , sespsis or predictors of poor outcomes . emergency surgery should be considered in psi patients with the ph is less than 7.3 , actual bicarbonate level is less than 20 , amylase is higher than 200 similarly , patients who are immunosuppressed with underlying liver disease , sepsis , and hypotension are all at a higher risk of mortality from pi . radiologically , the presence of portal venous gas is highly indicative of severe disease , and when present , mortality rates range from 37% to 75% ( 7 , 22 ) . in patients with symptomatic psi , laparotomy and laparoscopic exploration arkanolu et al . in a series of seven cases showed that safely performed ileal resection and anastomosis in 3 patients , underwent laparotomy in 2 patients , choledocotomy , drainage and cholecystectomy in a patient , and laparoscopic exploration in a patient . we performed to 2 patients ileal and cecal resection and anastomosis with antrectomy and gastroentorostomy . pneumatosis intestinalis is a rare condition that may be occur a sign of an underlying systemic disease . in psi patients with peritonitis mehmet aziret contributed in study concept , design , data collection and writing the paper .

introductionpneumatosis sistoides intestinalis ( psi ) is a rare condition with unknown origin , defined as the appearance of gas - filled cysts in the intestinal wall . it usually occurs due to respiratory infections , tumor or collagen disease , traumas , immunosuppression.presentation of casethree patients with psi were examined that followed up and treated in our clinic . the first patient was hospitalized for emergency treatment of previously diagnosed free - air under the diaphragm . he had a defense on physical examination and free - air was detected in x - ray and abdomen ct . we decided to laparatomy and peroperatively , stenotic pylorus with an abnormally increased stomach and gas - filled cysts were seen in the terminal ileum . antrectomy and gastrojejunostomy with partial ileum and cecum resection and end ileostomy were performed . the second patient underwent laparatomy because of intraperitoneal free - air and acute abdomen . partial ileum and cecum resection and ileotransversostomy were performed . the third patient with intraperitoneal free - air was treated with antibiotics , oxygen treatment and bowel rest.discussionpsi is usually asymptomatic . plain radiographs , usg , ct , upper gastrointestinal endoscopy , colonoscopy can use for diagnosis . treatment of psi depends on the underlying cause ; include elemental diet , antibiotics , steroids , hyperbaric oxygen therapy and surgery.conclusionin patients with asymptomatic and symptomatic psi are different treat . symptomatic psi can be safely treated antrectomy and gastrojejunostomy with partial ileum and cecum resection .

Introduction Case report 1 Case report 2 Case report 3 Discussion Conclusion Conflict of interest Funding Ethical approval Author contributions

the muscarinic receptors are metabotropic receptors that may be linked to plasma membrane k or ca ion channels [ 1 , 2 ] . they belong to the superfamily of rhodopsin - like , seven transmembrane domains , single - glycoprotein receptors that are connected by intra- and extracellular loops . muscarinic receptors initiate intracellular responses via interaction with gtp - binding proteins ( g - proteins ) , although activation of other signaling molecules has been reported [ 1 , 3 , 4 ] . there are five subtypes of muscarinic receptors , referred to as m1 to m5 , based on the order of their discovery , and according to the nomenclature proposed by caulfield and birdsall . m1-machr , m1achr , or machr1 for the m1 receptor . in this paper muscarinic receptor subtypes will be referred to as m1 , m2 , m3 , m4 , and m5 , according to iuphar and the mesh browser of the national library of medicine of the national institute of health , usa . molecular cloning revealed that the five muscarinic receptors are encoded by separate intronless human genes . the muscarinic receptor gene sequences have significant homologies with other members of this large super - family and across mammalian species . the seven hydrophobic transmembrane domains of the muscarinic receptors are highly conserved with an average of 66% identity . in contrast , their intracellular loops are less conserved , with the third intracellular loop being particularly variable and accommodating the binding domain of receptor subtypes . between the fifth and the sixth transmembrane regions , muscarinic receptors possess a large intracytoplasmic loop that exhibits high divergence between the different subtypes and is considered to be responsible for the g - protein - coupling selectivity [ 810 ] the name and gene location of the human m1 is on chromosome 11q13 ; m2 is on chromosome 7q31 - 35 ; m3 is on chromosome 1q43 ; m4 is on chromosome 11q12 - 112 ; m5 is on chromosome 15q26 [ 8 , 9 , 11 ] . as mentioned above , muscarinic receptors modulate different intracellular signal transduction pathways by coupling to multiple g proteins , which include stimulation of phospholipases c , a2 and d , camp degradation , cgmp production , attenuation of camp synthesis , and regulation of several ion channels [ 3 , 10 ] . this diversity in signaling is more complicated , since a single muscarinic receptor subtype is capable of activating more than one type of g protein in a single cell and , thus , is coupled to more than one effector complements of the cell [ 3 , 10 , 12 ] . muscarinic receptors can be divided into two groups according to their primary coupling efficiency to g - proteins . the first group of m2 and m4 muscarinic receptors couple to the pertusiss - toxin sensitive gi / o type proteins . the second group including m1 , m3 , and m5 can couple to gq/11-type proteins [ 3 , 5 ] . however , there is also evidence that muscarinic receptors couple to a wide range of signaling pathways , some of which are mediated by other types of g - proteins or other signaling mediators [ 13 , 14 ] . studies on animal and human cell lines as well as on tissues demonstrated that muscarinic receptors also act via activation of the extracellular signal - regulated kinases 1 and 2 ( erk1/2 ) that is referred to as mitogen - activated protein ( map ) kinase 1 . in human bronchial epithelial cells , it was demonstrated that various muscarinic receptor inhibitors including tiotropium ( m1 , m2 , and m3 antagonist ) , gallamine ( m2 antagonist ) , telenzepine ( m1 antagonist ) , and 4-diphenylacetoxy - n - methylpiperidine methiodide ( m3 antagonist ) downregulated acetylcholine - induced leukotriene b4 release via the activation of erk1/2 and nuclear factor - kappab ( nfb ) pathways . with respect to the involvement of muscarinic receptors in the regulation of inflammatory response , it has been reported that m2 and m3 receptors facilitate cigarette - smoke - extract - induced interleukin ( il)-8 secretion by in human airway smooth muscle cells via a protein kinase c - dependent activation of the inhibitor of ib and erk1/2 , which suggests a signaling pathway depicted in figure 2 . muscarinic receptors are expressed by tissue - forming cells in the airways , predominantly by smooth muscle , epithelium , and fibroblasts . in the human lung , the density of parasympathetic cholinergic innervation is greatest in the proximal airways and diminishes peripherally . the predominant role of acetylcholine released by the parasympathetic system is in the control of distal airway resistance and the release of mucus from submucosal glands , and from goblet cells in the airway epithelium . the distribution of muscarinic receptors in the human airway has been mapped by receptor autoradiography and in situ hybridization throughout the bronchial tree and is mainly restricted to muscarinic m1 , m2 , and m3 receptors [ 1820 ] , though m4 may also be involved . acetylcholine released by cholinergic nerves regulates airway smooth muscle tone and mucus secretion . in the human lung m1 subtype occurs not in the bronchus , but has been reported in human bronchial fibroblasts and bronchial epithelial cells . stimulation of m1 receptors in the human lung causes bronchoconstriction and plays a modulatory role in electrolyte and water secretion [ 18 , 23 ] . the presence of m2 receptors was reported in the human peripheral lung and the bronchus [ 20 , 24 ] . western blot analysis revealed the presence of m2 protein in human bronchial fibroblasts , epithelial cells , and smooth muscle cells . muscarinic m2 receptors are expressed by neurons , where they function as autoreceptors , limiting the release of acetylcholine from both preganglionic and parasympathetic nerve terminals of the lung [ 18 , 21 ] , of the human trachea , and of bronchi , but not of bronchioli . here the m3 receptor is the primary muscarinic receptor subtype that mediates contraction of bronchial and tracheal smooth muscle , even though it is expressed in these tissues at considerable lower levels ( about 1/4 ) than m2 . m3 receptor is expressed by the smooth muscle cells of the airways , by human bronchial fibroblasts , and by human bronchial epithelial cells , as well as in the human peripheral lung . the receptor predominantly occurs in the bronchus and its density decreases from the segmental to subsegmental bronchus and is abolished in lung parenchyma . stimulation of m3 receptors in the human lung , human central and peripheral airway smooth muscle , and in the human isolated bronchus causes bronchoconstriction and mucus secretion from submucosal glands [ 18 , 27 , 2931 ] . however , activation of m3 receptors on vascular endothelial cells also induces the synthesis of nitric oxide , which diffuses to adjacent vascular smooth muscle cells and causes vasodilatation . during the past decade , several investigators have demonstrated that the biosynthesis , release mechanisms , and muscarinic receptors of the cholinergic system are functionally expressed independently of cholinergic innervations . it is concluded from such evidence that acetylcholine is not merely a neurotransmitter and that it transcends the nervous system , which in relation to lung pathophysiology can modify the phenotypic and cell function of airway cells , including epithelial cells ( m1m4 ) , pulmonary vessel endothelial cells ( m1m5 ) , mesenchymal cells , such as smooth muscle fibers ( m2 , m3 ) and fibroblasts ( m2 > m1 > m3 > m4 ) , and lung - infiltrating immune cells , such as mononuclear leukocytes ( m1m5 ) , monocytes , and macrophages ( m1 , m2 , and m3 ) . the function of nonneuronal acetylcholine released by the airway epithelium may participate in airway smooth - muscle contraction , but this remains controversial . additionally , acetylcholine , either neuronal or nonneuronal , may modulate airway inflammation and tissue remodeling . for example , ensuing cellular effects in the airways following stimulation of m1 increased proliferation , while m4 activation increased migration and wound healing in epithelial cells . the pathophysiology of pulmonary obstructive diseases , such as chronic obstructive pulmonary disease ( copd ) and asthma , is associated with the stimulation of the parasympathetic system , resulting in increased bronchoconstriction and mucus secretion from airway submucosal glands in the human lung . since the early 70s , it has been established that it is the muscarinic receptor activity of acetylcholine that is involved in the pathophysiology of asthma and copd . muscarinic anticholinergic agents proved to be effective in the treatment of asthma and copd , since the vagal cholinergic tone appears to be a reversible component of airway narrowing . thus , inhalation of ipratropium bromide , which inhibits m1 , m2 , and m3 , was the first muscarinic inhibitor introduced for the treatment of patients with obstructive pulmonary diseases , followed by tiotropium bromide monohydrate that also binds to m1 , m2 , and m3 and has a longer duration of anticholinergic action . tiotropium has a considerably slower rate of dissociation from the m1 and the m3 receptors than from the m2 receptor , rendering kinetic selectivity of the drug for m1 and m3 receptors . thus , tiotropium is more effective , since it improves dyspnea and exercise capacity and reduces hyperinflation . in addition , there is evidence from animal and human studies of defect expression and/or stimulation of muscarinic receptors in the lungs of asthma and copd patients . it has been reported that m2 autoinhibitory receptors do not function normally in airways of some asthmatics . the loss of function of m2 receptors mediated lung hyperreactivity in antigen - challenged animals and proposed to be an important cause of airway hyperreactivity in asthma . the dysfunction of m2 autoinhibitory receptors in allergic asthma was proposed to be due to eosinophil - derived major basic protein , which acts as an allosteric antagonist of the m2 receptor , augmenting acetylcholine release , and this may modulate the cellular response associated with airway remodeling . in leukocytes and the bronchi of patients with cystic fibrosis it was shown that the content of acetylcholine is substantially reduced , leading to reduced vesicle storage and transport of nonneuronal acetylcholine . with respect to gene expression of muscarinic receptors , bronchoscopic evaluation of the mucosa in asthma patients revealed an increased expression of m3 receptor mrna in severe asthmatics compared to patients with mild - to - moderate asthma and significantly higher levels of m3 receptor mrna in patients with brittle asthma . a similar investigation revealed that there are significantly lower levels of the m3 receptor mrna in patients with copd as compared to asthma patients , and that m3 receptor mrna gene expression was significantly elevated in copd patients with bronchial hyperresponsiveness as compared with patients without bronchial hyperresponsiveness , indicating that different molecular mechanisms underlie the clinical heterogeneity of bronchoconstriction in severe asthma and copd . accumulating evidence over the past decade demonstrated that the pathology of asthma and copd , in addition to bronchoconstriction , is attributed to inflammation of the airways . the inflammation that occurs in asthma can be described as eosinophilic with an increase in th2 ( cd4 ) cells , whereas inflammation that occurs in copd is mainly neutrophilic with cd8 t cells predominating . both neuronal or nonneuronal acetylcholine and muscarinic pulmonary obstructive diseases are determined by cellular and structural changes of the airways , a process that was associated to chronic airway inflammation . airway remodeling in asthma and copd correlates with disease severity [ 48 , 49 ] and is characterized by mucus gland hypertrophy , goblet cell hyperplasia , and pulmonary vascular remodeling . specific cellular and structural changes in asthma include basement membrane thickening , subepithelial fibrosis , and thickening of the airway smooth muscle bundle , while in copd specific changes include peribronchial fibrosis and in severe stages of the disease increased airway smooth muscle mass . acetylcholine , neuronal or nonneuronal and muscarinic receptors appear to play an essential regulatory role in airway remodeling [ 21 , 52 , 53 ] . recent studies in human - volunteering asthma patients , however , demonstrated that cholinergic stimuli and allergen can induce a very fast remodeling of the airway epithelium and the underlying mesenchymal cells within 8 days . interestingly , all features of remodeling were prevented by an inhaled beta2-agonist , leading the authors to postulate that relaxation of the bronchi prevented remodeling . based on our earlier studies , we suggest a more direct inhibitory effect of the beta2-agonist on various extracellular matrix genes . airway epithelial cells contribute to airway remodeling by hypersecretion of mucous and proliferation , while airway mesenchymal cells contribute by means of proliferation , expression of contractile protein , and the release of components such as mediators , extracellular matrix protein deposition , and matrix metalloproteinase ( mmp ) secretion [ 21 , 55 ] . the hypersecretion of mucous by airway epithelial cells contributes to airway obstruction in chronic airway diseases . in vitro and in vivo studies on animal models of asthma and copd demonstrate the important role of acetylcholine in the regulation of mucus secretion . using human bronchus and cultured epithelial cells it was shown that the expression of muc5ac is increased in asthma and copd patients and can be induced by carbachol and cigarette smoke extract while being inhibited by aclidinium , a long - acting muscarinic antagonist , or atropine . animals studies show that tiotropium inhibits increased muc5ac expression and mucus gland hypertrophy in a guinea pig model of copd , as well as the allergen - induced mucus gland hypertrophy and muc5ac - positive goblet cell number . acetylcholine may also regulate the proliferative and profibrotic response of airway epithelial cells , either through the induction of mechanical strain or by an autocrine / paracrine mechanism required for the repair of the damaged airway epithelium . epithelial cell proliferation and the expression of transforming growth factor ( tgf)- ( profibrotic cytokine ) were increased in bronchial biopsy specimens of patients with mild asthma following repeated challenge with methacholine or house dust mite allergen . animal studies indicated that acetylcholine induces proliferation of epithelial cells in the rat trachea , mediated by muscarinic m1 receptors and of airway epithelial cells in monkeys . in the human lung , the stimulation of the m2 receptor induced cell proliferation of fibroblasts [ 44 , 64 ] and acetylcholine enhanced cell proliferation in cells isolated from copd patients , as compared to healthy nonsmokers , through a process involving erk1/2 and nfb phosphorylation . airway smooth muscle thickening is a characteristic pathology of asthma , and to a lesser extent of copd . accumulating evidence suggests that stimulation of muscarinic receptors is involved in the proliferation and maturation of airway smooth muscle cells . furthermore , muscarinic receptor activation enhanced the mitogenic effect of platelet - derived growth factor ( pdgf ) and egf on airway smooth muscle cells [ 66 , 67 ] . moreover , the expression of myosin light - chain kinase was augmented by carbachol in human airway smooth muscle cells exposed to cyclical mechanical strain and stimulation of muscarinic receptors further enhanced the tgf-1-induced expression of the contractile protein . in animal models of asthma and copd , tiotropium significantly inhibited airway smooth muscle remodeling and contractile protein expression in guineapigs [ 52 , 60 ] and smooth muscle thickening and the expression of tgf-1 in bronchoalveolar lavage fluid in an ovalbumine mouse model similar effects have been described for the selective m3 receptor antagonist bencycloquidium bromide , which inhibited ovalbumin - induced mrna expression of il-5 , il-4 , and mmp-9 , as well as lung tissue eosinophil infiltration , airway mucus production , and collagen deposition in lung tissues in a murine asthma model . the cell - type - specific expressions of muscarinic receptors and their effect on airway remodeling and inflammation is summarized in figure 3 . extracellular matrix molecules , such as collagenous proteins , matrix metalloproteases ( mmp ) , glycosaminoglycans ( gag ) , and proteoglycans play a key role in airway remodeling , inflammation , and emphysema [ 7276 ] . increased levels of mmp-1 , mmp-2 , and mmp-9 have been reported in the sputum and lung parenchyma of asthma or copd patients . hypoxia , which is associated with extracellular matrix remodeling in inflammatory lung diseases , such as fibrosis , copd , and asthma , upregulated the expression of mmp-1 , mmp-2 , and mmp-9 precursors without subsequent activation in human lung fibroblasts and pulmonary vascular smooth muscle cells . mmp-13 expression was increased only in fibroblasts and pdgf - bb inhibited the synthesis and secretion of all hypoxia - induced mmp via erk1/2 map kinase activation . same evidence indicates that muscarinic receptors mediate the expression of mmp in obstructive pulmonary diseases . tiotropium inhibited tgf--induced expression of mmp-1 and mmp-2 in human lung fibroblasts , but had no effect on tgf--induced timp-1 and timp-2 expression [ 79 , 80 ] . in contrast , bencycloquidium bromide , a selective m3 receptor antagonist , inhibited ovalbumin - induced expression of mmp-9 mrna in a murine asthma model , indicating that m1 and m3 receptors mediate profibrotic and inflammatory response via specific mmps . evidence for the involvement of muscarinic receptors in the homeostasis of mmp comes also from other tissues . in human colon cancer , the activation of the m3 receptors stimulated the expression of mmp-1 , mmp-7 , and mmp-10 , with subsequent transactivation of the epidermal growth factor receptor and proliferation . hypoxia and pdgf - bb induced synthesis of soluble collagen type i via erk1/2 and p38 map kinase in human lung fibroblasts and pulmonary vascular smooth muscle cells . in human lung fibroblasts stimulation of m2 receptors induced cell proliferation and collagen synthesis [ 44 , 64 ] . in a clinical trial , inhalation of methacholine induced airway remodeling in asthma patients , through the expression of tgf- and collagen type - i as shown in bronchial biopsies . treatment with tiotropium inhibited the increased peribronchial collagen deposition in a guinea pig copd model . they contribute to viscoelastic properties , regulate permeability and retention of plasma components within the matrix , inhibit vascular cell growth , affect hemostasis , platelet aggregation , and interact with lipoproteins and various growth factors . there are two main types of gag : the nonsulphated hyaluronic acid and the sulphated gag , heparan sulphate , heparin , chondroitin sulphate , dermatan sulphate , and keratan sulphate . with the exception of hyaluronic acid , gag are usually covalently attached to a protein core , forming overall structures referred to as proteoglycans . evidence for the involvement of muscarinic receptors in the homeostasis of gag comes from studies on various tissues , including the lung . in rat bladder , hyaluronic acid ameliorated h2o2-induced hyperactivity , possibly via the antioxidant activity and the inhibition of purinergic and muscarinic signaling pathway . in rat vascular smooth muscle cells of the aorta , m3 receptors were involved in heparin - dependent relaxation . in rabbits , acetylcholine - induced reactive oxygen species generation in myocytes and the intact heart was mediated via transactivation of egf receptors through mmp - dependent release of heparin - binding egf via muscarinic receptors . in mouse pancreatic inhaled heparin inhibited the bronchoconstriction induced by methacholine , even though contrary results have also been reported . muscarinic receptors and their intracellular molecular pathways comprise a major drug target in obstructive lung diseases . there is a need for further pharmacological exploitation of this crucial family of receptors as targets for more effective treatment of asthma and copd . this huge potential transcends the beneficiary effect of antimuscarinic agents on bronchoconstriction and expands to anti - inflammatory , antiproliferative , and antiremodeling effects . extracellular matrix molecules , such as gag and mmp may be valuable biomarkers to determine the effect of muscarinic receptor inhibitors in clinical studies investigating drugs with anti inflammatory and anti - remodeling effects in the human lung .

muscarinic receptors are expressed by most cell types and mediate cellular signaling of their natural ligand acetylcholine . thereby , they control numerous central and peripheral physiological organ responses to neuronal activity . in the human lung , muscarinic receptors are predominantly expressed by smooth muscle cells , epithelial cells , and fibroblasts . antimuscarinic agents are used for the treatment of chronic obstructive pulmonary disease and to a lesser extent for asthma . they are primarily used as bronchodilators , but it is now accepted that they are also associated with anti - inflammatory , antiproliferative , and antiremodeling effects . remodeling of the small airways is a major pathology in copd and impairs lung function through changes of the extracellular matrix . glycosaminoglycans , particularly hyaluronic acid , and matrix metalloproteases are among extracellular matrix molecules that have been associated with tissue inflammation and remodeling in lung diseases , including chronic obstructive pulmonary disease and asthma . since muscarinic receptors have been shown to influence the homeostasis of glycosaminoglycans and matrix metalloproteases , these molecules may be proved valuable endpoint targets in clinical studies for the pharmacological exploitation of the anti - inflammatory and antiremodeling effects of muscarinic inhibitors in the treatment of chronic obstructive pulmonary disease and asthma .

1. Muscarinic Receptors 2. Intracellular Signaling of Muscarinic Receptors 3. Functional Role of Muscarinic Receptor Subtypes in the Lung 4. The Functional Role of Nonneuronal Muscarinic Receptor Subtypes in the Lung 5. Muscarinic Receptors in Obstructive Pulmonary Diseases 6. Muscarinic Receptors and Tissue Remodeling in the Lungs 7. Muscarinic Receptor and Extracellular Matrix Molecules 8. Conclusion

schizophrenia is a severe disabling disorder that affects approximately 1% of the worldwide population , but its causes and pathogenesis remain not completely known.1,2 the neurodevelopmental model is , to date , the most widely accepted pathogenetic hypothesis of schizophrenia , although contributions from the neurodegenerative model are not entirely excluded.3 rapoport et al4 published selective review updates of recent clinical , epidemiological , brain imaging , and genetic studies in support of the hypothesis that schizophrenia is primarily a neurodevelopmental disorder . altered brain development in schizophrenia may be accounted for by the abnormal expression of genes that are essential for neurodevelopmental processes . such genetic mechanisms may significantly interact with prenatal and/or perinatal environmental insults to enhance the risk of developing schizophrenia.1 so , early neurodevelopmental anomalies may interact with typical brain maturation processes throughout childhood and adolescence , causing a wide range of relatively benign markers of developmental disruption.5 delayed childhood development may precede adult psychosis,6,7 and aberrant neurodevelopment may be even more salient in cases of schizophrenia with very early onset , but the rarity of childhood - onset schizophrenia has limited studies of such patients.8 childhood - onset schizophrenia , in fact , is a rare disorder with a prevalence rate of one child in 10,000 before the age of 12 years , and a remarkable increase around puberty and early adolescence.9,10 on the other hand , early - onset schizophrenia , in biological continuity with adult forms of the illness , continues to provide unique neurodevelopmental data , as probands are younger , and developmental brain changes are less influenced by the illness or its treatment . for these reasons , investigation of childhood and early - onset schizophrenia has become a major research area during recent years , thus contributing to a better understanding of schizophrenia at all ages.4,9 language abnormalities , motor impairment and poor coordination , delays in the onset of urinary continence , difficulty reading , and broadly defined premorbid social impairments , have been individually described in early - onset schizophrenia.810 however , studies to date on neurodevelopmental impairments in early - onset psychosis ( eop ) are few and not readily comparable , mainly due to the different methods used to intercept development abnormalities retrospectively , thus leading to heterogeneous results.810 several questions remain open , and a better understanding of the evolutionary trajectories of neurodevelopment would be important since these premorbid impairments are not specific to schizophrenia and may occur in other neurodevelopmental disorders , including attention deficit hyperactivity disorder , intellectual deficiency , and autism spectrum disorders.4 this study was based on the hypothesis that in patients with eop , we can define evolutionary trajectories characterized by specific clinical and neurobiological markers of premorbid neurodevelopmental impairment arising during the early years of life . the aim was to assess early childhood neurodevelopmental milestones , general intellectual ability , and neurological soft signs ( nss ) in a sample of patients with early - onset schizophrenia spectrum psychosis compared to a control sample of patients with migraine . we referred jointly to childhood - onset and adolescent - onset schizophrenia spectrum psychosis as early - onset psychosis , defined as the manifestation of psychotic symptoms before the age of 18 years . the study sample consisted of 36 patients of both sexes who were under 18 years of age . they were consecutively referred since 2007 , over a 5-year period , among the inpatients of the child neuropsychiatry unit , department of basic medical sciences , neurosciences and sense organs , university aldo moro of bari , italy . these patients were diagnosed as suffering from early - onset schizophrenia spectrum psychosis ( schizophrenia , schizophreniform disorder , schizoaffective disorder , psychosis not otherwise specified ) in accordance with the diagnostic and statistical manual for mental disorders , 4th edition text revision ( dsm - iv - tr ) criteria.11 patients were excluded if they had comorbid attention deficit hyperactivity disorder and autism spectrum disorder . the control sample consisted of 36 patients of both sexes , recruited among the inpatients of the same child neuropsychiatry unit with a diagnosis of migraine in accordance with the international classification of headache disorders ii edition ( ichd - ii ) . these patients were more easily recruitable for the control group instead of the healthy subjects , because they had already been included in our headache protocol . these individuals were selected based on age and sex , which were similar to those of the patients ( age within 12 months ) ; the control subjects were excluded if they had a history of current and lifetime major psychiatric disorder , as determined through the clinical assessment of psychopathological conditions and an anamnestic interview . the ethical committee of the hospital consortium policlinico of bari , italy approved the study . experienced child psychiatrists made a diagnosis of eop on the basis of the patient s clinical evaluations , information obtained from family members , and a review of the patient s past medical records . clinical diagnoses were supported using the italian version of the semistructured diagnostic interview , kiddie - schedule for affective disorders and schizophrenia present and lifetime version ( k - sads - pl ) , conducted by child psychiatrists trained in the use of the instrument . premorbid and actual functioning was assessed using the premorbid adjustment scale ( pas)12 and the children s global assessment scale ( cgas).13 both cases and controls were subjected to a comprehensive clinical assessment , including a physical , neurological , and psychological examination , as well as an instrumental evaluation by laboratory blood tests and electrocardiogram . at least one of the parents of both the case and control group subjects was interviewed to record early childhood neurodevelopmental milestones , using a modified version of the general developmental scale.14 developmental skills were examined by binary test items ( attained / failed ) , which were expected to be solved by a large majority of children at a given age . five areas of early development were assessed : motor ; speech ; sphincter control ; social skills ; and school learning . each broad area of impairment was scored as present or absent on the basis of the following items : delay in motor development , including first unsupported sitting ( > 8 months ) and/or walking ( > 18 months ) ; delay in speech / language development , including the first word other than mama / dada ( > 24 months ) and/or first meaningful two- or three - word phrases ( > 36 months ) ; problems with sphincter control , including enuresis ( if wetting at least once a week over the age of 5 years ) and encopresis ( if occurring at least once a week over the age of 4 years ) , for a minimum of 6 months ; and school learning disabilities ( reading , writing , and calculation ) , which were confirmed by either school report or information gathered from the child s parents . impaired social development between the ages of 06 years required a definite history of the lack of one or more gestures when communicating , the lack of reciprocal social communication , stereotyped or idiosyncratic use of language , abnormal prosody , the lack of imaginative / imitative play , the failure to regulate gaze / facial expression / posture in social communication , the failure to make friends and share interests , and the failure to seek comfort or share pleasure . from the hollis version of general developmental scale , we deleted the item any neurodevelopmental disorder ( eg , hyperkinesis , tics , autism , learning disabilities , iq < 70 ) because it maybe overinclusive , referring to clinical conditions that deserve specific attention in the field of neurodevelopmental disorders . therefore , we replaced the item reading difficulties with difficulties in school learning , to include disabilities in writing and arithmetic as well . the intelligence quotient ( iq ) was assessed separately , and patients with comorbid attention deficit hyperactivity disorder and autism spectrum disorder were excluded from the study group . the first choice was the wechsler intelligence scale for children revised ( wisc - r),15 which provides a measure of verbal performance and full - scale iqs . for patients with an abnormality of speech and language , according to the clinical judgment , we used a nonverbal test the leiter international performance scale revised ( leiter - r)16 which is a battery of visualization and reasoning . nss , with specific regard to subtle motor impairment , were assessed by asking the patients to perform some motor tasks for the examination of motor coordination and motor sequencing ; based on the normal or impaired execution of these motor tasks , each subject was evaluated for the presence or absence of subtle motor impairment . to assess the relations between eop and childhood developmental abnormalities , iq impairment , and subtle motor impairment , 22 tables were constructed , and a chi - squared test ( or fisher s exact test , when appropriate ) was calculated . when it was possible , the odds ratio ( or ) and its 95% confidence interval ( ci ) were calculated . the study sample consisted of 36 patients of both sexes who were under 18 years of age . they were consecutively referred since 2007 , over a 5-year period , among the inpatients of the child neuropsychiatry unit , department of basic medical sciences , neurosciences and sense organs , university aldo moro of bari , italy . these patients were diagnosed as suffering from early - onset schizophrenia spectrum psychosis ( schizophrenia , schizophreniform disorder , schizoaffective disorder , psychosis not otherwise specified ) in accordance with the diagnostic and statistical manual for mental disorders , 4th edition text revision ( dsm - iv - tr ) criteria.11 patients were excluded if they had comorbid attention deficit hyperactivity disorder and autism spectrum disorder . the control sample consisted of 36 patients of both sexes , recruited among the inpatients of the same child neuropsychiatry unit with a diagnosis of migraine in accordance with the international classification of headache disorders ii edition ( ichd - ii ) . these patients were more easily recruitable for the control group instead of the healthy subjects , because they had already been included in our headache protocol . these individuals were selected based on age and sex , which were similar to those of the patients ( age within 12 months ) ; the control subjects were excluded if they had a history of current and lifetime major psychiatric disorder , as determined through the clinical assessment of psychopathological conditions and an anamnestic interview . the ethical committee of the hospital consortium policlinico of bari , italy approved the study . experienced child psychiatrists made a diagnosis of eop on the basis of the patient s clinical evaluations , information obtained from family members , and a review of the patient s past medical records . clinical diagnoses were supported using the italian version of the semistructured diagnostic interview , kiddie - schedule for affective disorders and schizophrenia present and lifetime version ( k - sads - pl ) , conducted by child psychiatrists trained in the use of the instrument . premorbid and actual functioning was assessed using the premorbid adjustment scale ( pas)12 and the children s global assessment scale ( cgas).13 both cases and controls were subjected to a comprehensive clinical assessment , including a physical , neurological , and psychological examination , as well as an instrumental evaluation by laboratory blood tests and electrocardiogram . at least one of the parents of both the case and control group subjects was interviewed to record early childhood neurodevelopmental milestones , using a modified version of the general developmental scale.14 developmental skills were examined by binary test items ( attained / failed ) , which were expected to be solved by a large majority of children at a given age . five areas of early development were assessed : motor ; speech ; sphincter control ; social skills ; and school learning . each broad area of impairment was scored as present or absent on the basis of the following items : delay in motor development , including first unsupported sitting ( > 8 months ) and/or walking ( > 18 months ) ; delay in speech / language development , including the first word other than mama / dada ( > 24 months ) and/or first meaningful two- or three - word phrases ( > 36 months ) ; problems with sphincter control , including enuresis ( if wetting at least once a week over the age of 5 years ) and encopresis ( if occurring at least once a week over the age of 4 years ) , for a minimum of 6 months ; and school learning disabilities ( reading , writing , and calculation ) , which were confirmed by either school report or information gathered from the child s parents . impaired social development between the ages of 06 years required a definite history of the lack of one or more gestures when communicating , the lack of reciprocal social communication , stereotyped or idiosyncratic use of language , abnormal prosody , the lack of imaginative / imitative play , the failure to regulate gaze / facial expression / posture in social communication , the failure to make friends and share interests , and the failure to seek comfort or share pleasure . from the hollis version of general developmental scale , we deleted the item any neurodevelopmental disorder ( eg , hyperkinesis , tics , autism , learning disabilities , iq < 70 ) because it maybe overinclusive , referring to clinical conditions that deserve specific attention in the field of neurodevelopmental disorders . therefore , we replaced the item reading difficulties with difficulties in school learning , to include disabilities in writing and arithmetic as well . the intelligence quotient ( iq ) was assessed separately , and patients with comorbid attention deficit hyperactivity disorder and autism spectrum disorder were excluded from the study group . the first choice was the wechsler intelligence scale for children revised ( wisc - r),15 which provides a measure of verbal performance and full - scale iqs . for patients with an abnormality of speech and language , according to the clinical judgment , we used a nonverbal test the leiter international performance scale revised ( leiter - r)16 which is a battery of visualization and reasoning . nss , with specific regard to subtle motor impairment , were assessed by asking the patients to perform some motor tasks for the examination of motor coordination and motor sequencing ; based on the normal or impaired execution of these motor tasks , each subject was evaluated for the presence or absence of subtle motor impairment . at least one of the parents of both the case and control group subjects was interviewed to record early childhood neurodevelopmental milestones , using a modified version of the general developmental scale.14 developmental skills were examined by binary test items ( attained / failed ) , which were expected to be solved by a large majority of children at a given age . five areas of early development were assessed : motor ; speech ; sphincter control ; social skills ; and school learning . each broad area of impairment was scored as present or absent on the basis of the following items : delay in motor development , including first unsupported sitting ( > 8 months ) and/or walking ( > 18 months ) ; delay in speech / language development , including the first word other than mama / dada ( > 24 months ) and/or first meaningful two- or three - word phrases ( > 36 months ) ; problems with sphincter control , including enuresis ( if wetting at least once a week over the age of 5 years ) and encopresis ( if occurring at least once a week over the age of 4 years ) , for a minimum of 6 months ; and school learning disabilities ( reading , writing , and calculation ) , which were confirmed by either school report or information gathered from the child s parents . impaired social development between the ages of 06 years required a definite history of the lack of one or more gestures when communicating , the lack of reciprocal social communication , stereotyped or idiosyncratic use of language , abnormal prosody , the lack of imaginative / imitative play , the failure to regulate gaze / facial expression / posture in social communication , the failure to make friends and share interests , and the failure to seek comfort or share pleasure . from the hollis version of general developmental scale , we deleted the item any neurodevelopmental disorder ( eg , hyperkinesis , tics , autism , learning disabilities , iq < 70 ) because it maybe overinclusive , referring to clinical conditions that deserve specific attention in the field of neurodevelopmental disorders . therefore , we replaced the item reading difficulties with difficulties in school learning , to include disabilities in writing and arithmetic as well . the intelligence quotient ( iq ) was assessed separately , and patients with comorbid attention deficit hyperactivity disorder and autism spectrum disorder were excluded from the study group . the first choice was the wechsler intelligence scale for children revised ( wisc - r),15 which provides a measure of verbal performance and full - scale iqs . for patients with an abnormality of speech and language , according to the clinical judgment , we used a nonverbal test the leiter international performance scale revised ( leiter - r)16 which is a battery of visualization and reasoning . nss , with specific regard to subtle motor impairment , were assessed by asking the patients to perform some motor tasks for the examination of motor coordination and motor sequencing ; based on the normal or impaired execution of these motor tasks , each subject was evaluated for the presence or absence of subtle motor impairment . to assess the relations between eop and childhood developmental abnormalities , iq impairment , and subtle motor impairment , 22 tables were constructed , and a chi - squared test ( or fisher s exact test , when appropriate ) was calculated . when it was possible , the odds ratio ( or ) and its 95% confidence interval ( ci ) were calculated . the study sample consisted of 23 males and 13 females with a mean age at assessment of 13 years ( range : 717 years ) . there was no significant difference in the mean age of onset according to sex ( 10 years for males ; 11 years for females ) , with a range of onset ranging from 5 to 16 years in both cases . the control group consisted of 20 males and 16 females with a mean age of 12 years ( range : 717 years ) . the main sociodemographic and clinical features of the study and control samples are summarized in table 1 . childhood developmental abnormalities , totally considered , were significantly associated with eop ( or : 14.8 ; 95% ci : 460.9 ; p<0.001 ) , as can be seen in table 2 . specifically , subjects with eop had a higher rate of impaired social development ( p=0.001 ) , school learning difficulties ( p=0.04 ) , and enuresis ( p=0.0008 ) than control subjects ( figure 1 ) . significant differences were also observed between cases and controls with regard to the iq evaluation ( p<0.001 ) , showing an association between cognitive impairment and eop . in the study group , we found five individuals with borderline iq , nine with mild cognitive impairment , and five with medium cognitive impairment . three patients were unable to complete the iq testing because of their clinical conditions . in the control group , we found one case of mild cognitive impairment and two cases of medium cognitive impairment . patients with eop also had a significantly higher frequency of subtle motor impairments than the control subjects ( p=0.005 ) with reference to both motor coordination and motor sequencing . in this study , we examined the clinical markers of neurobiological impairment across different areas of childhood development in a sample of eop patients that were compared with a control group of patients with migraine . it is known that children who become schizophrenic later in life have more developmental problems than controls , but the following questions remain : which developmental factors are more specifically associated with the illness ? and , is it possible to define abnormal evolutionary trajectories that more typically predict the onset of psychosis ? our data suggested that childhood neurodevelopment in eop is characterized by a global impairment of functional and adaptive skills , rather than a specific limitation or delay in the age of achievement of some developmental landmarks , such as learning to stand , walk , or speak . one of the main findings of this study , in fact , is that an impairment of social skills evident before the age of 6 years was significantly associated with eop rather than migraine . this finding is shared by most of the previous studies conducted on childhood neurodevelopment in early - onset schizophrenia , which compared schizophrenia patients with nonpsychotic psychiatric subjects and healthy subjects as well.14,1720 we can infer by the results of our and previous studies that early premorbid social impairment could represent all of the markers of neurodevelopmental impairment that are more specifically associated with eop . our data also point out that the sample of eop had significant difficulties in school learning . this had already been suggested by other studies , where poor premorbid educational attainment might be a putative predictive factor for the development of schizophrenia spectrum disorders.21,22 this finding is in continuity with the decrease of functioning observed in the premorbid phase of eop , and it repurposes the need to define the level of cognitive functioning in these subjects . we found a significantly higher frequency of lower iq in patients with eop than control subjects ( p<0.001 ) , which was in accordance with the neuropsychological evidence for the association between cognitive impairment and eop.2326 cognitive impairment has been considered a core feature of schizophrenia;27 thus , the addition of cognitive impairment as a diagnostic criterion for schizophrenia in the fifth edition of the dsm ( dsm-5)28 was carefully considered . no change was made with respect to dsm - iv - tr because cognitive deficits have not been found to sufficiently distinguish schizophrenia from several other boundary disorders . while the nature of cognitive impairment at the time of diagnosis may not be discriminating of schizophrenia , the developmental pattern of declining cognition over the years prior to the onset of psychosis may be relevant for differential diagnosis.29 moreover , all the neurocognitive domains impaired in schizophrenia , such as verbal memory , working memory , motor function , attention , executive functions , and verbal fluency , have been related to adaptive and social skills , so that a recent and inspiring field of research in schizophrenia is represented by social cognition.27,30,31 in line with these considerations , our data showed an impairment of the neurodevelopmental trajectory , but only in the areas of social and cognitive skills . this finding recalls the idea of a clinical and neurobiological connection between these two domains , and supports the hypothesis that an impairment in social cognition , evident from the earliest stages of neurodevelopment , may be a specific marker of the premorbid phase of schizophrenia . we found no significant association between speech delays , motor development delays , and eop , suggesting that the age of achievement of some developmental landmarks is not associated with eop . with regard to the delay of motor development , some authors of previous studies found significantly higher rates of patients with motor impairment ( from 31% to 64% ) , including both delayed milestones and poor coordination , symptoms of restlessness , and abnormal repetitive movements.17,18 we found , instead , no statistically significant difference in the acquisition of specific milestones of postural motor development . our results pertaining to statistical significance and prevalence rates ( 3%7% ) were similar to those reported by vourdas et al32 and hollis,14 likely because we used structured methods of data collection . on the other hand , we found that subtle motor impairment , considered separately , was present in 46% of the sample of patients , and this was significantly different from the control group . it can thus be inferred that subtle motor abnormalities are most significantly associated with the development path of patients with eop , while clearly identifiable impairment in postural as suggested by heuser et al33 motor nss may be related to abnormalities in the frontal and parietal cortices and in the cerebellum of patients with first - episode psychosis . epidemiological and clinical studies conducted to date show that children with special needs , such as those with low iq , fragile x and rett syndromes , have higher rates of urinary incontinence than children without developmental physical or cognitive impairments.34,35 however , there is less evidence for delayed or impaired bladder control in childhood-/adolescent - onset schizophrenia.14,32 hyde et al36 highlighted that there is a heritable component to childhood enuresis in the families of patients with schizophrenia and , based on data obtained by volumetric brain magnetic resonance imaging scans and neuropsychological testing , it was suggested that a history of childhood enuresis may be a marker of frontal cortical maldevelopment , potentially related to genetic risk factors . in conclusion , although this study had limitations associated with a small sample size and the use of migraine patients as a control group , we propose that the contemporary presence of difficulties in social skills evident before the age of 6 years and a low iq score may be considered as an early manifestation of developmental trajectories that evolve into impairments in social cognition , and later into emerging schizophrenia . impairments in motor coordination and enuresis may be considered more generically associated with a genetic risk for developing this illness . cognitive deficits are significantly associated with poor premorbid adjustment and functional outcomes , there is a clear rationale for further research to clarify the relationship between neurocognition and social competence , as well as to examine the relationship between these specific domains and the negative symptoms of eop .

backgroundthe aim of this study was to assess the association between the clinical and neurobiological markers of neurodevelopmental impairments and early - onset schizophrenia spectrum psychosis.methodsa sample of 36 patients with early - onset schizophrenia spectrum psychosis was compared to a control sample of 36 patients with migraine . we assessed early childhood neurodevelopmental milestones using a modified version of the general developmental scale , general intellectual ability using the wechsler intelligence scale for children revised or leiter international performance scale revised for patients with speech and language abnormalities , and neurological soft signs with specific regard to subtle motor impairment.resultssubjects with early - onset psychosis had a higher rate of impaired social development ( p=0.001 ) , learning difficulties ( p=0.04 ) , enuresis ( p=0.0008 ) , a lower intelligence quotient ( p<0.001 ) , and subtle motor impairments ( p=0.005 ) than control subjects.conclusionwe suggest that neurodevelopment in early - onset psychosis is characterized by a global impairment of functional and adaptive skills that manifests from early childhood , rather than a delay or limitation in language and motor development . the current evidence is based on a small sample and should be investigated in larger samples in future research .

Introduction Methods Subjects Assessment Early childhood neurodevelopment IQ measures Subtle motor impairment Statistical analysis Results Discussion Conclusion

lifestyle is a recognizable behavioral pattern stemming in interaction between personal characteristics of an individual and his / her life circumstances which reflects social values , attitudes , and activities of a person or group . health care providers , such as doctors , nurses , dietitians , and administrators have significant effect in primary prevention , because they serve as health role models to others . health care staffs , specifically the physicians can play a major role in promoting a healthy lifestyle in general public , if they practice healthy habits themselves . physicians personal lifestyles , habits , and health behaviors have been shown that have positive effect on their patients . in addition , health behaviors of physicians are important markers which can reflect on public health . the main aim of this study was to assess the physicians lifestyle in order to formulate policies , to promote overall well - being in community . this cross - sectional descriptive study was carried out in ardabil , a province in iran during 201213 . all physicians were invited to take part in this study and have received required information about sample collection . of all registered doctors in medical council of ardabil city about 800 persons , after calculated the exact sample size by statistical methods , 239 healthy physicians from private clinics and teaching hospitals in ardabil city were selected by using simple random sampling technique . unhealthy physicians and incomplete questionnaire were excluded from the study , therefore after excluding 14 questionnaires and 225 subjects [ 117 ( 52% ) males and 108 ( 48% ) females ] entered in this study . the study before start was approved by medical ethical committee of the ardabil university of medical sciences . the questionnaires used were standardized and their validity and reliability has been established in domestic and foreign studies . anthropometric factors including measurement of weight and height was performed according to standard protocols by using standardized and zero calibrated instruments . height was measured in meters and weight was measured to the nearest 0.1 kg using portable soehnle digital scales with a range of 0 . bmi was calculated using dividing weight in kilograms by the square of height in meters . bmi between 25 and 29.9 is considered overweight and bmi of 30 or higher is considered obese . blood pressure ( bp ) was measured ( with alpk2 sphygmomanometer , japan ) ( after 10 minutes comfortable resting of participants , in duplicate and seated position according to a standard protocol ; the average of two measures of the first and fifth korotkoff phase was recorded as final systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) , respectively ) . individuals with sbp 140 mmhg and dbp 90 mmhg were considered hypertensive and other individuals is in normal range . physical activity was assessed using the office al iranian short - version of the international physical activity questionnaire ( ipaq ) which is available at www.ipaq.ki.se . the short form of ipaq used in our study had seven items , providing information on time spent on walking , vigorous - intensity , moderate - intensity physical activities , and sedentary activity during the previous 7 days . the metabolic equivalent of task minutes per week for each of walking , moderate - intensity , and vigorous - intensity activities were calculated as follows : walking = ( 3.3 ~ walking min ~ walking days ) ; moderate activity = ( 4.0 ~ moderate activity min ~ moderate activity days ) ; vigorous activity = ( 8.0 ~ vigorous activity min ~ vigorous activity days ) . furthermore , sufficient vigorous activity was computed on the basis of 3 or more days of vigorous - intensity activity of at least 20 min / day . likewise , sufficient moderate - intensity and walking activities were computed based on 5 or more days of moderate - intensity activity and walking of at least 30 minutes per day . physical activity levels were also classified into three categories : inactive , minimally active , and health - enhancing physically active , according to the scoring system provided by ipaq . food habits and usual dietary intake was assessed using a 56-item food frequency questionnaire . the student 's t - test and paired sample t - test were used to compare the means in independent groups and paired groups , respectively . descriptive statistics are presented as mean standard deviation . to evaluate differences between groups , this cross - sectional descriptive study was carried out in ardabil , a province in iran during 201213 . all physicians were invited to take part in this study and have received required information about sample collection . of all registered doctors in medical council of ardabil city about 800 persons , after calculated the exact sample size by statistical methods , 239 healthy physicians from private clinics and teaching hospitals in ardabil city were selected by using simple random sampling technique . unhealthy physicians and incomplete questionnaire were excluded from the study , therefore after excluding 14 questionnaires and 225 subjects [ 117 ( 52% ) males and 108 ( 48% ) females ] entered in this study . the study before start was approved by medical ethical committee of the ardabil university of medical sciences . the questionnaires used were standardized and their validity and reliability has been established in domestic and foreign studies . anthropometric factors including measurement of weight and height was performed according to standard protocols by using standardized and zero calibrated instruments . height was measured in meters and weight was measured to the nearest 0.1 kg using portable soehnle digital scales with a range of 0 . bmi was calculated using dividing weight in kilograms by the square of height in meters . bmi between 25 and 29.9 is considered overweight and bmi of 30 or higher is considered obese . blood pressure ( bp ) was measured ( with alpk2 sphygmomanometer , japan ) ( after 10 minutes comfortable resting of participants , in duplicate and seated position according to a standard protocol ; the average of two measures of the first and fifth korotkoff phase was recorded as final systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) , respectively ) . individuals with sbp 140 mmhg and dbp 90 mmhg were considered hypertensive and other individuals is in normal range . physical activity was assessed using the office al iranian short - version of the international physical activity questionnaire ( ipaq ) which is available at www.ipaq.ki.se . the short form of ipaq used in our study had seven items , providing information on time spent on walking , vigorous - intensity , moderate - intensity physical activities , and sedentary activity during the previous 7 days . the metabolic equivalent of task minutes per week for each of walking , moderate - intensity , and vigorous - intensity activities were calculated as follows : walking = ( 3.3 ~ walking min ~ walking days ) ; moderate activity = ( 4.0 ~ moderate activity min ~ moderate activity days ) ; vigorous activity = ( 8.0 ~ vigorous activity min ~ vigorous activity days ) . furthermore , sufficient vigorous activity was computed on the basis of 3 or more days of vigorous - intensity activity of at least 20 min / day . likewise , sufficient moderate - intensity and walking activities were computed based on 5 or more days of moderate - intensity activity and walking of at least 30 minutes per day . physical activity levels were also classified into three categories : inactive , minimally active , and health - enhancing physically active , according to the scoring system provided by ipaq . the student 's t - test and paired sample t - test were used to compare the means in independent groups and paired groups , respectively . descriptive statistics are presented as mean standard deviation . to evaluate differences between groups , of 225 cases , 67.6% ( 152 ) were general and 32.4% ( 73 ) were specialists . of all hypertensive cases , 66.7% were male , 61.1% were specialist , and 44.4% have non - normal bmi . [ table 1 ] the mean age of medical doctors was 43.51 7.35 years old ( 45.41 8.34 in males and 41.49 5.48 in females ) . surprisingly prevalence of hypertension in specialists of medical doctors was higher than general practitioners ( 15.1% vs 4.6% ) ( p < 0.009 ) . other results showed that 7 ( 40% ) of hypertensive practitioners and 180 ( 80% ) of all practitioners was aware of their hypertension and about 64.4% ( 145 ) of individuals were previously measured their bp under standard conditions ( twice every 15 min in a relaxed state ) . number of cigarettes smoked per day with males was significantly higher than females ( 34.5% vs 7.3 ) ( p < 0.001 ) . the results showed that most of cases with higher sbp were in age groups 4655 with 122 7.5 and most of cases with dbp were in age group 4655 with 79 4.5 . the rate of sbp significantly different in all age groups but the rate of dbp is similar in all age groups [ table 2 ] . there was statistical significant relation between age groups and bp based on chi - square test . about 115 ( 51.1% ) and 41 ( 18.2% ) of subjects were overweight and obese , respectively . and , there was no significant difference in obesity rate between age groups and both sexes based on chi - square test . also other results showed that 70.7% of male practitioners and 74.1% of female practitioners had regular 10-min walking each day , moderate activity of males was significantly higher than females , and severe activity of females was significantly higher than males ( p < 0.05 ) [ table 3 ] . there was no significant difference in mean scores of metabolic equivalent of task -minutes / week for walking between both sexes . the prevalence of physical inactivity in males and females was 47.4% and 38.6% , respectively . just one - fifth of medical doctors had vigorous physical activity for at least 20 min , 3 days a week . food frequency consumption of bread types , rice , macaroni , pies , biscuit , liquid fat , red meat , cream , butter , nuts , and sausage per week in overweight physicians were significantly more than normal weight physicians ( p < 0.05 ) , and food frequency consumption of coffee intake per week in hypertensive subjects was significantly higher than normotensive subjects ( p < 0.05 ) [ table 4 ] . the rate of inactivity rate in specialist with 46.6% was more than general practitioner [ table 5 ] . the prevalence of hypertension by exposure variables means of systolic and diastolic blood pressure by age group total means scores for all walking , moderate and vigorous physical activities by met - minutes / week the mean intake of food frequency status per week in medical doctors physical activity profile of ardabil 's medical doctors our findings showed that 8% of medical doctors were hypertensive , 21.3% smoker , 44% inactive , 51.1% overweight , and 18.2% obese , which all of these parameters are risks for health statues . study of bazargan et al . demonstrated that focus on creating healthy lifestyles of physicians specially controlling cardiovascular risk factors can benefit general population . in other words , physicians with impaired physical and mental health can have a direct impact on patient health care and safety . our data showed that about 70% of participants were overweight and obese which not similar to study by santos and sichieri . results of these studies are demonstrated that overweight and obesity are common problem of ardabil community and , as mentioned earlier , physicians can impact on public health . other studies are showing that high bmi and smoking are associated with increased hypertension too . also we found that , overall ; about 50% of our samples had a normal bmi regardless of their gender , which , this was similar to the finding of the previous studies . in our study , similar to studies in developing countries , the prevalence of overweight and obesity was higher in high level education groups . as improved physician health has been linked to more frequent and successful counseling with patients about lifestyle behaviors ; therefore , they should be encouraged to have a healthy lifestyle , unfortunately our study expands physician 's weak lifestyle and health behaviors and also obesity in ardabil city . also food frequency consumption of high carbohydrate ( i.e. , bread types , rice , macaroni , pies , biscuit , liquid fat , red meat ) , high - energy ( such as cream , butter , nuts , and sausage ) per week in overweight physicians were significantly more than normal weight physicians , and these results are consistent with similar studies among adults . we found that increased eating frequency of coffee intake per week in hypertensive subjects was significantly higher than normotensive subjects . similar to our finding , some studies have found a relationship between coffee intake and bp , whereas other investigations showed no positive or negative associations between hypertension and coffee drinking . coffee intake , smoking , low activity were risk factor for overweighting and obesity in physicians , while most of physicians emphasis on importance of healthy behaviors , few of them follow health lifestyle , and this factor can have a negative effect on society attitude about health . there was not any limitation in carrying out this study , only some of physicians tend to complete the questionnaires and we must replace them by new samples . because importance of lifestyle and quality of life studies between doctors and all health workers , doing more future studies about these themes is essential .

background and objective : few studies have been done on lifestyle of iranian physicians . as physicians have important role in health promotion , the main goal of the study was to assess the lifestyle of this influential group.materials and methods : a cross - sectional descriptive study was conducted on lifestyle of all registered physicians of ardabil hospitals , iran , 201213 . in this research , 225 physicians were selected , by using simple random sampling . demographic and lifestyle data were obtained by self - report using standard questionnaires , physical activity by official iranian short - version of the international physical activity questionnaire , and dietary intake by food frequency questionnaire . weight and height was performed according to standard protocols by using standardized and zero calibrated instruments . data were analyzed by inferential statistics using statistical package for the social sciences . 16 software.results:findings showed that 8% of participants were hypertensive , 21.3% smoker , 40%47% inactive , 51.1% overweight , and 18.2% obese . there was a significant relationship between blood pressure and self - reported lifestyle habits ( p < 0.05 ) . and 70.7% of males and 74.1% of females had regular 10-min walking each day and moderate activity of males was significantly higher than females ( p < 0.05 ) . food frequency weekly consumption of overweight and obese physicians were significantly higher than normal weight physicians ( p < 0.05).conclusion : few doctors follow a healthy lifestyle ; this may have a negative effect on society attitude about health .

Introduction Materials and Methods Participants Anthropometric and blood pressure measurements Physical activity and food habit assessment Results Discussion Conclusion Financial support and sponsorship Conflicts of interest

an anatomical configuration of the prostate termed the intravesical prostatic protrusion ( ipp ) can be measured as the vertical distance from the tip of the protruding prostate to the base of the bladder at the base of the prostate gland . the ipp can be measured noninvasively by transrectal ultrasonography ( trus ) and can predict voiding parameters for determining boo in men who present with luts . ipp may also be a useful predictor for clinical progression in men with benign prostatic hyperplasia ( bph ) [ 3 - 6 ] . ipp is insufficient for managing patients with bph / luts ; physicians know only that ipp is a significant predictor for boo , because most elderly people with bph / luts have some degree of ipp . hence , a novel means of gauging ipp is needed . the goal of this study was therefore to determine noninvasive parameters for predicting boo and defining the degree of ipp causing boo in a large population of korean men by analyzing the receiver operating characteristic ( roc ) curves for ipp , including the cutoff , and correlating ipp and other clinical variables with the results of a pressure - flow study . we performed a retrospective analysis of clinical outcomes of consecutive male korean patients aged 50 to 90 years with luts / bph who underwent transurethral resection of the prostate ( turp ) from may 2007 to december 2010 . patients with a known history of prostate or bladder carcinoma , urinary tract infection , renal impairment , bladder calculi , or neurological deficit were excluded . survey of the international prostate symptom score ( ipss ) and quality of life ( qol ) scores was performed for evaluation of baseline parameters . a physical examination included a digital rectal examination to screen for prostatic growth suspicious of malignancy and a neurological examination to exclude any neurological deficits or neurologically related bladder dysfunction . preoperative evaluation included urodynamic studies including pressure - flow studies and serum prostate - specific antigen ( psa ) . four urodynamic parameters including the postvoiding residual urine ( pvr ) , peak flow rate ( qmax ) , detrusor pressure at the peak flow rate ( pdetqmax ) , and maximal detrusor pressure during voiding ( pdetmax ) were statistical analyzed . urethral resistance was quantified by using the boo index ( booi ) , according to the formula booi = pdetqmax-2qmax . patients were classified into two groups by the boo : obstructed ( booi>40 ) and unobstructed ( booi40 ) . ipp was also measured as the vertical distance from the tip of the protruding prostate to the base of the bladder at the base of the prostate gland ( fig . roc curve analysis was performed to evaluate the diagnostic performance of ipp for predicting boo . a multivariate logistic regression was performed to identify strength and association between boo and ipp , compensating for factors such as age , ipss , qol , prostatic volume , and urodynamic parameters . of the 534 patients , 239 patients were included and 295 patients were excluded because of neurologic deficits , urinary tract carcinoma , or renal impairment . the mean age of the 239 included patients was 69.98.0 years ( meanstandard deviation ) . the mean ipss was 14.211.6 , the mean qmax was 9.56.8 ml / s , the mean pvr was 35.146.6 ml , and the mean degree of ipp was 3.424.71 mm . the number of patients in the unobstructed and obstructed groups was 193 and 46 , respectively . significant boo ( index>40 ) was associated with higher ipp ( 8.086.74 mm , p<0.001 ) compared with equivocal boo ( 1.983.11 mm , p<0.001 ) . the area under the roc curve ( auc ) for ipp was 0.759 ( 95% confidence interval [ ci ] , 0.657 to 0.861 ) ( fig . the aucs for total prostate volume , serum psa , qmax , and pvr were 0.746 , 0.572 , 0.384 , and 0.696 , respectively . boo was positively correlated with ipp , pvr , and prostate volume ( spearman 's rho=0.551 , 0.225 , and 0.391 , respectively ; p<0.001 for each parameter ) and was negative correlated with qmax ( spearman 's rho=-0.159 , p=0.026 ) . ipss and the qol index had poor predictive significance for boo ( positive predictive value of < 60% , p=0.624 and p=0.138 , respectively ) . qmax , pvr , prostatic volume , and ipp had significant predictive value for boo ( p=0.026 , p<0.001 , p<0.001 , and p<0.001 , respectively ) . however , qmax , pvr , and prostatic volume had lower positive predictive values ( 27% to 53% vs. 79% ) than did ipp , and the negative predictive value of qmax , pvr , and prostatic volume were similar to that of ipp ( 87% to 91% vs. 89% ) . logistic regression analysis revealed that ipp was a significant independent variable when other covariates including age were considered . the odds ratio of boo for ipp was 3.99 ( 95% ci , 1.46 to 10.8 ) ( table 2 ) . one of the important pathophysiologies in bph is boo , making evaluation of the severity and presence of boo important . there is no definite or reference standard for boo , except for pressure - flow study . however , the latter is too expensive ( especially in developing countries ) , is invasive , and is uncomfortable for patients . to avoid these drawbacks , the identification of new accurate methods that could substitute for the gold standard pressure - flow study at lower cost , expanded accessibility , and with relief of patient discomfort has become an important goal . other noninvasive clinical variables have no significant correlation with boo [ 5,8 - 10 ] . the ipss is a simple tool in the evaluation of benign prostatic enlargement , and a worsening score may warrant intervention . pvr may reflect the severity of boo , but the presence of bladder dysfunction confounds its value . in our study , ipss and qol score were not correlated with boo ( p=0.624 and p=0.138 , respectively ) . this was expected , because symptoms need not be related to obstruction . in addition , the urine flow rate and pvr had lower positive predictive values than did ipp . clinical data such as ipss , pvr , and flowmetry correlate mostly to lower urinary tract functional status rather than mechanical obstruction itself [ 8 - 10 ] . ipp aside from pressure - flow studies is a predictor of boo in bph / luts patients . ipp arises from the enlargement of the median and lateral lobes and causes a ball - valve type of obstruction , which thus disrupts the funneling effect of the bladder neck . the severity of ipp has been correlated with prostate volume , creating boo in patients with bph / luts . in the present study , the boo group displayed a greater ipp than did the equivocal boo group ( p<0.001 ) , and the degree of ipp correlated with boo ( r=0.551 ) . despite this analysis of the roc curve concerning the relationship between ipp and boo revealed a cutoff of 5.5 mm for ipp ( sensitivity of 66.7% and specificity of 80.5% ) : an ipp exceeding 5.5 mm was significantly associated with boo . but the finding that ipp exists in bph / luts patients can not completely explain the situation in patients in whom symptoms are intractable to treatment with alpha - blockers or turp . we think that a specific degree of ipp can be applied in the treatment of bph / luts patients . further studies are needed to define a cutoff of ipp as a causative criterion of boo . reported that roc curves of ipp yielded an auc of 0.858 and 0.758 , respectively , which are similar or greater than the presently found value . reported that the best cutoff was 7.5 mm ( sensitivity of 75.5% and specificity of 82.6% ) , which was greater than our cutoff of 5.5 mm . the patients in our study were korean males who presented with a complaint of luts . there could be a racial explanation for the difference in cutoff values between the prior and present studies . also , the study number ( n=239 ) was small ; studies with larger numbers are warranted . yet , the present study corroborates the findings of prior studies concerning the ipp cutoff for boo . we analyzed roc curves concerning the relationship between ipp and boo and between boo and other parameters ( prostate volume , psa , qmax , pvr ) . interestingly , the ipp displayed a greater auc value of 0.759 than did prostate volume ( 0.746 ) . the prevailing view is that ipp correlates with boo but does not have a significant value compared with prostate volume . the presently determined auc value for ipp indicates that the measurement of ipp has greater diagnostic value in boo than does prostate volume in evaluating prostate sonographic data . boo is dynamic and is influenced by the physical obstruction of the bladder and prostate , and we think that ipp measurement is needed in patients when prostate volume is not excessive , and that boo patterns may be useful . also , ipp measurements besides uroflowmetry , pvr , and psa likely are influential in diagnosing boo , in that in the present study ipp produced a larger auc than did psa , qmax , or pvr . these data support the view that ipp can predict boo , compared with qmax , pvr , and prostate volume , for bph / luts patients , and may have diagnostic predictive value similar to that of pressure - flow studies . also , by predicting boo and defining a specific ipp cutoff linked with the occurrence of boo , we suggest that the degree of ipp can be a guideline for further treatment in patients with bph / luts .

purposethe present study was done to define the degree of intravesical prostatic protrusion ( ipp ) causing bladder outlet obstruction ( boo ) in patients with benign prostatic hyperplasia ( bph)/lower urinary tract symptoms.materials and methodswe retrospectively evaluated 239 patients with bph , analyzing age , ipp , prostate volume , international prostate symptom score ( ipss ) , and the results from a pressure - flow study . urethral resistance was quantified by using the boo index ( booi ) , according to the formula booi = pdetqmax-2qmax ( where pdet is detrusor pressure at the peak flow rate and qmax is peak flow rate ) . boo was defined by a booi above 40 . patients with a booi below 20 were excluded . patients were classified into two groups ( obstructed and unobstructed groups ) by the booi . correlations were determined by logistic regression analysis , and receiver operating characteristic curves were plotted to estimate the optimal cutoff for ipp.resultsthere were significant differences in total prostate volume , postvoiding residual urine ( pvr ) , ipp , and qmax ( p<0.001 , p<0.001 , p<0.001 , and p=0.026 , respectively ) between the obstructed and unobstructed groups , but there were no significant differences in age ( p=0.653 ) , ipss total score ( p=0.624 ) , or quality of life score ( p=0.138 ) . ipp had a significant prognosis ( p<0.001 ) but was weakly correlated with prostate volume ( p=0.026 ) . the correlation coefficients between ipp and qmax , pvr , prostate volume , and boo were 0.551 , -0.159 , 0.225 , and 0.391 , respectively . for ipp , the area under the curve was 0.759 ( 95% confidence interval , 0.657 to 0.861 ) and the cutoff to indicate boo was 5.5 mm with 66.7% sensitivity and 80.5% specificity.conclusionsan ipp exceeding 5.5 mm was significantly associated with boo .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

approximately one - third of women will be affected by heavy or abnormal uterine bleeding ( aub ) at some time in their lives.1 aub risk factors include age , premenopausal leiomyomata , and endometrial polyps , and aub is a presenting symptom for the majority of women who undergo a hysterectomy.24 aub significantly diminishes quality of life , despite the fact that serious complications are rare.5 a common result of aub is anemia , which can cause weakness , fatigue , unexplained weight loss , mood swings , and impaired cognitive functioning.5 aub treatment can be pharmacological and/or surgical . options for surgical treatment include hysterectomy , myomectomy , uterine artery embolization , first - generation endometrial ablation methods ( including rollerball or laser ablation and transcervical resection of endometrium ) , and second - generation global endometrial ablation ( gea ) methods ( including laser intrauterine thermotherapy , cryoablation , and microwave , thermal balloon , or radiofrequency ablation ) . first - generation endometrial ablation methods require hysteroscopy to improve visualization and require general or regional anesthesia.4 second - generation methods can be performed in an outpatient office setting under local anesthesia , without the use of a hysteroscope , and require minimal cervical dilation ; they are faster and less technically demanding.4 both endometrial ablation and hysterectomy are effective , and are becoming more commonly used as first - line treatment . the choice between them is currently an informed tradeoff , with endometrial ablation methods having high patient satisfaction and a low complication rate , but sometimes requiring reintervention.4 surgical treatment with hysterectomy is both effective and permanent , but hysterectomy has greater morbidity and may have higher costs.4,6 the cost of these treatments is becoming a larger factor in their selection . as an outpatient treatment , gea may be viewed as a lower - cost alternative to hysterectomy by insurers such as medicaid , which currently insures about 12% of us women aged 1864 years.7 medicaid expansion under new federal mandates is expected to extend eligibility to an additional 7 million currently uninsured women aged 1964 years . treatment decisions for medicaid - insured women with aub will require data on the costs of surgical therapies in this population . the objective of this study is to describe aub treatment patterns among women enrolled in medicaid , and compare the direct health care costs of treating aub with hysterectomy or with gea . the study sample was selected from the truven health marketscan multi - state medicaid database . the database includes the medical , surgical , and prescription drug experience of nearly 31 million de - identified medicaid enrollees from multiple states , including records of inpatient services , inpatient admissions , outpatient services , and prescription drug claims , as well as information on long - term care and other medical care . all database records are de - identified and fully compliant with us patient confidentiality requirements , including the health insurance portability and accountability act ( hipaa ) of 1996 . a cohort of women who underwent gea or hysterectomy ( index event ) for the treatment of aub between january 1 , 2006 , and december 31 , 2010 were identified in the database . patients were selected using the following inclusion criteria : at least two claims on different dates with a diagnosis of aub ( icd-9-cm codes 626.2x , 626.4x , 626.6x , 626.8x or 627.0x ) in the 12 months prior to the index event , with at least one diagnosis specific to heavy or excessive bleeding ( icd-9-cm codes 626.2x or 627.0x ) ; aged 3055 years on the index date ; and at least 12 months of continuous health plan enrollment prior to ( pre - index period ) and following ( post - index period ) the index event . exclusion criteria included : diagnosis of menopause prior to the index date ; diagnosis of a primary cancer at any time during the study period ; hysterectomy or removal of a levonorgestrel - releasing intrauterine device during the pre - index period ; and indication of pregnancy or delivery in the 90 days prior to the index date . health insurance claims ( medical and prescription drug ) for the 12-month pre - index and post - index periods were evaluated for all patients who met the study selection criteria . records were examined to assess baseline demographic characteristics and pre - index clinical characteristics , as listed in table 1 , including the deyo - charlson comorbidity index.8 intervention - related costs were defined as treatment - related costs as well as the cost of complications and medical care and costs in the 30 days following index ; subsequent gynecological health care utilization and costs were captured for the remainder of the 12-month post - index period . health care costs were taken directly from the paid amount on fully adjudicated claims and included both the insurer and medicaid payment portions . health care costs were adjusted to 2011 dollars using the medical care component of the consumer price index.9 treatment - related complications were defined as the presence of any of the following events : cervical occlusion , cervical trauma ( including cervical lacerations and hematometra ) , uterine perforation , bowel perforation , fluid overload , pyometra , and device complications ; a second aub treatment within 30 days ; or an inpatient stay or emergency room visit within 2 days of the index procedure ( excluding patients with an inpatient index event ) . for gea patients , reintervention was defined as the presence of either a second gea intervention or hysterectomy at least 30 days after the index event . adjunctive pharmacotherapy was defined as the use of levonorgestrel - releasing intrauterine system , non - intrauterine system hormonal therapies , or tranexamic acid . among gea patients , adjunctive pharmacotherapy and reintervention were described for up to 3 years post - index for patients with available data . the costs of reintervention and adjunctive pharmacotherapy are included in the gynecology - related follow - up costs . standard statistical tests were used to compare patient characteristics and outcomes between gea and hysterectomy patients . chi - square tests were used for categorical measures and t - tests were used for continuous measures . the study sample was selected from the truven health marketscan multi - state medicaid database . the database includes the medical , surgical , and prescription drug experience of nearly 31 million de - identified medicaid enrollees from multiple states , including records of inpatient services , inpatient admissions , outpatient services , and prescription drug claims , as well as information on long - term care and other medical care . all database records are de - identified and fully compliant with us patient confidentiality requirements , including the health insurance portability and accountability act ( hipaa ) of 1996 . a cohort of women who underwent gea or hysterectomy ( index event ) for the treatment of aub between january 1 , 2006 , and december 31 , 2010 were identified in the database . patients were selected using the following inclusion criteria : at least two claims on different dates with a diagnosis of aub ( icd-9-cm codes 626.2x , 626.4x , 626.6x , 626.8x or 627.0x ) in the 12 months prior to the index event , with at least one diagnosis specific to heavy or excessive bleeding ( icd-9-cm codes 626.2x or 627.0x ) ; aged 3055 years on the index date ; and at least 12 months of continuous health plan enrollment prior to ( pre - index period ) and following ( post - index period ) the index event . exclusion criteria included : diagnosis of menopause prior to the index date ; diagnosis of a primary cancer at any time during the study period ; hysterectomy or removal of a levonorgestrel - releasing intrauterine device during the pre - index period ; and indication of pregnancy or delivery in the 90 days prior to the index date . health insurance claims ( medical and prescription drug ) for the 12-month pre - index and post - index periods were evaluated for all patients who met the study selection criteria . records were examined to assess baseline demographic characteristics and pre - index clinical characteristics , as listed in table 1 , including the deyo - charlson comorbidity index.8 intervention - related costs were defined as treatment - related costs as well as the cost of complications and medical care and costs in the 30 days following index ; subsequent gynecological health care utilization and costs were captured for the remainder of the 12-month post - index period . health care costs were taken directly from the paid amount on fully adjudicated claims and included both the insurer and medicaid payment portions . health care costs were adjusted to 2011 dollars using the medical care component of the consumer price index.9 treatment - related complications were defined as the presence of any of the following events : cervical occlusion , cervical trauma ( including cervical lacerations and hematometra ) , uterine perforation , bowel perforation , fluid overload , pyometra , and device complications ; a second aub treatment within 30 days ; or an inpatient stay or emergency room visit within 2 days of the index procedure ( excluding patients with an inpatient index event ) . for gea patients , reintervention was defined as the presence of either a second gea intervention or hysterectomy at least 30 days after the index event . adjunctive pharmacotherapy was defined as the use of levonorgestrel - releasing intrauterine system , non - intrauterine system hormonal therapies , or tranexamic acid . among gea patients , adjunctive pharmacotherapy and reintervention were described for up to 3 years post - index for patients with available data . the costs of reintervention and adjunctive pharmacotherapy are included in the gynecology - related follow - up costs . standard statistical tests were used to compare patient characteristics and outcomes between gea and hysterectomy patients . chi - square tests were used for categorical measures and t - tests were used for continuous measures . a total of 1,880 aub patients met the study inclusion and exclusion criteria . just over half of these women ( n=957 ) were treated with gea , while the remainder ( n=923 ) underwent hysterectomy . as shown in table 1 , the gea cohort was slightly younger than the hysterectomy cohort ( mean age 40.2 years versus 41.2 years , respectively , p<0.001 ) . approximately half of the study patients ( 53.4% ) were caucasian , 33.1% were african - american , and 2.3% were hispanic . patients were covered by network - based managed care plans ( 53.8% ) or comprehensive health plans ( 45.9% ) . compared with gea , a larger proportion of hysterectomy patients received their medicaid eligibility due to disability ( 46.4% versus 39.4% , p=0.003 ) . baseline mean deyo - charlson comorbidity index scores were slightly higher for gea patients , but not significantly so ( 0.69 versus 0.62 , p=0.223 ) . pre - index medication use was similar between the treatment cohorts ; over half of the patients used a prescription nonsteroidal anti - inflammatory drug ( 56% ) and nearly 70% used an antibiotic . very few patients used an oral contraceptive ( 5.3% ) during the pre - index period . hysterectomy patients were significantly more likely to have a diagnosis of uterine fibroids or polyps ( 55.5% versus 26.5% , p<0.001 ) , anemia ( 37.4% versus 27.3% , p<0.001 ) , or endometriosis ( 9.3% versus 2.7% , p<0.001 ) . gea and hysterectomy patients had a similar baseline prevalence of depression ( approximately 32% ) and thyroid disease ( approximately 11% ) . as shown in table 2 , the large majority ( 93% ) of gea procedures occurred in an outpatient setting , compared with 13% of hysterectomy procedures ( p<0.001 ) . expenditures for the intervention and 30-day follow - up care were significantly higher for hysterectomy ( $ 11,270 ) than for gea ( $ 3,958 , p<0.001 ) . gynecology - related costs for the remainder of the year were also higher for hysterectomy than for gea but not significantly so ( $ 63 per month versus $ 16 per month , p=0.111 ) . gynecology - related costs in the remainder of the year were more likely to come from an outpatient setting than an inpatient setting for gea patients ( 55% versus 45% ) , whereas for hysterectomy patients most costs were inpatient in nature ( 83% versus 17% ) . while hysterectomy patients were more likely to have a gynecology - related inpatient stay either at index or follow - up , the average length of stay among patients with a gynecology - related inpatient stay was similar between hysterectomy and gea patients ( 2.6 versus 2.5 days , p=0.74 ) . more than half ( 52% ) of hysterectomy patients had a treatment - related complication , compared with 36% of gea patients ( p<0.001 ) . women with complications after treatment had higher overall costs associated with treatments in both subgroups . among gea patients , patients with complications were 1.4 times more costly than gea patients without complications , while hysterectomy patients with complications were 1.3 times more costly than other hysterectomy patients without complications . reintervention and adjunctive pharmacotherapy in the 3 years following gea are reported in table 3 . among the 957 gea patients who met the study inclusion and exclusion criteria , 529 had 2 years of post - index continuous enrollment and 295 had 3years of post - index continuous enrollment . in the first year following gea , 10.6% of gea patients underwent reintervention ; 9.4% underwent hysterectomy and 1.1% underwent a second gea intervention . reintervention with either hysterectomy or a second gea was less common in the second ( 1.1% and 0.0% , respectively ) and third year ( 2.7% and 2.4% ) . adjunctive pharmacotherapy decreased over time , with 5.4% of patients using non - intrauterine system hormonal therapies in the first year , 1.1% in the second year , and 0.0% in the third year post - index . the current study describes aub - related treatment and follow - up costs for medicaid - enrolled women initiating either gea or hysterectomy . the total direct costs for treatment and 30-day follow - up were lower for gea ( $ 3,958 ) than for hysterectomy ( $ 11,270 , p<0.001 ) . gea patients were also less likely to have treatment - related complications ( 36% versus 52% , p<0.001 ) , which increased costs by 40% ( 30% in hysterectomy patients ) . these results suggest that gea may be a cost - saving alternative to hysterectomy for aub in the medicaid population . previous studies have suggested similar results in populations closely related to aub.10,11 in an analysis by jensen et al , costs incurred by patients with heavy menstrual bleeding , a type of aub , were compared with costs of patients without heavy menstrual bleeding . the average annual direct medical cost of patients with heavy menstrual bleeding was $ 5,816 , which was $ 2,533 higher than for their matched controls.11 after their first diagnosis of heavy menstrual bleeding , patients were followed for an average of 2.3 years . during that follow - up period , 27% did not receive any treatment for heavy menstrual bleeding . of the 73% who did previous studies have documented much higher costs for hysterectomy than for uterine - sparing procedures such as gea . in an analysis of women with newly diagnosed heavy menstrual bleeding , the mean cost of a hysterectomy episode in women aged 3539 years was $ 8,980 , compared with $ 5,288 for ablation ( these costs were $ 9,200 and $ 5,133 in women aged 4049 years).12 similarly , a cost analysis of women undergoing treatment for uterine fibroids reported adjusted direct medical costs of $ 10,269 for hysterectomy and $ 7,303 for endometrial ablation in the year after the intervention.13 in our study , reintervention rates following gea were more common in the first year , with 9.4% of gea patients undergoing hysterectomy and 1.1% undergoing a second gea intervention . reintervention with either hysterectomy or a second gea was less common in the second ( 1.1% and 0.0% ) and third year ( 2.7% and 2.4% ) . these reintervention rates are lower than previously reported estimates , which likely included more first - generation ablation techniques.14 however , the current analysis was consistent with other studies in describing higher complication rates for hysterectomy than for gea.15 this current analysis also followed patients for up to 3 years post - intervention ; however , only 2 and 3 years of follow - up were available on a subset of patients ( 55% and 31% , respectively ) and it is unclear if these patients differed substantively from patients with shorter follow - up . the results of this analysis may have broad implications in the context of current changes taking place in the medicaid program . specifically , these entail new federal mandates from the patient protection and affordable care act ( ppaca ) for medicaid expansion and the creation of state - based and federally - facilitated competitive marketplaces , or affordable insurance exchanges . the main goal of the ppaca is to provide a range of affordable insurance coverage options through medicaid and the new exchanges to reduce the number of uninsured in the usa.16,17 according to plan , the exchanges will achieve this goal by providing an opportunity for qualified individuals and small employers to evaluate and compare high - quality , affordable , and competitively priced private health plans.18,19 these exchanges will offer a new pathway to insurance coverage for many adults , effectively increasing the number of patients with government - sponsored health insurance.20 with women comprising the majority ( 69% ) of the adult medicaid population,21 changes in medicaid will matter to the population of low - income women with aub . presently , about 12% of women aged 1864 years rely on medicaid for their health care coverage.22 medicaid expansion under the ppaca is expected to expand eligibility to an additional 7 million previously uninsured women aged 1964 years and with incomes below 138% of the federal poverty level . it is expected that a substantial portion of these women will be childless adults who were not previously eligible for medicaid coverage and , like current medicaid enrollees , are more likely to be in poorer health than the general population.23 approximately 20% ( 1.4 million ) of newly eligible women in an expanded medicaid program will have aub , many of whom will go on to receive costly pharmacological and surgical treatments every year.2426 the results of our analyses indicate that the costs of aub intervention plus one month of follow - up ( combining patients with and without complications ) total approximately $ 4,000 for gea , considerably less than the $ 11,300 estimate for hysterectomy . supposing a conservative 1% annual treatment rate for aub with either gea or hysterectomy , medicaid s expanded coverage of many more aub patients is likely to require large outlays for these therapies . the high price of the inpatient hospital care required for hysterectomy will likely cause concern . as a result , state - based and federally facilitated exchanges may focus intently on pharmacological treatments and gea ( particularly in - office gea procedures ) as ways to reduce costs to medicaid , with a lasting impact on the treatment landscape for aub in the usa . first , although mortality is not a common outcome of aub and its treatments , data on mortality were not available in describing the complete picture of patient outcomes . second , administrative claims databases are developed and maintained for the purposes of facilitating health care payments . this suggests that comorbidities and indicators of severity might not be captured accurately because they are not used for payment generation . third , analysis of individual brands or types of gea or hysterectomy was prevented by the lack of specificity available in current procedure codes for gea and hysterectomy . finally , the study population consists of patients with medicaid ; therefore , the results may not represent all patients with aub , including the uninsured who may gain insurance coverage through the ppaca . the results of this analysis may have broad implications in the context of current changes taking place in the medicaid program . specifically , these entail new federal mandates from the patient protection and affordable care act ( ppaca ) for medicaid expansion and the creation of state - based and federally - facilitated competitive marketplaces , or affordable insurance exchanges . the main goal of the ppaca is to provide a range of affordable insurance coverage options through medicaid and the new exchanges to reduce the number of uninsured in the usa.16,17 according to plan , the exchanges will achieve this goal by providing an opportunity for qualified individuals and small employers to evaluate and compare high - quality , affordable , and competitively priced private health plans.18,19 these exchanges will offer a new pathway to insurance coverage for many adults , effectively increasing the number of patients with government - sponsored health insurance.20 with women comprising the majority ( 69% ) of the adult medicaid population,21 changes in medicaid will matter to the population of low - income women with aub . presently , about 12% of women aged 1864 years rely on medicaid for their health care coverage.22 medicaid expansion under the ppaca is expected to expand eligibility to an additional 7 million previously uninsured women aged 1964 years and with incomes below 138% of the federal poverty level . it is expected that a substantial portion of these women will be childless adults who were not previously eligible for medicaid coverage and , like current medicaid enrollees , are more likely to be in poorer health than the general population.23 approximately 20% ( 1.4 million ) of newly eligible women in an expanded medicaid program will have aub , many of whom will go on to receive costly pharmacological and surgical treatments every year.2426 the results of our analyses indicate that the costs of aub intervention plus one month of follow - up ( combining patients with and without complications ) total approximately $ 4,000 for gea , considerably less than the $ 11,300 estimate for hysterectomy . supposing a conservative 1% annual treatment rate for aub with either gea or hysterectomy , medicaid s expanded coverage of many more aub patients is likely to require large outlays for these therapies . the high price of the inpatient hospital care required for hysterectomy will likely cause concern . as a result , state - based and federally facilitated exchanges may focus intently on pharmacological treatments and gea ( particularly in - office gea procedures ) as ways to reduce costs to medicaid , with a lasting impact on the treatment landscape for aub in the usa . administrative claims databases are subject to several limitations . first , although mortality is not a common outcome of aub and its treatments , data on mortality were not available in describing the complete picture of patient outcomes . second , administrative claims databases are developed and maintained for the purposes of facilitating health care payments . this suggests that comorbidities and indicators of severity might not be captured accurately because they are not used for payment generation . third , analysis of individual brands or types of gea or hysterectomy was prevented by the lack of specificity available in current procedure codes for gea and hysterectomy . finally , the study population consists of patients with medicaid ; therefore , the results may not represent all patients with aub , including the uninsured who may gain insurance coverage through the ppaca . overall , the total treatment - related and indirect costs to the medicaid program were significantly higher for women undergoing hysterectomy compared with women undergoing gea in the year following the index event ( $ 11,270 versus $ 3,958 , p<0.001 ) . hysterectomy was nearly three times more costly than gea for the treatment of aub , with significantly higher rates of treatment - related complications . these results may be relevant to treatment decisions made by medicaid , which is expanding its coverage as a result of recent legislation , and soon will insure an estimated 1.4 million women with aub .

backgroundwomen with abnormal uterine bleeding ( aub ) may be treated surgically with hysterectomy or global endometrial ablation ( gea ) , an outpatient procedure . we compared the costs and clinical outcomes of these surgical procedures for aub among women in medicaid programs.methodsthe truven health marketscan medicaid multi - state database was used to identify medicaid women aged 3055 years with aub who newly initiated gea or hysterectomy ( index event ) during 20062010 . patients were required to have 12 months of continuous enrollment pre - index and post - index . baseline characteristics were assessed in the pre - index period ; health care utilization and costs ( 2011 usd ) , treatment complications , and reinterventions were assessed in the post - index period.resultsof 1,880 women who met the study criteria ( mean age 40.7 years ) , 53.4% were caucasian , 33.1% were african - american , and 2.3% were hispanic ; many ( 42.8% ) received their medicaid eligibility due to disability . similar proportions received gea ( 50.9% ) or hysterectomy ( 49.1% ) . at baseline , both groups also had similar deyo - charlson comorbidity scores ( 0.65 ) , and use of antibiotics ( 69.4% ) , nonsteroidal anti - inflammatory drugs ( 56.3% ) , and oral contraceptives ( 5.3% ) . more hysterectomy patients than gea patients had a treatment - related complication ( 52% versus 36% , respectively , p<0.001 ) . initial treatment costs were higher for hysterectomy ( $ 11,270 ) than for gea ( $ 3,958 , p<0.001 ) ; monthly gynecology - related costs in the remainder of the year were not significantly different for hysterectomy ( $ 63 ) and gea ( $ 16 , p=0.11).conclusionhysterectomy was nearly three times more costly than gea for initial treatment of aub , and associated with more treatment - related complications . these results may be informative in the context of new federal mandates for medicaid expansion , which are likely to focus on cost savings through use of outpatient treatments such as gea .

Introduction Materials and methods Data source Study population Analyses Results Discussion Implications for Medicaid expansion and affordable insurance exchanges Limitations Conclusion

coal is a combustible mineral of organic origin , composed mainly of carbon and hydrocarbons . the ranking of coal ( its hardness and age ) increases from peat to lignite , sub - bituminous to bituminous , and then to anthracitic . as its rank increases , the ratio of carbon to other chemicals and mineral contaminants increases . coal mine dust contains various minerals in small amounts , such as silica ( quartz ) , iron , aluminum , pyrite , kaolin , and mica . exposure to respirable mixed coal mine dust results in development of coal workers pneumoconiosis ( cwp ) , which is defined as the deposition of coal dust in the lungs , and the tissue s reaction to its presence . the risk of cwp seems to depend on the concentration and duration of exposure to coal dust . these non - palpable lesions appear as 14 mm in diameter , black areas distributed diffusely throughout the lung , but more so in the upper zone . microscopically , the macular lesion consists of focal collections of coal dust - laden macrophages at the division of respiratory bronchioles . it is also seen along the lymphatics within the secondary lobular septa , and beneath the visceral pleura . focal emphysema is a subtype of centriacinar emphysema , which is known to be a histopathologic sign of cwp . this process is mediated by antiproteases , secreted by coal dust - activated macrophages [ 24 ] , which , along with coal macules , forms the characteristic lesion of cwp . earlier cwp studies have shown that patients with p opacities have a lower diffusing capacity than those patients with q or r opacities . authors have attributed this to the development of focal emphysema in patients with p opacities . cwp is classified as a simple or complicated form , depending on the presence of progressive massive fibrosis ( pmf ) . clinically significant lung function impairment is believed to generally occur in complicated forms , predominantly in category b and c . in contrast , the present data suggest that simple cwp can also be associated with lung function impairment . moreover , it seems that this impairment has been principally due to emphysema and airflow obstruction , rather than underlying restrictive mechanisms . the primary goal of the present study was to test whether coal dust exposure is associated with emphysema and/or airflow obstruction in the absence of smoking history . we also investigated whether patients with p - type opacity have different traits regarding emphysema or airflow obstruction . this research was observational , as well as a hospital - based cross - sectional study . it was approved by the local ethics committee , although patient consent was not deemed necessary due to the study s retrospective nature . the paper was written according to the strengthening the reporting of observational studies in epidemiology ( strobe ) statement . all coal mine workers or ex - workers seeking compensation for possible pneumoconiosis were included in the study , and were evaluated by the occupational disease diagnosis council of bulent ecevit university , zonguldak , turkey from 1 january 2013 to 31 may 2015 . these miners were from the zonguldak coal basin , the major bituminous coal mine area in turkey . the occupational diseases diagnosis council was composed of all faculty members working in the department of pulmonary medicine of bulent ecevit university . the turkish institute for occupational health and safety certified that each author was an expert on pneumoconiosis ( on behalf of the international labour office , ilo ) . evaluation of pneumoconiosis was based on chest radiography , spirometry , and high - resolution computed tomography ( hrct ) , and is mandatory according to nssi regulations . the committee evaluated and classified each chest radiograph for perfusion , size , and shape of small and large opacities , according to the 1980 ilo . the perfusion evaluation was based on chest radiographs , as there are no validated standards for ct scans . the presence of the opacity perfusion subcategory , 1/0 or greater , or large opacity ( category large opacities were classified as follows : category a : sum of the greatest diameter of one or more opacities exceeding 1 cm but less than 5 cm . category b : sum of the greatest diameter of one or more opacities exceeding 5 cm , but their combined area not exceeding the equivalent of the right upper lung zone . category c : one or more opacities with combined area exceeding the equivalent of the right upper zone . five hundred thirty - two ( n=532 ) subjects were evaluated for compensation for pneumoconiosis . we excluded smokers , former smokers , anyone with a history of tuberculosis or lung malignancy , as well as any pneumoconiosis . the electronic database was able to collect demographics , spirometric , and procedural data , including these variables for each subject : age , tenure , smoking history , body mass index , fev1 , fvc , fef2575 , fev1/fvc , shape of small opacity , perfusion category , and potential large opacity . the perfusion of small opacities was scored on a linear scale as follows : 1/0=2 , 1/1=3 , 1/2=4 , 2/1=5 , 2/2=6 , 2/3=7 , 3/2=8 , and 3/3=9 . spirometric measurements were performed with the masterscreen pneumo - device ( flow - based ) at rest ( i.e. , while in a sitting position ) by a trained technician , according to guidelines of the american thoracic society and european respiratory society . spirometric predictive values were based on the european community for coal and steel reference values . all lung function data , except fev1/fvc values , are expressed as percentages of the predicted values . hrct scans were obtained with the activision 16-row ct scanner ( toshiba medical systems , otawara , japan ; 1.5 mm slice thickness , tube voltage 140 kv , tube current 175 ma ) . emphysema was quantified by the percentage of low - attenuation areas , using a hounsfield unit ( hu ) threshold < 950 and perc15 ( hu point below which 15% of the voxels are distributed ) . after determination of appropriate hrct scan data ( n=53 of n=57 ) for analysis in an electronic database , we decided to use a control group for emphysema comparability because ct densitometric analysis is affected by multiple factors . found the emphysema index ( % , sd ) to be 5.4 ( 66 ) and 30.2 ( 248 ) for non - smoking non - miners and never - smoking coal miners , respectively . we used this value for sample size calculation along with an allocation ratio ( n2/n1=5.7 ) . we accepted a 0.05 level for type 1 error ( ) and 0.2 for type 2 errors ( ) . nine age- and sex - matched control cases ( n=9 ) with no history of working in a mine or ever having smoked were chosen randomly from the radiology database . descriptive statistics of categorical variables are given as numbers or percentages ; continuous variables are given as medians ( interquartile range ) . the mann - whitney u - test was used to compare the medians of variables when appropriate . the relationship between emphysema and spirometric variables was evaluated with a partial correlation analysis , controlling for age and body mass index ( bmi ) . multiple linear regression analysis ( backward method ) was used to determine independent factors associated with the percent of emphysema and fev1/fvc , respectively . the following variables were included in the regression models : age , bmi , tenure , fev1/fvc , perfusion score and large opacity for percent of emphysema ( dependent variable ) and age , bmi , tenure ( underground working years ) , percent of emphysema , perfusion score and large opacity for fev1/fvc ( dependent variable ) , respectively . it was reported in previous studies that a - type large opacity shows similarity to simple cwp , rather than b- and c - opacities showing physiological impairment . the presence of large opacity was defined as category b or c and was entered into the regression analysis as indicator variables . statistical analysis was performed using spss version 18.0 for windows ( spss , ibm inc . , chicago , il ) and medcalc for windows , version 12.2.1.0 ( medcalc software , ostend , belgium ) . p values were two - sided , and values less than 0.05 were considered statistically significant . this research was observational , as well as a hospital - based cross - sectional study . it was approved by the local ethics committee , although patient consent was not deemed necessary due to the study s retrospective nature . the paper was written according to the strengthening the reporting of observational studies in epidemiology ( strobe ) statement . all coal mine workers or ex - workers seeking compensation for possible pneumoconiosis were included in the study , and were evaluated by the occupational disease diagnosis council of bulent ecevit university , zonguldak , turkey from 1 january 2013 to 31 may 2015 . these miners were from the zonguldak coal basin , the major bituminous coal mine area in turkey . the occupational diseases diagnosis council was composed of all faculty members working in the department of pulmonary medicine of bulent ecevit university . the turkish institute for occupational health and safety certified that each author was an expert on pneumoconiosis ( on behalf of the international labour office , ilo ) . evaluation of pneumoconiosis was based on chest radiography , spirometry , and high - resolution computed tomography ( hrct ) , and is mandatory according to nssi regulations . the committee evaluated and classified each chest radiograph for perfusion , size , and shape of small and large opacities , according to the 1980 ilo . the perfusion evaluation was based on chest radiographs , as there are no validated standards for ct scans . the presence of the opacity perfusion subcategory , 1/0 or greater , or large opacity ( category large opacities were classified as follows : category a : sum of the greatest diameter of one or more opacities exceeding 1 cm but less than 5 cm . category b : sum of the greatest diameter of one or more opacities exceeding 5 cm , but their combined area not exceeding the equivalent of the right upper lung zone . category c : one or more opacities with combined area exceeding the equivalent of the right upper zone . five hundred thirty - two ( n=532 ) subjects were evaluated for compensation for pneumoconiosis . we excluded smokers , former smokers , anyone with a history of tuberculosis or lung malignancy , as well as any pneumoconiosis . the electronic database was able to collect demographics , spirometric , and procedural data , including these variables for each subject : age , tenure , smoking history , body mass index , fev1 , fvc , fef2575 , fev1/fvc , shape of small opacity , perfusion category , and potential large opacity . the perfusion of small opacities was scored on a linear scale as follows : 1/0=2 , 1/1=3 , 1/2=4 , 2/1=5 , 2/2=6 , 2/3=7 , 3/2=8 , and 3/3=9 . spirometric measurements were performed with the masterscreen pneumo - device ( flow - based ) at rest ( i.e. , while in a sitting position ) by a trained technician , according to guidelines of the american thoracic society and european respiratory society . spirometric predictive values were based on the european community for coal and steel reference values . all lung function data , except fev1/fvc values , are expressed as percentages of the predicted values . hrct scans were obtained with the activision 16-row ct scanner ( toshiba medical systems , otawara , japan ; 1.5 mm slice thickness , tube voltage 140 kv , tube current 175 ma ) . emphysema was quantified by the percentage of low - attenuation areas , using a hounsfield unit ( hu ) threshold < 950 and perc15 ( hu point below which 15% of the voxels are distributed ) . after determination of appropriate hrct scan data ( n=53 of n=57 ) for analysis in an electronic database , we decided to use a control group for emphysema comparability because ct densitometric analysis is affected by multiple factors . found the emphysema index ( % , sd ) to be 5.4 ( 66 ) and 30.2 ( 248 ) for non - smoking non - miners and never - smoking coal miners , respectively . we used this value for sample size calculation along with an allocation ratio ( n2/n1=5.7 ) . we accepted a 0.05 level for type 1 error ( ) and 0.2 for type 2 errors ( ) . nine age- and sex - matched control cases ( n=9 ) with no history of working in a mine or ever having smoked were chosen randomly from the radiology database . descriptive statistics of categorical variables are given as numbers or percentages ; continuous variables are given as medians ( interquartile range ) . the mann - whitney u - test was used to compare the medians of variables when appropriate . the relationship between emphysema and spirometric variables was evaluated with a partial correlation analysis , controlling for age and body mass index ( bmi ) . multiple linear regression analysis ( backward method ) was used to determine independent factors associated with the percent of emphysema and fev1/fvc , respectively . the following variables were included in the regression models : age , bmi , tenure , fev1/fvc , perfusion score and large opacity for percent of emphysema ( dependent variable ) and age , bmi , tenure ( underground working years ) , percent of emphysema , perfusion score and large opacity for fev1/fvc ( dependent variable ) , respectively . it was reported in previous studies that a - type large opacity shows similarity to simple cwp , rather than b- and c - opacities showing physiological impairment . the presence of large opacity was defined as category b or c and was entered into the regression analysis as indicator variables . statistical analysis was performed using spss version 18.0 for windows ( spss , ibm inc . , chicago , il ) and medcalc for windows , version 12.2.1.0 ( medcalc software , ostend , belgium ) . p values were two - sided , and values less than 0.05 were considered statistically significant . all coal mine workers or ex - workers seeking compensation for possible pneumoconiosis were included in the study , and were evaluated by the occupational disease diagnosis council of bulent ecevit university , zonguldak , turkey from 1 january 2013 to 31 may 2015 . these miners were from the zonguldak coal basin , the major bituminous coal mine area in turkey . the occupational diseases diagnosis council was composed of all faculty members working in the department of pulmonary medicine of bulent ecevit university . the turkish institute for occupational health and safety certified that each author was an expert on pneumoconiosis ( on behalf of the international labour office , ilo ) . evaluation of pneumoconiosis was based on chest radiography , spirometry , and high - resolution computed tomography ( hrct ) , and is mandatory according to nssi regulations . the committee evaluated and classified each chest radiograph for perfusion , size , and shape of small and large opacities , according to the 1980 ilo . the perfusion evaluation was based on chest radiographs , as there are no validated standards for ct scans . the presence of the opacity perfusion subcategory , 1/0 or greater , or large opacity ( category a , b , or c ) was considered as evidence of cwp . large opacities were classified as follows : category a : sum of the greatest diameter of one or more opacities exceeding 1 cm but less than 5 cm . category b : sum of the greatest diameter of one or more opacities exceeding 5 cm , but their combined area not exceeding the equivalent of the right upper lung zone . category c : one or more opacities with combined area exceeding the equivalent of the right upper zone . five hundred thirty - two ( n=532 ) subjects were evaluated for compensation for pneumoconiosis . we excluded smokers , former smokers , anyone with a history of tuberculosis or lung malignancy , as well as any pneumoconiosis . the electronic database was able to collect demographics , spirometric , and procedural data , including these variables for each subject : age , tenure , smoking history , body mass index , fev1 , fvc , fef2575 , fev1/fvc , shape of small opacity , perfusion category , and potential large opacity . the perfusion of small opacities was scored on a linear scale as follows : 1/0=2 , 1/1=3 , 1/2=4 , 2/1=5 , 2/2=6 , 2/3=7 , 3/2=8 , and 3/3=9 . spirometric measurements were performed with the masterscreen pneumo - device ( flow - based ) at rest ( i.e. , while in a sitting position ) by a trained technician , according to guidelines of the american thoracic society and european respiratory society . spirometric predictive values were based on the european community for coal and steel reference values . all lung function data , except fev1/fvc values , are expressed as percentages of the predicted values . hrct scans were obtained with the activision 16-row ct scanner ( toshiba medical systems , otawara , japan ; 1.5 mm slice thickness , tube voltage 140 kv , tube current 175 ma ) . emphysema was quantified by the percentage of low - attenuation areas , using a hounsfield unit ( hu ) threshold < 950 and perc15 ( hu point below which 15% of the voxels are distributed ) . after determination of appropriate hrct scan data ( n=53 of n=57 ) for analysis in an electronic database , we decided to use a control group for emphysema comparability because ct densitometric analysis is affected by multiple factors . found the emphysema index ( % , sd ) to be 5.4 ( 66 ) and 30.2 ( 248 ) for non - smoking non - miners and never - smoking coal miners , respectively . we used this value for sample size calculation along with an allocation ratio ( n2/n1=5.7 ) . we accepted a 0.05 level for type 1 error ( ) and 0.2 for type 2 errors ( ) . nine age- and sex - matched control cases ( n=9 ) with no history of working in a mine or ever having smoked were chosen randomly from the radiology database . descriptive statistics of categorical variables are given as numbers or percentages ; continuous variables are given as medians ( interquartile range ) . the mann - whitney u - test was used to compare the medians of variables when appropriate . the relationship between emphysema and spirometric variables was evaluated with a partial correlation analysis , controlling for age and body mass index ( bmi ) . multiple linear regression analysis ( backward method ) was used to determine independent factors associated with the percent of emphysema and fev1/fvc , respectively . the following variables were included in the regression models : age , bmi , tenure , fev1/fvc , perfusion score and large opacity for percent of emphysema ( dependent variable ) and age , bmi , tenure ( underground working years ) , percent of emphysema , perfusion score and large opacity for fev1/fvc ( dependent variable ) , respectively . it was reported in previous studies that a - type large opacity shows similarity to simple cwp , rather than b- and c - opacities showing physiological impairment . the presence of large opacity was defined as category b or c and was entered into the regression analysis as indicator variables . statistical analysis was performed using spss version 18.0 for windows ( spss , ibm inc . , chicago , il ) and medcalc for windows , version 12.2.1.0 ( medcalc software , ostend , belgium ) . p values were two - sided , and values less than 0.05 were considered statistically significant . the study population consisted of 57 non - smoking men ( n=57 ) with cwp . the control group included 9 ( n=9 ) non - smoking men without any known pulmonary disease . the average age of subjects with cwp was 71 ( iqr , 6279 ) years . densitometric analysis was performed for 54 of 57 cases due to inappropriate hrct scan data in 3 cases . subjects were categorized into simple cwp ( 28 patients ) and complicated cwp ( pmf ) ( 29 patients ) groups . table 1 shows demographics , spirometry , and radiologic measurements in cwp and control subjects . percent of emphysema and perc15 values were significantly higher in the cwp group compared with the control group ( p<0.001 ) . there was also a significant difference in terms of emphysema and perc15 between simple cwp and the control group ( p<0.001 , p=0.001 ) , as well as between the pmf group and the control group ( p<0.001 , p<0.001 ) , respectively . spirometric evaluation revealed obstructive lung disease ( fev1/fvc<0.7 ) in the majority ( n=26 , 51% ) of patients with cwp . we used the global initiative for chronic obstructive lung disease , and defined 25/51 ( 49% ) subjects at stage 0 , 3 ( 6% ) at stage i ( mild ) , 16 ( 31% ) at stage ii ( moderate ) , 6 ( 12% ) at stage iii ( severe ) , and 1 ( 2% ) at stage iv ( very severe ) . the number and percent of subjects demonstrating the fev1/fvc ratio was less than 0.7 and that the fev1 was less than 80% of the predicted value was found to be 7 ( 26% ) in simple cwp and 16 ( 67% ) in the complicated cwp group . restrictive impairment in lung function ( fev1/fvc 0.70 and fvc < 80% as predicted ) was seen in 9 patients ( 18% ) , all of which had complicated cwp . correlation between spirometric findings and densitometric emphysema values was investigated . in patients with cwp , partial correlation analysis ( controlling for age and bmi ) revealed that the percent of emphysema and perc15 were correlated with fev1/fvc ( r=0.45 , p=0.002 , r=0.47 , and p=0.001 ) , and fef2575 ( r=0.36 , p=0.015 and r=0.56 , p<0.001 ) , respectively . fev1 was only significantly correlated with perc15 ( r=0.38 , p=0.01 ) , but no correlation was found between fvc and percent of emphysema or perc15 . in partial correlation analysis , a significant correlation ( p<0.05 ) was observed between tenure and percent of emphysema ( r=0.36 ) , perc15 ( r=0.39 ) , fev1/fvc ( r=0.35 ) , fef2575 ( r=0.38 ) , fev1 ( r=0.33 ) , but not with fvc ( r=0.18 ) . to evaluate factors associated with percent of emphysema and fev1/fvc , we performed multiple linear regression analyses . in the first model , factors associated with percent of emphysema were fev1/fvc and the presence of large opacity ( b and c ) ( =0.24 , p=0.009 and =3.97 , p=0.079 , respectively ) . other factors not retained in the models were age , bmi , tenure , and the perfusion score . in the second model , emphysema ( =0.51 , p=0.018 ) and tenure ( =0.63 , p=0.044 ) remained independently associated with fev1/fvc . other factors ( age , bmi , and perfusion score ) did not reach statistical significance in this model . last , we evaluated whether the p - type ( n=18 ) and other type of opacities ( q , r , s , t , u ) ( n=7 ) have different traits in terms of emphysema or airway obstruction in cases with simple cwp . regarding the topics of age , perfusion score ( median ) , and tenure , there were no differences between groups . percentage of emphysema and ratio of fev1/fvc were compared between groups using the mann - whitney u - test . although percentage of emphysema in subjects with p - type opacity was higher than it was in subjects with other type opacities , it was not statistically significant ( figure 2 ) . in contrast , there was a statistically significant difference in fev1/fvc between subjects with p - type opacities and other types ( figure 2 ) . the present study shows that cwp is closely associated with both emphysema and airflow obstruction in non - smoking subjects . the average percent of emphysema and percent of subjects with a fev1/fvc ratio was less than 0.7 , and was 15% and 51% , respectively , in subjects with cwp . in a multivariate analysis , the percent of emphysema was associated with fev1/fvc and the presence of large opacity ( category b and c ) . airflow obstruction ( fev1/fvc < 0.7 ) was associated with percent of emphysema and tenure . we attempted to exclude effects of cigarette smoking and concomitant pulmonary pathology on the lungs , which is a major advantage of our study . we did not find a correlation between ilo perfusion score and impairment of lung function or emphysema , which was similar to previous studies . additionally , in subjects with simple cwp , p - type opacities showed a higher percent of emphysema , although not significant , but a higher fev1/fvc value than other opacities . airflow obstruction , spirometrically , is diagnosed by decreased fev1/fvc , which is the result of decreased elastic recoil ( i.e. , emphysema ) of the lungs , promoting airflow and increased resistance ( airway wall thickening and bronchiolar obliteration ) of the airways , which limits airflow . concerning the relationship between cwp and emphysema , autopsy studies also have some limitations , such as the possibility of subjects with severe disease ( selection bias ) and a long interval between spirometry and death . additionally , the effect of smoking on the development of emphysema or airflow obstruction can not be eliminated , which is the most important problem in these studies [ 2022 ] . despite this , autopsy studies have strongly demonstrated that coal mine dust exposure is associated with emphysema , and its severity increases with increasing lung dust retention . the best evidence was reported by kumpel et al . , who found that coal mine dust exposure and cigarette smoking presented similar problems in the prediction of emphysema severity . furthermore , this study showed that the largest difference in emphysema severity was between non - smoking miners and non - smoking non - miners ( about 6-fold ) . they found that the average percent of emphysema in non - smoking miners was 30% . we found the percent of emphysema in non - smoking subjects with cwp to be 15% ( median , about 4-fold compared with the control group ) . our study showed that large opacities ( category b and c ) were associated with emphysema in the regression model . coal dust inhalation can initiate toxic and inflammatory processes in the airways and alveolar tissue . moreover , it is well - documented that the prevalence of chronic bronchitis is higher in coal miners . available evidence indicates that functional impairment is associated with small airways , rather than central or large airways . we found that the prevalence of airway obstruction ( fev1/fvc < 0.7 ) in subjects with simple cwp was 37% . similarly , kibelstis et al . reported that the prevalence of airway obstruction in non - smoking coal miners was 37.6% ( 10 ) . in our study , the percent of subjects with fev1/fvc ratio was less than 0.7 , and fev1 was less than 80% of the predicted value , or 26% in the simple cwp group and 67% in the pmf group , respectively . the relationship between the ilo perfusion score and lung function ( or emphysema ) is not clear . we did not find a significant relationship between the perfusion score and pulmonary function parameters , or the percent of emphysema . gevenois et al . suggest that the presence of micronodules in coal miners has no effect on lung function , and that it should only be considered as a marker of exposure . did not find an association between perfusion score and pulmonary function or blood gas parameters . on the other hand , it is also known that the presence of emphysema may reduce accuracy of reading of perfusion on chest radiographs . in our study , furthermore , morphologic changes at the level of respiratory bronchioles are not detected by conventional spirometry . previous reports noted that the shape of small opacities is associated with impairment of pulmonary function . it was shown that individuals with p opacities have a lower diffusing capacity than patients with q and r opacities . there is additional evidence that p - type opacity is associated with increased air space size , which is a result of development of focal emphysema . in our study , emphysema more common in individuals with p - type opacity than with other types , although this difference was not statistically significant . in contrast , other opacities were associated with airway obstruction ; obstructive disease of the small airways ( less than 1 mm in diameter ) are often not detected by conventional spirometry or measurements of airway resistance , and are ultimately responsible for less than 15% of total airway resistance . this may be a reason for normal spirometric findings in individuals with p - type opacity . pathologically , q and r opacities ( formerly micronodular and nodular , respectively ) were pathological palpable lesions . it was similarly demonstrated in previous reports that these lesions were associated with increased airway resistance . furthermore , in some studies , irregular opacities , which are considered to be related to tissue reaction to inhaled coal dust , were reported to be more strongly associated with impaired pulmonary function than were round opacities . in the group classified as other type opacities , the reason for impaired pulmonary function may be the combined effects of these opacities mentioned above . however , the limited number of cases in this group makes interpretation difficult . there are several limitations in our study , including a recall bias due to the retrospective design and small sample size , affected by strict inclusion criteria . a large proportion of the study population was composed of retired coal miners who had already applied for compensation , so selection bias was possible . underlying pulmonary disease ( e.g. , alpha 1-antitrypsin deficiency , asthma , or bronchial hyperreactivity ) may similarly change study outcomes . another limiting factor was that the pre - bronchodilator spirometric assessment was performed , which can influence prevalence of airway obstruction . the quantification of emphysema using ct densitometry can be influenced by multiple factors , including scanning protocol , scanner calibration , and variation in inspiratory level . exposure to dust varies widely from mine to mine , and , in certain places , within a given mine . multiple etiologic factors other than coal may be involved in individual cases , which makes causal interpretation difficult . there are several limitations in our study , including a recall bias due to the retrospective design and small sample size , affected by strict inclusion criteria . a large proportion of the study population was composed of retired coal miners who had already applied for compensation , so selection bias was possible . underlying pulmonary disease ( e.g. , alpha 1-antitrypsin deficiency , asthma , or bronchial hyperreactivity ) may similarly change study outcomes . another limiting factor was that the pre - bronchodilator spirometric assessment was performed , which can influence prevalence of airway obstruction . the quantification of emphysema using ct densitometry can be influenced by multiple factors , including scanning protocol , scanner calibration , and variation in inspiratory level . exposure to dust varies widely from mine to mine , and , in certain places , within a given mine . multiple etiologic factors other than coal may be involved in individual cases , which makes causal interpretation difficult . some authors have expressed doubt as to whether coal mine dust can cause clinically significant loss of lung function in the absence of complicated pneumoconiosis . however , available evidence indicates that the presence of simple cwp contributes significantly to the impairment of lung function in coal miners , which also seems to be related to obstructive lung disease . lifelong non - smokers offer a unique opportunity to evaluate the association between coal dust exposure and obstructive lung disease . despite all of these limitations , this study highlights the association of obstructive lung disease with coal dust exposure in non - smoking coal miners with pneumoconiosis .

backgroundaccumulating evidence shows that functional impairment in subjects with coal workers pneumoconiosis ( cwp ) is principally due to emphysema and airflow obstruction , rather than underlying restrictive mechanisms . however , cigarette smoking has remained a major confounder . the aim of this study was to assess whether coal dust exposure was associated with emphysema and/or airflow obstruction in the absence of smoking history.material/methodthe subjects evaluated for possible pneumoconiosis between 2013 and 2015 were retrospectively enrolled into this study . after excluding those with history of smoking , tuberculosis , or lung cancer , the study population was a total of 57 subjects . the emphysema severity and airflow obstruction were quantified by computed tomographic densitometry analysis and spirometry , respectively . for comparability regarding emphysema , 9 age- and sex - matched nonsmoker ( n=9 ) control subjects without known lung disease were randomly selected from a radiology database.resultsemphysema severity was significantly higher in the cwp group compared with the control group ( 15% vs. 4% , p<0.001 ) . the median percent emphysema and percentage of those with fev1/fvc < 0.7 was 13% and 37% in subjects with simple cwp and 18% and 67% in subjects with complicated cwp , respectively . percent emphysema and perc15 ( 15th percentile of the attenuation curve ) was correlated with fev1/fvc ( r=0.45 , r=0.47 ) and fef2575 ( r=0.36 , r=0.56 ) , respectively , but not with perfusion score . a linear regression analysis showed that factors associated with emphysema were fev1/fvc ( =0.24 , p=0.009 ) and large opacity ( =3.97 , p=0.079 ) , and factors associated with fev1/fvc were percent emphysema ( =0.51 , p=0.018 ) and tenure ( =0.63 , p=0.044).conclusionsour results support the observation that coal dust exposure is associated with emphysema and airflow obstruction , independent of smoking status .

Background Material and Methods Study design Participants and setting Data source and measurement Data analysis Results Discussion Limitations Conclusions

age - related macular degeneration ( amd ) is the leading cause of permanent , irreversible , central blindness ( scotoma in the central visual field makes impossible the following : reading and writing , stereoscopic vision , and recognition of colours and details ) in patients over the age of 50 in industrialized european and north american countries . late ( or advanced ) amd occurs in two distinct forms , and visual loss is caused by the geographic atrophic death of photoreceptors and retinal pigment epithelium ( rpe ) cells , the so - called dry amd , ga / amd , or by formation of the choroidal neovascular membrane ( cnv ) , as a result of pathological angiogenesis , the so - called exudative or wet amd , cnv / amd [ 13 ] . hypoxia , as well as hyperactivity of the complement system , together with the inflammatory process , leads to disturbances in the pro / antiangiogenic balance and rpe cells overexpression of proangiogenic vascular endothelial growth factor ( vegf ) , which plays a key role in the pathogenesis of cnv / amd [ 1 , 4 ] . amd is a complex degenerative and progressive disease of multifactorial etiology , and advanced age ( and its related physiological cell apoptosis and tissue involution ) and genetic predisposition are the strongest risk factors ; however , important are also other factors such as sex and environmental influences ( smoking cigarettes , heart and vascular disorders , hypertension , dyslipidemia / hypercholesterolemia , diabetes , obesity , improper diet , sedentary lifestyle , and phototoxic exposure ) [ 13 , 5 , 6 ] . despite intensive researches , the pathomechanism of amd has not yet been fully recognized , since the pathogenesis of amd is likely to involve the disruption of multiple physiological pathways ; however , according to many authors , inflammation and especially the complement system play key role in the etiopathogenesis of amd . inflammation is involved in both late forms of amd , that is , in ga / amd and cnv / amd [ 813 ] . the term parainflammation , first proposed by medzhitov , means tissue adaptive response to noxious stress or malfunction and has characteristics that are intermediate between basal and inflammatory states . it is not a classic form of inflammation caused by exogenous tissue injury or foreign antigen ( infection ) . parainflammation is a local low - grade / subclinical immune reaction , caused by endogenous inducers ( they may not be detectable by using common inflammatory biomarkers ) , to adapt tissue to harmful environment and to maintain their adequate functionality . in other words , parainflammation is a state between healthy homeostasis and chronic inflammation . however , if the noxious stress or malfunction is prolonged and not removed , then parainflammation progresses into a chronic condition , with classic well known and defined inflammatory pathways , and leads to tissue damage . oxidative stress is considered by many authors to be the main initial determinant for parainflammatory responses [ 79 ] . parainflammation means the activation of locally resident immune cells in the first instance and the induction of complement system , mainly its alternative pathway , in chronic conditions [ 79 ] . normal , adaptive parainflammation exists in the aging retina under physiologic conditions , as a protective response against low - grade noxious waste products accumulated in bruch 's membrane , and allows maintenance of retinal homeostasis [ 8 , 9 ] . however , prolonged period of retina exposition to stress and/or malfunction leads to chronic unbalanced and uncontrolled parainflammation process , due to either prolonged and aggressive tissue damage ( caused by extreme aging or unhealthy life style ) or decreased immune system ability to repair tissue ( caused by premature aging or genetic susceptibility ) . such chronic abnormal inflammatory process , or failure chronic para - inflammation [ 7 , 9 ] or pathophysiologic para - inflammatory response , leads to chronic detrimental inflammatory reactions and immunologic events [ 9 , 15 ] , promotes tissue damage , and contributes to the initiation of amd [ 9 , 16 , 17 ] , as well as playing an important role in amd progression [ 9 , 15 ] . according to many authors , amd is not classic ( regular ) inflammation but is a condition of chronic low - grade / subclinical degree of inflammation ( pathophysiologic parainflammation ) [ 8 , 9 ] , which plays role in every amd form . in the course of amd , chronic , pathophysiological parainflammatory process occurs in the macula and in paracentral retinochoroidal tissues and is related to the anatomy - activity complex consisting of the outer photoreceptors , rpe cells , bruch 's membrane , and choriocapillaris [ 79 ] . parainflammation is characteristic for tissues functionally dependent on nonproliferative cells and characterized by very high metabolism . photoreceptors , rpe , and ganglion cells are terminally differentiated ( postmitotic ) retinal cells , with limited or lack of ability to regenerate . neuroretina is a highly metabolic tissue , sensitive to noxious factors , and constantly exposed to light stimulation , which generates large amounts of oxidized materials . the major causes of tissue stress resulting in local triggers for parainflammation are oxidative stress and oxidative species , that is , reactive oxygen species ( ros ) and reactive nitrogen species ( rns ) , oxidized lipoproteins , advanced glycation endproducts ( ages ) , and apoptotic cells [ 79 ] . oxidative stress occurs when the production of ros and rns accelerates , or when the mechanisms involved in their rapid removal are impaired . in the retina , the inner segments of photoreceptors and rpe cells , both rich in mitochondria organelles , are the main source of oxidative and nitrative species in the pathophysiological conditions . sunlight exposure , mitochondrial respiration , phagocytosis of the outer photoreceptors segments by rpe cells , photosensitization of protoporphyrin , and lipofuscin phototoxicity , as well as unregulated choroidal blood flow , connected with increased fluctuations of tissue oxygen concentration , predispose the aging macula to oxidative stress and elevated mitochondrial ros and rns creation [ 19 , 20 ] . ros impair cells function by reacting with nucleic acids , proteins , and lipids ; they also induce production of proinflammatory cytokine and angiogenic signals and may lead to apoptosis of photoreceptors , rpe cells , and retinal ganglion cells in the aging retina . rns trigger damage of proteins and lipids , cause nitrosative stress in the aging retina , and may induce apoptotic death of photoreceptors . oxidative and nitrative species trigger parainflammation process directly , since oxidized or nitrated lipids and proteins stimulate inflammatory responses , as well as indirectly activating the tissue resident macrophages to express proinflammatory mediators . moreover , ron may alter vascular permeability and breakdown of blood - retina barrier , which promote release of proinflammatory factors . the retina , particularly the photoreceptor layer rich in unsaturated lipoproteins , is readily susceptible to oxidation by ros . lipid oxidation increases with the age and occurring oxidized low - density lipoproteins ( oxldl ) are particularly relevant to retinal parainflammation . oxldl bind to rpe cells and retinal microglia via scavenger receptors , that is , via cd36 , ldl - r , and newly described lectin - like oxidized lipoprotein receptor 1 ( lox-1 ) detected in rpe cells and in microglia [ 28 , 29 ] , as well as via the cd68 receptor detected in microglia . oxldl promote parainflammation activating lipid - laden monocytes and macrophages to secrete cytokines ( il-8 ) , growth factors ( tnf - alpha ) , and matrix metalloproteinases . the term advanced glycation endproducts(ages ) is a broad term which includes various proteins and lipids undergoing chemical reactions after exposure to sugars ; their physiological serum level is in the range of 210 lg / ml and increases with the age . ages promote inflammation in many general age - related diseases such as alzheimer 's disease , atherosclerosis , diabetes , osteoarthritis [ 3234 ] , and amd . ages are a constituent of drusen and accumulate in bruch 's membrane in the course of amd . they stimulate rpe cells to secrete different anti / proinflammatory factors , which trigger parainflammation state in rpe cells , that is , short - term adaptive rpe cell reaction on age stimulation ; however chronic rpe exposure to age favors deregulation of rpe function and leads to photoreceptors and rpe cells degeneration and atrophy . according to lin et al . , anti - inflammatory cytokines including il10 , il1ra , and il9 were overexpressed and proinflammatory cytokines including il4 , il15 , and ifn- were overexpressed , while other proinflammatory cytokines including il8 , mcp1 , and ip10 were underexpressed with age stimulation of human cultured rpe cells . strongly immunoreactive cxcl11 protein , associated with drusen , mediates chemotaxis between cells in inflammatory response . multifactorial antiviral protein rsad2 is also localized in drusen and outer retina , mediating inflammatory response , abnormal lipid accumulation , and atherosclerosis . extracellular receptors which bind ages on rpe cells include toll - like receptors ( tlrs ) , receptors for advanced glycation endproducts ( rages ) , and advanced glycation endproducts receptors ( agers ) . tlrs and rages activate inflammatory signals within the cells , and agers are important inhibitors of these signals . according to lin et al . , all the above cytokines are novel agent ( genes ) associated with amd pathogenesis and candidate marker of inflammatory events in the outer retina , and modulating their activity associated with specific pathways ( nf - jb and jak - stat pathways ) may be new therapeutic strategy to limit rpe dysfunction and photoreceptor loss in the course of amd . ages also induce secretion of proangiogenic cytokines by rpe cells ( upregulation of vegf in rpe cells after stimulation by ages ) . ( see below ) may lead to increased rpe exposure with ages and damage retinal tissue in the pathological angiogenesis process . in the aging retina , the cumulative effect of oxidative stress , endoplasmic reticulum stress , and accumulation of rpe byproducts lead to deposition of glycoproteins , lipids , and cellular debris , as well as creating clinically invisible basal deposits , that is , basal laminar deposits ( blamd ) and basal linear deposits ( blind ) . blamd are accumulated between the cytoplasmic and the basement membrane of rpe cells , and blind are stored in the extracellular matrix , between the rpe basement membrane and the internal collagen layer of the bruch 's membrane [ 4244 ] . basal deposits deepen the lipofuscinogenesis process in rpe cells , which means accumulation of lipofuscin ( age pigment ) developed from fully or partially digested outer segments of photoreceptors , as a result of the gradual failure of rpe liposomal enzymes [ 4244 ] . blamd and blind with diameters of over 2530 m turn into clinically visible macular and perimacular soft drusen ( sd ) , the hallmark of early amd , and a risk factor for developing advanced forms of amd during the next 5 to 10 years [ 45 , 46 ] , that is , ga / amd or cnv / amd [ 4749 ] . soft drusen are not only the side effect of dysfunction and degeneration of rpe cells [ 5 , 43 , 44 , 46 ] , but they indicate also a chronic , pathophysiological parainflammatory process in extracellular matrix . sd are the earliest diagnosable byproducts of local detrimental parainflammatory process and act as foci of chronic abnormal inflammation in bruch 's membrane [ 5 , 12 , 13 , 37 , 50 ] . they induceactive , nonspecific inflammatory cells reactions [ 46 , 51 ] and are biomarkers of immune - mediated processes in rpe / bruch 's membrane interface [ 5254 ] . inflammatory etiopathogenesis of human soft drusen confirms their content , which has been recognized during the course of numerous histochemical studies . soft drusen are composed of phospholipids , glycolipids , cholesterol , saturated and unsaturated fatty acids , carbohydrates , and approximately 130 various kinds of proteins , among which are apolipoproteins , vitronectin , clusterin , ubiquitin , fibronectin , integrins , extracellular matrix metalloproteinases , and their inhibitors [ 13 , 37 , 55 ] . soft drusen contain also numerous mediators of inflammation such as c - reactive protein ( crp ) [ 13 , 56 ] , adducts of the carboxyethylpyrrole protein ( cep ) , immunoglobulins ( igg ) , acute phase molecules ( vitronectin , amyloid p , and fibrinogen ) , and apolipoproteins as well as many complement - related proteins such as complement components ( c3a and c5a proteins ) , terminal complement complex ( c5 and c5b-9 proteins ) [ 54 , 56 ] , fluid - phase complement regulators ( complement factor h - cfh , vitronectin , and clusterin ) , and membrane - bound complement inhibitors ( complement receptor1-cr1 , also called cd35 and membrane cofactor protein - mcp , also called cd46 ) [ 61 , 62 ] . many complement - related proteins such as complement system activation products ( c3a , c5 , and c5b-9 proteins ) , complement activators ( cfh , cd35 , and cd46 proteins ) , and complement regulators have shown not only in drusen , but also in the capillary pillars of the choroid and within surgically removed choroidal neovascular membranes [ 37 , 54 , 62 , 63 ] , as well as in the human vitreous body . the complement system is a specific biochemical cascade and an integral element of inborn ( not specific ) immunological response , which supports ( complements ) the host antibodies ability to destroy pathogens , promotes removal of apoptotic cells and immune complexes , and enhances an individual adaptive immune response . its deregulation or dysfunction leads to various autologous diseases [ 65 , 66 ] and is implicated in the etiopathogenesis and progression of amd [ 63 , 65 ] . the complement system consists of more than 40 proteins and regulators synthesized by the liver and circulating in the blood as inactive precursors or proproteins . the classical pathway is triggered by antigen - antibody complexes , the alternative pathway is activated by binding to membranes of pathogen or host cell , and lectin pathway is triggered by binding serum lectin to mannose residues ( polysaccharides ) on microbial surfaces . the alternative and lectin pathways are called nonspecific immune response pathways , since they do not require the presence of a typical pathogen and act without the presence of antibodies . activation of these pathways results in a proinflammatory response including generation of cell - killing terminal complement complex ( tcc ) , also called the membrane - attack complex ( mac ) , which mediate aggressor 's cells lysis , after the spontaneous hydrolysis of the c3 factor ( classic pathway ) or the c3b factor ( alternative pathway ) , release of chemokines to attract inflammatory cells to the site of damage , and enhancement of capillary permeability [ 63 , 67 , 68 ] . the complement system is continuously activated at low levels in the normal eye , but intraocular complement regulators ( cfh , cd35 , and cd46 proteins ) tightly regulate its spontaneous activation to maintain activity at a level that promotes elimination of pathogens and waste products without damaging healthy tissue , and leukocytes , lymphocytes , and macrophages additionally support the complement system in cleaning the extracellular matrix [ 67 , 68 ] . with the age , the longstanding photooxidation process and oxidized albuminous components of lipofuscin deregulate complement system and play pivotal role in the activation of classic and alternative pathways , which leads to the chronic immune inflammatory process in the macula in the course of amd [ 37 , 57 , 69 ] . oxidized components of lipofuscin , that is , isolevuglandin , bis - retinoid pyrimidine a2e , and carboxyethylpyrrole protein ( cep ) , and its adducts ( generated in blood as a result of cep conjugation with circulating plasma albumin ) are present in lipofuscin of rpe cells , in soft drusen , and in systemic circulation [ 37 , 57 , 69 ] . these strongly immunogenic factors constitute a target for the immune system and stimulate the production of antibodies ( cep protein ) and endogenic , autoantibodies ( cep protein adducts ) [ 37 , 57 ] , which lead to autologous immune - mediated inflammation , strong attack of both the drusen material and its own rpe cells , cells apoptosis , and finally maculae damage [ 63 , 67 , 68 ] . in amd , the complement system activation is not only an effect , but also an evidence of local , strong , and chronic deregulation of immune system in bruch 's membrane and complement system pathognomonic role has been confirmed in both : dry amd and wet - amd [ 7073 ] . autologous inflammation mediated by overactive alternative pathway is especially strong in amd patients with the y402h gene polymorphism , since it causes hypofunction or lack of the protective complement factor h ( cfh ) . cfh is a serum suppressor of alternative complement activity in plasma , in host cells and tissue , and in sites of tissue inflammation . cfh is produced locally in the eye and accumulated in the interphotoreceptor matrix , in rpe cells and the sub - rpe space , in soft drusen , and in the choroids [ 61 , 74 ] . cfh binds to antigen epitopes , the part of an antigen recognized by antibodies , b cells , or t cells , and protects against oxidative stress . cfh is involved in inhibiting the inflammatory response mediated via c3b ( the alternative pathway of complement ) both by acting as a cofactor for cleavage of c3b to its inactive form c3bi and by weakening the active complex which is formed between c3b and factor b . cfh slows down the cascade of reactions connected with an increase in tcc , especially within the range of the alternative pathway [ 61 , 76 , 77 ] , and the mutation in cfh ( tyr402his ) reduces the ability of cfh to regulate the alternative pathway permitting it to run uncontrolled [ 61 , 68 , 74 ] . the cfh - y402h gene polymorphism reduces also the cfh affinity for c - reactive protein ( crp ) and results in the high levels of unbound crp , which facilitate chronic inflammation [ 78 , 79 ] . c - reactive protein ( crp ) is a nonspecific serum biomarker for acute phase of subclinical inflammation . crp induces innate immune response to infection and/or tissue injury , since crp activates complement directly , via alternative pathway , and indirectly , via crp - cfh interaction , which improve cfh ability to inhibit complement . there is a significant inverse correlation between the crp and cfh levels in patients with advanced amd and cfh gene polymorphism , which means that high level of crp and insufficient level of cfh at rpe / bruch 's membrane / choroid complex may lead to uncontrolled complement activation and macula damage . moreover , crp serum level > 3 mg / l is related to a double amd risk in comparison with crp concentration < 1 mg / l , and elevated serum crp level together with homozygous cfh - y402h polymorphism leads to 19.3 risk ratio of amd and to 6.8 risk ratio of amd progression . according to what is above some authors state that crp has a direct responsibility in macular damage via complement mediated mechanisms , especially in cases with cfh - y402h gene polymorphism , and crp is a risk factor for developing amd [ 83 , 84 ] . however , the full mechanism of crp influence on amd is not understood , and some authors contradict the relation between crp and amd [ 85 , 86 ] . apart from complement factor h , also other serum suppressors of the complement system activity are important , including the family of factor h - like proteins 15 , c4-binding proteins , and surface protein such as membrane decay - accelerating factor cd55 , membrane cofactor protein cd46 , and receptors of the complement system cr1 , cr2 , and cr3 [ 64 , 87 ] . the strong support for the immunoinflammatory model of amd pathogenesis is the highly significant correlations between amd and polymorphism of genes encoding proteins directly involved in the complement alternative pathway activity . polymorphism of genes encoding complement factor h , complement component 3 , complement factor i , and cfh - related genes-2 - 4 - 5 increases amd risk , and polymorphism of genes encoding complement factor b , complement component 2 , and cfh - related genes-1 - 3 reduces risk of amd development . microglial cells are specialized tissue macrophages resident in the retina and are responsible for detecting noxious stimuli in local microenvironment . microglial activation is triggered by different endogenous factors such as oxidized protein and lipid , as well as ages . in the aging retina , microglial activation is associated with the breaking down of the outer blood retinal barrier . activated microglial cells ( large cell body and short dendrites are morphological signs of activation ) migrate from the neuroretina into the subretinal space , that is , into potential space between outer segment of photoreceptors and the apical surface of the rpe cells in order to digest ( phagocyte ) their accumulated deposits in bruch 's membrane [ 30 , 91 , 92 ] . microglial activation and coexisting parainflammation process allow the maintenance of local retinal homeostasis and is a form of protective response against low - grade noxious elements in the aging retina [ 9 , 93 ] . however , in cases of prolonged stress and/or increased immunological responses or loss of mechanisms of their sufficient control , chronic microglial activation and chronic parainflammation state induce proapoptotic events and cause photoreceptor cells injury . patients with cnv / amd have autoantibodies to various antigens such as alpha beta crystallin , alpha - actinin , amyloid , c1q , chondroitin , collagen i , collagen iii , collagen iv , elastin , fibronectin , heparan sulfate , histone h2a , histone h2b , hyaluronic acid , laminin , proteoglycan , vimentin , and vitronectin systemically expressed . many of the above proteins are the building elements of extracellular matrix , that is , the 24 m thick bruch membrane , which acts as a frame for the metabolically active rpe cells and a physical barrier for their passage and that of the endothelial vessels and regulates the diffusion of nutrient molecules between the retina and the choroid . collagens type i and type iii ( fibrillar ) , elastin , fibronectin , and proteoglycans ( mainly heparan sulfate proteoglycans ) are the main structural components of internal / outer collagenous and elastic layer of bruch 's membrane , and collagen type iv ( nonfibrillar ) , laminins , and proteoglycans ( mainly heparan sulfate proteoglycans ) are the main structural components of rpe and choriocapillaris basement membranes . the presence of such numerous autoantibodies against main structural elements of bruch 's membrane indicates strong immunological process in extracellular matrix , which may facilitate penetration of neovascular membrane through choroidal / retinal interface under neuroretina in exudative amd . sera of patients with amd indicate also the presence of other autoantibodies , that is , autoantibodies against aldolase c ( aldoc ) and pyruvate kinase m2 ( pkm2 ) . aldoc and pkm2 are enzymes associated with glucose metabolism and are specific for dry and wet amd . autoantibodies against aldoc / pkm2 indicate activity disorders stimulated by the immunological inflammatory condition , which in turn may deepen the structural changes in the bruch membrane . according to morohoshi anti - pkm2 igg antibodies mediate inflammation by activation of the classic pathway of complement cascade ( when they bind their cognate antigen and form immune complexes ) or by binding fcc receptors ( fccrs ) on effector cells such as macrophages . the fccri , fccriia , fccriic , fccriiia , and fccriiib are the activating receptors , and fccriib is the inhibitory receptor in humans . activated / inhibited fccrs activate / inhibit function of effector cells , that is , their capability of phagocytosis , cytokine production , and degranulation . it implicates a possible role for antibody - mediated inflammation in early and advanced amd largely dependent on the presence and activity of fcc receptors . it is favored by higher expression of intercellular adhesion molecule-1 ( icam-1 ) in the macula , as compared to the peripheral retina . elevated white blood cell counts correlate with the presence of soft drusen and cnv / amd , as well as possibly with ga / amd , which indicates the relationship of systemic circulating , nonspecific chronic inflammatory cells with the pathogenesis of early and advanced amd . monocytes after migration from bloodstream into the subendothelial space differentiate into mature tissue resident macrophages . their recruitment into inflamed tissues mediates chemokines ( signaling proteins ) released by damaged or infected cells . locally produced monocyte chemoattractant protein ( mcp)-1 ( ccl2 ) is one of the key chemokines which regulates migration and infiltration of monocyte . mcp-1/ccl2 interact with the chemokine receptors type 1 and 2 ( cx3cr1 and ccr2 , resp . ) macrophages induce granulomatous inflammatory reactions in bruch 's membrane [ 46 , 51 , 108 ] . they were found in the choroids of amd patients , in degraded areas of bruch 's membrane [ 110 , 111 ] , and in surgically excised cnv membranes . anti - inflammatory macrophages maintain balance of waste products in the aging bruch membrane and remove sd and other deposits . proinflammatory macrophages maintain parainflammation state of amd lesions and promote the development of late stages of disease [ 113 , 114 ] . m2 macrophages act mainly as scavenger and anti - inflammatory factor ; since they digest proinflammatory agents such as ages , complement complexes , oxidized membrane , and dead cells , removal of apoptotic cell debris limits inflammation through inhibiting the production of inflammatory cytokines [ 113 , 114 ] . failure in recruitment m2 macrophages , caused by inhibition of receptor ccr2 , essential for scavenging deposits leads to increased rpe cells exposure to ages , which may stimulate rpe cells to secrete proangiogenic cytokines , including vegf . in late amd , m2 macrophages represent proangiogenic activity and facilitate cnv formation , since they may express proangiogenic cytokines ( including vegf ) , growth factors , and reactive oxygen species negatively affecting the rpe and choriocapillaris , and they may promote proteolysis of bruch 's membrane as well . they also serve as scaffold for cnv development [ 113 , 114 , 116 ] . etiopathogenesis of amd involves the disruption of many physiological pathways , but chronic pathophysiological parainflammation plays a key role in amd development . normal , adaptive parainflammation process exists in the aging retina under physiologic conditions , but unbalanced and uncontrolled parainflammation leads to detrimental chronic inflammatory response and causes early and advanced amd forms . chronic pathophysiological parainflammation is mediated by many factors and stimulated by complement system , especially its alternative pathway , which is carried out in bruch 's membrane , and leads to early and advanced , exudative amd forms . chronic pathophysiological parainflammation connected with microglial activation carried out also in retinal / choroidal interface leads to advanced atrophicans form of amd . chronic pathophysiological parainflammation connected with autoantibodies and formation of immune complexes carried out in bruch 's membrane lead to early and advanced amd .

the products of oxidative stress trigger chronic low - grade inflammation ( pathophysiological parainflammation ) process in amd patients . in early amd , soft drusen contain many mediators of chronic low - grade inflammation such as c - reactive protein , adducts of the carboxyethylpyrrole protein , immunoglobulins , and acute phase molecules , as well as the complement - related proteins c3a , c5a , c5 , c5b-9 , cfh , cd35 , and cd46 . the complement system , mainly alternative pathway , mediates chronic autologous pathophysiological parainflammation in dry and exudative amd , especially in the y402h gene polymorphism , which causes hypofunction / lack of the protective complement factor h ( cfh ) and facilitates chronic inflammation mediated by c - reactive protein ( crp ) . microglial activation induces photoreceptor cells injury and leads to the development of dry amd . many autoantibodies ( antibodies against alpha beta crystallin , alpha - actinin , amyloid , c1q , chondroitin , collagen i , collagen iii , collagen iv , elastin , fibronectin , heparan sulfate , histone h2a , histone h2b , hyaluronic acid , laminin , proteoglycan , vimentin , vitronectin , and aldolase c and pyruvate kinase m2 ) and overexpression of fcc receptors play role in immune - mediated inflammation in amd patients and in animal model . macrophages infiltration of retinal / choroidal interface acts as protective factor in early amd ( m2 phenotype macrophages ) ; however it acts as proinflammatory and proangiogenic factor in advanced amd ( m1 and m2 phenotype macrophages ) .

1. Introduction 2. Chronic Low-Grade Inflammation (Pathophysiological Parainflammation) Model of AMD Etiopathogenesis 3. Summary

these fractures are difficult to treat and operative treatment is usually recommended for favorable outcome [ 14 ] . these are associated with high energy trauma ( in the youngsters ) and osteoporotic bones ( in the elderly ) and are frequently comminuted and intra - articular . current generation of distal femoral locking compression plates is precontoured based on the average bony anatomy of the adult population and they form a fixed angled construct . the pull - out strength of locking screws is higher than the conventional screws and is particularly useful in osteoporotic bones . these plates are designed to apply in minimally invasive fashion to preserve local biology and avoid problems with fracture healing and infection [ 6 , 7 ] . the purpose of this study was to evaluate functional outcome , fracture healing , and the complications of distal femoral intra - articular fractures using locking compression plates . we reviewed 46 consecutive distal femoral fractures treated with distal femoral locking compression plates between 2009 and 2012 . ao synthes distal femoral locking compression plate and zimmer periarticular distal femoral locking plate were used in 19 and 27 patients , respectively . there were 36 men and 10 women with mean age of 35 years ( range 2072 ) . inclusion criteria were as follows : ( i ) type c distal femoral fractures and ( ii ) patients older than 18 years of age . pathological fractures and types a and b distal femoral fractures were excluded from the study . 10 patients had intra - articular fractures involving both condyles ( 33c-1 ) , 12 patients had metaphyseal comminution ( 33c-2 ) , and 24 patients had articular comminution ( 33c-3 ) . 13 patients with multiple injuries and open fractures were treated within 3 wks after temporary application of external fixator . all surgeries were carried out at our tertiary care level 1 trauma centre with patient placed supine on the radiolucent table . appropriate - sized distal femoral locking plate was slid in distal to proximal direction submuscularly over lateral aspect of distal femur . usually we prefer minimum length of the plate which is three times the fracture comminution segment . first a standard 4.5 mm cortical screw was placed in the femoral diaphysis and tightened lightly . for proximal fixation 3 or 4 bicortical screws one or more partially threaded cancellous screws were used in intercondylar region whenever required to achieve compression ( figure 2 ) . small incision was given towards the end of the plate to confirm approximation of plate to the bone . suction drain was used in 20 patients and was removed after 24 to 48 hrs . static quadriceps exercises with active hip and knee mobilization were started from 1st postoperative day . follow - up radiographs were taken after 6 wks , 12 wks , 6 months , 9 months , and 12 months after surgery . gradual weight bearing was started based on the evidence of bridging callous on follow - up radiographs . radiological union was defined as the presence of cross trabeculation on both ap and lateral radiographs . clinical and functional outcomes were assessed using the knee society score ( table 2 ) . one case required autogenous iliac crest bone grafting and the other case required bone grafting along with refixation using longer plate due to breakage of proximal screws with broken proximal screws ( figure 3 ) . one patient required arthroscopic arthrolysis with implant removal due to severe restriction of movement and hardware prominence . no cases of infection and rotational or angular deformity more than 5 degrees was noted ( table 1 ) . 2 patients had limb length discrepancy of < 2 cm and no treatment was needed . at the latest follow up rom > 120 is noted in 32 patients , 90120 in 10 patients , and 7090 in 2 patients . to maintain the fracture biology and to minimize the soft tissue trauma , minimally invasive plating techniques have been developed for the fixation of distal femoral fractures . the main goals of the above - mentioned techniques are to maintain the important anatomy and to promote early fracture healing . locking plate systems such as the liss liss has a lower risk of early implant loosening than the dynamic condylar screw and promotes early mobilization [ 15 , 16 ] and rapid healing without bone grafting with low risk of infection [ 15 , 16 ] and less blood loss . the lcp differs from the liss in that the lcp has combination holes and does not have a jig . pain over lateral aspect of the distal femur following fixation with liss has been attributed to the jig . previous studies have demonstrated successful early results and relatively low complication rates using minimally invasive plating techniques for the fractures of distal femur [ 12 , 17 , 18 ] . we have used minimally invasive plate fixation technique using standard lateral approach for the fixation of simple intra - articular fractures ( c1 ) . however , more extensive approaches are needed for fixation of complex intra - articular fractures ( c2/c3 ) . in these fractures this articular block was fixed to the femoral shaft using indirect plate fixation technique . in our series of 44 patients , there were no cases of infection and varus / valgus alignment of more than 5 degrees . we noted 2 cases of nonunion , out of which one case required autogenous iliac crest bone grafting and the other case required bone grafting along with refixation using longer plate due to breakage of proximal screws . one case required arthrolysis with implant removal due to severe restriction of rom and hardware prominence . as reviewed by miclau et al . . relatively low rate of bone grafting in our series is probably due to improved surgical technique with better soft tissue handling . early experience with the liss for distal femoral fractures in multicentric study in europe demonstrated a 20% incidence of varus / valgus deformity greater than 5 degrees . our relatively low incidence of deformity is probably because of improved surgical expertise along with better understanding of fracture anatomy . limitations of the study include ( i ) all the drawbacks pertaining to its retrospective nature and ( ii ) small sample size . strength of our study is that we included only intra - articular fractures ; most of these were complex which require adequate surgical expertise . the results of our study suggest that use of standard lateral approach for simple intra - articular distal femoral fractures ( c1 ) and transarticular / minimally invasive techniques for complex intra - articular fractures ( c2/c3 ) results in improved exposure of the knee joint and better union rates with low incidence of bone grafting

background . intra - articular fractures of distal femur present a huge surgical challenge . the aim of this study is to evaluate functional outcome , fracture healing , and the complications of distal femoral intra - articular fractures using locking compression plates . material and methods . we reviewed 46 distal femoral fractures treated with distal femoral locking compression plates between 2009 to 2012 . there were 36 men and 10 women with mean age of 35 years ( range 2072 ) . more than half of the patients were of type c3 ( ao classification ) and had been caused by high energy trauma with associated injuries . results . 2 patients were lost to follow - up . of the remaining 44 patients , the mean follow - up period was 25 months ( range 1836 ) . the mean time for radiological union was 12 weeks ( range 1018 ) except 2 patients which had gone for nonunion . at the latest follow up rom > 120 is noted in 32 patients , 90120 in 10 patients , and 7090 in 2 patients . 38 patients ( 86% ) had good / excellent outcome . conclusion . use of standard lateral approach for simple intra - articular distal femoral fractures ( c1 ) and transarticular / minimally invasive techniques for complex intra - articular fractures ( c2/c3 ) results in improved exposure of the knee joint and better union rates with low incidence of bone grafting .

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

previously described nod.2m.hhd mice ( 17 ) are maintained by sibling matings at the jackson laboratory . some experiments used an n10 backcross generation nod stock congenically expressing the cd45.2 rather than the cd45.1 variant leukocyte marker . nod.2m.hhd mice in which antigen - presenting cells ( apcs ) specifically express an mhc class ii promoter ( h2-e)driven mouse proinsulin 2 transgene ( nod.2m.hhd-pi ) were generated by crossing nod.2m.hhd with previously described nod.pi mice ( 27 ) . the institutional animal care and use committee at the jackson laboratory approved all animal experiments . synthetic peptides ins1/2 a210 ( ivdqcctsi ) , ins1 b515 ( hlcgphlvea ) , igrp228236 ( fgidllwsv ) , igrp265273 ( vlfglgfai ) , and flu16 ( flu mp5866 ) ( gilgfvftl ) were purchased from mimotopes pty , melbourne , australia . monoclonal antibodies specific for cd11c ( clone n418 ) , cd8 ( clone 536.72 ) , cd45.1 ( clone a201.7 ) , and cd45.2 ( clone 1042.1 ) were purchased from bd biosciences , san jose , ca and ebiosciences , san diego , ca . ecdi peptide - coupled splenocyte ( peptide - sps ) treatment was carried out as previously described ( 28 ) . briefly , spleens were removed from syngeneic female mice , collagenase d treated ( roche diagnostics , manheim , germany ) , and the erythrocytes lysed . the splenocytes were incubated with ecdi ( 150 mg/3.2 10 cells [ calbiochem , la jolla , ca ] ) , and as indicated , a mixture of ins and/or igrp peptide(s ) ( 1 mg / ml each ) on ice for 1 h , hand - shaking every 10 min . the peptide - sps were washed , centrifuged , filtered to remove cell clumps , and resuspended in pbs . nod.2m.hhd female mice ( 46 weeks old ) received 50 10 peptide - sps in 200 l pbs by intravenous injection . controls consisted of nod.2m.hhd female mice treated with splenocytes bearing the ecdi - coupled hla - a2.1 binding but diabetes - irrelevant flu16 peptide . diabetes onset was defined by a urinary glucose value of 3 as assessed with ames diastix ( bayer , diagnostics division , elkhart , nj ) . mice remaining free of overt diabetes through 26 weeks posttreatment were killed and their pancreata fixed in bouin s solution and sectioned at three nonoverlapping levels . islets ( at least 20 per mouse ) were individually scored as follows : 0 , no lesions ; 1 , peri - insular leukocytic aggregates , usually periductal infiltrates ; 2 , < 25% islet destruction ; 3 , > 25% islet destruction ; and 4 , complete islet destruction . an insulitis score for each mouse was obtained by dividing the total score for each pancreas by the number of islets examined . interferon ( ifn)- enzyme - linked immunospot ( elispot ) assays were performed as previously described ( 17 ) . spots were counted using an automated elispot reader system ( immunospot ; ctl analyzers , cleveland , oh ) . numbers of ifn- or il-10 spots were normalized to the indicated numbers of cd8 t - cells . nod.2m.hhd mice treated every 5 weeks with peptide - sps were primed in a rear footpad 2 days after the third treatment with a cocktail containing 20 g each of the hla - a2.1restricted ins1/2 a210 , ins1 b515 , igrp228236 , and igrp265273 peptides emulsified in 50 l of complete freund s adjuvant . ten days after priming , the mice were killed , the popliteal lymph nodes were isolated and dispersed by collagenase d treatment , and cd8 t - cell reactions to each individual peptide were determined by ifn- elispot analyses . splenic cd8 t - cells were enriched from nod.2m.hhd mice by the previously described magnetic bead based negative selection approach ( 29 ) and incubated in vitro at a concentration of 5 10/ml with 5 10/ml ins- and/or igrp - sps . the next day , the cd8 t - cells were recovered and assessed by ifn- elispot analyses for reactivity against hla - a2.1restricted ins and igrp peptides . starting at 3 weeks of age , nod.2 m .hhd mice were treated with repeated intraperitoneal injections of a soluble mixture containing 25 g of each peptide ( ins a210 , ins b515 , igrp228236 , and igrp265273 ) . the log - rank test was used to compare diabetes incidence curves and the nonparametric unpaired test to compare antigen - reactive cd8 t - cell numbers between different treatment groups . previously described nod.2m.hhd mice ( 17 ) are maintained by sibling matings at the jackson laboratory . some experiments used an n10 backcross generation nod stock congenically expressing the cd45.2 rather than the cd45.1 variant leukocyte marker . nod.2m.hhd mice in which antigen - presenting cells ( apcs ) specifically express an mhc class ii promoter ( h2-e)driven mouse proinsulin 2 transgene ( nod.2m.hhd-pi ) were generated by crossing nod.2m.hhd with previously described nod.pi mice ( 27 ) . the institutional animal care and use committee at the jackson laboratory approved all animal experiments . synthetic peptides ins1/2 a210 ( ivdqcctsi ) , ins1 b515 ( hlcgphlvea ) , igrp228236 ( fgidllwsv ) , igrp265273 ( vlfglgfai ) , and flu16 ( flu mp5866 ) ( gilgfvftl ) were purchased from mimotopes pty , melbourne , australia . monoclonal antibodies specific for cd11c ( clone n418 ) , cd8 ( clone 536.72 ) , cd45.1 ( clone a201.7 ) , and cd45.2 ( clone 1042.1 ) were purchased from bd biosciences , san jose , ca and ebiosciences , san diego , ca . ecdi peptide - coupled splenocyte ( peptide - sps ) treatment was carried out as previously described ( 28 ) . briefly , spleens were removed from syngeneic female mice , collagenase d treated ( roche diagnostics , manheim , germany ) , and the erythrocytes lysed . the splenocytes were incubated with ecdi ( 150 mg/3.2 10 cells [ calbiochem , la jolla , ca ] ) , and as indicated , a mixture of ins and/or igrp peptide(s ) ( 1 mg / ml each ) on ice for 1 h , hand - shaking every 10 min . the peptide - sps were washed , centrifuged , filtered to remove cell clumps , and resuspended in pbs . nod.2m.hhd female mice ( 46 weeks old ) received 50 10 peptide - sps in 200 l pbs by intravenous injection . controls consisted of nod.2m.hhd female mice treated with splenocytes bearing the ecdi - coupled hla - a2.1 binding but diabetes - irrelevant flu16 peptide . diabetes onset was defined by a urinary glucose value of 3 as assessed with ames diastix ( bayer , diagnostics division , elkhart , nj ) . mice remaining free of overt diabetes through 26 weeks posttreatment were killed and their pancreata fixed in bouin s solution and sectioned at three nonoverlapping levels . islets ( at least 20 per mouse ) were individually scored as follows : 0 , no lesions ; 1 , peri - insular leukocytic aggregates , usually periductal infiltrates ; 2 , < 25% islet destruction ; 3 , > 25% islet destruction ; and 4 , complete islet destruction . an insulitis score for each mouse was obtained by dividing the total score for each pancreas by the number of islets examined . interferon ( ifn)- enzyme - linked immunospot ( elispot ) assays were performed as previously described ( 17 ) . spots were counted using an automated elispot reader system ( immunospot ; ctl analyzers , cleveland , oh ) . numbers of ifn- or il-10 spots were normalized to the indicated numbers of cd8 t - cells . nod.2m.hhd mice treated every 5 weeks with peptide - sps were primed in a rear footpad 2 days after the third treatment with a cocktail containing 20 g each of the hla - a2.1restricted ins1/2 a210 , ins1 b515 , igrp228236 , and igrp265273 peptides emulsified in 50 l of complete freund s adjuvant . ten days after priming , the mice were killed , the popliteal lymph nodes were isolated and dispersed by collagenase d treatment , and cd8 t - cell reactions to each individual peptide were determined by ifn- elispot analyses . splenic cd8 t - cells were enriched from nod.2m.hhd mice by the previously described magnetic bead based negative selection approach ( 29 ) and incubated in vitro at a concentration of 5 10/ml with 5 10/ml ins- and/or igrp - sps . the next day , the cd8 t - cells were recovered and assessed by ifn- elispot analyses for reactivity against hla - a2.1restricted ins and igrp peptides . starting at 3 weeks of age , nod.2 m .hhd mice were treated with repeated intraperitoneal injections of a soluble mixture containing 25 g of each peptide ( ins a210 , ins b515 , igrp228236 , and igrp265273 ) . the log - rank test was used to compare diabetes incidence curves and the nonparametric unpaired test to compare antigen - reactive cd8 t - cell numbers between different treatment groups . cd8 t - cells were enriched from pooled spleens of 9- to 16-week - old nod.2m.hhd mice and cocultured in vitro with syngeneic splenocytes bearing an ecdi - coupled cocktail of the hla - a2.1restricted ins1/2 a210 , ins1 b515 , igrp228236 , and igrp265273 -cell autoantigens ( ins / igrp - sps ) and assessed the next day for responsiveness to each individual epitope by ifn- elispot analyses . compared with those exposed to the control flu16 peptide ( flu - sps ) , cd8 t - cells from nod.2m.hhd mice cocultured with ins / igrp - sps displayed significantly decreased responses to restimulation by igrp epitopes ( fig . levels of reactivity to the hla - a2.1restricted ins peptides was low among control cd8 t - cells exposed to flu16 and not further influenced by previous coculture with ins / igrp - sps ( fig . coincubation with syngeneic splenocytes bearing an ecdi cross - linked mixture of the two ins peptides ( ins - sps ) did not diminish cd8 t - cell responsiveness to ins or igrp epitopes ( fig . however , preincubation with igrp - sps did significantly diminish the ability of cd8 t - cells from nod.2m.hhd mice to respond to restimulation by igrp but not ins epitopes ( fig . il-10 elispot analyses also indicated that regardless of preincubation conditions , no cd8 t - cells from nod.2m.hhd mice responding to ins or igrp stimulation produced this immunosuppressive cytokine . on the basis of these collective data , we subsequently assessed syngeneic ins / igrp - sps as a possible diabetes intervention approach in nod.2m.hhd mice . hla - a2.1restricted -cell antigenic peptides that are ecdi cross - linked to syngeneic splenocytes significantly diminish responsiveness by cognate cd8 t - cells from nod.2m.hhd mice . a : cd8 t - cells from nod.2m.hhd mice were incubated with syngeneic flu - sps or ins / igrp - sps ( bearing the complete mixture of igrp265273 , igrp228236 , ins1/2 a210 , and ins1 b514 peptides ) . the next day , recovered cd8 t - cells were cocultured for 48 h with 1 mol / l of each of the individual ins or igrp peptides , and antigen reactivity was assessed by elispot analyses of ifn- production . b : cd8 t - cells from nod.2m.hhd mice were incubated with syngeneic flu - sps , igrp - sps ( mixture of igrp265273 and igrp228236 ) , or ins - sps ( mixture of ins1/2 a210 and ins1 b514 ) . t - cell reactivity to individual igrp or ins peptides was then determined as described above . p values are based on comparison with flu - sps . * p < 0.01 , * * p < 0.05 ; p values ins - sps compared with igrp - sps , * * * p < 0.01 , # # p < 0.001 . initial analyses found that repeated injections of a soluble mixture of the four ins and igrp peptides ( 25 g each ) did not inhibit diabetes development in nod.2 m .hhd mice ( data not shown ) . thus , given the in vitro results shown in fig . 1 , we assessed whether a single intravenous injection of ins / igrp - sps given at 46 weeks of age could protect nod.2m.hhd female mice from diabetes development . diabetes development was inhibited in mice treated with the ins / igrp - sps , compared with controls receiving flu - sps ( fig . nod.2m.hhd mice were injected with syngeneic splenocytes separately bearing either a mixture of the two ecdi cross - linked ins ( ins - sps ) or igrp ( igrp - sps ) peptides . because one injection of ins / igrp - sps initially afforded a partial diabetes protective effect , treatments with ins - sps and igrp - sps were repeated at 5-week intervals . compared with controls receiving flu - sps , only igrp - sps and not ins - sps exerted a diabetes protective effect in nod.2m.hhd mice ( fig . treatments with syngeneic splenocytes separately bearing either the ecdi - coupled igrp228236 or igrp265273 peptide failed to significantly inhibit diabetes development in nod.2m.hhd mice ( fig . thus , splenocytes must bear a combination of the ecdi - coupled igrp228236 and igrp265273 peptides in order to elicit robust diabetes protective effects in nod.2m.hhd mice . furthermore , although both ins / igrp - sps and igrp - sps treatment could inhibit overt diabetes development in nod.2m.hhd mice , neither intervention significantly suppressed insulitis levels ( fig . nod.2m.hhd female mice were injected intravenously at 46 weeks of age with ins / igrp - sps ( bearing the ecdi - linked mixture of igrp265273 , igrp228236 , ins1/2 a210 , and ins1 b514 peptides ) . syngeneic splenocytes bearing a mixture of ecdi - coupled igrp , but not ins peptides , inhibit diabetes but not insulitis development in nod.2m.hhd mice . beginning at 46 weeks of age , nod.2m.hhd female mice were injected intravenously at 5-week intervals with either igrp - sps , ins - sps , igrp265273-sps , or igrp228236-sps . igrp - sps developed diabetes at a significantly lower rate ( p = 0.04 ) than flu - sps treated controls . b : percentage of nod.2m.hhd mice in each treatment group with the indicated range of insulitis scores . we assessed whether the diabetes protective effect of ins / igrp - sps treatment was associated with altered in vivo responsiveness of either hla - a2.1restricted ins or igrp autoreactive cd8 t - cells in nod.2m.hhd mice . two days following a third ins / igrp - sps or flu - sps treatment given at 5-week intervals , nod.2m.hhd mice were primed in the footpad with a cocktail of the four ins and igrp peptides . ten days postpriming , the mice were killed and cd8 t - cells within the draining popliteal lymph nodes were assessed for reactivity to each individual peptide by ifn- elispot analyses . all mice were still nondiabetic at the time of analysis . in ins / igrp - sps treated nod.2m.hhd mice , cd8 t - cell responses to both the hla - a2.1restricted igrp228236 and igrp265273 peptides but neither ins epitope were significantly decreased ( p = 0.03 and 0.004 ) ( fig . 4 ) . treatment with igrp - sps but not with ins - sps also showed significantly decreased cd8 t - cell responses to both the igrp228236 and igrp265273 peptides ( p = 0.05 and 0.002 ) ( fig . these results further indicate that in the context of the human hla - a2.1 class i variant , autoreactive cd8 t - cells recognizing the igrp228236 and/or igrp265273 peptides are of significant pathogenic importance during diabetes development in nod.2m.hhd mice . ins / igrp - sps and igrp - sps inhibit in vivo responses of hla - a2.1restricted igrp but not ins autoreactive cd8 t - cells . beginning at 46 weeks of age , nod.2m.hhd female mice were injected intravenously with ins / igrp - sps , igrp - sps , ins - sps , or flu - sps at 5-week intervals . two days after the third treatment , mice were footpad primed with a mixture of the four ins / igrp peptides . ten days after priming , cd8 t - cells within the draining popliteal lymph nodes were cocultured for 48 h with 1 previous studies have indicated that ( pro)insulin is a key autoantigen for diabetes development in standard nod mice ( 27,3033 ) . this was partly demonstrated by studies showing that mhc class ii promoter driven transgenic expression of proinsulin-2 in apcs of nod mice inhibits insulitis and diabetes development ( 27,30 ) . hence , we used such a transgenic approach as an alternative means to test whether ( pro)insulin may also be an autoantigen of pathogenic importance in nod.2m.hhd mice . nod.2m.hhd mice expressing the previously described ( 27 ) proinsulin-2 transgene in apcs ( designated nod.2m.hhd-pi ) were generated and assessed for diabetes development . 5a , compared with nontransgenic controls , the rate of type 1 diabetes development was somewhat retarded in nod.2m.hhd-pi female mice but did not quite achieve statistical significance ( p = 0.08 ) by kaplan - meier analyses . however , as assessed by analyses , the cumulative frequency of diabetes development by 35 weeks of age was significantly lower ( p < 0.005 ) in nod.2m.hhd-pi mice than in nontransgenic controls . insulitis levels were also significantly lower in nod.2m.hhd-pi mice than in nontransgenic controls ( fig . 5b ) . baseline and primed levels of cd8 t - cell responses to the hla - a2.1restricted ins1/2 a210 or ins1 b514 epitopes were found not to differ in nod.2m.hhd-pi mice and nontransgenic controls ( data not shown ) . these findings do not eliminate the possibility that like those targeting igrp epitopes , hla - a2.1restricted insulin autoreactive cd8 t - cells are also important pathogenic contributors to diabetes development in nod.2m.hhd mice . however , these apc transgenic expression studies indicate that even if they do not represent autoantigens recognized by pathogenic hla - a2.1restricted cd8 t - cells , ( pro)insulin derived epitopes are important targets of at least diabetogenic cd4 t - cells in nod.2m.hhd mice . insulitis and diabetes development in nod.2m.hhd-pi mice . a : incidence curve of diabetes development in nod.2m.hhd-pi female mice and nontransgenic littermates . the rate of diabetes development did not quite achieve a statistical difference ( p = 0.08 ) by kaplan - meier analyses . the cumulative frequency of diabetes development by 35 weeks of age was significantly different ( p < 0.005 ) as assessed by analyses . b : histological grading of insulitis in pancreas sections of 35-week - old female mice ( nod.2m.hhd-pi mice , n = 14 ; nod.2m.hhd mice , n = 16 ) . it has been previously reported that splenocytes bearing ecdi - coupled proteins or peptides do not directly induce tolerogenic responses by cd4 t - cells in an efficient manner , but rather do so indirectly following their uptake and processing by host - type apc ( 21 ) . if this is also the case for inducing cd8 t - cell tolerance , we reasoned it should remain possible to inhibit diabetes development in the hla - a2.1expressing nod.2m.hhd stock by treatments with splenocytes from totally mhc class i deficient nod.2 m mice bearing the complete cocktail of ecdi cross - linked ins and igrp epitopes ( designated ins / igrp-2 m sps ) . unlike those receiving flu-2 m sps , diabetes development was completely abrogated in ins / igrp-2 m sps - treated nod.2m.hhd mice ( fig . ins / igrp - sps do not have to share host - type mhc class i identity to attenuate diabetes development in nod.2m.hhd mice . beginning at 46 weeks of age nod.2m.hhd mice were injected intravenously at 5-week intervals with ins / igrp-2 m sps or flu-2 m sps . b : ins / igrp-2 m sps inhibit in vivo responses of hla - a2.1restricted igrp but not ins autoreactive cd8 t - cells in nod.2m.hhd mice . two days after the third treatment , mice were footpad primed with a mixture of the four ins / igrp peptides . ten days after priming , cd8 t - cells within the draining popliteal lymph nodes of nondiabetic mice were cocultured for 48 h with 1 mol / l of each of the individual ins or igrp peptides , and antigen reactivity was assessed by elispot analyses of ifn- production . two days after a third ins / igrp-2 m sps or flu-2 m sps treatment given at 5-week intervals , nod.2m.hhd mice were primed in the footpad with a cocktail of the four ins and igrp peptides . ten days postpriming , cd8 t - cells within the draining popliteal lymph nodes were assessed for reactivity to each individual peptide by ifn- elispot analyses . cd8 t - cell responses to both the hla - a2.1restricted igrp228236 and igrp265273 peptides but again not the ins epitopes were significantly decreased in ins / igrp-2 m sps - treated nod.2m.hhd mice ( fig . these collective results indicate that in order to induce cd8 t - cell tolerance and to elicit diabetes protective effects in nod.2m.hhd recipients , donor cells bearing ecdi cross - linked hla - a2.1 restricted igrp autoantigenic peptides do not also have to express the relevant host - type mhc class i variant . the results described above also indicated that rather than directing inducing tolerogenic responses inhibiting diabetogenic cd8 cell activity in nod.2m.hhd mice , ins / igrp - sps or igrp - sps instead do so in an indirect manner dependent on their uptake by host - type apc . to directly test this possibility , we compared the ability of ecdi - treated or untreated donor cells that were also labeled with the carboxyfluorescein diacetate succinimidyl ester ( cfse ) tracker dye to be taken up by host - type antigen - presenting dendritic cells ( dcs ) . donor- and recipient - type cells were also distinguished by respective expression of the cd45.2 versus cd45.1 pan - leukocyte marker variant . there was a significantly greater uptake by host - type splenic cd8 + dcs of ecdi - treated than untreated donor cells that had been intravenously infused 1 day earlier ( fig . this likely explains why fewer ecdi - treated than untreated donor cells could be detected in the recipients , but with the apoptotic status of the former also allowing them to be more efficiently engulfed by host - type dcs . these results indicate the ability of donor cells bearing ecdi - coupled igrp peptides to elicit diabetes protective effects likely entails their uptake by host - type dcs that then display the autoantigenic epitopes to cd8 t - cells in a tolerance - inducing manner . more efficient uptake by host - type splenic cd8 + dcs of ecdi - treated than untreated donor cells . splenocytes from nod.cd45.2 congenic mice were labeled with the cfse tracker dye and then subsequently treated or not with ecdi before being infused intravenously into standard nod cd45.1-expressing recipients . the following day , viable host - type splenic dcs were assessed by flow cytometry for comparative uptake of cfse - labeled ecdi - treated or untreated donor cells . a : depiction of gating strategy to assess uptake by viable ( propidium iodide negative ) host - type cd45.1-expressing splenic dc subsets of cfse - labeled donor - type leukocytes that had or had not been treated with ecdi . b : mean proportions sem of host - type splenic cd8 + dcs that had taken up cfse - labeled ecdi - treated or untreated donor - type leukocytes ( n = 3/group ) . cd8 t - cells were enriched from pooled spleens of 9- to 16-week - old nod.2m.hhd mice and cocultured in vitro with syngeneic splenocytes bearing an ecdi - coupled cocktail of the hla - a2.1restricted ins1/2 a210 , ins1 b515 , igrp228236 , and igrp265273 -cell autoantigens ( ins / igrp - sps ) and assessed the next day for responsiveness to each individual epitope by ifn- elispot analyses . compared with those exposed to the control flu16 peptide ( flu - sps ) , cd8 t - cells from nod.2m.hhd mice cocultured with ins / igrp - sps displayed significantly decreased responses to restimulation by igrp epitopes ( fig . levels of reactivity to the hla - a2.1restricted ins peptides was low among control cd8 t - cells exposed to flu16 and not further influenced by previous coculture with ins / igrp - sps ( fig . coincubation with syngeneic splenocytes bearing an ecdi cross - linked mixture of the two ins peptides ( ins - sps ) did not diminish cd8 t - cell responsiveness to ins or igrp epitopes ( fig . however , preincubation with igrp - sps did significantly diminish the ability of cd8 t - cells from nod.2m.hhd mice to respond to restimulation by igrp but not ins epitopes ( fig . il-10 elispot analyses also indicated that regardless of preincubation conditions , no cd8 t - cells from nod.2m.hhd mice responding to ins or igrp stimulation produced this immunosuppressive cytokine . on the basis of these collective data , we subsequently assessed syngeneic ins / igrp - sps as a possible diabetes intervention approach in nod.2m.hhd mice . hla - a2.1restricted -cell antigenic peptides that are ecdi cross - linked to syngeneic splenocytes significantly diminish responsiveness by cognate cd8 t - cells from nod.2m.hhd mice . a : cd8 t - cells from nod.2m.hhd mice were incubated with syngeneic flu - sps or ins / igrp - sps ( bearing the complete mixture of igrp265273 , igrp228236 , ins1/2 a210 , and ins1 b514 peptides ) . the next day , recovered cd8 t - cells were cocultured for 48 h with 1 mol / l of each of the individual ins or igrp peptides , and antigen reactivity was assessed by elispot analyses of ifn- production . b : cd8 t - cells from nod.2m.hhd mice were incubated with syngeneic flu - sps , igrp - sps ( mixture of igrp265273 and igrp228236 ) , or ins - sps ( mixture of ins1/2 a210 and ins1 b514 ) . t - cell reactivity to individual igrp or ins peptides was then determined as described above . p values are based on comparison with flu - sps . * p < 0.01 , * * p < 0.05 ; p values ins - sps compared with igrp - sps , * * * initial analyses found that repeated injections of a soluble mixture of the four ins and igrp peptides ( 25 g each ) did not inhibit diabetes development in nod.2 m .hhd mice ( data not shown ) . thus , given the in vitro results shown in fig . 1 , we assessed whether a single intravenous injection of ins / igrp - sps given at 46 weeks of age could protect nod.2m.hhd female mice from diabetes development . diabetes development was inhibited in mice treated with the ins / igrp - sps , compared with controls receiving flu - sps ( fig . nod.2m.hhd mice were injected with syngeneic splenocytes separately bearing either a mixture of the two ecdi cross - linked ins ( ins - sps ) or igrp ( igrp - sps ) peptides . because one injection of ins / igrp - sps initially afforded a partial diabetes protective effect , treatments with ins - sps and igrp - sps were repeated at 5-week intervals . compared with controls receiving flu - sps , only igrp - sps and not ins - sps exerted a diabetes protective effect in nod.2m.hhd mice ( fig . treatments with syngeneic splenocytes separately bearing either the ecdi - coupled igrp228236 or igrp265273 peptide failed to significantly inhibit diabetes development in nod.2m.hhd mice ( fig . thus , splenocytes must bear a combination of the ecdi - coupled igrp228236 and igrp265273 peptides in order to elicit robust diabetes protective effects in nod.2m.hhd mice . furthermore , although both ins / igrp - sps and igrp - sps treatment could inhibit overt diabetes development in nod.2m.hhd mice , neither intervention significantly suppressed insulitis levels ( fig . nod.2m.hhd female mice were injected intravenously at 46 weeks of age with ins / igrp - sps ( bearing the ecdi - linked mixture of igrp265273 , igrp228236 , ins1/2 a210 , and ins1 b514 peptides ) . syngeneic splenocytes bearing a mixture of ecdi - coupled igrp , but not ins peptides , inhibit diabetes but not insulitis development in nod.2m.hhd mice . beginning at 46 weeks of age , nod.2m.hhd female mice were injected intravenously at 5-week intervals with either igrp - sps , ins - sps , igrp265273-sps , or igrp228236-sps . a : mice were monitored for diabetes development . only mice receiving igrp - sps developed diabetes at a significantly lower rate ( p = 0.04 ) than flu - sps treated controls . b : percentage of nod.2m.hhd mice in each treatment group with the indicated range of insulitis scores . we assessed whether the diabetes protective effect of ins / igrp - sps treatment was associated with altered in vivo responsiveness of either hla - a2.1restricted ins or igrp autoreactive cd8 t - cells in nod.2m.hhd mice . two days following a third ins / igrp - sps or flu - sps treatment given at 5-week intervals , nod.2m.hhd mice were primed in the footpad with a cocktail of the four ins and igrp peptides . ten days postpriming , the mice were killed and cd8 t - cells within the draining popliteal lymph nodes were assessed for reactivity to each individual peptide by ifn- elispot analyses . in ins / igrp - sps treated nod.2m.hhd mice , cd8 t - cell responses to both the hla - a2.1restricted igrp228236 and igrp265273 peptides but neither ins epitope were significantly decreased ( p = 0.03 and 0.004 ) ( fig . 4 ) . treatment with igrp - sps but not with ins - sps also showed significantly decreased cd8 t - cell responses to both the igrp228236 and igrp265273 peptides ( p = 0.05 and 0.002 ) ( fig . these results further indicate that in the context of the human hla - a2.1 class i variant , autoreactive cd8 t - cells recognizing the igrp228236 and/or igrp265273 peptides are of significant pathogenic importance during diabetes development in nod.2m.hhd mice . ins / igrp - sps and igrp - sps inhibit in vivo responses of hla - a2.1restricted igrp but not ins autoreactive cd8 t - cells . beginning at 46 weeks of age , nod.2m.hhd female mice were injected intravenously with ins / igrp - sps , igrp - sps , ins - sps , or flu - sps at 5-week intervals . two days after the third treatment , mice were footpad primed with a mixture of the four ins / igrp peptides . ten days after priming , cd8 t - cells within the draining popliteal lymph nodes were cocultured for 48 h with 1 previous studies have indicated that ( pro)insulin is a key autoantigen for diabetes development in standard nod mice ( 27,3033 ) . this was partly demonstrated by studies showing that mhc class ii promoter driven transgenic expression of proinsulin-2 in apcs of nod mice inhibits insulitis and diabetes development ( 27,30 ) . hence , we used such a transgenic approach as an alternative means to test whether ( pro)insulin may also be an autoantigen of pathogenic importance in nod.2m.hhd mice . nod.2m.hhd mice expressing the previously described ( 27 ) proinsulin-2 transgene in apcs ( designated nod.2m.hhd-pi ) were generated and assessed for diabetes development . as shown in fig . 5a , compared with nontransgenic controls , the rate of type 1 diabetes development was somewhat retarded in nod.2m.hhd-pi female mice but did not quite achieve statistical significance ( p = 0.08 ) by kaplan - meier analyses . however , as assessed by analyses , the cumulative frequency of diabetes development by 35 weeks of age was significantly lower ( p < 0.005 ) in nod.2m.hhd-pi mice than in nontransgenic controls . insulitis levels were also significantly lower in nod.2m.hhd-pi mice than in nontransgenic controls ( fig . 5b ) . baseline and primed levels of cd8 t - cell responses to the hla - a2.1restricted ins1/2 a210 or ins1 b514 epitopes were found not to differ in nod.2m.hhd-pi mice and nontransgenic controls ( data not shown ) . these findings do not eliminate the possibility that like those targeting igrp epitopes , hla - a2.1restricted insulin autoreactive cd8 t - cells are also important pathogenic contributors to diabetes development in nod.2m.hhd mice . however , these apc transgenic expression studies indicate that even if they do not represent autoantigens recognized by pathogenic hla - a2.1restricted cd8 t - cells , ( pro)insulin derived epitopes are important targets of at least diabetogenic cd4 t - cells in nod.2m.hhd mice . insulitis and diabetes development in nod.2m.hhd-pi mice . a : incidence curve of diabetes development in nod.2m.hhd-pi female mice and nontransgenic littermates . the rate of diabetes development did not quite achieve a statistical difference ( p = 0.08 ) by kaplan - meier analyses . the cumulative frequency of diabetes development by 35 weeks of age was significantly different ( p < 0.005 ) as assessed by analyses . b : histological grading of insulitis in pancreas sections of 35-week - old female mice ( nod.2m.hhd-pi mice , n = 14 ; nod.2m.hhd mice , n = 16 ) . it has been previously reported that splenocytes bearing ecdi - coupled proteins or peptides do not directly induce tolerogenic responses by cd4 t - cells in an efficient manner , but rather do so indirectly following their uptake and processing by host - type apc ( 21 ) . if this is also the case for inducing cd8 t - cell tolerance , we reasoned it should remain possible to inhibit diabetes development in the hla - a2.1expressing nod.2m.hhd stock by treatments with splenocytes from totally mhc class i deficient nod.2 m mice bearing the complete cocktail of ecdi cross - linked ins and igrp epitopes ( designated ins / igrp-2 m sps ) . unlike those receiving flu-2 m sps , diabetes development was completely abrogated in ins / igrp-2 m sps - treated nod.2m.hhd mice ( fig . ins / igrp - sps do not have to share host - type mhc class i identity to attenuate diabetes development in nod.2m.hhd mice . beginning at 46 weeks of age nod.2m.hhd mice were injected intravenously at 5-week intervals with ins / igrp-2 m sps or flu-2 m sps . b : ins / igrp-2 m sps inhibit in vivo responses of hla - a2.1restricted igrp but not ins autoreactive cd8 t - cells in nod.2m.hhd mice . two days after the third treatment , mice were footpad primed with a mixture of the four ins / igrp peptides . ten days after priming , cd8 t - cells within the draining popliteal lymph nodes of nondiabetic mice were cocultured for 48 h with 1 mol / l of each of the individual ins or igrp peptides , and antigen reactivity was assessed by elispot analyses of ifn- production . two days after a third ins / igrp-2 m sps or flu-2 m sps treatment given at 5-week intervals , nod.2m.hhd mice were primed in the footpad with a cocktail of the four ins and igrp peptides . ten days postpriming , cd8 t - cells within the draining popliteal lymph nodes were assessed for reactivity to each individual peptide by ifn- elispot analyses . cd8 t - cell responses to both the hla - a2.1restricted igrp228236 and igrp265273 peptides but again not the ins epitopes were significantly decreased in ins / igrp-2 m sps - treated nod.2m.hhd mice ( fig . these collective results indicate that in order to induce cd8 t - cell tolerance and to elicit diabetes protective effects in nod.2m.hhd recipients , donor cells bearing ecdi cross - linked hla - a2.1 restricted igrp autoantigenic peptides do not also have to express the relevant host - type mhc class i variant . the results described above also indicated that rather than directing inducing tolerogenic responses inhibiting diabetogenic cd8 cell activity in nod.2m.hhd mice , ins / igrp - sps or igrp - sps instead do so in an indirect manner dependent on their uptake by host - type apc . to directly test this possibility , we compared the ability of ecdi - treated or untreated donor cells that were also labeled with the carboxyfluorescein diacetate succinimidyl ester ( cfse ) tracker dye to be taken up by host - type antigen - presenting dendritic cells ( dcs ) . donor- and recipient - type cells were also distinguished by respective expression of the cd45.2 versus cd45.1 pan - leukocyte marker variant . there was a significantly greater uptake by host - type splenic cd8 + dcs of ecdi - treated than untreated donor cells that had been intravenously infused 1 day earlier ( fig . this likely explains why fewer ecdi - treated than untreated donor cells could be detected in the recipients , but with the apoptotic status of the former also allowing them to be more efficiently engulfed by host - type dcs . these results indicate the ability of donor cells bearing ecdi - coupled igrp peptides to elicit diabetes protective effects likely entails their uptake by host - type dcs that then display the autoantigenic epitopes to cd8 t - cells in a tolerance - inducing manner . more efficient uptake by host - type splenic cd8 + dcs of ecdi - treated than untreated donor cells . splenocytes from nod.cd45.2 congenic mice were labeled with the cfse tracker dye and then subsequently treated or not with ecdi before being infused intravenously into standard nod cd45.1-expressing recipients . the following day , viable host - type splenic dcs were assessed by flow cytometry for comparative uptake of cfse - labeled ecdi - treated or untreated donor cells . a : depiction of gating strategy to assess uptake by viable ( propidium iodide negative ) host - type cd45.1-expressing splenic dc subsets of cfse - labeled donor - type leukocytes that had or had not been treated with ecdi . b : mean proportions sem of host - type splenic cd8 + dcs that had taken up cfse - labeled ecdi - treated or untreated donor - type leukocytes ( n = 3/group ) . our results indicate that treatment with ins / igrp - sps ( bearing an ecdi cross - linked mixture of the ins1/2 a210 , ins1 b515 , igrp228236 , and/or igrp265273 peptides ) inhibits diabetes development in humanized we found igrp - sps to be more effective than ins - sps in inhibiting diabetes development in nod.2m.hhd mice . furthermore , the inhibition of diabetes development in nod.2m.hhd mice by ins / igrp - sps treatment was associated with an attenuation of igrp but not ins - specific autoreactive cd8 t - cell responses . treatment with igrp228236 or igrp265273 single peptide - sps did not significantly inhibit diabetes development in nod.2m.hhd mice . this indicated that cd8 t - cell tolerance must be established to both the hla - a2.1restricted igrp228236 and igrp265273 epitopes in order to elicit diabetes - protective effects in nod.2m.hhd mice . in standard nod mice , insulin appears to be an earlier target than igrp of cd4 and cd8 t - cells initiating diabetes development ( 3133 ) . it is unclear why ins / igrp - sps or ins - sps treatment did not alter levels of hla - a2.1restricted cd8 responses against the ins1/2 a210 or ins1 b514 epitopes in nod.2m.hhd mice . however , nod.2m.hhd mice transgenically expressing proinsulin-2 in apcs were largely protected from insulitis and diabetes development . these apc transgenic studies also indicated that even if diabetes development in nod.2m.hhd mice does not require hla - a2.1restricted cd8 responses targeting ( pro)insulin epitopes , an important component of their disease susceptibility still entails cd4 t - cell responses against this pancreatic -cell antigen . it has been previously reported that although peptide - sps can directly induce cd4 t - cell tolerance induction processes in an inefficient manner , they do so more efficiently through an indirect mechanism involving their uptake and processing by host - type apcs ( 25 ) . because of these alternative mechanisms , mhc compatibility between splenotype donor and host is not required in order to induce cd4 t - cell tolerance to ecdi - coupled antigens , although syngeneic donor cells are more efficient at doing so . we found ins / igrp peptides ecdi coupled to completely mhc class i deficient donor splenocytes strongly inhibited igrp - specific cd8 t - cell responses and diabetes development in nod.2m.hhd mice . hence , donor / host mhc class i compatibility is not required to efficiently induce cd8 t - cell tolerance to ecdi - coupled self - antigenic peptides . these findings indicate that igrp - sps inhibit diabetes development in nod.2m.hhd mice by inducing cd8 t - cell tolerance through an indirect host - type apc - dependent pathway . indeed , other data indicate that host - type dcs more efficiently take up donor ecdi - treated than untreated leukocytes . a review by luo et al . ( 21 ) discusses efforts by the immune tolerance network to develop a clinical trial using ecdi insulin - coupled peripheral blood lymphocytes as a possible diabetes intervention in humans . our current results indicate that the use of humanized nod.2m.hhd mice and other related strains may facilitate the development of clinically translatable peptide - based therapies for diabetic patients . humanized mouse resources make it possible to determine which autoantigenic peptides when ecdi cross - linked to autologous leukocytes are most likely to attenuate hla - a2.1restricted cd8 t - cell responses that recent evidence ( 1016 ) indicates may be important for diabetes development in many human patients .

objectivetype 1 diabetes can be inhibited in standard nod mice by autoantigen - specific immunotherapy targeting pathogenic cd8 + t - cells . nod.2mnull.hhd mice expressing human hla - a2.1 but lacking murine major histocompatibility complex class i molecules develop diabetes characterized by cd8 t - cells recognizing certain autoantigenic peptides also targeted in human patients . these include peptides derived from the pancreatic -cell proteins insulin ( ins1/2 a210 and ins1 b514 ) and islet - specific glucose-6-phosphatase catalytic subunit related protein ( igrp265273 and igrp228236 ) . hence , nod.2mnull.hhd mice represent a model system for developing potentially clinically translatable interventions for suppressing diabetogenic hla - a2.1restricted t - cell responses.research design and methodsstarting at 46 weeks of age , nod.2mnull.hhd female mice were injected intravenously with syngeneic splenocytes to which various admixtures of the four above - mentioned peptides were bound by the cross - linking agent ethylene carbodiimide ( ecdi).resultstreatment with such cells bearing the complete cocktail of ins and igrp epitopes ( designated ins / igrp - sps ) significantly inhibited diabetes development in nod.2mnull.hhd recipients compared with controls receiving splenocytes coupled with an irrelevant hla - a2.1restricted flu16 peptide . subsequent analyses found syngeneic splenocytes bearing the combination of the two ecdi - coupled igrps but not ins peptides ( igrp - sps or ins - sps ) effectively inhibited diabetes development in nod.2mnull.hhd mice . this result was supported by enzyme - linked immunospot ( elispot ) analyses indicating combined ins / igrp - sps diminished hla - a2.1restricted igrp but not ins autoreactive cd8 + t - cell responses in nod.2mnull.hhd mice.conclusionsthese data support the potential of a cell therapy approach targeting hla - a2.1restricted igrp autoreactive cd8 t - cells as a diabetes intervention approach in appropriate human patients .

RESEARCH DESIGN AND METHODS Mice. Peptides and antibodies. ECDI peptide-coupled cell treatment. Assessment of diabetes and insulitis development. Enzyme-linked immunospot assays, CD8 T-cell culture, and in vivo T-cell priming. In vitro assessment of peptide-SP effects on diabetogenic CD8 T-cells. Soluble peptide treatment. Statistical analyses. RESULTS Splenocytes bearing ECDI-coupled peptides ablate IGRP-specific CD8 T-cell responses in vitro. Syngeneic splenocytes bearing ECDI-coupled autoantigenic IGRP, but not INS peptides, inhibit diabetes development in NOD. INS/IGRP-SPs and IGRP-SPs inhibit in vivo responses of HLA-A2.1restricted IGRP but not INS autoreactive CD8 T-cells in NOD. Proinsulin is a pathogenic autoantigen in NOD. INS/IGRP-SPs do not have to share host MHC class I identity to tolerize IGRP-specific CD8 T-cells and to attenuate diabetes development in NOD. DISCUSSION

a long segmental bone defects repair is one of the challenging problems in orthopaedic surgery . although allogenic bone grafts are a current major option [ 13 ] , this technique is associated with problems of significant failure rates , poor mechanical properties , and immunological rejection . porous materials are of significant importance for bone tissue engineering applications because of the good biological fixation to surrounding tissue through bone tissue . porous titanium and titanium alloys have been investigated as they provide favourable mechanical properties with an elastic modulus closed to that of natural bone under a load bearing condition . surface characteristics of porous titanium are important determinants in its scaffold properties since the surface condition of titanium has been reported to play a critical role in bone formation associated with superior osteoblast adhesion and subsequent cell behaviors [ 610 ] . recent studies have raised a concern that degradation of biological ability with increase in adsorption of organic impurities on titanium - based biomaterials [ 6 , 7 ] . this reduces hydrophilicity , adsorption of cell - binding proteins , and subsequent cell functions . titanium - based biomaterials therefore need to be fabricated as clean surface without particular contamination such as titanium carbide . there are a number of approaches in fabrication of porous titanium and titanium alloys such as sintering loose titanium powder or fibers , slurry sintering , and also rapid prototyping [ 3 , 4 , 11 , 12 ] . initial surface contamination of sintered porous titanium would be unavoidable event as this is generally processed within the carbon graphite molds under a high thermal pressure [ 3 , 4 ] . this study aimed to develop a contamination - free porous titanium scaffold by a plasma - activated sintering within an originally developed tin - coated graphite molds . the surfaces of porous titanium sheets with or without a coated graphite mold were characterized . the cell adhesion property of the porous titanium sheet was also evaluated in this study . jis grade ii titanium ( ks-50 , kobe steel , tokyo , japan ) block was used as starting material . narrow titanium fibers were made by being turned with diameter approximetly 0.4 - 0.5 mm . the surface of graphite molds were coated with thickness of 1.0 mm tin by presintering under vacuum with a condition of 1 mpa predisplacement and was processed by plasma - activated sintering under a vacuum at pressure of 20 mpa at 3800 a for 15 sec . elemental titanium fibers were filled in the 30 mm 1.0 mm graphite mold with or without a tin coating . the titanium fibers were subjected to the predisplacement with 1 mpa pressure in the graphite molds . the porous titanium sheet 30 mm 0.5 mm was processed by plasma - activated sintering under a vacuum at pressure of 20 mpa , at 3800 a for 15 sec . the surface topographies of the coatings on the specimens were then observed by sem ( s-2360n , hitachi , tokyo , japan ) . the crystalline phases of the titanium samples before the tests were detected by tf - xrd ( xrd-6100 , shimadzu , kyoto , japan ) with cuk radiation . the xrd was operated at 40 kv and 40 ma with a scanning speed of 0.02/4 s and a scanning range of 2060. 10 10 0.5 mm polished titanium plates and titanium sheets were subjected to cell culture . an osteoblastic cell line , mc3t3-e1 , was obtained from the riken cell bank ( tsukuba , japan ) . cells were cultured in minimal essential medium ( gibco ) containing 10% fetal bovine serum ( gibco ) and 1% antibiotic ( penicillin , gibco ) under a 5% co2 atmosphere at 37c . cells were suspended in the medium at 1 10 cells / ml and used for experiments . a 1-ml quantity of floating cells was cultured onto titanium samples at 37c under 5% co2 for 1 d. a cell - counting kit ( dojindo , kumamoto , japan ) was used for the measurement of cell adhesion . after incubation , each specimen was moved to another well and washed 3 times with pbs ( gibco ) to remove nonadherent cells . adherent cells were mixed with 1 ml of medium and 100 l of reagent solution . after 1 hr of incubation , the absorbance at 450 nm was measured . the number of adherent cells was calculated from the activity of the original cell suspension . adherent cells on each specimen after 1 hr of cultivation were dehydrated after being washed with pbs . the cells were fixed with 3.7% formaldehyde in pbs and permeabilized by treatment with 0.1% triton x-100 ( sigma , tokyo , japan ) in pbs for 1 min . the cells were then incubated for 3 hrs in a rhodamine - conjugated phalloidin solution . after the cells were washed with water , stress fiber formation and cell morphology were observed with the use of a fluorescence microscope ( e-600 , nikon , tokyo , japan ) . the appropriateness of the hypothesis of homogeneous variances was investigated by means of bartlett 's test . as shown in figure 1 , the porous titanium sheet was structured under the high thermal pressure . the titanium sheet processed with tin coating was observed to be clean whereas a particular contamination was observed on the titanium sheet processed without tin coating . the xrd analysis revealed that the titanium sheet prepared without tin coated graphite mold showed distinctive tic peaks while the sheet with coated graphite mold showed tic peak ( figure 2 ) . the peaks attributable to rutile tio2 were detectable on the titanium sheet prepared with tin coated graphite mold . the peaks attributable to tio2 were not detectable on titanium sheet processed without tin coating . the number of adherent cells on titanium sheet processed with tin coating was significantly ( p = .002 ) higher than that without tin coating after 1 d ( figure 3 ) . adherent cells on the titanium sheet with tin coating had begun to show stress fibers and widely extend , while the stress fiber formation and cell extension on titanium sheet without tin coating were not distinctive ( figure 4 ) . the plasma - activated sintering is a rapid sintering method associated with self - heating phenomena within the powder . this is capable of sintering metal or ceramic powders rapidly to its full density at a relatively lower temperature compared to the conventional furnace sintering methods . the carbon graphite mold has been employed in the plasma - activated sintering due to its electroconductive property and thermostability [ 3 , 4 , 11 , 13 ] . the direct heating of graphite mold and the large spark pulse current provide a very high thermal efficiency . the peak of tic was detected on the titanium sheet processed with graphite mold without tin coating . since the titanium fiber elements were directly in contact with the carbon graphite mold during processing ; a particular contamination such as tic is unavoidable event in this condition . alternatively , the tic peak was not detectable on the titanium sheet processed within the tin - coated carbon graphite mold . this modified plasma - activated sintering with the tin - coated graphite mold would be useful to fabricate the contamination - free titanium sheet . recent study suggested that adsorption of organic impurities on titanium surface are responsible for reducing the initial cells adhesion , subsequent proliferation002c and differentiation [ 68 ] . amount of carbon absorbed on the titanium sheet seems to be an important part in determining the initial affinity level for osteoblasts and new bone formation . the present study demonstrated that number of adherent cells on the modified titanium sheet was much greater than that of bare titanium plate . additionally , stress fiber formation and the extension of the cells were observed on the titanium sheets . the initial adhesion of cells induces stress fiber formation , phosphorylation of focal adhesion kinase , and activation of other intracellular signal transduction molecules thereby affecting cell proliferation , differentiation and new bone formation . thus , the contamination - free surface of modified titanium would be useful for new bone generation at a segmental bone defect in comparison with the unmodified sintered porous titanium . in conclusion , the tin - coated carbon graphite mold is a new method for processing a contamination - free porous titanium sheet . this modified titanium sheet is expected to be a new tissue engineering material in orthopedic bone repair .

this study aimed to develop a contamination - free porous titanium scaffold by a plasma - activated sintering within an originally developed tin - coated graphite mold . the surface of porous titanium sheet with or without a coated graphite mold was characterized . the cell adhesion property of porous titanium sheet was also evaluated in this study . the peak of tic was detected on the titanium sheet processed with the graphite mold without a tin coating . since the titanium fiber elements were directly in contact with the carbon graphite mold during processing , surface contamination was unavoidable event in this condition . the tic peak was not detectable on the titanium sheet processed within the tin - coated carbon graphite mold . this modified plasma - activated sintering with the tin - coated graphite mold would be useful to fabricate a contamination - free titanium sheet . the number of adherent cells on the modified titanium sheet was greater than that of the bare titanium plate . stress fiber formation and the extension of the cells were observed on the titanium sheets . this modified titanium sheet is expected to be a new tissue engineering material in orthopedic bone repair .

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

women are among the most important foundations of the family and society ; and the health of the community depends to a large extent , on meeting the needs of this group . menopause is the cessation of menstruation resulting from loss of ovarian follicular function , which is a recognized physiological event in women 's lives . in addition to hormonal changes particularly reduction of estrogen levels with biological , psychological and social changes , a woman who has not experienced menstrual period within at least 12 months retrospectively is deemed to have entered the phase of menopause and the postmenopausal period starts from this moment . depending on the disorder of ovarian function and therefore , lack of estrogen in the postmenopausal period , symptoms such as hot flashes , irritability , sleeping disorders , fatigue , anxiety and loss of concentration are observed in the early period . risk of coronary artery disease and incidence rate of osteoporosis increase due to loss of protective effects of estrogen in the later period . if we assume menopause age as beginning at 50 , women spend one - third of their lives with the pathological changes they are exposed to in the postmenopausal period . earlier , the world health organization had reported that by 2003 there would be 1.2 billion women at and over age of 50 . therefore , although the menopause seems like a natural process , it is a period that must definitely be followed and treated . one of the aims of health for all in the 21st century is the improvement of the quality of life . as a physiological event , menopause has complications that influence women 's quality of life . several measures such as hormone therapy and non - pharmacological therapies are recommended to reduce menopausal symptoms and improve quality of life in this period . hormonal replacement treatment ( hrt ) is used as a standard therapy in menopausal treatment . the effect of exercise in preventing the postmenopausal symptoms as another aspect of treatment is widely accepted today . postmenopausal symptoms can be prevented significantly by encouraging the women over middle age to gain the habit of exercising regularly . prior research has showed that postmenopausal women living in cities are more sedentary . due to unbalanced and single type nourishment , weight gain is a common phenomenon in those living in rural areas . the weight increase , which depends on age , inactivity and hormone drugs used in menopause , is minimized by regular exercise and the burning of excess calories . in the literature , there are studies that research the effects of exercise on muscle strength , bone density and on the postmenopausal symptoms in the women in postmenopausal period . in addition , the studies in which the effects of exercises on quality of life is researched , is insufficient . in accordance , the objective of our study was to conduct an exercise program of 6 weeks and determine the changes on physical fitness levels and quality of life on postmenopausal women . our study is aimed to determine the effects of exercise on quality of life with a controlled exercise program of 8 weeks appropriate to the postmenopausal period . the inclusion criteria of this study were : to have entered into the menopause naturally ( subjects stated that they had no operation during their lives)aged 45 - 65 old , to have been taking hrt for at least 1 year ( subjects received combined continuous 0.62 mg per day conjugated estrogen ( ce ) + 2.5 mg per day medroxy - progesterone acetate ( map)to have no health problems that may prevent from doing exercise and to have no high cardiac risk to have entered into the menopause naturally ( subjects stated that they had no operation during their lives ) aged 45 - 65 old , to have been taking hrt for at least 1 year ( subjects received combined continuous 0.62 mg per day conjugated estrogen ( ce ) + 2.5 mg per day medroxy - progesterone acetate ( map ) to have no health problems that may prevent from doing exercise and to have no high cardiac risk exclusion criteria of this study were : postmenopausal women who exercise its check list was incomplete . information about this study was provided to the subjects . before being accepted into the study , it was explained to them that , if they preferred not to continue , they could withdraw from the study any time . postmenopausal women were randomly allocated by means of a random number table to one of two groups . forty women in the first group were included into the 8-week aerobic exercise program and 40 women in the second group formed the non - exercise control group . the women in second group continued to take hrt and were not included in any exercise program . six different sub - sections of nph included different numbers of questions with different scoring ( physical mobility , pain , sleep , energy , social isolation and emotional reactions ) . each sub - section 's maximum cumulative score total should be 100 ( worst health ) and minimum 0 ( best health ) . the subjects were included in the exercise program for 8-week period with the frequency of 5 times a week . the program started with warming - up exercises consisting of stretching exercises and the 5-min low - intensity walking ; warming - up exercises were followed by a program starting with 20 min and increasing up to 40 min at the end of 8 weeks . this program included the exercise detailed below : bicycle for lower extremity endurance training for 5 to 15 min ; exercise on the step board for lower extremity endurance for 2 to 6 min ; posture exercises across a mirror for neck and back muscles ( 10 times ) ; strengthening exercises with isoflex bands ; free weights for back and abdominal muscles , pectoral , scapular muscles , and for upper - lower extremity muscles ; balance exercise with trampoline for balance training ; and flexibility exercise for pectoral , hamstring , gastro - cnemius - soleus , and back muscles . the exercise program was completed with cooling exercises consisting of 5-min low - intensity walk on the treadmill . the exercise program with sub - maximal intensity was applied to the cases and a borg scale was used in determining the intensity of the exercise . exercise intensity was regulated by progressively increasing the repeating number , duration and endurance amounts of the exercises with the borg scale follow - up . a paired sample t - test was used to compare the differences between before and after exercise at the same group and an independent sample t - test was used to compare the differences between the exercise and the control groups . anova test was used for more than 2 groups and chi - square test respectively for continuous and categorical data . for all analyses , postmenopausal women were randomly allocated by means of a random number table to one of two groups . forty women in the first group were included into the 8-week aerobic exercise program and 40 women in the second group formed the non - exercise control group . the women in second group continued to take hrt and were not included in any exercise program . six different sub - sections of nph included different numbers of questions with different scoring ( physical mobility , pain , sleep , energy , social isolation and emotional reactions ) . each sub - section 's maximum cumulative score total should be 100 ( worst health ) and minimum 0 ( best health ) . the subjects were included in the exercise program for 8-week period with the frequency of 5 times a week . the program started with warming - up exercises consisting of stretching exercises and the 5-min low - intensity walking ; warming - up exercises were followed by a program starting with 20 min and increasing up to 40 min at the end of 8 weeks . this program included the exercise detailed below : bicycle for lower extremity endurance training for 5 to 15 min ; exercise on the step board for lower extremity endurance for 2 to 6 min ; posture exercises across a mirror for neck and back muscles ( 10 times ) ; strengthening exercises with isoflex bands ; free weights for back and abdominal muscles , pectoral , scapular muscles , and for upper - lower extremity muscles ; balance exercise with trampoline for balance training ; and flexibility exercise for pectoral , hamstring , gastro - cnemius - soleus , and back muscles . the exercise program was completed with cooling exercises consisting of 5-min low - intensity walk on the treadmill . the exercise program with sub - maximal intensity was applied to the cases and a borg scale was used in determining the intensity of the exercise . exercise intensity was regulated by progressively increasing the repeating number , duration and endurance amounts of the exercises with the borg scale follow - up . a paired sample t - test was used to compare the differences between before and after exercise at the same group and an independent sample t - test was used to compare the differences between the exercise and the control groups . anova test was used for more than 2 groups and chi - square test respectively for continuous and categorical data . for all analyses , when these two groups were evaluated according to their physical characteristics , it was observed that they were homogeneous . as shown in table 1 , the average age of the subjects was 54.05 4.78 years with a range of 46 - 61 . furthermore , the number of years that they were menopausal , and the number of years that they were under hrt therapy was questioned as menopausal information and the answers were recorded . it was observed that these two groups were similar also in terms of menopausal characteristics . baseline demographic characteristics of subject as shown in table 2 , there was no statistically significant difference according to nhp indicating an improvement in the quality of life before exercise . mean quality of life scores in the exercise and control groups before exercise results from the survey [ see table 3 ] in the exercise group show significant decreases in the scales of all the items indicating that the quality of life had been improved according to six sub - items of the nhp . no significant changes were found in the quality of life in the control group at the end of 8 weeks . at the end of the 8 weeks , there was a statistically significant difference in the exercise group according to nhp indicating an improvement in the quality of life . menopause is a natural process that is seen in an average age of 50 , and women spend almost one - third of their lives in the postmenopausal period . the quality of life worsens in the postmenopausal period as a result of the physical , psychological and social problems that start with menopause . it is emphasized in many studies that in order to achieve the best results from hrt , this therapy should be combined with an exercise program . there are many studies indicating the negative effects of postmenopausal symptoms on quality of life . luis cobero , the president of spain gynecology and obstetrics association , had explained that the life qualities of postmenopausal women improve not only with hrt , but also with modifications in their diets , modifications in lifestyles , and regular physical activity . gzeloglu applied nhp in order to establish the effects of osteoporosis in 50 postmenopausal women and reported that quality of life impairment increased with the duration of postmenopausal period . gzeloglu emphasized that the reliability of nhp scale was rather high , and it could be used to evaluate the life quality of postmenopausal women . one intervention study concluded that fitness level and quality of life of postmenopausal women could be improved by a regular controlled exercise program of six weeks . elavsky found that physical activity and menopausal symptoms were related to physical self - worth and positive effect , and in turn , greater levels of physical self - worth and positive effect were associated with higher levels of menopause - related quality of life . it was found that life quality improved for all the sub - items of nhp in exercise group . we concluded that quality of life could be improved with regular and controlled exercise of 8 weeks in postmenopausal women . when it is considered that our cases were not geriatric , our study has the properties of a protective treatment for these cases . the long - term effects of exercise on quality of life of our cases can be evaluated provided that the habit of exercising can be achieved by these individuals as a lifestyle . studies that contain the data from this same group in subsequent years will be necessary for such an evaluation .

background : one of the most critical stages of women 's lives is menopause and one of the aims of health for all in the 21st century is the improvement of the quality of life.aim and objective : the aim of this study was to determine the effect of exercise on quality of life in postmenopausal women.materials and methods : this study was designed by a randomized - controlled trial . eighty volunteer postmenopausal women who experienced the menopause period naturally and have been taking hormone replacement treatment ( hrt ) were divided into two groups randomly ( exercise group n = 40 , control group n = 40 ) . the nottingham health profile ( nhp ) was used to assess quality of life in both groups before and after 8 weeks . the exercise group participated in an exercise program , which was composed of sub - maximal aerobic exercises for an 8-week period 5 times a week . quality of life in two groups was compared at the end of 8 weeks.results:the results showed that there was a statistically significant difference in the exercise group for the nhp indicating an improvement in the quality of life ( p < 0.05).conclusion : we concluded that quality of life on postmenopausal women could be improved with a regular and controlled exercise program of 8 weeks . thus , implementing appropriate educational programs to promote the quality of life in postmenopausal women is recommended .

INTRODUCTION MATERIALS AND METHODS Design Statistical analysis RESULTS DISCUSSIONS CONCLUSION

the following active materials were employed in this work : li4ti5o12 ( d50 1.9 m ) from mti corporation , licoo2 ( 710 m ) from sigma aldrich , and anatase tio2 ( < 10 nm ) from sachtleben chemie . all components of the electrolyte , namely ethylene carbonate and dimethyl carbonate and lipf6 , and metallic lithium were purchased from sigma aldrich ( battery grade ) . the oxygen - annealed li4ti5o12 samples ( o_lto ) were obtained by treatment commercially available li4ti5o12 ( p_lto ) in flowing o2 ( 100 vol % ) at 650 c for 120 min . the o2-annealed sample ( o_lto ) was then treated in flowing h2 ( 5 vol % in ar ) at 650 c for 90 min to obtain hydrogen - annealed li4ti5o12 ( h_lto ) . the bet surface areas were determined by nitrogen physisorption using a tristar ii 3020 from micromeritics . high resolution transmission electron microscopy ( hrtem ) analyses were carried out with a field emission gun microscope fei tecnai f20 , working at 200 kv and with a point - to - point resolution of 0.19 nm . electron energy loss spectroscopy ( eels ) spectra were obtained in a gatan quantum detector coupled to the f20 microscope . x - ray power diffraction ( xrd ) analyses were carried out on a bruker axs d8 advance x - ray diffractometer with cuk1 radiation ( =1.5406 ) . the uv / vis spectra were recorded in diffuse reflectance mode in a perkinelmer lambda 950 uv / vis spectrometer equipped with a 150 mm int . the chemical composition of the surface of the lto was analyzed by x - ray photoelectron spectroscopy ( xps ) using a phi instrument model 5773 multitechnique with a1k radiation ( 1486.6 ev ) . the fluid electrodes were prepared by mixing the active materials and carbon additive in the electrolyte ( ec : dmc in 1 m lipf6 ) by magnetic stirring for overnight . the fluid electrode was flowed through the cell for , at least , 1 h before starting the experiments to further homogenize the suspension . for the suspensions , 2.5 g and 0.125 g of active and conductive additive , respectively , were added into 12 ml for tio2 and licoo2 and into 6 ml for lto . the poor electrical conductivity and low surface area of lto forced us to increase the concentration of particles in the suspension to obtain decent performances . for solid electrodes , a slurry containing 75:15:10 wt % of active material , conductive additive and poly(vinylidene difluoride ) ( pvdf ) binder ( sigma aldrich ) was prepared by dispersing in n - methyl pyrrolidone ( nmp ) ( sigma aldrich ) and mixing for overnight using magnetic stirring . the slurry was doctor - bladed onto copper foil ( schlenk ) and dried at 60 c for 10 h in air , resulting in an active material loading of about 3 mg cm . 10 mm disk electrodes were punched out using a commercially available hole punch ( hoffmann ) and dried overnight at 80 c in a vacuum oven the electrochemical cells for the characterization of both solid and fluid electrode were two - electrode configuration and were assembled in an ar - filled glove box ( o2<1 ppm and h2o<1 ppm ) . celgard 2500 and ec : dmc in 1 m lipf6 served as separators and electrolyte , respectively . the electrochemical cell employed for testing fluid electrodes the dimensions of the channel were 7541 mm and the current collector was metallic copper . electrochemical measurements were performed with a bio - logic vmp-3 ( bio logic sas ) . films containing 90 % of lto and 10 % of poly(vinylidene difluoride ) ( pvdf ; binder ) were prepared on copper foil by the doctor blade technique . 45 micrometer , which was measured with a digital external micrometer ( holex 421505 ) . discs of 12 mm diameter were cut and the electrical conductivity was estimated by 2 probe measurement using a multimeter ( uni - t ut70a ) . the measurements for electrical conductivity were carried out using the same cell employed for the electrochemical evaluation of fluid electrode , with the exception of the separators and the metallic lithium . the electrical resistivity of the suspension was then estimated with multimeter ( silver electronics ut33d ) knowing the section and the length of the cell ( 3 cm1 mm ) . note that the objective of these measurements was to compare the electrical conductivities of the three suspension ( p_lto , o_lto and h_lto ) , rather than obtaining the exact values . to avoid changing the cell configuration factor for the different samples , the cell was not disassembled between the three experiments but it was vigorously cleaned with pure dmc . the solid electrode prepared for the measurement of the electrical conductivity of the active material ( 90:10 lto / binder ) were also employed for eis . a typical three - electrode swagelok cell using metallic lithium as counter and reference electrode was used . whatman and ec / the cell was assembled in an argon - filled glove - box , then taken outside the glove - box and tested with a bio - logic vmp-3 ( bio logic sas ) . the state of charge was tuned galvanostatically at 0.1 c. the eis were performed in a frequency range of 100 khz0.020 hz with an amplitude of 20 mv . the following active materials were employed in this work : li4ti5o12 ( d50 1.9 m ) from mti corporation , licoo2 ( 710 m ) from sigma aldrich , and anatase tio2 ( < 10 nm ) from sachtleben chemie . all components of the electrolyte , namely ethylene carbonate and dimethyl carbonate and lipf6 , and metallic lithium were purchased from sigma aldrich ( battery grade ) . the oxygen - annealed li4ti5o12 samples ( o_lto ) were obtained by treatment commercially available li4ti5o12 ( p_lto ) in flowing o2 ( 100 vol % ) at 650 c for 120 min . the o2-annealed sample ( o_lto ) was then treated in flowing h2 ( 5 vol % in ar ) at 650 c for 90 min to obtain hydrogen - annealed li4ti5o12 ( h_lto ) . the bet surface areas were determined by nitrogen physisorption using a tristar ii 3020 from micromeritics . high resolution transmission electron microscopy ( hrtem ) analyses were carried out with a field emission gun microscope fei tecnai f20 , working at 200 kv and with a point - to - point resolution of 0.19 nm . electron energy loss spectroscopy ( eels ) spectra were obtained in a gatan quantum detector coupled to the f20 microscope . x - ray power diffraction ( xrd ) analyses were carried out on a bruker axs d8 advance x - ray diffractometer with cuk1 radiation ( =1.5406 ) . the uv / vis spectra were recorded in diffuse reflectance mode in a perkinelmer lambda 950 uv / vis spectrometer equipped with a 150 mm int . the chemical composition of the surface of the lto was analyzed by x - ray photoelectron spectroscopy ( xps ) using a phi instrument model 5773 multitechnique with a1k radiation ( 1486.6 ev ) . the fluid electrodes were prepared by mixing the active materials and carbon additive in the electrolyte ( ec : dmc in 1 m lipf6 ) by magnetic stirring for overnight . the fluid electrode was flowed through the cell for , at least , 1 h before starting the experiments to further homogenize the suspension . the reservoirs were constantly stirred during the electrochemical characterization . for the suspensions , 2.5 g and 0.125 g of active and conductive additive , respectively , were added into 12 ml for tio2 and licoo2 and into 6 ml for lto . the poor electrical conductivity and low surface area of lto forced us to increase the concentration of particles in the suspension to obtain decent performances . for solid electrodes , a slurry containing 75:15:10 wt % of active material , conductive additive and poly(vinylidene difluoride ) ( pvdf ) binder ( sigma aldrich ) was prepared by dispersing in n - methyl pyrrolidone ( nmp ) ( sigma aldrich ) and mixing for overnight using magnetic stirring . the slurry was doctor - bladed onto copper foil ( schlenk ) and dried at 60 c for 10 h in air , resulting in an active material loading of about 3 mg cm . 10 mm disk electrodes were punched out using a commercially available hole punch ( hoffmann ) and dried overnight at 80 c in a vacuum oven the electrochemical cells for the characterization of both solid and fluid electrode were two - electrode configuration and were assembled in an ar - filled glove box ( o2<1 ppm and h2o<1 ppm ) . celgard 2500 and ec : dmc in 1 m lipf6 served as separators and electrolyte , respectively . the electrochemical cell employed for testing fluid electrodes the dimensions of the channel were 7541 mm and the current collector was metallic copper . electrochemical measurements were performed with a bio - logic vmp-3 ( bio logic sas ) . films containing 90 % of lto and 10 % of poly(vinylidene difluoride ) ( pvdf ; binder ) were prepared on copper foil by the doctor blade technique . 45 micrometer , which was measured with a digital external micrometer ( holex 421505 ) . discs of 12 mm diameter were cut and the electrical conductivity was estimated by 2 probe measurement using a multimeter ( uni - t ut70a ) . the measurements for electrical conductivity were carried out using the same cell employed for the electrochemical evaluation of fluid electrode , with the exception of the separators and the metallic lithium . the electrical resistivity of the suspension was then estimated with multimeter ( silver electronics ut33d ) knowing the section and the length of the cell ( 3 cm1 mm ) . note that the objective of these measurements was to compare the electrical conductivities of the three suspension ( p_lto , o_lto and h_lto ) , rather than obtaining the exact values . to avoid changing the cell configuration factor for the different samples , the cell was not disassembled between the three experiments but it was vigorously cleaned with pure dmc . the solid electrode prepared for the measurement of the electrical conductivity of the active material ( 90:10 lto / binder ) were also employed for eis . a typical three - electrode swagelok cell using metallic lithium as counter and reference electrode was used . whatman and ec / the cell was assembled in an argon - filled glove - box , then taken outside the glove - box and tested with a bio - logic vmp-3 ( bio logic sas ) . the state of charge was tuned galvanostatically at 0.1 c. the eis were performed in a frequency range of 100 khz0.020 hz with an amplitude of 20 mv . as a service to our authors and readers , this journal provides supporting information supplied by the authors . such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors

the semi - solid flow battery ( ssfb ) is a promising storage energy technology featured by employing semi - solid fluid electrodes containing conductive additive and active li - ion battery materials . the state of art anode material for ssfb is li4ti5o12 ( lto ) . this work shows that lto improves drastically the performance in fluid electrode via hydrogen annealing manifesting the importance of the electrical conductivity of the active material in ssfbs . on the other hand , the properties of fluid electrodes allow the contributions of ionic and electrical resistance to be separated in operando . the asymmetric overpotential observed in li4ti5o12 and tio2 is proposed to originate from the so - called electron bottleneck mechanism based on the transformation from electrically insulator to conductor upon ( de-)lithiation , or vice versa , which should be considered when modelling , evaluating or designing advanced materials based on li4ti5o12 , tio2 or others with insulating - conducting behavior materials .

Experimental Section Materials and thermal treatment Structural characterization Suspension preparation Electrochemical characterization Electrical conductivity of active material Electrical conductivity test of suspensions Electrochemical impedance spectroscopy (EIS) Supporting Information

the animal ethics committee and animal care and use committee of the national institute of animal health approved the study . five holstein calves 23 months of age were intracerebrally injected with 1 ml of 10% ( w / v ) brain homogenates prepared from the medulla oblongata of bse / jp24 . in our earlier report , experimentally challenged cattle appeared to display clinical signs indicative of bse at 11 months postinoculation ( mpi ) ( 9 ) . animals were sequentially euthanized before and after the onset of clinical signs ( cattle identification codes 8515 and 496 at 10 and 12 mpi , respectively ) and at the terminal stage of the disease ( cattle identification codes 528 , 1061 , and 5566 at 16 mpi ) . we provisionally categorized the adrenal gland as nerve tissue because of the presence of chromaffin cells in the medulla of the gland . western blot analysis for prpres was performed on obex tissue samples as described previously by using anti - prp monoclonal antibody t2 ( 9 ) . prpres was detectable in all obex samples obtained 10 , 12 , and 16 mpi , suggesting that transmission of l - type bse prions to these animals was successful . dilution of the protease - treated brain sample and analysis of western blot results showed that the detection threshold for prpres was 1.25 g of brain tissue equivalent ( data not shown ) . a variety of nerve and lymphoid tissue samples were investigated for accumulation of prpres by western blot analysis by using phosphotungstic acid precipitation , as described previously ( 10 ) ; examples of cattle tissue samples obtained 10 and 16 months mpi ( codes 8515 and 1061 , respectively ) are shown in figure 1 . in cattle at the preclinical stage , prpres was detectable in all tested ganglia and barely detectable in the vagus nerve and vagosympathic trunk . in cattle at the terminal stage , prpres was barely detectable in the forelimb nerves ( suprascapular nerve , brachial nerve plexus , median nerve , and radial nerve ) , whereas substantial amounts of prpres were present in other nerve tissues except for facial and hypoglossal nerves ( table ) . a broader nerve tissue distribution of prpres was observed in cattle at 16 mpi than at 10 and 12 mpi . contrary to what we found in nerve tissues , we detected no prpres from tests performed on lymphoid tissues obtained from any of the 5 cattle studied . western blot analysis of a protease - resistant form ( prpres ) of a normal cellular prion protein in nerve tissue samples obtained from cattle 10 ( a ) and 16 ( b ) months postinoculation ( cattle identification codes 8515 and 1061 , respectively ) . the nerve tissues tested are shown above the lanes : 1 , trigeminal ganglia ; 2 , pituitary gland ; 3 , anterior cervical ganglion ; 4 , facial nerve ; 5 , hypoglossal nerve ; 6 , cranial mesenteric ganglia ; 7 , vagus nerve ( cervical part ) ; 8 , stellate ganglia ; 9 , adrenal gland ; 10 , phrenic nerve ; 11 , vagus nerve ( pectoral part ) ; 12 , vagosympathic trunk ( pectoral part ) ; 13 , vagosympathetic trunk ( lumbar part ) ; 14 , accessory nerve ; 15 , suprascapular nerve ; 16 , brachial nerve plexus ; 17 , median nerve ; 18 , radial nerve ; 19 , sciatic nerve ; 20 , tibial nerve , 21 , middle cervical ganglion . molecular mass standards ( kda ) are indicated on the left of each panel . * prpres , prion protein resistant ; bse , bovine spongiform encephalopathy ; mpi , months postinoculation ; + , positive for prpres ; , negative for prpres . infectivity of selected nerve tissues ( including the obex , sciatic nerve , adrenal gland , brachial nerve plexus , and vagus nerve ) obtained from cattle euthanized at 10 , 12 , and 16 mpi ( codes 8515 , 498 , and 5566 , respectively ) was analyzed by intracerebral injection into mice transgenic for bovine prion protein , as described previously ( 11 ) . as a negative control , mice were injected with cells from the brainstem of a normal cow . the presence of prpres in the brains of all mice used in the experiment was determined by western blot analysis . infectivity was detected in all nerve tissues tested , regardless of the presence of detectable prpres ( figure 2 ) . inocula from selected tissues obex ( a ) , sciatic nerve ( b ) , adrenal gland ( c ) , branchial nerve plexus ( d ) , and vagus nerve cervical part ( e)were prepared from cattle euthanized at 10 ( code 8515 , circle ) , 12 ( code 498 , square ) , and 16 ( code 5566 , triangle ) months postinoculation were and inoculated intracerebrally into mice transgenic for bovine prion protein . ratios above graph indicate number of prion - diseased mice / number of inoculated mice at 500 d postinoculation . we report accumulation of l - type atypical bse prions in peripheral nerve tissues sampled from intracerebrally challenged cattle . our study demonstrated that almost all of the peripheral nerve tissues tested became prpres positive in a time - dependent manner , whereas no prpres was detectable in lymphoid tissues , even in cattle with fatal atypical bse . our results suggest the possibility that , like classical bse prions , l - type bse prions propagated in the central nervous system and were spread centrifugally by nerve pathways ( 11,12 ) . in italy , l - type bse prions have been characterized in detail by using cattle challenged intracerebrally . however , prpres was not detected in their peripheral tissues , including the peripheral nerves ( 13 ) . the reason for the discrepancy in prpres detection is unclear . in view of the similarities between the l - type and bse / jp24 prion characteristics ( 9 ) , this discrepancy may result from differences in the methods used for prpres detection . we detected infectivity in the nerve tissue samples ( including samples from the obex , sciatic nerve , adrenal gland , brachial nerve plexus , and vagus nerve ) obtained 10 , 12 , and 16 mpi . on the basis of the incubation time of 223 25 ( mean sd ) days in mice injected with a 1,000-fold dilution of the obex homogenate , infectious titers in peripheral nerve tissues appeared to be 1,000 lower than those estimated in the obex during endpoint titration of infectivity . our results demonstrate that l - type atypical bse prions can be distributed in the peripheral nerve tissues of intracerebrally challenged cattle . these findings are useful for understanding l - type bse pathogenesis and accurately assessing the risks associated with this disease . investigations of prion distribution in cattle that have been orally challenged with l - type bse prions are critical .

we recently reported the intraspecies transmission of l - type atypical bovine spongiform encephalopathy ( bse ) . to clarify the peripheral pathogenesis of l - type bse , we studied prion distribution in nerve and lymphoid tissues obtained from experimentally challenged cattle . as with classical bse prions , l - type bse prions accumulated in central and peripheral nerve tissues .

The Study Conclusions

some hematologic dyscrasias have been associated with thymoma . about 5% of patients with thymoma develop pure red cell aplasia ( prca ) . other hematologic complications occur far less frequently and include autoimmune hemolytic anemia , pernicious anemia , peripheral neutropenia , agranulocytosis , thrombocytopenia and hypogamma - globulinemia . prca disappear after thymectomy but reappear after a latency period even without recurrence of thymoma . aplastic anemia is a rare complication of thymoma and is properly documented in only a few cases . as thymoma - associated hematologic dyscrasias are likewise supposed to be of immunologic origin , we report here a case of very severe aplastic anemia following thymectomy , which was successfully treated with immunosuppressants such as cyclosporine a and g - csf . a 60-year - old woman who complained of cough and dyspnea visited the pulmonary department in april 2000 . chest radiography and computed tomography ( ct ) showed a large mass in the anterior mediastinum , compatible with a thymoma ( figure 1 ) . about sixteen months after the operation , a non - productive cough and high fever developed . a routine hematologic feature was as follows ; hb 5.9 g / dl ; hct 16.9% ; wbc 0.810/l , absolute neutrophil count 0.210/l ; platelets 8610/l . there was no recent antecedent exposure to chemicals and drugs , and serology for hepatitis a , b , c , parvovirus and hiv infection was negative . cytomegalovirus and epstein - barr virus serology was consistent with previous viral exposure . with a high suspicion of aplastic anemia , multiple bone marrow aspirations and biopsy histopathological examination of the bone marrow disclosed extremely low marrow cellularity with increased fatty areas . the patient received transfusions of platelet concentrate and empiric antibiotic therapy for fever and neutropenia during her hospital course . after her neutropenic fever subsided , oral cyclosporine a ( 5 mg / kg / day ) was administered . three months after this treatment , she is clinically stable with no transfusion requirement and without any signs of infection . peripheral blood counts were as follows : hb 10.3 g / dl ; hct 30.2% ; wbc 3.510/l , absolute neutrophil count 1.910/l ; platelets 16410/l . from then to march , 2002 , the patient remained in complete remission with low doses of cyclosporine alone . thymoma is the most common tumor of the anterior mediastinum and is often associated with autoimmune diseases , including myasthenia gravis , polymyositis , hypogammaglobulinemia and various cytopenia . in fact , the overall long - term survival of patients with thymoma is poor once cytopenias occur . although thymectomy is indicated in thymomas with blood dyscrasia , a permanent hematologic remission can be expected in less than 30% of patients with thymoma - associated prca . often , the immunoassociated symptoms like prca disappear after thymectomy but reappear after a latency period , even without recurrence of thymoma , and there is no codified therapy for surgically resistant or relapse cases . in aplastic anemia , thymectomy may fail to resolve aplastic cytopenias as in the case presented by liozon et al . pl et al presented a patient with aplastic cytopenia - associated thymoma in whom complete remission was achieved with surgical removal of the thymoma . alg and cyclosporin , often successful in primary as well as secondary prca and idiopathic aa , should be proposed in unresponsive cases . reported that the combination of octreotide and prednisone produced a complete clinical response in a patient with malignant thymoma and pure red cell aplasia . aplastic anemia is a rare complication of thymoma and is extremely infrequent after thymectomy . in large series , the incidence of aa with thymoma is 01.4% to our knowledge , this is the fourth report of aplastic anemia following thymectomy many pathogenetic mechanisms have been suggested to explain the bone marrow failure seen in aplastic anemia . especially , the role of suppressor t lymphocytes is considered important in the development of aplastic anemia . similarly , thymoma associated with aplastic anemia seems to be caused by the suppression of the bone marrow due to an unbalanced regulation and the inhibitory effects of t lymphocytes on bfu - e and cfu - e as demonstrated in previous reports . because immune mechanisms appear to play an important part in the maintenance and/or progression of thymoma - associated aplastic anemia , this is the rationale for the use of immunosuppressive therapy as one option in the treatment of the disease . cyclosporine a has been associated with good responses in cases of thymoma - associated prca and aplastic anemia , so we decided to treat the patient with cyclosporine a and g - csf . thus , our case seems to be an example for the development of very severe aa , following surgical removal of a thymoma , in which an immunosuppressive agent like cyclosporine a has been efficient .

aplastic anemia is a rare complication of thymoma and is extremely infrequent after thymectomy . we present a case of a 60-year - old woman with very severe aplastic anemia appearing sixteen months after thymectomy for a thymoma . she underwent thymectomy for a thymoma in april 2000 . preoperative examination revealed no hematologic abnormality . about sixteen months after the operation , she was readmitted because of pancytopenia with cough and fever . bone marrow aspiration revealed a very severe hypoplasia in all the three cell lines with over 80% fatty tissue , and chest ct revealed no recurrence of thymoma . her aplastic anemia had responded to cyclosporine a and granulocyte - colony stimulating factor ( g - csf ) .

INTRODUCTION CASE DISCUSSION

coagulase negative staphylococci ( cons ) are a major component of the normal flora of human skin and the oral flora found on mucous membranes ( 1 , 2 ) . these gram positive bacteria have been found to be the third most common causative agent of nosocomial infections ( 1 , 2 ) . in addition to their virulence , cons possess the ability to survive on synthetic medical devices and equipment and also on various surfaces in hospitals for weeks to months ( 3 ) . while this species of gram - positive bacteria have been reported to colonize patients and hospital staff , they can also be introduced or transmitted on to the body through the use of contaminated equipment and devices such as intravenous catheters , prosthetic heart valves , orthopaedic implants and others ( 4 , 5 ) . cons are a heterogeneous group of bacteria , consisting of approximately 40 species , of which , several species have been recognized as potential pathogens to humans ( 6 ) . the most common species isolated from human specimens that result in disease are s. epidermidis , s. haemolyticus , s. hominis , and s. saprophyticus . other species such as s. warneri , s. lugdunensis , s. capitis , s. simulans , s. cohnii , s. saccharolyticus , and s. xylosus have been considered as significant opportunistic pathogens but rarely isolated ( 7 , 8) . cons species have been recognised as etiologic agents of a wide variety of infections especially in immune - compromised patients , patients with indwelling or implanted foreign bodies ( 9 ) . they can play an important role in bacteraemia ( 10 ) , central nervous system shunt infection , intravascular catheter - related infections , endocarditic ( 11 ) , urinary tract infections ( 12 ) , surgical site infections ( 13 ) , endophthalmitis , foreign body infection , peritonitis and wound , bone and joint infections as well as infections in neonates ( 14 , 15 ) . distinction between a clinically significant pathogenic infection and contaminating cons isolates is difficult and remains a major challenge for clinicians ( 8 , 16 ) . moreover , the susceptibility pattern of cons has varied to antimicrobial agents between species and countries due to wide use of antibiotics for prophylaxis or therapy especially in hospitalized patients ( 17 , 18 ) . the main aim of this study was to determine the most common cons species isolated from different clinical specimens in the northern province of jordan and to evaluate the susceptibility pattern of these species . specimens were collected from all patient populations and consisted of inpatient hospitalized patients as well as outpatient samples . the type of specimen obtained for analysis consisted of the following : blood culture , abscess fluid , catheter tip , urine , wound culture , respiratory samples , nasal samples , body fluids , urethral samples , eye cultures , cerebro - spinal fluid , tissue samples , semen , ear , and cervical swabs . the seven hospital wards from which samples were obtained included the intensive care unit ( icu ) , nursery intensive care unit ( nicu ) , emergency department ( ed ) , department of surgery , internal medicine , paediatrics , and the outpatient clinics ( opd ) . clinical specimens were collected using standard collection techniques ( 9 , 13 ) , and inoculated on appropriate bacteriological media , including 10% sheep blood agar , chocolate agar , thioglycollate , macconkey agar media . inoculated plates were incubated aerobically at 37 c for 1824 hours while blood cultures were analysed using an automated blood culture system ( bact / alert 3d . identification of isolated cons was made according to the standard methods for any potentially clinically significant growth appearing on the culture media based on quantity , feature of growth , source and site of specimens ( 19 ) . primary identification was initiated using basic microbiological features and biochemical characteristics such as colony morphology , gram staining , catalase and coagulase tests ( 19 ) . final identification and antibiotic susceptibility testing of the bacteria isolated from clinical specimens was performed using different auto - analyzing systems . bauer disk diffusion technique according to clinical and laboratory standards institute ( clsi ) guidelines on mueller - hinton agar ( 20 , 21 ) . in vitro antibiotic susceptibility patterns of all isolated cons was determined against 18 antibiotics ( g ) : penicillin , ampicillin ( 30 ) , amoxicillin - clavulanic acid ( 30 ) , cefazolin ( 30 ) , trimethoprim - sulfa - methoxazole ( 1.25/23.75 ) , oxacillin ( 30 ) , gentamicin ( 120 ) , erythromycin ( 15 ) , ceftriaxon ( 30 ) , ciprofloxacin ( 5 ) , norfloxacin ( 5 ) , nitrofurantoin ( 300 ) , linezolid ( 30 ) , levofloxacin ( 5 ) , tetracycline ( 30 ) , clindamycin ( 2 ) , rifampicin ( 5 ) and vancomycin ( 30 ) . all antibiotic medication was purchased from oxoid microbiology products , thermo scientific ( hampshire , uk ) . statistical analysis consisted of descriptive statistics where data were expressed as a number and corresponding percentage as appropriate . based on standard manual and automated methods , 223 specimens showed the presence of cons species , which were isolated and identified from various clinical specimens collected from in patients and out - patients of different ages and sexes during the study period . of the fifteen samples sources , the most common source of cons species was blood culture ( n=69 , 30.9% ) isolates , followed by abscesses ( n=36 , 16.2% ) , catheter tip ( n=34 , 15.2% ) , urine ( n=27 , 12.2% ) and wound ( n=14 , 6.3% ) ( table 1 ) . the remaining ten sources , when combined together , accounted for 43(19.2% ) of all obtained isolates . samples were obtained from seven different hospital wards and showing a varied distribution of cons sources as shown in ( table 2 ) . ten species of cons were isolated and identified during the study from various body sites , the most common species isolated from all specimens were staphylococcus epidermidis and s. haemolyticus which comprised 122 ( 54.7% ) and 52 ( 23.4% ) , respectively . species identification showed the presence of s. hominis in 13 ( 5.8% ) isolates followed by s. lugdunensis in 9 ( 4% ) , s. capitis in 8(3.6% ) and s. saprophyticus in 7 ( 3.1% ) . four other less common isolates of cons consisted of s. auricularis , s. warneri , s. simulans and s. xylosus collectively represented the remaining 12 ( 5.4% ) ( fig.1 ) . analysis of each individual isolated species of cons showed a variable distribution of all species among the different sources of clinical specimens and the different inpatient and outpatient hospital wards ( table 3 ) . s. epidermidis , the most common species of cons isolated , was isolated from all types of specimens and was present on all hospital wards . however , the majority of the bacteria were found growing in blood cultures ( 34% ) and on catheter tips ( 21% ) . despite the ubiquitous presence of this species on all wards , the majority was found in icu ( 46% ) isolates while having a high presence in specimens received from opd clinics ( 21% ) and the nicu ( 17% ) . distribution of cons isolates in clinical specimens and hospital wards nasal swab , body fluids , urethral , eye , csf , tissue , semen , ear , cervical swab surgary , internal wards the second most commonly isolated cons species was s. haemolyticus . the majority of these bacteria were isolated from blood ( 25% ) and abscess ( 11% ) specimens although they were also identified to be in high presence on catheter tips ( 10% ) and in urine specimens ( 10% ) ( table 3 ) . s. haemolyticus was found to be dominant in icu isolates ( 52% ) and present in high number of samples received from opd clinics ( 31% ) when compared to the other hospital wards . the other eight species of cons had a sporadic distribution among the sources of the individual specimens and between the various hospital wards ; their distribution patterns can be seen in table 3 . in general , the species showed high sensitivity to vancomycin ( 100% ) , linozolid ( 98.2% ) , rifampin ( 95.5% ) and nitrofurantin ( 92.8% ) . meanwhile , the lowest sensitivity rate was observed with ampicillin ( 1.8% ) , penicillin ( 2.7% ) , ceftriaxon ( 22% ) , cefazolin ( 22.4% ) , amoxicillin - clavulanic acid ( 24.2% ) and erythromycin ( 24.2% ) . the most commonly isolated species , s. epidermidis , showed a similar susceptibility pattern as described above for all cons ( table 4 ) . susceptibility pattern of cons of the most common isolated species , specimen types and hospital wards . results of susceptibility testing of the most common specimen sources of cons ( blood culture , abscess , catheter tip , and urine culture ) and the most common hospital wards where specimens were obtained ( icu , opd , nicu , paediatrics ) using the set of antibiotics can be seen in ( table 4 ) . cons obtained from blood cultures were susceptible to linezolid ( 100% ) and vancomycin ( 100% ) followed by rifampin ( 94% ) , and tetracyclin ( 84% ) . antibiotics with the least activity against the gram positive bacteria were ampicillin ( 1.4% ) and penicillin ( 2.9% ) . specimens obtained from abscess , catheter tips , and urine cultures mimicked the susceptibility that was described from blood cultures although specimens from catheter tips had additional antibiotics which were ineffective at eradicating infection . in addition to ampicillin and penicillin , catheter tip cons infections had very low susceptibility to amoxicillin - clavulonic acid , cefazolin , ceftriaxone , and oxacillin . furthermore , susceptibility pattern of cons species according to the hospital wards showed similar results as seen when examining susceptibility for all species of cons . however , two hospital locations , the icu and paediatric wards , showed high rates of resistance to amoxicillin , clavulonic acid , cefazolin , ceftriaxone , and oxacillin , similar to isolates cultured from catheter tips . coagulase - negative staphylococci ( cons ) are a group of micro - organisms that have largely been considered as contaminants because of the role in the normal flora . recently , some studies have implicated this group as one of the more important causative agents of human infections , especially in immune - compromised patients , premature newborns and patients with indwelling medical devices ( 4 , 5 ) . the various species of cons are one of the most commonly isolated bacteria from clinical specimens in a clinical microbiology laboratory ( 22 ) . they are the most common agents of nosocomial bloodstream infections as well as other type of infections either in healthy individuals or hospitalized patients ( 23 ) . we have demonstrated that the highest percentage of cons species isolates were found in blood cultures and in patients located in the icu , which is consistent with other similar studies ( 4 , 5 , 24 ) . our results also show that while the most common source of cons species isolation was in blood cultures ( 30% ) , other sources including abscess , catheter tip , and urine cultures combined for a total of 43% . unsurprisingly , the icu accounted for 41% , or the majority , of all cons isolates obtained by laboratories . however , an interesting finding was that the outpatient clinics ( 27% ) accounted for slightly less than the adult icu and more than the nursery icu . this high number can possibly be explained by looking further into the population of patients being seen in the outpatient departments . for instance , urinary tract infections and wound infections are high among out - patients population , which could potentially clarify the increased use of microbiology specimens to manage these outpatient conditions . another possible reason for the opd specimens having such a high percentage of cons species isolates may be related to poor sample collection techniques and/or multi - sample sources and clinics ( 8 , 11 ) . increase use of central venous catheters ( cvc ) and other indwelling vascular devices in hospitals , especially in icu patients , create a potential for increased rates of infection . the possibility of colonization of these catheters and/or medical devices with cons as a result of skin contamination of patients and staff may explain the high percentage of isolates obtained from icus and the high number of infected catheter tips cultures ( 4 , 2527 ) . due to these circumstances , cons species must be considered as an important causative agent of nosocomial bacteraemia and catheter related infection ( 1 , 28 ) . in our study , s. epidermidis is the major isolated species followed by s. haemolyticus , and both were isolated from all clinical specimens at all studied hospital wards . our results are consistent with studies performed in other countries ( 8 , 2833 ) . cons species are one of the most common causative agents of sepsis and nosocomial infections for newborns and infants . they have been isolated from various medical devices used in these units , from nicu environment , and paediatric wards ( 14 , 31 ) . the percentage of isolated cons from the nicu and paediatric wards in our study is 9.3% and 7.3% , respectively , which is consistent with other findings while some of the inter - study differences are possibly due to the isolated species , the type of infection and clinical case of the patients ( 10 , 14 , 28 , 33 ) . different antibiotics were used to evaluate the susceptibility pattern of isolated cons according to recommendations of clsi 2009 guidelines for gram positive bacteria ( 21 ) . these four antibiotics may play an important role in the treatment and prevention of nosocomial infections of cons . however , cons species showed remarkable resistance to ampicillin , penicillin and other types of antibiotics listed in ( table 4 ) . while bacteria continue to acquire resistance to antibiotics , selection of the appropriate agents our results from the specimens obtained from northern population of jordan are consistent with the results observed by other institutions and studies ( 18 , 28 , 31 , 32 , 3436 ) . we demonstrated that the predominant isolated cons species were s. epidermidis and s. haemolyticus , which makes the susceptibility pattern of these two species closely resembling the susceptibility pattern of all other isolated cons . long hospitalization periods , extensive use of antibiotics and the ability of these organisms to create multi - layered bio - films on artificial surfaces are the possible reasons of a high resistance rate in general and to oxacillin specifically ; especially in the species that were isolated from catheter tips and blood cultures ( 11 , 16 , 18 , 27 , 30 , 34 , 36 ) . the low resistance rate of isolated cons bacteria from urine samples may be due to presence of a high number of s. saprophyticus species ( 9 , 12 , 28 , 32 ) . on the other hand , the high resistance rate of species that were isolated from the icu and the paediatric wards may be due to the patients clinical case , age and type of causative agent identified ( 24 , 29 , 31 , 37 ) . the most common cons isolates in the northern province of jordan were found to be s. epidermidis and s. haemolyticus and varied in percentage according the specimen that was obtained . while this group of organisms shows high susceptibility to vancomycin , rifampin , and linezolid , high rates of resistance to amoxicillin and penicillin and other variability exists and warrants further evaluation . infections originating in blood cultures , catheter tips , or from patients in the icu or paediatric wards should undergo careful susceptibility testing because of the decreased efficacy of additional antibiotics in these situations .

background : coagulase negative staphylococci ( cons ) are one of the most common bacteria found on human skin and on mucous membranes as a component of normal flora . the presence of cons in clinical specimens is frequently associated with an infectious aetiology or contamination.objectives:we aimed to evaluate cons species distribution and susceptibility patterns in specimens obtained from clinics and hospitals in the northern area of jordan.methods:standard identification methods showed the presence of cons in 223 specimens at different local hospitals . susceptibility testing was performed using 18 antibiotics in accordance with the clinical and laboratory standards institute ( clsi ) recommendations.results:staphylococcus epidermidis and s. haemolyticus were found to be the most common species isolated from all specimens representing 122 ( 54.7% ) and 52 ( 23.4% ) of all cons species , respectively . antibiotic susceptibility testing of cons species revealed their sensitivity to vancomycin , linozolid , rifampin and nitrofurantin , while showing a highly resistant pattern to ampicillin , penicillin , ceftriaxone , cefazolin , amoxicillin - clavulanic acid and erythromycin . some variation of the susceptibility pattern of cons species were identified in specimens isolated from the icu and paediatric hospital wards as well as from clinical specimens of urine , blood and catheter tips.conclusion:the most common cons isolates were found to be s. epidermidis and s. haemolyticus with variable percentages according to the specimen source . moreover , a high susceptibility cons to vancomycin , rifampin , and linezolid showed resistance to amoxicillin and penicillin .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

the chemistry of carbenes has seen an explosive growth both as complexes for metals and as organocatalysts , but our knowledge of the chemistry of the derived enetetramines is much less advanced . we have recently shown that unactivated alkyl and aryl halides are reduced by enetetramines , acting as very strong neutral , ground - state , organic electron donors . thus benzimidazole - based donor 1(4,5 ) was found to react efficiently via set ( single electron transfer ) with unactivated aryl as well as alkyl iodides , e.g. , 3 and 5 , respectively , leading to the cyclization products 4 and 6 derived from the corresponding initial aryl and alkyl radicals ( scheme 1 ) . substrates such as 5 showed that alkyl carbanions were not intermediates ( carbanion formation would be expected to lead to p - methoxystyrene and cyclohex-2-en-1-ol ) , but these products were not detected . more recently the imidazole - derived donor , 2 , was shown to be an even more powerful reagent than 1 , featuring double - electron transfer to iodoarenes , leading to cyclization of the aryl anion derived from ester 7 to form ketone 8 and reduced product 9 . the impressive reducing power of reagents such as 1 and 2(10 ) is ascribed both to the ability of nitrogen atoms to stabilize positive charge on an adjacent carbon , and to the aromatization energy that is gained , following electron donation , on forming their radical - cations 11 and 13 , respectively , and their dications 12 and 14 , respectively . comparison with the simpler enetetramine , tdae 10 , shows that 10 is a weaker electron donor ; oxidation does not lead to aromatic stabilization energy . the greater reactivity of 2 , compared to 1 , relates to the greater aromatization energy associated with formation of the bis - imidazolium salt 14 from precursor 2 , compared to forming bis - benzimidazolium salt 12 from precursor 1 . similar arguments can apply to the radical cations 13 and 11 , as depicted in scheme 2 . this paper now reports and investigates the unprecedented formation of aliphatic aldehydes by extrusion of a single carbon atom from the imidazolium salt - derived enetetramine 2 when it reacts with alkyl and appropriate aryl halides . this new reactivity came to light upon reacting alkyl halides with enetetramine 2 ( scheme 3 ) . donor 2 ( 1.5 equiv ) was prepared in situ by deprotonation of salt 15(7,8 ) and subsequently reacted with 5-phenyliodopentane 16 . when a neutral workup was used , a trace of aldehyde was noted in the nmr spectra of the crude reaction products . turning to acidic workup afforded aldehyde 17 in 19% yield . to explore whether an aldehyde might be obtained from an alkyl bromide , substrate 18 the conditions of the reaction were then studied to maximize the yield of the aldehyde . an excess of donor 2 ( 5 equiv , concentrated solution ) led to isolation of aldehyde 19 in 61% yield following acidic workup . again , the yield of the aldehyde was significant only when the reaction workup featured acidic conditions , suggesting that the aldehyde required to be liberated from a protected form during workup . the origin of the aldehyde carbonyl carbon in products 17 and 19 was now addressed . this could happen by conversion of the alkyl halide to the corresponding alkyl anion following transfer of two electrons from donor 2 . to test this possibility , reaction of the corresponding alkyl anion with dma should now afford the ketone , 20 , following workup . however , no ketone was formed , and , instead , aldehyde 17 was again isolated ( 32% yield ) after further purification by column chromatography . this clearly shows that the aldehyde product can arise from a pathway that does not involve dmf and hence really must involve the bisimidazolylidene 2 . this still left the possibility that reaction in dmf as solvent could form aldehyde by two routes , in one of which the aldehyde carbon was derived from 2 , while in the other it derived from dmf . [ it will be shown later with substrate 47 that aldehyde does not derive from dmf at all , within the limits of detection ] . looking for mechanistic routes for cc bond formation between the substrate 21 and molecule 2 , three options present themselves , all involving reaction at the central carbons of 2 ( scheme 4 ) . thus 2 could behave simply as a nucleophile , undergoing sn2 reaction with the alkyl halide substrates . the driving force for regioselective reaction at the central carbons is the aromaticity of the resulting imidazolium ring in 24 . an alternative route to the same product features electron transfer from the donor 2 to the alkyl halide 21 . the third possibility is that an alkyl anion 23 is formed from the substrate following transfer of two electrons as suggested above ; 23 would then be well placed to couple to dication 14 . the path to 24 will be discussed later in this article , but first a mechanism needs to be proposed to explain how such an intermediate could lead to aldehydes ( scheme 5 ) . the ultimate fate of the carbene group is not clear from the products that we isolate , but for the formation of the aldehyde , a possible route would feature the reduction of the imidazolium unit of 25 to radical 26 by electron transfer , followed by hydrogen atom abstraction to give the imidazoline 27 . the best yields of aldehydes were obtained when multiple equivalents of bisimidazolylidene 2 were used in the reaction , and when the reaction featured acidic workup conditions . the need for multiple equivalents of donor 2 would be consistent with the need for a reduction step ( 25 to 26 ) ; excess of donor 2 would facilitate this electron transfer . similarly , the improved yields of aldehydes isolated from these reactions when using acidic workup conditions would be explained by the need to hydrolyze imidazoline 27 . alternatively ( we thank a reviewer for this suggestion ) , 25 could undergo deprotonation to form enediamine 29 . the deprotonation might be carried out intramolecularly by the carbene carbon in 25 , which , from a model , might be feasible . within 29 , nucleophilic attack by the enediamine upon the imidazolium salt would afford 30 , and hydrolysis of this compound on acidic workup would lead to formation of aldehyde - acid 31 , which could undergo decarboxylation to give the observed aldehydes . to see if evidence in favor of either route could be found , the simple 1,3-dimethyl-2-(3-phenylpropyl)-1h - imidazolium iodide 33 was prepared and treated with donor 2 . in the reductive route if this happens , then other imidazolium salts , such as 33 , should also be reduced by 2 . however , no aldehyde was formed in the reaction of iodide 33 with donor 2 , suggesting that aldehydes do not arise from a reductive route . the alternative route to aldehydes 28 goes through hydrolysis of imidazolium salt 30 during the workup procedure . if that imidazolium salt can be hydrolyzed to an acid , then imidazolium salt 33 should also afford an acid , i.e. , 35 . the reaction of imidazolium salt 33 with 2 , followed by acidic workup did indeed afford acid 35 but in low yield ( 2% ) . compound 30 is relatively more activated than 33 for hydrolysis , and so it is possible that it would afford higher yields of acid 31 ( and subsequently aldehyde 28 ) than seen here for acid 35 . thus , the best working hypothesis is that the aldehydes 28 arise from the hydrolysis and decarboxylation route . the favored route to aldehydes discussed above , relies on iminium saltenediamine conversions between 25 , 29 , and 30 . we envisaged that 37 , an analogue of 25 , featuring a leaving group ( or ) to the imidazolium salt would lead to further reactions that would signal its role as an intermediate ( see scheme 6 ) . deprotonation of 37 would afford the enediamine 38 ; this compound would then be primed to expel the alkoxide group ro , thereby affording imidazolium cation 39 . in turn , this could be deprotonated in the basic medium to yield 40 , from which expulsion of ro could be expected . the isolation of alcohol(s ) roh , roh could therefore be an important pointer in support of alkylimidazolium intermediates , such as 25 in scheme 5 . while alcohol roh could also arise through other mechanisms , it is difficult to think of an alternative way in which roh would be produced , and hence we set particular importance on the isolation of this alcohol in the test reactions below . the hindered alkyl halide substrates 45ah were selected and prepared as shown in scheme 7 . reaction of initial substrate 45a very clearly led to mixtures of the corresponding two alcohols , phenylpropanol + benzyl alcohol ( 1.5:1 ) . sensing that the deviation from an expected 1:1 ratio might be due to volatility of the benzyl alcohol , substrate 45b was reacted with 2 and afforded a 1:1 mixture of phenylpropanol + phenylbutanol as judged by nmr , in accordance with the hypothesis in scheme 6 . however , the separation of the alcohols was challenging on the scale of the experiment , and so 45ch employed r = me to facilitate isolation of roh . clearly , the alcohol roh is isolated in high yield from these reactions , completely consistent with scheme 6 . the aliphatic substrates 16 and 18 do not allow us to distinguish between the three routes shown in scheme 4 . on the other hand , reactions of donor 2 with substrates 45 do provide key information . the outcomes of these reactions are consistent with both the direct sn2 mechanism and/or with the intermediacy of aliphatic radicals from set to the alkyl halides . however , they are inconsistent with formation of alkyl anions , since the developing carbanion represented by 46 should force the -elimination of the alkoxide in a concerted reaction affording 44 , but no 44 was formed . to gain further insight , iodide 47 afforded two products , 48 and 49 , in 42% and 13% yields , respectively ( see scheme 8) . the same products 48 ( 42% ) and aldehyde 49 ( 5% ) were afforded . the aldehyde showed no labeling with deuterium in nmr or mass spectrometry this supports our previous statement that the aldehyde products do not incorporate the formyl group of dmf ] . similarly , iodide 50 gave reduced product 51 ( 40% ) and aldehyde 52 ( 5% ) . for both reactions , the major product is likely to arise from transfer of two electrons to the substrate to form the aryl anion , consistent with our previous results for donor 2 . as in the experiments with the previous substrates in this article , isolation of aldehyde was dependent on the use of acid in the reaction workup . the two aldehydes 49 and 52 , as minor products , can not arise from sn2 reaction and so must result from the trapping of an alkyl radical or an alkyl anion . although the formation of alkyl anions by direct reduction of alkyl halides ( via alkyl radicals ) is rejected above following the experiments with substrates 45 , alkyl anions might arise indirectly by cyclization of aryl anions , and aryl anions are known to form when donor 2 reacts with aryl iodides . to probe that point , substrate 53 was prepared and reacted with donor 2 in dmf ( scheme 9 ) . this afforded three products in a combined yield of 70% : the reduced product 54 ( 27% ) , the cyclized product 55 ( 8% ) and the alcohol 56 ( 35% ) ( scheme 9 ) . formation of product 54 sees two electrons being transferred in quick succession from donor 2 to substrate 53(8 ) to form anion 58 ( scheme 10 ) . if anion 58 could cyclize to give aliphatic anion 62 , this would lead to formation of alcohol 56 as well as vinylindoline 63 . alcohol 56 arises through initial and rapid cyclization of aryl radical 57 to form 59 ( scheme 10 ) . hydrogen abstraction by 59 then leads to 55 . alternatively , trapping of 59 with radical - cation 13 affords salt 60 , which acts as the source of alcohol 56 following the pathway proposed in scheme 6 . no products that derived from the electrophile 61 were detected , signaling its high reactivity . in summary , aliphatic aldehydes are formed on reaction of bisimidazolylidene 2 with both aliphatic and aryl iodides as well as alkyl bromides . an sn2 mechanism may play a role in aldehyde formation from the aliphatic substrates , but the aldehydes formed from the aryl substrates arise via a radical mechanism . this indicates that some aryl radicals cyclize before a second electron can be received from donor 2 .

reaction of imidazolylidene - derived enetetramine 2 with aliphatic iodides and bromides ( and with aryl iodides bearing alkene - containing side - chains in the ortho - position ) leads to formation of aliphatic aldehydes through an unprecedented extrusion of a one - carbon unit from the enetetramine . an intermediate 2-alkylimidazoline 24 is proposed , where the alkyl group derives from the substrate ; this imidazoline undergoes further reaction in situ to afford the observed aldehydes on acidic workup . modified substrates were designed and prepared to probe the chemistry of the alkylimidazoline adducts and provided extensive information on the chemistry of the adducts .

Introduction Results and Discussion

supplementary material is available for this article at 10.1007/s13659 - 013 - 0057 - 0 and is accessible for authorized users .

four new diterpenoids , laxiflorins s - v ( 14 ) , bearing four different types , and one known compound , laxiflorin o ( 5 ) , were isolated from isodon eriocalyx var . laxiflora . compound 1 was the first example of ent - kauranoids bearing a unique c24 carbon framework and compound 4 was the first example of 3,4-seco - ent - abietane diterpenoids from the isodon genus . their structures were determined by spectroscopic methods ( uv , ir , ms , nmr ) . electronic supplementary materialsupplementary material is available for this article at 10.1007/s13659 - 013 - 0057 - 0 and is accessible for authorized users .

Electronic Supplementary Material Electronic supplementary material

hearing disturbance is the third most common chronic disease in elderly individuals , following arthritis and high blood pressure.1 ) hearing disturbance also occurs in younger individuals , due to their frequent exposure to noise.2 ) hearing aids improve hearing by amplifying sound , thus promoting the ability of patients to communicate with other people . the korea centers for disease control and prevention recently announced that the prevalence of bilateral hearing disturbance in individuals over age 12 years was 4.5% , increasing to 25.9% in individuals over age 65 years . after age 50 years , the rate of bilateral hearing disturbance triples every 10 years , being 2.9% for persons in their 50 s , 12.1% for persons in their 60 s , and 31.7% for persons aged 70 years and over . however , only 11.3% of individuals aged 65 years with unilateral or bilateral hearing disturbance use hearing aids , with the rate being much lower in females than in males ( 7.2% vs. 17.0%).3 ) the national statistics service of korea has estimated that 11.8% of the total population in 2012 were aged 65 years and that this percentage will increase to about 24.3% in 2030.4 ) thus , interest in and demand for hearing aids for recovery from hearing disturbance are expected to increase . nevertheless , despite improvements in hearing aid performance , design , and adaptation , the current hearing aid usage rate is still quite low , with an estimated 20% of patients prescribed hearing aids returning or declining the devices.5 ) to elucidate the reasons that patients refuse and/or return properly prescribed hearing aids , we analyzed hearing aid usage and the reasons for return in younger ( < 65 years ) and elderly ( 65 years ) adults . of the 1318 patients who visited the department of otolaryngology , kyunghee university school of medicine hearing aid clinic and were prescribed hearing aids from january 2000 to november 2012 , 81 ( 6.14% ) returned their hearing aids within 3 months , including 36 patients aged < 65 years and 45 aged 65 years . using retrospective chart analysis and phone surveys , we compared reasons for returning hearing aids in the two groups . the characteristics of patients with returning hearing aid were subdivided into patient related , hearing related , and hearing aid related factors . patient related factors included patient age , gender , period of hearing disturbance , and main symptom at the time of hospital visit ( hearing disturbance , ringing , or fullness in the ears ) . hearing related factors included the results of pure tone audiometry at the time of hearing aid prescription , speech discrimination test , dynamic range , and degree of hearing disturbance , defined as mild ( 26 - 40 db ) , moderate ( 41 - 55 db ) , moderately severe ( 56 - 70 db ) , severe ( 71 - 90 db ) , and profound ( > 90 db ) . hearing aid related factors included hearing aid location : completely in the canal ( cic ) , in the canal ( itc ) , in the ear ( ite ) , behind the ear ( bte ) , and open type bte . for analysis , reasons for hearing aid return were subdivided into 2 groups , hearing aid problems and patient problems . hearing aid problems included ineffective hearing aids , noise ( noisiness , vibration ) , discomfort , and bad sound quality , whereas patient problems included uselessness ( e.g. hearing recovery , ill health , death ) , financial situation , and difficulties managing the hearing aids . all statistical analyses were performed using the statistical package for social science 18.0 ( spss inc . , parameters were compared using the chi - square test and student 's t - test , as warranted . of 1318 hearing aid users , 81 ( 6.14% ) , of mean age 65.314.5 years , returned their hearing aids . mean ages in the younger and elderly adults were 52.611.4 years and 75.46.5 years , respectively . of the 81 patients , 33 were male and 48 were female , including 17 males and 19 females in the younger adult group and 16 males and 29 females in the elderly group . although females over age 65 years returned more hearing aids than females under age 65 years , this difference was not statistically significant ( p=0.288 ) . the overall duration of hearing disturbance was 7.412.2 years , 8.814.5 years in younger adults and 6.310.1 years in elderly patients ( p=0.373 ) . the main symptom at first appearance at the hospital was hearing disturbance , observed in 72 of the 81 patients ( 88.8% ) , including 32 of 36 ( 88.8% ) younger and 40 of 45 ( 88.8% ) elderly adults . thirty - five patients ( 43.2% ) , 19 younger ( 52.7% ) and 16 elderly ( 35.5% ) individuals , reported ringing in the ears , whereas 17 ( 20.9% ) , 9 younger ( 25% ) and 8 elderly adults ( 17.7% ) , reported ear fullness ( table 1 ) . we found that the overall spondee recognition threshold in all 81 patients was 59.014.9 db , 63.314.0 db in younger and 55.614.7 db in elderly adults ( p=0.019 ) . mean overall speech discrimination ability was 74.413.8% , 75.612.1% for younger and 73.316.1% for older adults , mean overall most comfortable loudness ( mcl ) was 82.2814.9 , 85.2219.1 for younger and 79.339.3 for older adults ( p=0.419 ) , mean overall uncomfortable loudness ( ucl ) was 102.399.2 , 104.1111.2 for younger and 100.677.0 for older adults ( p=0.443 ) ; and mean overall dynamic range was 49.317.4 db , 49.121.0 db for younger and 49.614.2 db for older patients ( p=0.948)(table 2 ) . assessment of the degree of hearing disturbance in the 81 patients who returned their hearing aids found mild , moderate , moderately severe , severe , and profound deafness in 8 ( 9.8% ) , 30 ( 37% ) , 25 ( 30.8% ) , 17 ( 20.9% ) , and 1 ( 1.2% ) patients , respectively . these degrees of hearing disturbance were observed in 0 ( 0% ) , 14 ( 38.8% ) , 12 ( 33.3% ) , 9 ( 25% ) , and 1 ( 2.8% ) , respectively , of the younger group , and in 8 ( 17.7% ) , 17 ( 35.5% ) , 13 ( 28.8% ) , 8 ( 17.7% ) , and 0 ( 0% ) , respectively , of the older group ( table 3 ) . types of hearing aids returned included 22 cic type , 41 itc type , 3 ite type , 6 bte type , and 9 open type . in the younger group , these types were returned by 10 , 19 , 0 , 3 , and 4 patients , respectively , whereas , in the older group , these types were returned by 12 , 22 , 3 , 3 , and 5 patients , respectively ( table 4 ) . the most frequent hearing aid problem was ineffectiveness , reported by 26 patients ( 32.0% ) , 10 in the younger and 16 in the elderly group . other hearing aid problems included noise , reported by 19 patients ( 23.4% ) , 7 in the younger adult and 12 in the elderly group ; wearing discomfort , cited by 14 patients ( 17.2% ) , 9 in the younger and 5 in the elderly group ; and poor sound quality , mentioned by 3 patients ( 3.7% ) , 1 in the younger and 2 in the elderly group . patient problems included management difficulty in 8 patients ( 9.8% ) , 1 in the younger and 7 in the elderly group ( 2 male and 5 female patients ) ; financial difficulty in 6 patients ( 8.6% ) , 5 in the younger and 1 in the elderly group ; and uselessness ( e.g. hearing recovery , poor health , death ) in 5 patients ( 6.1% ) , 3 in the younger and 2 in the elderly group ( table 5 ) . hearing loss results in partial severance of communication via speaking and language and information exchange , resulting in considerable inconvenience in daily life and negative mental and/or emotional effects . patients who experience hearing loss can therefore benefit substantially from hearing aids . the degree of noise in modern society has increased the number of hearing - impaired individuals . moreover , people are living to an older age , increasing the population of elderly individuals . thus , the demand for hearing aids is expected to increase dramatically in the future . in korea , 7.2% of the total population in 2000 was over 65 years of age , and this percentage is expected to increase to 14.5% by 2018.6 ) in 2011 , it was estimated that only 13% of individuals with hearing disorders who required hearing aids had been prescribed hearing aids , fewer than the 25% in countries such as the usa and other european countries.7,8,9 ) nevertheless , the market for hearing aids has grown continuously , due to increases in the population of elderly individuals and their income level , as well as to the development of medical devices , and this market is expected to increase in the next few decades . despite advances in hearing aid development and technology , 3% to 16% of patients prescribed hearing aids return the devices.10 ) factors associated with adaptation to hearing aids include younger age , strong desire for rehabilitation,11 ) and mild deafness . regular visits to the hospital for hearing aid adaptation and adjustment have been associated with good hearing rehabilitation.12 ) adaptation to and benefits from hearing aids have been associated with degree of education , hearing aid type and price , battery price , wearing hearing aids daily for a certain period of time , and horizontal shape of the hearing aid.13,14,15,16,17 ) we found , however , that , despite hearing aid prescription , consultation , and fitting processes by experts , 81 of 1318 ( 6.14% ) patients prescribed hearing aids returned their devices within 3 months . females were more likely to return hearing aids than males ( 59.2% vs. 40.8% ) , especially when females and males over age 65 years were compared . elderly women may be less familiar with the operation of their devices and be less involved in social life than men with hearing aids , making elderly women less sure about the necessity of wearing hearing aids . these findings suggest that attention should be paid to hearing aid prescription and management in women , especially elderly women . in general age has been found to correlate with the severity of deafness , making the threshold higher in elderly patients due to hearing aid failure . in contrast , we found that the spondee recognition threshold was higher in the younger than in the elderly group . this may have been due to our selection of itc type rather than bte or ite type hearing aids for patients with severe deafness , due to the better appearance of the itc type . other characteristics of the 2 groups were similar , including speech discrimination ability , ucl , mcl , and dynamic range . the return rate by both groups was highest for individuals with moderate hearing loss , followed by individuals with moderately severe and severe hearing loss . when we assessed hearing aid related factors , we found that the rates of return of each hearing aid type were similar in the two groups . this may have resulted from our consideration of patient preference , including allowing the patient to make the decision on hearing aid type . in evaluating the reasons for return of hearing aids , we found that ineffectiveness of the device was the most frequent reason , accounting for 32.0% of returns , the highest percentage in both groups . noises , including acoustic feedback , occlusion effect , over - amplification , surrounding noise , and sound transformation , accounted for 23.4% of returns . our finding , that hearing aid return was due in large part to hearing aid ineffectiveness and hearing aid noises , indicates that hearing recovery in patients with hearing disturbance is limited and that hearing aid technology is not sufficiently developed to result in a normal hearing level . these patients usually do not adapt easily to new technology , have less sensitive sensations in their hands , find handling small hearing aids difficult , delay repairing their hearing aids , and are unfamiliar with handling hearing aids , despite repeated consultations and education . whereas 52.4% of patients hesitated to purchase hearing aids due to inconvenience , 51.2% were reluctant for financial reasons . we found , however , that few elderly individuals returned their hearing aids for financial reasons , since many of their healthcare expenses were paid by their children . in contrast , most of the younger adults likely had to pay these expenses themselves . hearing aid return ratios may be reduced by adequate medical consultation , including a diagnosis of the reason for deafness , proper hearing tests to determine the type and degree of deafness , and to choose the optimal hearing aid for hearing improvement . follow - up measures after hearing aid prescription should include a continuous hearing rehabilitation service . periodic consultations and education may help solve problems and minimize any inconvenience , allowing patients to adapt to hearing aids . although hearing aid return rates were similar in younger and elderly adults , but the reasons showed different tendency . financial considerations were cited more by younger adults , while difficulties in managing hearing aids were cited more frequently by elderly adults . patients in both groups , however , reported that the most frequent reasons for return were inadequate hearing improvement and inconvenience wearing the hearing aid due to noise amplification .

background and objectivesincreases in older aged populations and exposure to complicated noise environments have increased the number of hearing - impaired patients , creating greater demands for hearing aids . we have assessed the reasons that individuals rejected wearing and returned properly prescribed hearing aids , as well as differences in individual factors between younger and elderly adults.subjects and methodsof 1138 patients for whom hearing aids were prescribed at kyung hee university medical center hearing aid clinic , 81 ( 6.14% ) returned their hearing aids , including 36 patients aged < 65 years and 45 aged 65 years . patient - related , hearing - related , and hearing aid - related factors were assessed by retrospective chart analysis and phone survey and compared in the two groups.resultsthe primary symptoms reported by the 81 patients who returned their hearing aids were hearing disturbance , ringing , and fullness in the ear , in that order and in both groups . the rate of hearing aid return was similar in elderly females and males ( p=0.288 ) . the spondee recognition threshold was significantly higher in younger than in elderly adults ( 63.314.0 db vs. 55.614.74 db , p=0.019 ) , but the hearing aid return rate was highest in patients with moderate hearing loss in both groups . in evaluating the reasons for return of hearing aids , we found that ineffectiveness of the device was the most frequent reason , accounting for 32.0% of returns , the highest percentage in both groups , with the most frequent patient problem caused by management difficulty in elderly and financial difficulty in younger adults.conclusionsthe reasons for hearing aid return were different in two groups . financial considerations were cited more by younger adults , while difficulties in managing hearing aids were cited more frequently by elderly adults . patients in both groups , however , reported that the most frequent reasons for return were inadequate hearing improvement and inconvenience wearing the hearing aid due to noise amplification .

Introduction Subjects and Methods Results Discussion Conclusion

approximately 10% of children seen in primary care develop signs of failure to thrive ( ftt ) . children who do not respond to treatment for inadequate caloric intake or have a suspected organic cause get further laboratory diagnosis to identify the 1% of cases with ftt that result from diagnosable disease . in addition to inadequate caloric intake and caloric absorption , excessive caloric consumption due to hyperthyroidism , malignancy , and chronic disease such as severe heart failure ( hf ) must be considered . elevated n - terminal pro brain natriuretic peptide ( n - bnp ) levels were found in children with severe hf due to congenital heart disease or dilated cardiomyopathy [ 1 , 2 ] . from 2005 to 2008 we included n - bnp measurements in our laboratory program for infants with ftt in an attempt to use a laboratory test to identify infants who suffer from severe hf . surprisingly we found significantly elevated n - bnp in infants with ftt without any evidence for cardiac disease . the purpose of this study was to compare n - bnp levels in infants with ftt with infants with severe hf and healthy controls from recently published n - bnp values . consecutive infants who were admitted to our hospital with ftt and bodyweight below the tenth percentile from 2005 to 2008 were included . those infants with an organic cause of ftt like short bowel syndrome or endocrinologic disease the remaining infants of the caloric deprivation ( cd ) group suffer from failure to thrive due to inadequate caloric intake and inappropriate type or volume of feeding . the infants with severe hf due to congenital heart disease were taken together in the heart failure group . normal n - bnp values shown in figure 1 were taken from the literature . for statistical analysis shown in table 2 , we received the original data for healthy infants from norozi et al . and koch et al . . peripheral venous blood samples were obtained from all participants , after a rest for at least 15 minutes . the subjects were in the supine position during blood sampling , which was typically done between 8:30 a.m. and 11:00 a.m. the blood samples were immediately placed on ice and subsequently centrifuged at 5000 rpm for 10 minutes . in all children n - bnp was determined using immunoassay ( elecsys 2010 , roche , germany ) . we excluded all infants with organic cause for ftt like short bowel syndrome or endocrinologic disease . each patient had normal c - reactive protein values and therefore we could exclude significant inflammation . n - bnp serum concentrations are markedly elevated in the first days of life in healthy neonates , probably due to circulatory changes in the perinatal period . the mann - whitney test was used for comparison of n - bnp values of our patients and the original data from healthy infants published by norozi et al . and koch et al . . the data analyses were performed using excel 2000 ( microsoft , usa ) and prism ( graphpad software inc . demographic and height and weight characteristics for the three study groups are summarized in table 1 . 26 patients were included in our study , the cd group contained 15 patients , and the hf group contained 11 patients . there is no significant statistical difference in age , bodyweight , body length , gestational age , and head circumference in the cd group and the hf group . the cd group had a significantly lower postnatal weight gain 73 ( 0147 ) g / week at home mostly caused by inadequate breast feeding at home , compared with the weight gain during hospital stay 225 ( 100500 ) g / week . further laboratory investigations show no significant difference for haemoglobin , white blood cells , alt , and creatinine values in infants with hf compared to the infants with ftt . the hf group included 7 infants prior to cardiac surgery ( ventricular septal defects or atrioventricular septal defects ) . four infants suffered from severe hf more than 2 weeks after cardiac surgery ( transposition of the great arteries , single ventricle ) . seven infants with severe hf received a medical therapy with digoxin , diuretics , angiotensin converting enzyme inhibitors , and/or beta - blocker . our results show that infants with ftt had significantly elevated n - bnp values compared with the healthy infants 115 ( 151121 ) pg / ml versus 530 ( 1193150 ) pg / ml , p < .001 . n - bnp values in the cd group are not significantly different from values of infants with severe hf 673 pg / ml , ( 408 35000 ) pg / ml . as shown in figure 1 n - bnp of our healthy infants group brain natriuretic peptide is secreted mainly by cardiac myocytes of the ventricles and to a lesser degree of the atria . however , several recent studies demonstrate an inverse relationship between body mass index and n - bnp levels were completed in adults with and without severe hf . in this study we present significant n - bnp elevations in a group of 15 infants with cd . these infants only suffer from an inadequate caloric intake without any evidence for severe hf , congenital heart disease , and liver or kidney disease . there was no evidence of diastolic dysfunction on the echocardiograms , indicated by normal mitral valve inflow profiles . in our study the age of the remaining infants was in good accordance with the reference group of the literature . the median n - bnp level of our infants with ftt 530 ( 1193150 ) pg / ml ) is comparable to children with ross class iii hf ( 744 pg / ml ) . our heart failure group included only infants with severe hf with a very high n - bnp level of 40835000 pg / ml . these data are in good accordance with data from a pilot study regarding n - bnp in patients with hf due to ventricular septal defects showing that the perioperative change in n - bnp was most closely correlated with improved weight gain . also it is well known from adults with severe hf that cachectic patients have the highest n - bnp levels . although the reason for the relationship between body mass index and n - bnp is unknown , the increased concentration of the natriuretic peptide receptor - c clearance receptor on adipocyte cells has led some to postulate that increased clearance is the reason for lower n - bnp levels in obese patients . however , evidence against this hypothesis comes from das et al . , who found that n - bnp was correlated with greater lean mass but not greater fat mass . this may represent preservation of the physiological response to change in body fat but might also suggest that adiponectin plays a role in the pathogenesis of cachexia . the correlation between bnp and adiponectin also raises the possibility that the former might increase the secretion of the latter . in an experimental setting tsukamoto could show that administration of bnp over 14 days in rats increased the plasma adiponectin concentration and concluded that bnp enhances adiponectin production in adipocytes , and bnp is a potential regulator of adiponectin production . ohara et al . found that the plasma adiponectin level increased along with an increase of plasma bnp in healthy subjects independently of other confounding factors and they concluded that adiponectin reflects cardiac function . for example , the authors who published the reference values of n - bnp serum concentrations in healthy neonates and children did not give any information about the bodyweight of their study group . this may be the reason for their high range of reference values ( 51121 pg / ml ) in so - called healthy infants between 2 and 12 months . we urgently need reference values from infants with a well - documented normal nutritional state . further studies may delineate whether measuring n - bnp levels are useful for the initial evaluation or ongoing management of patients with ftt due to either inadequate caloric intake or severe hf . the pathophysiological consequence of elevated natriuretic peptides in children with ftt with regard to volume regulation , blood pressure , and cardiovascular prognosis remains speculative . our observation reveals new horizons for the explanation of well - known cardiovascular risk factors like the enhanced cardiovascular mortality in patients with low birth weight or ftt during infancy ( barker hypothesis ) . the link between low birth weight and ftt in infancy , catch - up growth in early childhood , and the higher risk of death from coronary heart disease in adults is important but yet not well understood . longitudinal n - bnp measurements in these patient groups may help for a better pathophysiological understanding of barkers hypothesis and risk stratification in infants with low birth weight and fft . a statistical problem is based upon the high range of n - bnp values in healthy infants especially in the first four weeks of life . due to the nature of our retrospective study we were not able to measure adiponectin in our patients , which could be very helpful for interpretation of enhanced n - bnp , especially if there were a correlation between the level of adiponectin and n - bnp . additional limitations of the study include the fact that there are a relatively small number of patients , and that data were obtained at a single time point without additional follow - up values .

background . brain natriuretic peptide and its inactive fragment n - terminal pro - bnp ( n - bnp ) are reliable markers of ventricular dysfunction in adults and children . we analyzed the impact of nutritional state on n - bnp levels in infants with failure to thrive ( ftt ) and in infants with severe heart failure ( hf ) . the purpose of this study was to compare n - bnp levels in infants with ftt with infants with severe hf and healthy controls . methods . in a retrospective cohort study , we compared n - bnp levels from all consecutive infants with ftt and bodyweight below the tenth percentile ( caloric deprivation ( cd ) group ) to infants with severe hf . reference values from infants between 2 and 12 month were taken from the literature and healthy infants . results . our results show that infants with ftt ( n = 15 ) had significantly ( p < .001 ) elevated n - bnp values compared with the healthy infants ( n = 23 ) , 530 ( 1193150 ) pg / ml versus 115 ( 151121 ) pg / ml . n - bnp values in this cd group are comparable to the median value of infants with severe hf ( n = 12 ) 673 ( 40811310 ) pg / ml . there is no statistical significant difference in age . conclusion . nutritional state has an important impact on n - bnp levels in infants with ftt . we could show comparable levels of n - bnp in infants with ftt and infants with severe hf .

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

although novel treatment protocols have been developed , the prognosis for colon cancer remains poor , with a 5-year survival rate lower than 40% . patients with colon cancer mainly die of metastasis and recurrence ( 2 ) . despite numerous experimental and clinical studies on the pathogenesis of colon cancer , its mechanisms have not yet been elucidated , though they are of great clinical significance for the diagnosis and treatment of this cancer . tumor necrosis factor receptor - associated factor 6 ( traf6 ) has been reported to be highly expressed in many tumor tissues ( 3 ) , but it is still of value to detect its expression in patients with colon cancer and to clarify its clinical significance ( 4 , 5 ) . the aim of this study was to detect the expression levels of traf6 protein in tumor and paracancerous tissues using immunohistochemical methods , to identify correlations between the clinicopathological characteristics and prognosis of colon cancer , and to provide evidence for future prognostic determinations and target therapies . a total of 135 patients with primary colon cancer , who underwent surgery at our hospital from february 2008 to march 2013 , were selected . informed consent was obtained from each patient , and the study protocol conformed to the ethical guidelines of the 1975 declaration of helsinki . patients who had received radiotherapy , chemotherapy , or biological immunotherapy before surgery , or who were lost to follow - up , were excluded . the sample size equation is : where n is the sample size , z is the confidence interval ( for a level of confidence of 95% in this study , z = 1.96 ) , p is the probability value , and e is the error value . tumor and paracancerous tissues resected during surgical procedures were immersed in neutral formalin at 4c for 2 days , and embedded with paraffin into 6-m - thick sections ( leica biosystems , usa ) , which were then deparaffinized with dimethyl benzene and hydrated with ethanol solutions at gradient concentrations . after antigen retrieval ( beijing zhongshan golden bridge biotechnology co. , ltd . , china ) and blocking with rabbit immune serum ( beijing zhongshan golden bridge biotechnology co. , ltd . , china ) , the sections were incubated with traf6 antibody ( santa cruz , 1 : 50 ) in a 37c incubator for 2 hours , washed with pbs , incubated with secondary antibody for 1 hour , washed again with pbs , and incubated with horseradish peroxidase - labeled streptavidin ( beijing zhongshan golden bridge biotechnology co. , ltd . , afterwards , the sections were washed with pbs , colored with dab ( beijing zhongshan golden bridge biotechnology co. , ltd . , pbs was used to replace primary antibody as the negative control . with regard to standards for the determination of immunohistochemical staining , traf6 was determined to be positively expressed in the case of brownish - yellow precipitates in the cytoplasm . the procedure was completed by two experienced pathologists . by using the double - blind method , 15 fields of vision were selected for each section with a high - powered lens ( 400 ) , and the ratio of the number of traf6-positive cells to 100 average cells was calculated as the positive rate . positive results of immunohistochemical staining were determined according to the following two aspects : 1 ) percentage of traf6-positive cells : < 1% , 0 ; 1% - 10% , 1 point ; 10% - 30% , 2 points ; 30% - 60% , 3 points ; > 60% , 4 points , and 2 ) staining results of traf6-positive cells : negative , 0 ; yellowish , 1 point ; yellow , 2 points ; brownish yellow , 3 points . for the determination of results , tumor and paracancerous tissues resected during surgical procedures were immediately stored in liquid nitrogen , from which total rna was extracted by using trizol reagent according to the kit s instructions . upstream primer for traf6 : 5-gaatcacttggcacgacactt-3 , downstream primer for traf6 : 5-gagtttccattttggcagtca-3 and length of amplified fragment : 227 bp . upstream primer for gapdh : 5-accacagtccatgccatcac-3 , downstream primer for gapdh : 5-tgctgtagccaaattcgttg-3 and length of amplified fragment : 450 bp . rt - pcr was performed under the following conditions : reverse transcription at 50c for 30 minutes , reaction at 95c for 15 minutes ( cycling was started ) , denaturation at 94c for 30 d , annealing at 57c for 30 seconds , and extension at 72c for 30 seconds , for a total of 30 cycles . after final extension at 72c for another 10 minutes , the pcr products were subjected to agarose gel electrophoresis . the numerical data were compared by test , and the survival rates were estimated by the kaplan - meier method . a total of 135 patients with primary colon cancer , who underwent surgery at our hospital from february 2008 to march 2013 , were selected . informed consent was obtained from each patient , and the study protocol conformed to the ethical guidelines of the 1975 declaration of helsinki . patients who had received radiotherapy , chemotherapy , or biological immunotherapy before surgery , or who were lost to follow - up , were excluded . the sample size equation is : where n is the sample size , z is the confidence interval ( for a level of confidence of 95% in this study , z = 1.96 ) , p is the probability value , and e is the error value . tumor and paracancerous tissues resected during surgical procedures were immersed in neutral formalin at 4c for 2 days , and embedded with paraffin into 6-m - thick sections ( leica biosystems , usa ) , which were then deparaffinized with dimethyl benzene and hydrated with ethanol solutions at gradient concentrations . after antigen retrieval ( beijing zhongshan golden bridge biotechnology co. , ltd . , china ) and blocking with rabbit immune serum ( beijing zhongshan golden bridge biotechnology co. , ltd . , china ) , the sections were incubated with traf6 antibody ( santa cruz , 1 : 50 ) in a 37c incubator for 2 hours , washed with pbs , incubated with secondary antibody for 1 hour , washed again with pbs , and incubated with horseradish peroxidase - labeled streptavidin ( beijing zhongshan golden bridge biotechnology co. , ltd . , afterwards , the sections were washed with pbs , colored with dab ( beijing zhongshan golden bridge biotechnology co. , ltd . , pbs was used to replace primary antibody as the negative control . with regard to standards for the determination of immunohistochemical staining , traf6 was determined to be positively expressed in the case of brownish - yellow precipitates in the cytoplasm . the procedure was completed by two experienced pathologists . by using the double - blind method , 15 fields of vision were selected for each section with a high - powered lens ( 400 ) , and the ratio of the number of traf6-positive cells to 100 average cells was calculated as the positive rate . positive results of immunohistochemical staining were determined according to the following two aspects : 1 ) percentage of traf6-positive cells : < 1% , 0 ; 1% - 10% , 1 point ; 10% - 30% , 2 points ; 30% - 60% , 3 points ; > 60% , 4 points , and 2 ) staining results of traf6-positive cells : negative , 0 ; yellowish , 1 point ; yellow , 2 points ; brownish yellow , 3 points . for the determination of results , tumor and paracancerous tissues resected during surgical procedures were immediately stored in liquid nitrogen , from which total rna was extracted by using trizol reagent according to the kit s instructions . upstream primer for traf6 : 5-gaatcacttggcacgacactt-3 , downstream primer for traf6 : 5-gagtttccattttggcagtca-3 and length of amplified fragment : 227 bp . upstream primer for gapdh : 5-accacagtccatgccatcac-3 , downstream primer for gapdh : 5-tgctgtagccaaattcgttg-3 and length of amplified fragment : 450 bp . rt - pcr was performed under the following conditions : reverse transcription at 50c for 30 minutes , reaction at 95c for 15 minutes ( cycling was started ) , denaturation at 94c for 30 d , annealing at 57c for 30 seconds , and extension at 72c for 30 seconds , for a total of 30 cycles . after final extension at 72c for another 10 minutes , the pcr products were subjected to agarose gel electrophoresis . the numerical data were compared by test , and the survival rates were estimated by the kaplan - meier method . the he staining results for the tumor and paracancerous tissues are shown in figure 1 . significantly more traf6 protein was expressed in tumor tissues than in paracancerous tissues . in tumor tissues ( figure 1 a - c ) , traf6 protein was mainly expressed in the cytoplasm , showing dark brown particles . meanwhile , tumor stromal cells were also positively stained . in paracancerous tissues ( figure 1 d - f ) , however , traf6 protein was mainly scattered in the cytoplasm , exhibiting light brown particles . in the meantime , traf6 was weakly positively expressed in the cytoplasm of normal tissues ( positive rate : 33.3% ) , which was significantly less than in tumor tissues ( positive rate : 66.7% ; p < 0.05 ) ( table 1 ) . a - c , traf6 expression in tumor ; and d - f , paracancerous tissues . a and d ( 100 ) : he staining ; b and e ( 200 ) , c and f ( 400immunohistochemical staining . tumor and paracancerous tissues all expressed traf6 mrna . the traf6 mrna expression level in tumor tissues ( 15.21 2.43 ) was significantly higher than in paracancerous tissues ( 4.23 1.13 ; p < 0.01 ) ( figure 2 ) . the positive expression rate of traf6 was not correlated with the age or gender of the patients ( p > 0.05 ) , whereas it was correlated with dukes staging , degree of differentiation , and lymphatic metastasis ( p < 0.05 ) . traf6 was highly expressed in patients with later stages , lower degrees of differentiation , and lymphatic metastases ( table 2 ) . the expression of traf6 was negatively correlated with the 5-year survival rate ( figure 3 ) ; the patients in whom more traf6 was expressed survived longer . the he staining results for the tumor and paracancerous tissues are shown in figure 1 . significantly more traf6 protein was expressed in tumor tissues than in paracancerous tissues . in tumor tissues ( figure 1 a - c ) , traf6 protein was mainly expressed in the cytoplasm , showing dark brown particles . meanwhile , tumor stromal cells were also positively stained . in paracancerous tissues ( figure 1 d - f ) , however , traf6 protein was mainly scattered in the cytoplasm , exhibiting light brown particles . in the meantime , traf6 was weakly positively expressed in the cytoplasm of normal tissues ( positive rate : 33.3% ) , which was significantly less than in tumor tissues ( positive rate : 66.7% ; p < 0.05 ) ( table 1 ) . a - c , traf6 expression in tumor ; and d - f , paracancerous tissues . a and d ( 100 ) : he staining ; b and e ( 200 ) , c and f ( 400immunohistochemical staining . tumor and paracancerous tissues all expressed traf6 mrna . the traf6 mrna expression level in tumor tissues ( 15.21 2.43 ) was significantly higher than in paracancerous tissues ( 4.23 1.13 ; p < 0.01 ) ( figure 2 ) . the positive expression rate of traf6 was not correlated with the age or gender of the patients ( p > 0.05 ) , whereas it was correlated with dukes staging , degree of differentiation , and lymphatic metastasis ( p < 0.05 ) . traf6 was highly expressed in patients with later stages , lower degrees of differentiation , and lymphatic metastases ( table 2 ) . the expression of traf6 was negatively correlated with the 5-year survival rate ( figure 3 ) ; the patients in whom more traf6 was expressed survived longer . as a member of the traf family , traf6 consists of 530 amino acids , including a highly conservative traf6 domain at the c terminus and an activated coiled - coil domain at the n terminus . in particular , the activated domain comprises a ring finger and five zinc fingers ( 6 ) . out of all of the trafs , traf6 is the only member that directly binds to interleukin receptor - associated kinases , nf-b receptor activator , and cd40 . through the signaling pathways mediated by membrane surface receptors , such as lps , il-1 , tlr4 , and cd40 , traf6 is able to finally activate nf-b , activator protein 1 , and pkb / akt . since traf6 can activate several signaling pathways simultaneously , it plays a crucial role in the growth , proliferation , differentiation , and death of cells ( 7 ) . it was previously reported that traf6 dominantly controlled the onset , progression , invasion , and metastasis of tumors ( 3 , 8 , 9 ) . traf6 has also controlled the proliferation and invasion of tumor cells in lung cancer and esophagus cancer tissues by regulating the activity of nf-b ( 3 , 10 ) , and it has been shown that inhibiting the activity of traf6 in multiple myeloma cells could suppress their proliferation and promote apoptosis ( 11 ) . the expression of akt was shown to be significantly elevated in colon cancer tissues ( 12 ) , and inhibiting the activity of the akt enzyme could promote the apoptosis of tumor cells and suppress their proliferation ( 13 ) . moreover , nf-b is highly expressed in 40% of colon cancer tissues , which is closely associated with the infiltration and metastasis of these tissues . however , inhibiting the activity of nf-b could suppress the proliferation of these cells . therefore , we postulated that traf6 , as the key enzyme for the activation of akt and nf-b , may be highly expressed in colon cancer tissues . compared with paracancerous tissues , the expression of traf6 in tumor tissues was significantly increased , suggesting that traf6 may play a critical role in the onset , development , invasion , and metastasis of colon cancer . patients with dukes stage c or d , and those with lymphatic metastases , had significantly higher traf6 expression levels than those with stage a or b disease , and those without colon cancer . in addition , the 5-year survival rate of the patients with positive traf6 expression was significantly lower than in those with negative expression . hence , it is of value to detect the expression level of traf6 in colon cancer tissues in clinical practice . with regard to the mechanism behind the regulatory effect of traf6 on the onset and progression of colon cancer cells , sun et al . found that after binding to hif-1 , traf prevented it from degradation by polyubiquitination linked with lys63 ( 14 ) . as a result , hif-1 protein was destabilized and induced to accumulate in cells , which controlled the proliferation of tumor cells and angiogenesis by regulating the expression of many genes . although primarily the effects of traf6 on colon cancer have been studied , this disease s pathogenesis , and the possibility of treating it by down - regulating the expression of traf6 , should be further evaluated in animal models in order to provide valuable theoretical evidence for colon cancer therapy using traf6 interventions . in summary , traf6 was abnormally expressed in colon cancer tissues , which was closely associated with the prognosis . targeted traf6 therapy , as a novel protocol , may be effective for mitigating colon cancer .

background : tumor necrosis factor receptor - associated factor 6 ( traf6 ) has been reported to be highly expressed in many tumor tissues , but it is still of value to detect its expression in patients with colon cancer and to clarify its clinical significance.objectives:to study the expression and clinical significance of traf6 in patients with colon cancer.patients and methods : a total of 135 patients with colon cancer who underwent surgery at our hospital from february 2008 to march 2013 were selected . the expression levels of traf6 protein in tumor and paracancerous tissues were detected with the immunohistochemical method to evaluate their clinical significance.results:the positive expression rate of traf6 in tumor tissues was 66.7% , which was significantly higher than in normal paracancerous tissues ( 18.5% ) . the positive expression rate of traf6 was significantly correlated with dukes staging , degree of differentiation , and lymphatic metastasis ( p < 0.05 ) , but not gender or age ( p > 0.05 ) . the expression level of traf6 was negatively correlated with the 5-year survival rate ; patients with high traf6 expression levels had significantly decreased survival compared to those with low levels ( p < 0.05).conclusions : traf6 plays an important role in the onset of colon cancer ; therefore , drugs targeting it may have great clinical significance in the treatment of this disease .

1. Background 2. Objectives 3. Patients and Methods 3.1. General Information 3.2. Immunohistochemical and HE Staining 3.3. Detection of TRAF6 mRNA Expression in Tumor and Normal Tissues by RT-PCR 3.4. Statistical Analysis 4. Results 4.1. TRAF6 Expression in Tumor and Paracancerous Tissues 4.2. RT-PCR Results 4.3. Correlation Between TRAF6 Expression and Clinicopathological Characteristics, Staging of Colon Cancer 4.4. Correlation Between TRAF6 Expression and 5-Year Survival Rate 5. Discussion

the calcifying odontogenic cyst ( coc ) is an uncommon odontogenic lesion , which was described by gorlin et al . in 1962 as a distinct entity . since then , literature shows a large controversy regarding terminology and classification in spite of the currently acceptation of gorlin 's original designation by the world health organization ( who ) in 1971 . coc is thought to represent a non - neoplastic lesion , but it has a potential for continuous growth . a lot of confusion and disagreement is present in the terminology and classification of coc . some investigators have considered coc as a tumor with a tendency for marked cyst formation . this concept , called monistic by toida has led some researchers to substitute the terms calcifying ghost cell odontogenic tumor or approach has been suggested that coc can contain two entities : cyst : calcifying ghost cell odontogenic cystneoplasm ( benign : calcifying ghost cell odontogenic tumor ; or malignant : malignant calcifying ghost cell odontogenic tumor ) ; and combined lesion : each of the categories described above associated with odontoma , ameloblastoma , or other odontogenic lesions . cyst : calcifying ghost cell odontogenic cyst neoplasm ( benign : calcifying ghost cell odontogenic tumor ; or malignant : malignant calcifying ghost cell odontogenic tumor ) ; and combined lesion : each of the categories described above associated with odontoma , ameloblastoma , or other odontogenic lesions . the calcifying cystic odontogenic tumor ( ccot ) represents less than 2% of all odontogenic tumors and cysts . the intraosseous ccot ( iccot ) is an unilocular or multilocular destructive radiolucent lesion that may contain irregular calcifications . ccot is usually an asymptomatic slow growing swelling of the jaws or gingival tissues , depending upon whether the lesion is intra or extraosseous . it has been reported to be found between the 1 and 9 decades of life , with a highest incidence occurring in the 6 - 7 decades and tends to appear with equal frequency amongst males and females . clinically it appears as a localized or pedunculated gingival mass with no distinctive features and radio graphically it shows no or only superficial bone involvement with surface erosion . the histological features of eccot are similar to the iccot characteristics , and include an epithelial lining of varied thickness , composed of a distinct columnar ( and occasionally cuboidal ) basal layer , with cells that contain darkly staining nuclei , polarized away from the basement membrane and palisading , similar to the ameloblasts . within the epithelial lining , there is an irregular collection of cells , including sheets of stellate reticulum and eosinophilic cells so called ghost cells . next to the basal layer , irregular or dysplastic dentin and osteoid can also be found . the present paper reports a case of an eccot in a 17-year - old male with the brief review of relevant literature . a 17-year - old male patient reported with a chief complaint of swelling in relation to the right region of the lower jaw , since 4 months . on intra - oral examination , a pinkish dome - shaped , oval nodular growth was seen measuring about 1 1 cm in diameter , in relation to 48 region . the overlying mucosa was normal with superficial blood vessels , giving a pinkish appearance [ figure 1 ] . intra - oral periapical radiograph ( iopa ) shows no osseous involvement , but vertically impacted 48 [ figure 2 ] . based on these features a differential diagnosis of hyperplastic or reactive lesions arising from the gingival and eruption cyst was made . clinical appearance of the growth radiograph intra - oral periapical radiograph in relation to 48 excisional biopsy under local anesthesia was performed . microscopic examination showed a cyst lined by an epithelium with numerous ghost cells and few calcified masses . the basal cells were cuboidal to columnar with darkly staining nuclei , polarized away from the basement membrane , and showed palisading appearance similar to ameloblasts [ figure 3 ] . basal cells are cuboidal to columnar in shape showing hyperchromatism , few showing reverse polarity with palisading appearance ( h and e , 10 ) coc or gorlin cyst was described first by gorlin and pretarious as a definite pathologic entity . a rare developmental odontogenic lesion ( less than 2% of jaw cysts ) is described as having features of both cystic and solid neoplasm . the latest who classification 2005 of odontogenic tumors [ table 1 ] renamed the cystic type of this entity as ccot with four subtypes . world health organization calcification ( 2005 ) of calcifying cystic odontogenic tumor and dentinogenic ghost cell tumor the term dentinogenic ghost cell tumor was retained for neoplastic type . most cases of ccot arise centrally from within the bone , but occasionally they may occur peripherally . eccot has been reported to be found between the 1 and 9 decades of life , with a highest incidence occurring in the 6 - 7 decades , and tends to appear with equal frequency amongst males and females . it is usually present as a slowly enlarging asymptomatic mass with smooth surface in the gingiva or alveolar mucosa . the clinical differential diagnosis of eccot includes common gingival growths of non - neoplastic nature and other peripheral odontogenic tumors . the most common locations included the incisor - cuspid and the premolar regions , with only a few lesions located in the molar - retromolar region . the size of the lesion ranged from 0.5 to 4 cm in greatest diameter and the majority of cases were less than 1.5 cm . clinically , a variety of diagnosis are made by clinicians , the most common being the fibroma or fibrous hyperplasia . other differential diagnoses are gingival cyst of adult and peripheral ameloblastoma . however , currently the origin of eccot is not known but regarding to the histogenesis of eccot two major sources of origin may be considered . the lesion which are located entirely within connective tissue of gingiva and separated from the surface epithelium by a band of connective tissue ( just like present case ) probably arise from remnants of the dental lamina whereas other lesions appear arising from the surface epithelium . radio graphically , it has been reported that eccot appears as a radiolucent area with scattered amounts of calcification . microscopically four variants of eccot like ccot were distinguished : type 1 ( simple cystic),type 2 ( odontoma associated),type 3 ( ameloblastomatous proliferating),type 4 ( eccot associated with benign odontogenic tumors other than odontoma ) . type 1 ( simple cystic ) , type 2 ( odontoma associated ) , type 3 ( ameloblastomatous proliferating ) , type 4 ( eccot associated with benign odontogenic tumors other than odontoma ) . type 1 is the most common pattern characterized by cystic epithelial lining in which a well - defined basal layer of columnar cells and overlying layers resemble stellate reticulum of enamel organ with masses of ghost cells , which may be located within the epithelial lining or fibrous capsule . the presence of ghost cells in ccot is not pathognomonic , having been described in ameloblastoma , ameloblastic fibroma , ameloblastic fibro - odontoma , and odontomas . sometimes variable quantities of dentinoid material are laid down adjacent to epithelial lining . in some instances , the microscopic differential diagnosis , especially type 1 and 3 must be made with peripheral ameloblastoma . both lesions share the presence of prominent elongated basal cells and stellate reticulum zone , but the presence of ghost cells is rare in ameloblastoma . eccot has a less aggressive behavior than the intraosseous counterpart and a simple excision biopsy is curative . follow - up treatment ranged from 9 months to 60 months , only one case of recurrence could be observed . in summary , the current paper presents a case of eccot ( type 1 ) in an 17-year - old male patient , located in the posterior alveolar mucosa in relation to 48 region , measuring about 1 1 cm in diameter . it should be included by the general dentists in the differential diagnosis of other oral hyperplastic / reactive lesions or cystic lesion arising from the gingiva or alveolar mucosa due to its clinical similarities . more data are needed to draw conclusion relative to any differences in their nature and behavior .

odontogenic cysts comprise a diverse group of exceptional lesions derived from epithelial elements of the tooth - forming apparatus . calcifying cystic odontogenic tumor is a rare odontogenic lesion , which represents about 2% of all odontogenic tumors and cysts . it may occur in a central ( intraosseous ) or peripheral ( extraosseous calcifying cystic odontogenic tumor [ eccot ] ) location . eccot in contrast to central , tends to affect the older patients , commonly located in the anterior lower jaw , is less aggressive and the recurrence is rare . here , we report clinico - pathological and radiographic features of eccot located in relation to 48 ( posterior lower jaw ) measuring about 1 cm in the greatest diameter in a 17-year - old male .

I C D

all - ceramic restorations have gained more popularity due to their high esthetic , high improvement in their fracture strength and good biocompatible properties . recently , yttria - stabilized tetragonal zirconia polycrystals ( y - tzp ) have been introduced to the dental professionals . these materials have to be fabricated in cad / cam ( computer - aided design / computer - aided manufacturing ) procedures that have been investigated under in vivo conditions.1 the partially stabilized zirconia shows high fracture strength and structural reliability compared to glass - ceramics when fabricated into prostheses framework . however , due to their low translucency of the light , all zirconia frameworks have to be veneered with glass - ceramics or porcelain for esthetic reasons . these veneering materials have to directly face with chewing force and moisture , resulting in cracks or chipping.2 chipping of a veneering material is a typical failure of all types of ceramic covered dental prostheses . these chipping problems have been reported with porcelain fused to metal ( pfm ) restorations . various factors that might have an effect on chipping have been proposed , such as adhesion between framework and veneering , veneering thickness , and supporting morphology of the finish line.2 nowadays , frameworks for all - ceramic crown design by cad / cam have been based upon empirical machine guidelines rather than clinical scientific data . most of all cad / cam systems , the frameworks of the crowns are design to arbitrary thicknesses of 0.4 to 0.6 mm . like porcelain fused to metal restorations , zirconia frameworks should be designed to provide the appropriate veneering porcelain thickness and support to minimize internal stress , reduce mechanical failures , and optimize esthetics of the veneering porcelains.4 the objective of this study is to compare the failure load and failure characteristics of two different zirconia framework designs of premolar crowns when subjected to static loading . cobalt - chromium casting master die was replicated from prepared preformed plastic maxillary right second premolar for all ceramic crowns . the preparation was prepared leaving a 0.8 mm deep chamfer finishing line ; 1.5 mm of occlusal reduction ; 6 occlusal convergence angle . the master die was scanned using the ineos blue scanner ( sirona , long island city , ny , usa ) . scanned data were computed and then designed for all - ceramic crown framework using the cerec 3d software ( sirona , long island city , ny , usa ) . first , the 0.5 mm thick framework ( ev ) including 0.5 mm thick crown margin was prepared ( fig . second , cutback design was prepared as same as that of metalceramic crowns to obtain uniform , adequate thickness and support of 0.8 mm thick of the veneering porcelain ( cb ) with the same crown margin 0.5 mm . subsequently , zirconia frameworks ( 10 frameworks / each group ) were fabricated from pre - sintered zirconia ingot ( incoris zi , sirona , long island city , ny , usa ) by a cerec inlab mc xl machine ( sirona , long island city , ny , usa ) . after milling , the framework was removed from the machine and final sintering in an infire htc speed furnace ( sirona , long island city , ny , usa ) . the framework was veneered with vita vm9 porcelain ( vident , brea , ca , usa ) using brushing technique to derive the anatomical crown shape . the veneering porcelain was constructed under vacuum - former sheet to obtain the same total thickness of the crown . all crowns were calibrated by measuring at references point on buccal and lingual incline of the buccal cusp . several pilot specimens ( 4 specimens ) were cut at the reference points to measure the real thickness of both zirconia framework and veneering ceramic . the thickness of veneering ceramic of the ev design crowns was 1.4 0.06 mm whereas those of cb design crowns was 0.8 0.05 mm , and those of zirconia frameworks were 0.5 0.04 mm and 1.1 0.07 mm respectively . before cementation , the internal walls of the crowns and the metal dies were cleaned with water steam . each crown was cemented using resin cement ( relyx u-100 , 3 m espe , st . paul , mn , usa ) onto the metal die , using finger pressure then placed under a 2 kg load for 15 minutes . all specimens were subjected to the universal testing machine ( instron model 5566 , instron corp , norwood , ma , usa ) . the cylinder rod with rounding head , radius 5 mm , was placed on the lingual incline plane of the buccal cusp of the crown . therefore , the load was applied at 45 degree to the long axis of the crown at a crosshead speed of 0.5 mm / min until crown fracture occurred . all specimens were examined under an optical light microscope ( nikon mm-11 , nikon corp , tokyo , japan ) and a scanning electron microscope ( jsm-5410lv , jeol ltd . , cohesive failure : fracture occurred within veneering materials more than 80%adhesive failure : fracture occurred along the zirconia and veneering materials interface more than 80%failure through the zirconia framework cohesive failure : fracture occurred within veneering materials more than 80% adhesive failure : fracture occurred along the zirconia and veneering materials interface more than 80% failure through the zirconia framework the load to failure data was recorded in newtons ( n ) . the difference between the even thickness framework design and cutback framework design of all ceramic crowns were examined using independent sample t - test at confidence interval of 95% ( =.05 ) . the mean and standard deviations of the failure load and failure characteristics of the crowns are shown in table 1 . n while those of cb design showed a bit higher at 1450.4 175.7 n. the t - test showed the significant differences in the failure load among the tested groups ( p<.05 ) . under visual examination , 2 ) , however cohesive or adhesive failure could not be classified by visual inspection . the results in table 1 showed that all ev design crown found cohesive failure while the cb design crown found majority failure through the zirconia framework . all of the crown fracture was found in splitting into a buccal and a lingual half pattern . the fracture lines ran from the position of the loading into the mesio - distal orientation towards the crown margin ( fig . the cutback framework design is based on what has been proposed previously for porcelain fused to metal restorations to overcome the porcelain chipping experienced at that times.5,6 clinically , delamination and minor chip - off fractures of the veneering ceramic were found as the major reason for failures of zirconia fixed restoration.1,7,8 one of the reasons for porcelain fracture is improper framework design . the improper framework design causes the improper support for the porcelain veneer layer and also the nonappropriate thickness of the veneering layer . the modification of the framework design by creating an appropriate support and allowing the proper veneering thickness has been proved to reduce the porcelain chipping rates.4 although there is no control group to compare the result , a clinical study on zirconia modified framework design on fixed partial dentures showed high survival rates in 3 years that can be speculated on the potential of the framework design for providing better porcelain support during function9 . in this study , a significant higher failure load was found for crowns with cutback design with optimal cusp support than the crowns with even thickness design . this result conformed to the previous studies that a framework with anatomical shape crown ( cut back framework ) withstood significantly higher loads before fracture than did crowns with even thickness framework.10 they explained the differences in fracture load by the thicker layer of veneering ceramic on crowns with even thickness framework . the larger the volume of ceramic , the larger the size of the flaw population and the higher the risk of prevalence of critical flaw.11,12 however , the failure characteristic in this study seems contradictory to the bonfante et al . 's study.13 they predominantly found smaller veneer layer fracture on the crowns with cutback design framework than the crowns with even thickness framework . while in this study the cutback design framework crowns predominantly found failure through the zirconia framework whereas all the even thickness framework crowns found only veneer fracture . this may be due to the thick veneering ceramic ( 1.4 mm ) with poor support is acting as the force defender of the crown , leading to veneering ceramic fracture before the loads are high enough to affect the framework . for cutback design crowns , the design creates an appropriate support and appropriate thickness of veneering ceramic ( 0.8 mm ) . the difference of both studies is the position of the load applied onto the crown . in this study , the force was applied on the buccal cusp with 45 degree to the long axis of the crown instead of the central fossa of the occlusal surface . in this study , the force was applied 45 degrees to the long axis of the tooth because clinically , the shear force is the frequent force that occurred on this tooth . the alignment of this tooth is generally inclined buccally at an average 9.5 degrees to the occlusal plane.14 however , the chewing force is not occurred vertically but sliding contact motion . theoretically , the maximum angle that can cause the highest force is 45 degree.15 as a consequence , it was assumed that this angle should cause the maximum load in the clinical application . the force was applied on the lingual incline plane of buccal cusp of premolar crown due to the clinical situation , the shear force occurs on this cusp . maxillary premolar has been found that non - functional cusps of restored teeth have a fracture more frequently than functional cusps.16 according to the studies of farah and craig,17 craig et al.,18 and nally et al.,19 the stress distribution on the porcelain shows isochromatic shearing - stress trajectories radiating from the edges of the punch when loaded in compression . therefore , fracture in porcelain would start at these trajectories running mesio - distal orientation , leading to crown fracture found in splitting into half pattern in this study . the employment of the single load to failure tests to understand the performance of dental materials has its limitations . in clinic , ceramic failure occurs from cumulative damage and slow crack growth20 when it is subjected to cyclic chewing force , approximately 380 n in the premolar area.21 this amount of force is much lesser than the failure force in this study . thus , the results in this study need to be interpreted with caution . in order to obtain more clinically relevant results , further research is necessary , including fatigue loading prior to static load tests for zirconia crowns . within the limitation of this study , the conclusions are as follows different framework designs have influence on the failure load and failure characteristics of all ceramic zirconia crowns . the cutback zirconia design for all ceramic crowns is a promising way to reduce veneer chipping failures .

purposethis in vitro study aimed to compare the failure load and failure characteristics of two different zirconia framework designs of premolar crowns when subjected to static loading.materials and methodstwo types of zirconia frameworks , conventional 0.5 mm even thickness framework design ( ev ) and 0.8 mm cutback of full contour crown anatomy design ( cb ) , were made for 10 samples each . the veneer porcelain was added on under polycarbonate shell crown made by vacuum of full contour crown to obtain the same total thickness of the experiment crowns . the crowns were cemented onto the cobalt - chromium die . the dies were tilted 45 degrees from the vertical plane to obtain the shear force to the cusp when loading . all crowns were loaded at the lingual incline of the buccal cusp until fracture using a universal testing machine with cross - head speed 0.5 mm / min . the load to fracture values ( n ) was recorded and statistically analyzed by independent sample t-test.resultsthe mean and standard deviations of the failure load were 1,170.1 90.9 n for ev design and 1,450.4 175.7 n for cb design . a significant difference in the compressive failure load was found ( p<.05 ) . for the failure characteristic , the ev design was found only cohesive failures within veneering porcelain , while the cb design found more failures through the zirconia framework ( 8 from 10 samples).conclusionthere was a significant difference in the failure load between two designs , and the design of the framework influences failure characteristic of zirconia crown .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

following thyroid cancer , gastric cancer ( gc ) is the second most common cancer in korea . recently , as the korean society is aging , there has been an increasing number of elderly patients diagnosed with gc . despite this , there is little data available on the clinicopathological characteristics of gc in elderly patients , particularly in those who are very elderly . in korea , the life expectancy is more than 80 years and the most common cause of death is cancer . according to the statistics released by statistics korea , approximately 12% of cancer - related deaths although the 5-year survival rate of gc has been increasing over the past two decades due to early detection by screening endoscopy , little is known about gc screening in the very elderly . the safety of endoscopic treatment is comparable to surgery in elderly patients [ 4 - 8 ] . endoscopic resections ( ers ) are also performed on elderly patients with comorbid heart and lung diseases . therefore , the strategy of gc treatment varies in elderly patients with age - associated comorbidities . age is a clinically important factor for determining the treatment modality , and clinicians are often cautious when managing elderly patients with gc because of their age - related performance status [ 11 - 13 ] . this study investigated the clinicopathological characteristics of gc in extremely old ( over 85 years ) patients who received treatment or conservative observations . from june 1998 to july 2014 , a total of 358 patients over 85 years old were diagnosed with gc at severance hospital ( seoul , korea ) . for the present analysis , the following patients were excluded ( 1 ) those who had no subsequent follow - up visits after diagnosis , ( 2 ) those who had a significant comorbidity that could affect the mortality , and ( 3 ) those who had been treated at another hospital during the follow - up period . the patients were evaluated in terms of their general information , such as age , sex , and treatment method . the variables investigated also included the macroscopic classification , histological classification , comorbidity , postoperative mortality , and clinical outcome . as categorized by the treatment modality for gc , 81 patients underwent treatment ( treatment group ) , such as an endoscopic resection , surgery , chemotherapy , or radiotherapy , and 89 patients received conservative observation ( conservative group ) . therefore , the patients were divided roughly into two groups : those with macroscopically early gcs ( cancer confined to the mucosa or submucosa ) and those with macroscopically advanced gc ( cancer with invasion extending through the muscularis propria ) . the institutional review board ( irb ) of severance hospital approved this study ( irb approval number : 4 - 2014 - 0464 ) . the chi - square test and fisher exact test were used to compare the clinicopathological characteristics between the treatment - modality groups . follow - up data on all patients were obtained from the korea national health insurance service database . the median follow - up period was 17.8 months ( range , 0.1 to 172.4 months ) . all statistical analyses were performed using spss ver . 20.0 for windows ( ibm co. , armonk , ny ) . from june 1998 to july 2014 , a total of 358 patients over 85 years old were diagnosed with gc at severance hospital ( seoul , korea ) . for the present analysis , the following patients were excluded ( 1 ) those who had no subsequent follow - up visits after diagnosis , ( 2 ) those who had a significant comorbidity that could affect the mortality , and ( 3 ) those who had been treated at another hospital during the follow - up period . the patients were evaluated in terms of their general information , such as age , sex , and treatment method . the variables investigated also included the macroscopic classification , histological classification , comorbidity , postoperative mortality , and clinical outcome . as categorized by the treatment modality for gc , 81 patients underwent treatment ( treatment group ) , such as an endoscopic resection , surgery , chemotherapy , or radiotherapy , and 89 patients received conservative observation ( conservative group ) . therefore , the patients were divided roughly into two groups : those with macroscopically early gcs ( cancer confined to the mucosa or submucosa ) and those with macroscopically advanced gc ( cancer with invasion extending through the muscularis propria ) . the institutional review board ( irb ) of severance hospital approved this study ( irb approval number : 4 - 2014 - 0464 ) . the chi - square test and fisher exact test were used to compare the clinicopathological characteristics between the treatment - modality groups . follow - up data on all patients were obtained from the korea national health insurance service database . the median follow - up period was 17.8 months ( range , 0.1 to 172.4 months ) . all statistical analyses were performed using spss ver . 20.0 for windows ( ibm co. , armonk , ny ) . the sex or histologic type was similar in the two groups . on the other hand , older age , macroscopically advanced cancer , and upper - middle located cancer were significantly more common in the conservative group ( table 1 ) . in the treatment group , 21 patients underwent er , 48 patients underwent gastrectomy , and 12 patients underwent chemotherapy or radiotherapy . of the 21 patients who received er , 20 had lesions included in the absolute indication and one patient had lesions included in the expanded indication . of the 48 patients who received surgery , 46 underwent a curative gastric resection ( subtotal gastrectomy , n=43 ; total gastrectomy , n=3 ) and two received palliative gastrectomy . the curative resection rate was 18/21 ( 85.7% ) for er and 46/48 ( 95.8% ) for gastrectomy . in the conservative observation group , 38/89 patients ( 42.7% ) refused the recommended treatment . in the overall study cohort , the most common concomitant disease was hypertension ( 32.9% ) . eighty - two patients ( 48.2% ) had one or more comorbid diseases and 37 patients ( 21.8% ) had more than two comorbid diseases . one hundred forty - three patients ( 84.1% ) visited the hospital with symptoms and 27 patients ( 15.9% ) were diagnosed with gc during a health check - up . the most common initial presentation was abdominal pain , which was present in 69 patients ( 40.6% ) . thirty - seven patients ( 21.8% ) had alarm signs ( weight loss , signs and symptoms of upper gastrointestinal bleeding , and anemia ) , and the presence of an alarm sign was significantly more common in the conservative observation group than in the treatment group . on the other hand , a postoperative complication ( bleeding ) occurred in one patient who underwent a gastrectomy ; this patient died three days after surgery . the overall survival rate in the treatment group was significantly higher than that in the conservative group ( fig . furthermore , the disease - specific mortality was significantly lower in the treatment group than in the conservative group ( fig . the 81 patients in the treatment group and the 89 patients in the conservative group were divided into early gc and advanced gc subgroups , as shown in fig . the overall survival rate was significantly higher in the treatment group than in the conservative group . for patients with early gc , the overall survival rate was also significantly higher in the treatment group than in the conservative group ( p < 0.001 ) ( fig . similarly , the disease - specific mortality in the treatment group was significantly lower than that in the conservative group , regardless whether early or advanced gc had been diagnosed ( p < 0.001 ) ( fig . subgroup analysis was performed on the overall survival and disease specific mortality between the conservative observation group who refused the recommended treatment and the treatment group . the overall survival rate of the treatment group was significantly higher than the conservative observation group who refused the recommended treatment and the disease specific mortality rate was significantly lower ( fig . the patients in the treatment group and the conservative observation group who refused the recommended treatment were divided into early gc and advanced gc subgroups . the overall survival rate was significantly higher and the disease - specific mortality rate was significantly lower in the treatment group than in the conservative observation group who refused the recommended treatment , regardless whether early or advanced gc had been diagnosed ( fig . the overall survival rate of the er and surgery group was similar and the disease - specific mortality of the er and surgery group was also similar ( data not shown ) . seven factors ( clinical course , age , sex , histologic type , macroscopic classification , alarm sign , and concomitant disease ) were evaluated by univariate analyses to investigate the associations of the clinicopathological characteristics with the prognosis . significant differences were observed for the clinical course ( p < 0.001 ) , age ( p=0.027 ) , histologic type ( p=0.031 ) , alarm sign ( p < 0.001 ) , and macroscopic classification ( p the same seven factors were then analyzed by multivariate analysis , which revealed the clinical course ( p < 0.001 ) , alarm sign ( p=0.009 ) , and macroscopic classification ( p < 0.001 ) to be independent prognosis factors ( table 3 ) . the sex or histologic type was similar in the two groups . on the other hand , older age , macroscopically advanced cancer , and upper - middle located cancer were significantly more common in the conservative group ( table 1 ) . in the treatment group , 21 patients underwent er , 48 patients underwent gastrectomy , and 12 patients underwent chemotherapy or radiotherapy . of the 21 patients who received er , 20 had lesions included in the absolute indication and one patient had lesions included in the expanded indication . of the 48 patients who received surgery , 46 underwent a curative gastric resection ( subtotal gastrectomy , n=43 ; total gastrectomy , n=3 ) and two received palliative gastrectomy . the curative resection rate was 18/21 ( 85.7% ) for er and 46/48 ( 95.8% ) for gastrectomy . in the conservative observation group , 38/89 patients ( 42.7% ) refused the recommended treatment . in the overall study cohort , the most common concomitant disease was hypertension ( 32.9% ) . eighty - two patients ( 48.2% ) had one or more comorbid diseases and 37 patients ( 21.8% ) had more than two comorbid diseases . one hundred forty - three patients ( 84.1% ) visited the hospital with symptoms and 27 patients ( 15.9% ) were diagnosed with gc during a health check - up . the most common initial presentation was abdominal pain , which was present in 69 patients ( 40.6% ) . thirty - seven patients ( 21.8% ) had alarm signs ( weight loss , signs and symptoms of upper gastrointestinal bleeding , and anemia ) , and the presence of an alarm sign was significantly more common in the conservative observation group than in the treatment group . on the other hand , a postoperative complication ( bleeding ) occurred in one patient who underwent a gastrectomy ; this patient died three days after surgery . the overall survival rate in the treatment group was significantly higher than that in the conservative group ( fig . 1a ) . furthermore , the disease - specific mortality was significantly lower in the treatment group than in the conservative group ( fig . the 81 patients in the treatment group and the 89 patients in the conservative group were divided into early gc and advanced gc subgroups , as shown in fig . the overall survival rate was significantly higher in the treatment group than in the conservative group . for patients with early gc , the overall survival rate was also significantly higher in the treatment group than in the conservative group ( p < 0.001 ) ( fig . similarly , the disease - specific mortality in the treatment group was significantly lower than that in the conservative group , regardless whether early or advanced gc had been diagnosed ( p < 0.001 ) ( fig . subgroup analysis was performed on the overall survival and disease specific mortality between the conservative observation group who refused the recommended treatment and the treatment group . the overall survival rate of the treatment group was significantly higher than the conservative observation group who refused the recommended treatment and the disease specific mortality rate was significantly lower ( fig . the patients in the treatment group and the conservative observation group who refused the recommended treatment were divided into early gc and advanced gc subgroups . the overall survival rate was significantly higher and the disease - specific mortality rate was significantly lower in the treatment group than in the conservative observation group who refused the recommended treatment , regardless whether early or advanced gc had been diagnosed ( fig . the overall survival rate of the er and surgery group was similar and the disease - specific mortality of the er and surgery group was also similar ( data not shown ) . seven factors ( clinical course , age , sex , histologic type , macroscopic classification , alarm sign , and concomitant disease ) were evaluated by univariate analyses to investigate the associations of the clinicopathological characteristics with the prognosis . significant differences were observed for the clinical course ( p < 0.001 ) , age ( p=0.027 ) , histologic type ( p=0.031 ) , alarm sign ( p < 0.001 ) , and macroscopic classification ( p < 0.001 ) ( table 2 ) . the same seven factors were then analyzed by multivariate analysis , which revealed the clinical course ( p < 0.001 ) , alarm sign ( p=0.009 ) , and macroscopic classification ( p < 0.001 ) to be independent prognosis factors ( table 3 ) . as the society ages , clinicians are increasingly being confronted with the challenges of treating elderly patients . in korea , the number of patients diagnosed with gc at extremely old ages has been increasing . several reports have shown that older patients tend to receive less treatment for gcs than younger patients [ 14 - 18 ] . when clinicians make the decision to treat cancer , they consider the cancer stage , the patient s performance status , deterioration of the patient s mental status , and concomitant disease together ; they do not rely on the patient s age alone . generally , the prognosis of elderly patients has been thought to be worse than that of younger patients because of concomitant disease . on the other hand , deaths due to other concomitant diseases amounted to 34%-37% of the total deaths in > 80-year - old patients with gc . investigated patients with gc who were older than 80 years and reported that the survival rate after surgical treatment was significantly higher than that for conservative observation , regardless of the patient s performance status and irrespective of whether his or her mental status had deteriorated . the macroscopic classification of the case ( e.g. , early or advanced cancer ) was an important prognostic factor . treatments are recommended for gc , even for older patients with age - associated comorbidities that are under control . in a study of the natural history of early gc , tsukuma et al . even patients with early gc usually die within 3 years in the absence of treatment . on the other hand , katai et al . reported that when a gastrectomy had been performed safely by specialists , the survival rate of elderly patients with early gc was not significantly different from that of the general population . therefore , the treatment of early gc should not be discouraged in very elderly patients . in this study , patients in the treatment group , those with early gc , and those without an alarm sign showed higher overall survival rates than the others . compared to gastrectomy , er is a less invasive treatment for gc and has many advantages . these advantages include preservation of the stomach , which increases the quality of life and reduces the length of hospitalization compared to gastrectomy . furthermore , er - related complications result from the difficulties encountered when performing the resection endoscopically , which are associated with the tumor size and location , rather than age . several reports have shown that er is a safe and feasible treatment for gc in elderly patients . reported that the results of an endoscopic mucosal resection in patients older than 80 years were outstanding , and were the same as those obtained via gastrectomy . therefore , elderly patients with earlier - stage cancer should receive less - invasive treatments . as the techniques of preoperative and perioperative management have improved , the outcomes of gastric surgery in the elderly have also shown favorable results . similarly , the overall survival rate of the patients in the present study s treatment group was significantly higher than that in the conservatively observed group . in this study , advanced gc accounted for 72.9% of cases . the percentage of patients with advanced cancer was significantly higher in the conservative observation group than in the treatment group . therefore , it is important to recognize cancers at the earlier stage to provide a better prognosis in elderly patients . in a previous study , in the present study , 88 patients ( 51.8% ) did not have any concomitant disease . furthermore , 143 patients ( 84.1% ) presented with symptoms and 37 patients ( 21.8% ) showed an alarm sign . therefore , elderly patients who are being followed up for concomitant disease also need to be checked for gastrointestinal disease . when patients have symptoms , the clinicians need to consider further evaluations . this study was retrospective and could not investigate exactly how the decision was made as to whether to treat the patients or not . in addition , the tnm classification of the patients could not be evaluated because the staging of the patients in the conservative group could not be investigated accurately . in this study , however , the overall survival of the patients with advanced gc was significantly lower than in the early gc chronological age alone should not be used as a strategy of determining if treatment will be administered for gc . patients with early gc or lower - risk preexisting comorbidities should not be discouraged from treatment , even if they are older than 85 years .

purposegastric cancer is the third - leading cause of cancer - related death in korea . as the korean population is ageing , the number of extremely old patients with this disease is increasing . this study examined the clinicopathological characteristics of gastric cancer in extremely old ( over 85 years ) patients who received treatment or conservative observations and compared the treatment outcomes according to the treatment modality.materials and methodsa total of 170 patients over 85 years of age were diagnosed with gastric cancer . of these , 81 underwent treatment for gastric cancer and 89 received conservative observations . the clinicopathological characteristics of the treatment and conservative groupswere compared.resultsthe mean age of the patients was 86.5 years . the conservative group included significantly more patients with older ages , macroscopically advanced cancer and upper - middle located cancer . the overall survival rate of the treatment group was significantly higher than that of the conservative group . the disease - specific mortality rate was significantly lower in the treatment group than in the conservative group . multivariate analysis revealed the clinical course , alarm sign , and macroscopic classification to be independent prognosis factors.conclusionby itself , the chronological age should not be used as a strategy to determine whether treatmentwill be administered for gastric cancer . patients who have early gastric cancer or lower - risk preexisting comorbidities should not be discouraged from treatment , even if they are older than 85 years .

Introduction Materials and Methods 1. Patients 2. Statistical analysis Results 1. Clinicopathological characteristics 2. Clinical outcomes 3. Survival 4. Effects of clinicopathological characteristics on prognosis Discussion Conclusion

a 19-year - old man injured his left foot in a motorcycle accident . during the fall , the foot was forcibly twisted . a clinical examination showed that the foot was supinated and severely contaminated on the anterolateral side . initial radiographs of the ankle revealed complete absence of the talus and fractures of the lateral malleolus and anterior process of the calcaneus ( fig . the patient was shifted to the operating room , and wound irrigation and debridement were performed , followed by internal fixation of the lateral malleolus and calcaneus . because of the void created by the absent talus , a temporary antibiotic impregnated polymethyl - methacrylate cement spacer was inserted in the talar space , and a triplanar external fixator was placed across the ankle joint ( fig . , the extruded talus was found in a trash basket near the site of the accident and was transferred to the hospital . it was heavily contaminated and scratched with soil and rubbish . we thoroughly cleaned the talus by pulsatile lavage in the operating room , and it was kept frozen . a reverse fasciocutaneous flap from the calf muscle was used for coverage of a palm - sized soft tissue defect of the anterolateral aspect of the ankle . the cement spacer was removed 4 months later , and tibiocalcaneal fusion with bone grafts was performed instead of reimplantation of the frozen talus . we packed the defect tightly with a cancellous bone graft from the anterior superior iliac spine and a frozen allograft of the femoral head . antibiotic - impregnated cement beads were inserted after removing the bone grafts and aggressive curettage of the talar space . compressive tibiocalcaneal fusion using an ilizarov external fixator and an additional autogenous iliac bone graft were reperformed ( fig . 3 ) . we achieved fusion 21 months after the injury followed by proximal tibial lengthening with the ilizarov external fixator for the 4 cm leg length discrepancy . the patient is walking with full weight - bearing without any aids for regular activities of daily living 8 years after the injury . solid fusion between the tibia , calcaneus , and navicular was observed on a lateral radiograph ( fig . 4 ) . a 45-year - old man sustained a fall from 5 m while rock - climbing . the patient sustained a pantalar dislocation with a > 10 cm sized transverse open wound on the medial aspect of the ankle ( fig . initial radiographs showed complete absence of the talus and a tip fracture of both malleoli . the totally extruded talus was found on site but was severely contaminated with gravel , soil , and mud . we had anticipated an inevitable avascular necrosis of the talus , as it was completely deprived of a blood supply ; therefore , we planned to perform an initial subtalar fusion expecting revascularization from the calcaneus followed by ankle arthroplasty . the talus was temporarily fixed using multiple kirschner wires after massive wound debridement and irrigation because of the high risk for infection . a primary subtalar fusion was performed 4 days later , with reimplantation of the extruded talus and internal fixation with cannulated screws . 6 ) . radiographs taken 6 months after the trauma , showed osteolysis on the distal tibial articular surface and loosening of the screws ( fig . 7 ) . with suspicion of infection , curettage of the talar body was performed and an antibiotic impregnated cement spacer was inserted . however , the biopsy result revealed granulation tissue without significant neutrophil infiltration , with necrotic bone and marrow fibrosis . the ankle was fused using multiple screws , and a femoral head allograft was used to fill the talar defect 12 months after the trauma . two years after the injury , the preserved talonavicular joint made mid - foot motion possible and tibio - talar - calcaneal fusion was achieved ( fig . the patient has had a good functional outcome with the ability to enjoy a 4 hour course of tracking twice per week . a 19-year - old man injured his left foot in a motorcycle accident . during the fall , the foot was forcibly twisted . a clinical examination showed that the foot was supinated and severely contaminated on the anterolateral side . initial radiographs of the ankle revealed complete absence of the talus and fractures of the lateral malleolus and anterior process of the calcaneus ( fig . the patient was shifted to the operating room , and wound irrigation and debridement were performed , followed by internal fixation of the lateral malleolus and calcaneus . because of the void created by the absent talus , a temporary antibiotic impregnated polymethyl - methacrylate cement spacer was inserted in the talar space , and a triplanar external fixator was placed across the ankle joint ( fig . , the extruded talus was found in a trash basket near the site of the accident and was transferred to the hospital . it was heavily contaminated and scratched with soil and rubbish . we thoroughly cleaned the talus by pulsatile lavage in the operating room , and it was kept frozen . a reverse fasciocutaneous flap from the calf muscle was used for coverage of a palm - sized soft tissue defect of the anterolateral aspect of the ankle . the cement spacer was removed 4 months later , and tibiocalcaneal fusion with bone grafts was performed instead of reimplantation of the frozen talus . we packed the defect tightly with a cancellous bone graft from the anterior superior iliac spine and a frozen allograft of the femoral head . antibiotic - impregnated cement beads were inserted after removing the bone grafts and aggressive curettage of the talar space . compressive tibiocalcaneal fusion using an ilizarov external fixator and an additional autogenous iliac bone graft were reperformed ( fig . 3 ) . we achieved fusion 21 months after the injury followed by proximal tibial lengthening with the ilizarov external fixator for the 4 cm leg length discrepancy . the patient is walking with full weight - bearing without any aids for regular activities of daily living 8 years after the injury . solid fusion between the tibia , calcaneus , and navicular was observed on a lateral radiograph ( fig . a 45-year - old man sustained a fall from 5 m while rock - climbing . the patient sustained a pantalar dislocation with a > 10 cm sized transverse open wound on the medial aspect of the ankle ( fig . initial radiographs showed complete absence of the talus and a tip fracture of both malleoli . the totally extruded talus was found on site but was severely contaminated with gravel , soil , and mud . we had anticipated an inevitable avascular necrosis of the talus , as it was completely deprived of a blood supply ; therefore , we planned to perform an initial subtalar fusion expecting revascularization from the calcaneus followed by ankle arthroplasty . the talus was temporarily fixed using multiple kirschner wires after massive wound debridement and irrigation because of the high risk for infection . a primary subtalar fusion was performed 4 days later , with reimplantation of the extruded talus and internal fixation with cannulated screws . additional reduction and fixation of the fractured medial malleolus 6 ) . radiographs taken 6 months after the trauma , showed osteolysis on the distal tibial articular surface and loosening of the screws ( fig . 7 ) . with suspicion of infection , curettage of the talar body was performed and an antibiotic impregnated cement spacer was inserted . however , the biopsy result revealed granulation tissue without significant neutrophil infiltration , with necrotic bone and marrow fibrosis . the ankle was fused using multiple screws , and a femoral head allograft was used to fill the talar defect 12 months after the trauma . two years after the injury , the preserved talonavicular joint made mid - foot motion possible and tibio - talar - calcaneal fusion was achieved ( fig . the patient has had a good functional outcome with the ability to enjoy a 4 hour course of tracking twice per week . a total talar extrusion associated with a high energy injury is rare and is frequently complicated by soft tissue injuries and fractures . this type of injury is prone to infection , avascular necrosis , secondary arthritis , and poor functional outcome . there is also controversy about the initial surgical treatment . both primary talectomy and salvage of the talus have been recommended , but a primary talectomy in cases in which the talus has been extruded through the wound and contaminated has a high likelihood of infection . smith et al.9 ) and assal and stern1 ) reported that a totally extruded talus should be managed with thorough cleaning and debridement of the wound followed by reimplantation . if the extruded talus is available and replaced , the survival of the talus probably depends on avoiding infection and avascular necrosis , and the existence of residual soft tissue attachment retaining a blood supply to the talus . some authors have reported that reimplantation of an extruded talus with some retaining soft tissue attachments has relatively good clinical results.7,8,9 ) however , in cases without any soft tissue attachments , the result of reimplantation of a likely avascular and contaminated talus , which may act as a sequestrum , is questionable . in contrast , reimplantation and wound closure can make it difficult to perform appropriate wound care that may require debridement and cleaning . only nine cases in six reports have documented complete extrusion of the talus without any soft tissue attachments.1,2,4,5,6,9 ) reimplantations were performed in five of the nine cases , and no subsequent infection or avascular necrosis were detected in the replaced talus . only three cases developed osteoarthritis ( table 1 ) . these results seem to be contradictory to our conception and are unusually excellent , even compared with those after fractures of the talar body or neck . the causes of the poorer results were considered failure of infection control in case 1 and progressive bone resorption in case 2 . palomo - traver et al.7 ) reported that the talus should be replaced unless it is totally extruded or grossly contaminated . it was suggested that impaired circulation related to trauma of the surrounding vascular and lymphatic structures leads to a nonhealing condition and subsequent infection ; thus , predisposing the patient to acute and chronic bone infection.4,10 ) the cause of infection even after talectomy in case 1 seemed to be the unrepairable , contaminated open wound and the dead space created by the absent talus that made it difficult to control the infection . in case 1 , we were worried about infection , and the talus was not available for reimplantation , whereas the talus in case 2 was contaminated but was available , and we replaced it after a thorough wash . we expected that avascular necrosis of the talus was inevitable and planned a primary subtalar fusion and revision ankle arthroplasty . we achieved the subtalar fusion , but had to perform massive debridement for unclearly progressive osteolysis around the ankle joint and then conducted a tibio - talar fusion with an allograft . we presumed that the repeated debridement and cleaning of the wound prevented the infection , but the cause of osteolysis may have been due to poor viability of the deep frozen talus . both of our patients were treated without initial reimplantation to prevent infection . nevertheless , they had complications such as infection and unclearly progressive osteolysis and a long period of disability before achieving fusion . it is a reasonable option to replace an extruded talus as soon as possible after thorough debridement unless the talus is contaminated , because an open wound usually occurs from inside to outside .

total talar extrusion without a soft tissue attachment is an extremely rare injury and is rarely reported . appropriate treatment remains controversial . we describe the long - term outcomes of two patients who had complete talar extrusion without remaining soft tissue attachment treated with arthrodesis . both of our patients had complications such as infection and progressive osteolysis . we suggest reimplantation of the extruded talus after thorough debridement as soon as possible as a reasonable option unless the talus is contaminated or missing , because an open wound may arise from inside to outside .

CASE REPORT Case 1 Case 2 DISCUSSION

sudden cardiac death is a major cause of mortality in the industrialized world , and ventricular fibrillation ( vf ) is the leading cause of sudden cardiac death . an electrical wavebreak in the heart tissue may lead to vf . according to the so - called restitution hypothesis , rate - dependent alteration of action potential duration ( apd ) and refractoriness ( r ) the changes in apd that accompany the changes in rate , reflect the dependence of apd on the preceding diastolic interval ( di ) , a relationship characterized by the electrical restitution curve . all of these mean that the modeling and simulating of restitution property in the cardiac cell is very important . cellular automata ( ca ) may be considered as a method for modeling discrete dynamic systems . a ca consists of a discrete system of lattice sites ( cells ) having various initial values . these cells evolve in discrete time steps , as each cell assumes a new state based on the rules , that is , the states of its local neighborhood and a finite number of previous time steps . the neighborhood is described by specifying the set of cells that are the neighbors of a given cell . ( a ) von neumann ; ( b ) moore a ca lattice may be 1-d or multidimensional . for 2-d ca , if a cell which is still in the refractory state is re - stimulated by an adequate large stimulus ( s2 in figure 2 ) , the generated apd ; apdn+1 will be shorter than that in the normal case , apdn , that is , when the cell is stimulated after complete refractory . illustration of the restitution property , apd and di the action potential duration restitution property refers to the relationship between apdn+1 and the previous diastolic interval din , as shown in figure 2 . di is defined by the duration between the end of the cell 's re - polarization and the next applied stimulation [ figure 2 ] . the plot of apdn+1 versus din is called the restitution curve [ figure 3 ] . a sample restitution curve restitution curve may be explained as a function f : apdn+1 = f(din ) ( 1 ) experimental measurements and observations confirmed that the restitution curve may be fitted by a sigmoid function like : there are different methods for experimental plotting of the restitution curve , like the s1-s2 standard and the dynamical restitution method . action potential duration is usually explained as apd95 or apd90 , apd95 is defined as the time distance between the s2 stimulus ( beginning of ap ) and 95% of the cell 's full repolarization . in this regarding figure 2 , di is defined as follows : di=(s1-s2 interval)-(apd95 caused by last s1 ) ( 3 ) figure 1 illustrates two types of neighborhoods that are generally used in literature . in this article a model based on ca this article is organized as follows : section 2 reviews the restitution property . in section 3 the proposed automata model is introduced , as also the method of implementation of the restitution property in the proposed model . two state variables are assigned to each cell ; one for the action potential variable ( apmn ) and the other for the refractory variable ( rmn ) , where m , n , and t denote the column number , row number , and the time step , respectively . the variable smn stands for the stimulus applied to the cell , which is in the coordinate ( m , n ) in time t. table 1 summarizes the description of the other parameters . definition of parameters used in the proposed model the transition rule ( automata rule ) in the proposed model is defined as follows : i.if smn is greater than the excitation threshold ; and apmn = rmn = 0 , the cell in position mn ; cellmn will be excited in the next time step ; t+1 and apmn = apup and rmn=0ii.if smn < and rmn = apmn = 0 , the cell will not be stimulated and stays in its previous ( rest ) stateiii.if rmn , + apmn 0 , the cellmn will take the path in the state space , as shown in figure 4 . notice that rmn starts decreasing when the apmn reaches its maximum value after a complete evolution in the state space the rmn and apmn will return to their rest states again if smn is greater than the excitation threshold ; and apmn = rmn = 0 , the cell in position mn ; cellmn will be excited in the next time step ; t+1 and apmn = apup and rmn=0 if smn < and rmn = apmn = 0 , the cell will not be stimulated and stays in its previous ( rest ) state if rmn , + apmn 0 , the cellmn will take the path in the state space , as shown in figure 4 . notice that rmn starts decreasing when the apmn reaches its maximum value after a complete evolution in the state space the rmn and apmn will return to their rest states again evolution of the cellmn state variables ; apmn and rmn in the state space . see the text for transition rules the parameter values in the model are set so that the time course of ap and r in a single cell are as depicted in figure 5 . here the continued and dashed lines show ap and r , respectively . referring to figure 5 , it is clear that the general morphology of action potential and refractory in a heart cell are preserved . ( a ) time course of ap and r as implemented in the model ; ( b ) a real ap time course for the r state variable , five phases ( 0 , 1 , 2 , 3 , 4 ) are assumed . in phase 0 phase 1 stands for r when the cell has just entered the rest state . in this phase , r continues decreasing and a strong enough stimulus can re - excite the cell . in phase 4 , r reaches its minimum value ; 0 . here , the cell is in rest and normal stimulus can excite it and generate an ap with normal duration , that is , a normal apd . if a cell is excited during phases 2 or 3 , the restitution property will require that the apd is shorter than normal . the main aim of this research was the simulation of restitution in a single cell , so a lattice of a single cell without any neighbor was considered . the parameter rdown was considered as a time - dependent parameter , as : in eq . 4 , t denotes time and is an adjustable coefficient . the model was implemented by matlab the model parameters were adjusted so that by stimulating a single cell , the outcome of the ap and r morphology was generally similar to a ventricular cell . the outcome is depicted on the left side of figure 6 . referring to figure 6 , ap and r morphology are reserved as well as the restitution property in figure 6 , three stimuli are applied to a cell , at times 0 , 40 , and 70 milliseconds . the time interval between the first and second stimulus is long enough . in other words ; di1 is normal and the cell has enough time for full recovery , and this leads to a normal apd2 . the right hand side of figure 6 indicates that the proposed model can simulate the restitution property , that is , the third apd ( apd3 ) is shorter than the first and second one . after assurance about the installation of the restitution property in the model , its validity should be justified . if the proposed model is precise , it should simulate each of these curves by the adjustment of its parameters . three experimental restitution curves in this research the adjustment of the model was done via the regulation of n , , and apdmax . the normalized mean square error ( nmse ) criterion was used for fitness comparison of the restitution curves . at first , the guinea pig restitution curve of figure 7 was tested . comparison of the guinea pig restitution curve with the simulated curve created by the model . the proposed model can simulate the guinea pig restitution curve second , as an attempt for the model validity justification , the rabbit 1 restitution curve of figure 7 was examined . comparison of the rabbit 1 restitution curve with the simulated curve created by the model . it is clear that the proposed model can simulate the rabbit 1 restitution curve and finally , the rabbit 2 restitution curve of figure 7 was investigated . the outcome is illustrated in figure 10 . here also , it is clear that the proposed model can simulate the restitution curve of rabbit 2 , when its parameters are adjusted as shown in the table of figure 10 . comparison of the rabbit 2 restitution curve with the simulated curve created by the model . a cellular automata - based model was proposed for simulating restitution . for the validation test of the model , the restitution curves that were simulated by the model were evaluated against the experimental results of the validated sources . in this research , the parameters , n , and apdmax were used for model regulation . the results showed that the proposed model could not only simulate restitution properly , but could also be regulated for simulating restitution curves of different kinds of cardiac cells . on account of the low computational load of the automata - based cell models , the model would be very useful for simulating propagation of electrical waves in the ventricular tissue and investigation of the wave break and fibrillation , which would form the matter of the authors future researches . he is with the department of biomedical engineering and the research laboratory of biomedical signals and sensors , iran university of science and technology ( i.u.s.t . ) , tehran , iran . fateme pourhasanzade is a phd student in biomedical engineering department ; iran university of science and technology ( i.u.s.t . ) , tehran , iran .

ventricular fibrillation is the cause of the most sudden mortalities . restitution is one of the specific properties of ventricular cell . the recent findings have clearly proved the correlation between the slope of restitution curve with ventricular fibrillation . this ; therefore , mandates the modeling of cellular restitution to gain high importance . a cellular automaton is a powerful tool for simulating complex phenomena in a simple language . a cellular automaton is a lattice of cells where the behavior of each cell is determined by the behavior of its neighboring cells as well as the automata rule . in this paper , a simple model is depicted for the simulation of the property of restitution in a single cardiac cell using cellular automata . at first , two state variables ; action potential and recovery are introduced in the automata model . in second , automata rule is determined and then recovery variable is defined in such a way so that the restitution is developed . in order to evaluate the proposed model , the generated restitution curve in our study is compared with the restitution curves from the experimental findings of valid sources . our findings indicate that the presented model is not only capable of simulating restitution in cardiac cell , but also possesses the capability of regulating the restitution curve .

INTRODUCTION AIMS MATERIALS AND METHODS RESULTS CONCLUSIONS BIOGRAPHIES

therefore , living organisms persistently explore their environment for extending their boundaries into favorable regions . one fundamental difference we are taught early on between motile animal and nonmotile plant cells is the way in which they carry out their boundary extension . motile cells achieve whole - body displacement or locomotion , which allows them to leave resource - depleted domains behind while moving on to new areas . plant cells , however , become rooted to a spot and are only able to extend outwards from it in response to different stimuli . two major conditions have to be met for achieving locomotion ; a flexible bounding layer of the cell , which allows for rapid change in its shape , and the ability to retract one end of the cell while extending the other ( small et al . , 2002a ) . plant cells do not fulfill these requirements because they are encased in a relatively rigid cellulosic cell wall and , clearly , they do not have rear - end retractability . however , the cell wall of actively expanding plant cells is actually quite labile in contrast to its later , rigid nature in a mature , fully expanded cell ( mathur , 2004 ) . further , though lacking rear - end retractability , the forward extension of a plant cell , like that of an animal cell , also involves membrane protrusion ( vidali et al . , 2001 ) recent cell - biological and molecular evidence from higher plants suggests that , despite their sessile and wall - encased nature , plant cells possess a core machinery similar to the one required for forward motility of animate cells . although usually used in the context of crawling amoeboid locomotion , here i use the term leading edge for both animal and plant cells as the part of a cell that extends or leads . though descriptions vary from cell to cell , the generalized leading edge of an amoeboid cell comprises a 25-m - wide veil - like , organelle free , cytoplasmic extension called the lamellipodium . the leading edge has an actin - rich zone comprising of a fine f - actin mesh followed by a microtubule - rich region ( etienne - manneville , 2004 ) ( fig . 1 a ) . a few pioneering microtubules do extend into the f - actin mesh ( small et al . schematic depiction of similar intracellular zonation at the leading edge of animal and plant cells . ( a ) a generalized motile animal cell with a rear end and leading edge . enlarged view of the lamellipodium displays an organelle - free area near the absolute edge , followed by an actin - rich zone with dynamic , rapidly polymerizing actin . the microtubular zone usually lies behind the actin - packed zone , though pioneering microtubules make their way into the actin zone . ( b ) a seedling depicting two of the regions where diffuse-growing cells , such as hypocotyl cells and tip-growing cells such as root hairs are found . ( c ) plant cells exhibiting diffuse growth have large expanding vacuoles that press the cytoplasm into a thin layer against the plasma membrane . a fine f - actin meshwork lying below the plasma membrane is followed by cytoplasmic microtubules on its inner side . ( d ) plant cells that extend by tip - focused growth are characterized by an apical accumulation of vesicles in an organelle - free zone . a fine actin meshwork with its distal region interspersed by dynamic microtubules follows . a vacuole occupies the rest of the nongrowing , mature part of the tubular cell . note that the conserved region ( blue speckled ) of rho - gtpase / actin interaction leading to a fine f - actin mesh in each cell type has been based on the localization of rac in live animal cells ( kraynov et al . , 2000 ) and atrop localizations for different plant cells ( fu et al . , 2001 , 2002 ; a very similar intracellular zonation is seen in plants ( fig . 1 , b d ; mathur , 2004 ) . for plant cells , two kinds of expansion modes are recognized ; one called tip - growth , where the growth processes is limited to a small region that extends to form a tubular structure , and the second , designated diffuse growth , where the process of growth is dispersed over a large area of the cell ( mathur , 2004 ) . tip - growing cells ( fig . 1 , b and d ) are best typified by elongating root - hair and pollen tubes and , like lamellipodia , exhibit an apical region with a stretched plasma membrane , followed by a organelle - depleted clear zone ( vidali and hepler , 2001 ) . a fine , labile f - actin zone is defined next and leads into a dense f - actin region where the filaments become progressively bundled ( fu et al . , 2001 ; ketelaar et al . , extend only to the edge of the fine f - actin mesh ( sieberer et al . a large vacuole fills the rest of the cell ( carol and dolan , 2002 ) . by comparison , the volume occupied by vacuoles is quite large in diffuse - growing cells so that the cytoplasm is compressed into a thin layer against the plasma membrane ( fig . 1 , b and c ; mathur et al . although this thin cytoplasmic layer has not allowed intracellular zonation in these cells to be appreciated as clearly as in tip - growing cells , a fine cortical mesh , underlying cytoplasmic f - actin bundles ( fu et al . , 2002 ; mathur et al . , 2003b ) , and microtubules ( saedler et al . , 2004a ) 1 c ) . the apparent similarity in zonal relationship between the plasma membrane and cytoskeletal elements in the region of active protrusive growth between animal and plant cells ( fig . 1 ) extends to the molecular mechanisms responsible for the creation of a leading edge . a considerable body of information exists about the multiple protein complexes and regulatory molecules that get activated to create the leading edge in an animal cell ( kraynov et al . , 2000 ; three key interacting components members of the rho / rac / cdc42 superfamily of gtpases , and actin and microtubule cytoskeletons although rho - gtpase loops interact with and regulate both actin and microtubule cytoskeletons and microtubules provide directionality to the leading edge , it is actin dynamics that play a major role in membrane protrusion ( small et al . cytoskeleton regulation in animal cells involves cdc42/rac - gtpase triggered activation of a suppressor of camp receptor from dictyostelium ( scar)/wiskott - aldrich syndrome protein family verprolin - homologous protein ( wave ) complex . this complex in turn activates a 7-subunit actin - related protein ( arp2/3 ) complex to enhance actin polymerization ( millard et al . , 2004 ; the final outcome is a fine dendritic mesh of filamentous actin seen at the leading edge ( fig . 1 ) . plants , too , possess a unique subfamily of rho - family gtpases , called rho - related gtpase of plants ( rop ; vernoud et al . , 2003 ) , whose members localize to areas of active growth ( fu et al . , 2001 , 2002 ; fig . 1 , c and d ) and play numerous roles , including that of actin cytoskeleton regulation ( gu et al . , further , different components of a putative scar / wave - like complex ( millard et al . , 2004 ; vartiainen and machesky , 2004 ) , namely scar - related proteins ( frank et al . , 2004 ) , nap125/gnarled ( brembu et al . , 2004 ; deeks et al . , 2004 ; el - din el - assal et al , 2004 ; li et al . , 2004 ; zimmermann et al . , 2004 ) , pir121/klunker / pirogi ( basu et al . , 2004 ; brembu et al . , 2004 ; li et al . , 2004 ; saedler et al . , 2004b ) , hspc300/brick1 ( frank and smith , 2002 ) , and ab1 - 1like proteins ( deeks et al . , 2004 ) , have been cloned from arabidopsis . at least one rop , atrop2 , has been shown to interact with pir121 , strongly suggesting that a scar / wave - like pathway mediates the rop finally , homologues of the different subunit of the arp2/3 complex have been cloned from plants and in some cases shown to be interchangeable with their animal orthologues ( le et al . , 2003 ; li et al . , 2003 ; mathur et al . , 2003a , b ; el - assal et al . , 2004 ; saedler et al . are believed to affect microtubule plus - end growth by removing a stearic hindrance that allows cytoplasmic microtubules localized access to cortical domains ( rodriguez et al . , 2003 ) . in plants , studies on actin microtubule interactions are just beginning ( mathur , 2004 ) . however , observations of an aberrant aggregation and stabilization of cytoplasmic microtubules in actin - compromised cells suggests an actin control over microtubules very similar to that observed in animal cells ( saedler et al . , 2004a ) . alternatively , microtubule control over the actin cytoskeleton has also been demonstrated in drosophila by providing evidence that eb1 , a microtubule plus end binding protein , mediates the delivery of drhogef2 , an activator of rho1 to cortical domains for its actomyosin - related functions in cell retraction ( rogers et al . , 2004 ) . though not yet demonstrated , similar microtubule rop interactions could be envisaged in plants too , as several microtubule plus end binding proteins ( bisgrove et al . , 2004 ) as well as a putative rop - gef , spike1 , have been cloned from arabidopsis . interestingly , cells of the spike1 mutant display an aberrant microtubule cytoskeleton ( qiu et al . , 2002 ) . numerous other modulators ( table i ) involved in fine - tuning of the core elements and their interactions appear to be conserved between animals and plants ( goode et al . , 2000 ; rodriguez et al . , 2003 ; wasteneys and yang , 2004 ) ( table i ) . adf , actin depolymerizing factor ; eb1 , microtubule plus end binding protein ; rop , rho - like proteins of plants ; gef , guanine nucleotide exchange factor ; gap , gtpase - activating protein ; gdi , guanine nucleotide dissociation inhibitor . compromised function of the three key molecular components , as mentioned in the previous section , has major implications for both motility and cell shape . a brief comparison of cellular phenotypes resulting from defects in the core elements underscores the commonalities . actin - dependent lamellipodium protrusion and cell motility get greatly attenuated in animal cells with an aberrant actin cytoskeleton ( etienne - manneville , 2004 ) . likewise , cell expansion is significantly reduced in actin - compromised plant cells ( baluska et al . , 2001 ) . this is especially apparent in arabidopsis mutants such as nap125/gnarled , pir121/klunker / pirogi ( brembu et al . , 2004 ; deeks et al . , 2004 ; el - assal et al . , 2004 ; li et al . , 2004 ; saedler et al 2004 ) , and hspc300/brick1 ( mutant in maize ; frank and smith , 2002 ) , whose respective gene products possibly feed into the arp2/3 complex regulatory pathway . mutations in different subunit homologues of the putative plant arp2/3 complex such as arp2/wurm , arp3/distorted1 , arpc2/distorted2 , and arpc5/crooked also exhibit similar phenotypes ( for review see mathur , 2005 ) . each of these mutants exhibits aberrant f - actin organization and characteristic cellular deformations resulting from abnormal , random expansion . however , as mentioned earlier in this paper , actin cytoskeleton activity is heavily dependent upon the stimulation provided by rho - gtpases . animal cells with aberrant rho - gtpase activity display numerous defects in pseudopod extension and chemotaxis ( chung et al . , 2000 ; raftopoulou and hall , 2004 ) . overexpression of rops in plants results in increased fine f - actin meshworks and a swollen cell morphology , suggesting an overall reduction in growth polarization ( molendijk et al . , 2001 ; fu et al . , 2002 ; jones et al . , 2002 ) . because loss of polarity is a characteristic feature of microtubule defective cells too , in both animal ( small et al . , 2002b ) and plant cells ( mathur , 2004 ) these observations again point to a conserved , intimate relationship between rho - family gtpases , and the actin and microtubule cytoskeletons . although the similarities described above emphasize conserved mechanisms for membrane protrusion between animal and plant cells , they also reflect the profound impact the mechanism of boundary extension has on the survival ability of an organism . for motile cells , loss of motility leads to an inability to move to resource - rich areas and ultimately leads to death . consequently , many animal mutants for the core elements display lethal phenotypes . for the nonmotile but photosynthetic ( and therefore relatively self - sufficient ) plant cells , a change in cell shape does not lead to death directly . as a result , many comparable plant mutants , though misshapen and underweight ( el - assal et al . , 2004 ; li et al . , 2004 ) , are able to grow and complete their life cycle , giving the erroneous impression that the core genes are not as vital for plants as for animals . however , when grown in a population of siblings , the inability of mutant plants to extend properly becomes a severe handicap that affects their survival ability . a comparison of key cell biological and molecular features in two apparently disparate processes , namely , forward motility of animal cells and extension growth of plant cells , reveals a core machinery that has been conserved by both motile and nonmotile life forms for achieving boundary extension ; an essential requirement for survival . given this realization , future studies on boundary extension mechanisms of different life forms should prove exciting , not only for uncovering the extent of similarities , but also for discovering the range of cell biological and molecular variations , and the adaptations and innovations introduced by different organisms for surviving in their specialized niches .

locomotion clearly sets plants and animals apart . however , recent studies in higher plants reveal cell - biological and molecular features similar to those observed at the leading edge of animal cells and suggest conservation of boundary extension mechanisms between motile animal cells and nonmotile plant cells .

Boundary extension: a necessity for living organisms Boundary extension requires a leading edge Creating the leading edge Defects at the leading edge: mutant phenotypes Conclusions and perspectives

one - host cattle tick , rhipicephalus ( boophilus ) microplus ( canestrini 1887 ) , is an economically important ectoparasite of livestock and creates major problem for milk producers in tropical and subtropical countries including india . it causes severe economic losses by blood loss , reduction in weight gain , and direct damage to skin and hides and also by serving as a vector of various economically important infectious diseases . it is the most prevalent tick infesting all age groups of domestic livestock in various agroclimatic zones of punjab state , india [ 2 , 3 ] . the global losses due to ticks and tick borne diseases ( ttbds ) were estimated to be between us$ 13.9 and 18.7 billion annually while in india the cost of controlling ttbds has been estimated to be us$ 498.7 million / annum . the control of this parasite is mostly based on the large scale repeated use of chemical acaricides , namely , organophosphates ( op ) , synthetic pyrethroids ( sp ) , amidines , and macrocyclic lactones ( ml ) . pereira et al . reported that to control r. ( b. ) microplus , it is necessary to consider that only 5% of parasites are located on the host , so the remaining 95% remain in the environment . accordingly , several studies [ 810 ] have emphasized that the successful control of a tick population is related not only to the efficacy of an acaricide but also to the deleterious effects that these active agents cause over tick populations in the field , especially over the reproductive parameters of engorged r. ( b. ) microplus females . in india , about 60% of livestock is reared by small and marginal farmers and use of various op compounds ( diazinon and malathion ) is very common for the control of livestock and poultry pests . op compounds are also used against agriculturally important pests and for mass eradication of mosquito larvae in their breeding places . a number of studies have shown development of op resistance in r. ( b. ) microplus [ 12 , 13 ] particularly in punjab state [ 14 , 15 ] . however , data on effect of malathion ( op ) on the reproductive parameters of ticks indicating its overall tick control efficacy besides causing tick mortality is currently lacking . based on these observations , the present study aimed to evaluate deleterious effects of malathion on the reproductive parameters of engorged r. ( b. ) microplus females that had detached from naturally infested cattle . punjab state is located in the northwest region of india which extends from the latitudes 29.30n to 32.32n and longitudes 73.55e to 76.50e . it covers a geographical area of 50,362 km and lies between altitudes 180 and 300 m above sea level . average rainfall in state is 565.9 mm ranging from 915 mm in north to 102 mm in south with moderately humid climate . fully engorged r. ( b. ) microplus adult female ticks were collected from the dairy sheds of nineteen districts ( amritsar , barnala , bathinda , faridkot , fatehgarh sahib , ferozepur , gurdaspur , hoshiarpur , jalandhar , kapurthala , ludhiana , mansa , moga , muktsar , pathankot , patiala , rupnagar , sangrur , and sbs nagar ) of punjab , india . the ticks were collected in separate vials , closed with muslin cloth to allow air and moisture exchange , brought to the entomology laboratory , department of veterinary parasitology , college of veterinary sciences , gadvasu , ludhiana , and kept at 28 1c and 85 5% relative humidity ( rh ) . technical grade ( 97.9% ) malathion ( accustandard inc . , usa ) was used to prepare the stock solution of 10,000 ppm in methanol . for the bioassay , different concentrations of malathion ( 1250 , 2500 , 5000 , 10000 , and 20000 ppm ) were prepared in distilled water from the stock solution and tested against the various field isolates of r. ( b. ) microplus . briefly , 120 engorged females for each isolate were randomly separated into groups of ten ( 10 ) . a dose - dependent response study was conducted by immersing adult ticks for two minutes in various concentrations of malathion . two replicates , with 10 engorged females per replicate , were performed for each concentration . after immersion , the ticks were dried on filter paper with the help of paper towels and placed in sterile petri dishes for complete drying . afterwards , the ticks were weighed , were transferred to individual glass tubes covered with muslin cloth , and were kept in incubator maintained at 28 1c and 85 5% rh . the adult ticks which survived the exposure of drug laid eggs which were allowed to hatch to larvae under similar conditions of incubation . the ticks which did not oviposit even after 14 days posttreatment were considered dead and the following parameters were compared : weight of engorged female.egg mass weight ( emw ) laid by the live ticks , recorded at 14 days after treatment.reproductive index ( ri ) = egg mass weight / engorged female weight.percentage inhibition of oviposition ( % io ) = [ ( ri control ri treated)/ri control 100].hatching percentage of eggs . egg mass weight ( emw ) laid by the live ticks , recorded at 14 days after treatment . dose response data of ri and % io were analyzed by probit method using graphpad prism version 4.0 , san diego , ca , usa . the data were statistically analyzed using a one - way analysis of variance ( anova ) with group multiple comparisons by tukey 's test ( graphpad prism 4 ) . the effect of exposure of increasing concentrations of malathion on reproductive parameters of engorged r. ( b. ) microplus ticks , namely , egg mass weight ( emw ) , reproductive index ( ri ) , percentage inhibition of oviposition ( % io ) , and hatching percentage , was studied by ait . the regression graph of these reproductive parameters of treated ticks was plotted against log values of progressively increasing concentrations of malathion ( table 1 ) . a negative dose - dependent slope was recorded for mean emw in all tick isolates because with the increasing concentrations of malathion the surviving ticks laid significantly ( p > 0.05 ) fewer eggs . consequently , the mean ri of treated ticks showed a decreasing dose - dependent response and a negative slope was recorded . results thus indicate that although the increase in concentration of malathion may have not caused one hundred per cent mortality in ticks , the surviving ticks showed a significant decrease ( p > 0.05 ) in their efficiency to convert their live weight into egg mass . also , a dose - dependent significant increase ( p > 0.05 ) in the mean % io of treated ticks along with a positive slope was recorded . the effect of malathion on the average emw of treated ticks was recorded to be dose dependent and the decrease was more pronounced at higher concentrations . complete cessation of egg laying was recorded at 5000 ppm in one isolate , 10000 ppm in four isolates , and 20000 ppm in six isolates , whereas eight isolates laid eggs even upon exposure to the highest concentrations ( table 2 ) . similarly , details of the effect of malathion on the ri of treated ticks are presented in table 3 . the values of ri ranged from 0.0 to 0.33 in ticks exposed to the recommended concentration of malathion ( 5000 ppm ) used in field conditions . the values of % io ranged in 4.468.6 , 25.276.2 , 35.6100.0 , 45.7100.0 , and 71.4100.0 in groups treated with 1250 , 2500 , 5000 , 10000 , and 20000 ppm of malathion , respectively ( table 4 ) . the hatching percentage of eggs was determined by visual estimation and a dose - dependent effect was recorded . a low hatching percentage was recorded in eggs laid by all malathion treated female ticks in comparison to control ticks treated with distilled water . complete inhibition of hatching was recorded in eggs laid by all ticks treated with concentrations of 10000 ppm and above ; however , the survival of the hatched larvae was not affected by malathion treatment and was similar to control group . the absence of studies conducted with the malathion that was used in the present study , regarding its effects on reproductive parameters of fully engorged r. ( b. ) microplus females , does not allow a comparison with our obtained results . most trial studies using chemical acaricides are conducted to detect the resistance of r. ( b. ) microplus to these compounds using in vitro methodologies , such as the adult immersion test ( ait ) , larval packet test ( lpt ) , or the larval immersion test ( lit ) , recognized by the food and agriculture organization as a standard for the evaluation of efficacy or resistance [ 1821 ] . further , in the present study , technical grade malathion was selected over commercial formulation for the bioassay because commercial products are prepared with many proprietary ingredients and it is difficult to assess the responses due to active ingredients . for the preparation of stock solution , technical grade malathion was dissolved in 100% methanol and the working concentrations were prepared using water . the use of organic solvents facilitates the adsorption of compound over the surface area of target biological materials and possibly enhances penetration of active ingredients of the acaricide across the exoskeleton . extensive experience in the field has led to suggestions that the use of strictly managed , uninterrupted , short - interval treatments at recommended concentrations is a reliable means of avoiding or delaying resistance . however , it has also been proposed that intermittent use of high concentration acaricides to kill ticks with resistant alleles may provide a basic means of delaying resistance . increased concentration has been used successfully in controlling ddt- , op- , and sp - resistant strains of r. ( b. ) microplus . this also helped to prolong the life of op acaricides , but the potential host toxicity and chemical residue problems now need to be reconsidered before an increased concentration could be used for resistance management . however , use of increased concentrations for treatment of animals shed for the elimination of off - the - host stages of the ticks could be beneficial in tick control as they constitute around 95% of total tick population . several studies [ 810 ] have emphasized the successful control of a tick population , due to the deleterious effects that active agents cause over tick populations in the field , especially over the reproductive parameters of engorged r. ( b. ) microplus females . this would further reduce the number of treatments of the animals and would lead to low residual effects in the milk and meat products thus benefitting the end user health . based on this finding , the results that we obtained in this present study regarding the reproductive parameters of fully engorged rhipicephalus ( boophilus ) microplus females might be sufficient to reduce the number of chemical treatments administered to cattle .

the present study was aimed at evaluating effects of malathion on the various reproductive parameters , namely , egg mass weight ( emw ) , reproductive index ( ri ) , percentage inhibition of oviposition ( % io ) , and hatchability percentage of eggs of rhipicephalus ( boophilus ) microplus ( canestrini 1887 ) females from 19 districts of punjab , india . the effect on various parameters was found to be dose dependent and more discernible upon exposure to higher concentrations . complete cessation of egg laying was recorded in tick isolates on exposure to 5000 ppm and above . the values of % io ranged in 4.468.6 , 25.276.2 , 35.6100.0 , 45.7100.0 , and 71.4100.0 in groups treated with 1250 , 2500 , 5000 , 10000 , and 20000 ppm of malathion , respectively . a low hatching % was recorded in eggs of all treated female ticks in comparison to control treated with distilled water and complete inhibition of hatching was recorded at 10000 ppm and above . however , the survival of the hatched larvae was not affected and was similar to control group . the results of the current study can be of immense help in formulation and implementation of effective tick control measures .

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

anaplastic lymphoma kinase ( alk ) is a receptor tyrosine kinase of the insulin receptor superfamily , and in mice and humans , the normal expression of alk is largely restricted to the brain and nervous system [ 14 ] . mice deficient in alk appear to have no overt developmental abnormalities [ 58 ] ; however , behavioural abnormalities have been noted in these mice . alk - deficient mice perform better on tests of cognitive ability and display less anxiety than their wild - type littermate controls [ 6 , 7 ] . behavioural tests also demonstrated increased alcohol consumption and altered sensitivity to alcohol in alk - deficient mice compared to wild - type littermates . intriguingly , single - nucleotide polymorphisms ( snps ) in alk have been identified in humans that correlate with decreased response to alcohol . a correlation between alk snps and schizophrenia has also been noted in a japanese study . in drosophila melanogaster , the jelly belly protein ( jeb ) has been characterized as an alk ligand [ 1012 ] . in mammals , there does not appear to be a jeb homologue but two ligands for alk have been described , pleiotrophin and midkine . however , there is not complete agreement regarding whether these are indeed alk stimulating ligands [ 15 , 16 ] . more recently , perez - pinera and colleagues proposed an alternative mechanism by which pleiotrophin could be stimulating alk signalling . in their model , the binding of pleiotrophin to its known receptor , receptor tyrosine phosphatase / ( rptp / ) , relieves the inhibitory dephosphorylation of alk by rptp / , thereby turning on alk signalling . dependence receptors induce apoptosis in their nonliganded state , but suppress apoptotic signalling in response to ligand binding . alk - positive anaplastic large cell lymphomas ( alk+ alcl ) are a distinct subset of non - hodgkin lymphomas with a t or null cell immunophenotype recognized by the world health organization classification scheme for hematological neoplasms [ 58 , 59 ] . these lymphomas express the cd30 ( ki-1 ) surface antigen , but the morphologic identification of alk+ alcl can be challenging , as the cytologic features of the tumor cells can be highly variable from case to case . nevertheless , the identification of the so - called hallmark cells , which are characterized by a horseshoe- or kidney - shaped nucleus and a prominent perinuclear golgi body , can facilitate the diagnosis [ 58 , 59 ] . regarding the pathobiology of alk+ alcl , several groups in the late eighties and early nineties noted that these lymphomas possessed a recurrent chromosomal translocation , the t(2;5)(p23;q35 ) translocation [ 6064 ] . in 1994 , it was demonstrated that this translocation generates a fusion gene between a previously uncharacterized tyrosine kinase on chromosome 2 , and the nucleophosmin ( npm ) gene on chromosome 5 [ 20 , 21 ] . this kinase was termed alk owing to its association with alcl and the expression of this kinase led to the identification of what is now considered to be a clinically distinct entity , alk+ alcl . in addition to npm , several other alk translocation partners have since been identified in alk+ alcl [ 23 , 24 , 27 , 2931 , 33 , 39 , 42 , 43 , 45 ] . these cancers include a portion of inflammatory myofibroblastic tumours ( imt ) [ 25 , 32 , 34 , 43 , 44 , 46 , 55 , 65 ] , nonsmall cell lung carcinomas ( nsclc ) [ 28 , 49 , 54 , 57 ] , diffuse large b - cell lymphomas ( dlbcl ) [ 22 , 3537 , 47 , 48 , 51 ] , colon cancers [ 50 , 56 ] , breast cancers , renal cell carcinomas [ 26 , 52 , 53 ] , and extramedullary plasmacytomas . two papers also reported detecting tropomyosin 4- ( tpm4-)alk fusion protein expression in some cases of esophageal squamous cell carcinoma [ 40 , 41 ] . moreover , it has very recently been established that inhibitors of alk are effective at treating patients with alk+ alcl and other malignancies expressing alk fusion proteins [ 67 , 68 ] . although not a focus of this paper , alk has been reported to be highly expressed in breast cancer , and alk gene amplifications and activating mutations have been identified in familial and sporadic neuroblastoma [ 7075 ] and thyroid cancer . the t(2;5)(p23;q35 ) translocation generates a fusion gene termed npm - alk whose transcription is under the control of npm regulatory sites . npm is a ubiquitously expressed protein that is predominately found in the nucleolus , but can shuttle between the cytoplasm and nucleus . npm is multifunctional and regulates several cellular activities including transcription , ribosome biogenesis , and the shuttling of proteins between the nucleus and cytoplasm . the npm - alk fusion gene consists of the first four exons of npm which encodes for the first 117 amino acids of the npm protein , and the alk portion of the fusion includes the exons encoding for the intracellular tail and kinase domain of alk . importantly , the npm part of the fusion includes the npm dimerization / oligomerization domain [ 80 , 81 ] . as we will discuss in the next section , this domain is critically important for npm - alk activity , and the presence of a dimerization / oligomerization domain is a common feature of other alk fusion partners . an essential role for the npm portion of npm - alk was first demonstrated by experiments showing that deletion of the entire npm region of npm - alk generated a protein incapable of transforming nih 3t3 cells . similarly , bischof et al . demonstrated that npm truncation or deletion mutants were not tyrosine phosphorylated and were unable to transform fischer rat 3t3 cells . since npm has been reported to form hexamers and other oligomers [ 82 , 83 ] , researchers examined whether npm could be providing an oligomerization domain in npm - alk . indeed , gel filtration and sucrose gradient experiments demonstrated that npm - alk forms oligomeric complexes in an npm - dependent manner . moreover , npm - alk can dimerize with endogenous npm , and it is believed that this accounts for why some npm - alk is observed in the nucleus . the basic domain of echinoderm microtubule - associated protein - like 4 ( eml4 ) also functions as a dimerization domain in eml4-alk , and this is likely mediated by a coiled - coil motif within the basic domain . most other alk fusion partners possess known dimerization / oligomerization domains that are postulated to mediate dimerization / oligomerization of the fusion proteins ( table 2 ) . msn - alk ( a fusion between moesin and alk ) appears not to have an oligomerization domain and is postulated to be activated through the colocalization of msn - alk fusion proteins to cellular membranes . thus , dimerization , oligomerization , or colocalization of alk fusion proteins appears to be a common and necessary requirement for these oncoproteins to signal . npm - alk activates downstream signalling events that promote proliferation , prevent apoptosis , and enhance migration in alk+ alcl ( reviewed in [ 5 , 85 , 86 ] ) . we will focus on the stat3 , mek / erk , and pi3k / akt pathways , as much is known about the role these pathways play in alk+ alcl pathogenesis . in particular , we will discuss the cellular processes these pathways regulate in this lymphoma , and how they are regulated by npm - alk signalling . members of the signal transducer and activator of transcription ( stat ) family of transcription factors are activated by interferon , cytokine , and growth factor receptor signalling . the tyrosine phosphorylation of stats by tyrosine kinases , particularly the janus kinases ( jaks ) , facilitates the dimerization of stats . this allows the stats to translocate to the nucleus and promote the transcription of genes involved in proliferation , cell survival , and the immune response [ 87 , 88 ] . in alk+ alcl , the activation of stat3 has been strongly implicated in the pathogenesis of this lymphoma ( figure 1 ) . stat3 is activated in alk+ alcl cell lines and patient samples [ 8991 ] , as well as cells isolated from npm - alk transgenic mice [ 92 , 93 ] , as measured by its phosphorylation on tyrosine 705 . the inhibition of stat3 in alk+ alcl cell lines , either through the overexpression of a dominant - negative stat3 construct or decreasing stat3 expression using antisense oligonucleotides , resulted in decreased proliferation and the induction of apoptosis . stat3 was also required for npm - alk to transform mouse embryo fibroblasts , and for the continued survival of t - cell lymphomas induced in mice by the expression of an npm - alk transgene . stat3 exerts its biological effects in alk+ alcl through regulating the expression of multiple genes . microarray studies performed by piva and colleagues demonstrated that knockdown of stat3 altered the expression of ~1500 genes in a variant of the sup - m2 alk+ alcl cell line . importantly , stat3 functions both as an activator and repressor of transcription , and approximately 60% of the stat3-regulated genes identified by piva et al . were repressed by stat3 . those genes found to be upregulated by stat3 include : genes that promote proliferation such as cyclin d1 , cyclin d3 , c - myc , icos , c / ebp [ 9397 ] ; those that promote survival such as bcl - xl , survivin , bcl-2 , mcl-1 , bcl2a1 , c / ebp [ 90 , 93 , 97 , 98 ] ; others including cd30 , pd - l1 , timp-1 , socs3 , hif1 , twist1 , il10 , and il2r chain [ 94 , 95 , 99104 ] . stat3 is also responsible for repressing the expression of t - cell genes that are commonly not expressed in alk+ alcl including cd3 , zap-70 , lat , and slp-76 , and it appears to do so in part through the upregulation of dna methyltransferases ( dnmts ) . dnmts methylate cpg motifs in promoter regions of genes , and this blocks the binding of some transcription factors and facilitates the recruitment of methyl - c binding proteins to these promoters . methyl - c binding proteins can then recruit histone deacetylases and methyltransferases that convert promoter regions into transcriptionally inactive heterochromatin . zhang and colleagues demonstrated that stat3 also promotes the binding of dnmts 13 to the il2r promoter in order to repress il2r gene expression . silencing il2r chain expression appears to be critical in alk+ alcl as re - introduction of the il2r into alk+ alcl cell lines resulted in decreased npm - alk expression and reduced viability . this study also demonstrated that stat3 enhances dnmt1 expression through the suppression of the dnmt1-targeting microrna , mir-21 . stat3 is also responsible for the epigenetic silencing of stat5a in alk+ alcl , which prevents stat5a from repressing npm - alk expression and thereby interfering with npm - alk signalling . given the importance of stat3 transcriptional activity in alk+ alcl , it is not surprising that many mechanisms contribute to the activation of stat3 in this lymphoma . stat3 [ 89 , 109 ] and jak3 have both been shown to coimmunoprecipitate with npm - alk , and several studies have shown that npm - alk promotes the tyrosine phosphorylation of stat3 [ 89 , 90 , 92 , 93 ] . however , there is not complete agreement regarding whether stat3 tyrosine phosphorylation is jak3 dependent [ 94 , 110 ] , or whether stat3 is tyrosine phosphorylated in a jak3-independent manner , possibly through direct tyrosine phosphorylation by npm - alk . the serine / threonine phosphatase pp2a has also been implicated in positively regulating stat3 activity in alk+ alcl , as inhibition of pp2a activity with calyculin a was demonstrated to reduce stat3 tyrosine phosphorylation . stat3 signalling is also likely enhanced in this lymphoma due to the fact that alk+ alcl cell lines do not express the stat3 inhibitor , pias3 . moreover , the shp-1 tyrosine phosphatase is often silenced by dna methylation in alk+ alcl [ 112 , 113 ] , and this is likely due in part to the recruitment of dnmts and histone deacetylases to the shp-1 promoter by stat3 . silencing shp-1 in alk+ alcl is important as shp-1 negative regulates npm - alk signalling through either the direct or indirect dephosphorylation of npm - alk , jak2 , and stat3 [ 114116 ] , and the targeting of npm - alk for proteasomal degradation [ 115 , 116 ] . cytokine signalling also plays a role in regulating stat3 activity in alk+ alcl . signalling through the il9 , il21 , and il22 receptors has been shown to promote stat3 activation in this lymphoma , and much of this may be due to autocrine signalling . furthermore , the expression of the il22r1 subunit of the il22 receptor is promoted by npm - alk , demonstrating a link between npm - alk and cytokine signalling in this lymphoma . since the il9 and 21 receptors utilize the il2r common chain , these findings still need to be reconciled with the results of zhang and colleagues which found that the il2r chain is silenced in alk+ alcl . signalling mediated by the extracellular signal - regulated kinases 1 and 2 ( erk1 and 2 ) promotes proliferation , survival , differentiation , and migration . these serine / threonine kinases are activated by many growth factor receptors through a well - defined kinase cascade . this kinase cascade is initiated by the activation of the ras gtpase , which activates the raf-1 serine / threonine kinase . raf-1 then activates the dual specificity kinases , mapk / erk kinases 1 and 2 ( mek1 and 2 ) , which phosphorylate and activate the erks . the erk pathway is activated in alk+ alcl cell lines and patient samples [ 122 , 123 ] and plays a central role in promoting cell proliferation and suppressing apoptosis in this cancer ( figure 2 ) . treatment with the mek1/2 inhibitor , u0126 , was found to reduce proliferation [ 123125 ] and enhance apoptosis [ 124 , 125 ] in alk+ alcl cell lines . reduced proliferation was also evident when the karpas 299 alk+ alcl cell line was treated with erk1 and/or 2 sirna . however , only the silencing of erk1 in these cells was found to increase apoptosis . two important downstream mediators of mek / erk signalling in alk+ alcl are the serine / threonine kinase , mammalian target of rapamycin ( mtor ) , and the junb transcription factor . the mtor pathway has been demonstrated to be activated in alk+ alcl patient samples , as measured by phosphorylation of mtor [ 125 , 126 ] and downstream targets of mtor signalling [ 123 , 125127 ] . marzec and colleagues found that treatment of the su - dhl-1 alk+ alcl cell line with mek inhibitors or erk1/2 sirna resulted in reduced phosphorylation of the ribosomal s6 protein ( rps6 ) . rps6 is a downstream target of mtor signalling , and phosphorylation of rps6 promotes cell growth . the p70 s6 kinase ( p70s6k ) , which is activated by mtor and phosphorylates rps6 , is also inhibited in su - dhl-1 cells treated with u0126 , but surprisingly not in the sr-786 alk+ alcl cell line . mek / erk signalling was postulated to activate mtor through inhibition of the tuberous sclerosis complex ( tsc ) . tsc is a gtpase - activating protein that inhibits mtor through inactivating the rheb gtpase . the notion that mek / erk signalling inhibits tsc in alk+ alcl is supported by the finding that treatment of su - dhl-1 cells with mek inhibitors resulted in decreased phosphorylation of tsc2 on inhibitory serine 1798 . the activation of mtor and the phosphorylation of mtor substrates , eukaryotic initiation factor 4e - binding protein-1 ( 4e - bp1 ) and p70s6k , has also been demonstrated to be dependent on pi3k and akt activity in alk+ alcl . phosphorylation of 4e - bp1 by mtor results in the dissociation of 4e - bp1 from eukaryotic initiation factor 4e ( eif4e ) , which allows eif4e to initiate translation . however , the importance of the pi3k / akt pathway in the activation of mtor in alk+ alcl has been questioned . treatment of alk+ alcl cell lines with the mtor inhibitor , rapamycin , resulted in reduced proliferation [ 123 , 125127 , 129 ] and the induction of apoptosis [ 126 , 127 ] . sirna - mediated knockdown of mtor was similarly found to reduce proliferation and enhance apoptosis in alk+ alcl cell lines . decreased proliferation as a result of mtor inhibition is at least in part due to the dephosphorylation of the retinoblastoma ( rb ) protein , decreased cyclin a expression , and increased expression of the cyclin - dependent kinase inhibitors , p27 and p21 . increased apoptosis in response to rapamycin treatment is likely due to decreased expression of the antiapoptotic proteins bcl-2 , bcl - xl , mcl-1 , and c - flip . inhibition of mtor was also demonstrated to reduce the size of npm - alk - expressing murine tumours in immunocompromised mice . the transcription of junb is also promoted by mek signalling in alk+ alcl cell lines [ 122 , 123 ] , through the ets-1 transcription factor . interestingly , mtor signalling also contributes to enhanced junb translation in alk+ alcl cell lines through the targeting of junb mrna to ribosome - rich polysomes . junb is an ap-1 family transcription factor that is highly expressed in alk+ alcl cell lines and patient samples [ 134136 ] and has been shown to promote the proliferation of the karpas 299 alk+ alcl cell line . junb also influences phenotypic characteristics of this lymphoma through promoting the transcription of cd30 [ 122 , 137 ] and the granzyme b serine protease . cd30 signalling also activates mek / erk / junb signalling in this lymphoma to further promote cd30 expression . the activation of raf-1 , mek , and erk in alk+ alcl cell lines is dependent on npm - alk activity [ 124 , 139 ] , and the ectopic expression of npm - alk has also been demonstrated to induce the activation of these proteins [ 123 , 124 , 140 , 141 ] . npm - alk can activate ras when ectopically expressed in the jurkat t leukemia cell line , and the expression of a dominant negative n17 ras decreased npm - alk - dependent nf - at / ap-1 luciferase activity in jurkat cells . furthermore , treatment of the su - dhl-1 alk+ alcl cell line with the ras inhibitor , fti-277 , resulted in increased apoptosis and decreased proliferation . the ras activator , son of sevenless ( sos ) , has been argued to be recruited to npm - alk via the adapter protein grb2 by molecules such as shc , shp2 , and insulin receptor substrate-1 ( irs-1 ) [ 80 , 141143 ] . ras activation has also been proposed to occur through a plc-dependent activation of ras guanyl nucleotide - releasing protein ( rasgrp ) . while raf-1 is activated by npm - alk , it does not appear to be required for erk activation in alk+ alcl cell lines . another map3k , cot / map3k8 , may be the primary activator of mek in this lymphoma . treatment of the su - dhl-1 cell line with cot sirna or a cot inhibitor decreased erk and mtor activation and reduced cellular proliferation . whether cot is regulated by npm - alk the phosphatidylinositol 3-kinase ( pi3k)/akt pathway regulates cell growth , differentiation , apoptosis , metabolism , and migration [ 144 , 145 ] . pi3k is composed of two subunits , a regulatory p85 subunit and a catalytic p110 subunit , and this enzyme phosphorylates inositol phospholipids on the 3 position of the inositol ring [ 144 , 145 ] . these lipids , in turn , activate a number of pleckstrin homology ( ph ) domain - containing proteins ; most notably the serine / threonine kinase akt [ 144 , 145 ] . signalling through the pi3k pathway promotes cell survival and proliferation in alk+ alcl ( figure 3 ) . treatment of alk+ alcl cell lines or ba / f3 cells ectopically expressing npm - alk with pi3k inhibitors induces apoptosis and reduces proliferation [ 146 , 147 ] . pi3k inhibitors also inhibit the transformation of rat-1 fibroblasts by npm - alk , and a dominant negative p85 subunit unable to associate with the p110 subunit was demonstrated to inhibit the ability of npm - alk - expressing ba / f3 cells to form colonies in methylcellulose . the akt substrate , glycogen synthase kinase-3 ( gsk-3 ) , is an important target of npm - alk signalling in alk+ alcl . phosphorylation of gsk-3 on serine 9 by akt inhibits gsk-3 activity , and in alk+ alcl this has been argued to be important for preventing gsk-3 from phosphorylating , and targeting for degradation , the antiapoptotic protein mcl-1 and the positive cell cycle - regulator , phosphatase cdc25a . furthermore , this study showed that phosphorylation of gsk-3 on serine 9 correlated with elevated cdc25a levels in alk+ alcl patient tumour biopsies . a separate study also demonstrated that npm - alk promotes cdc25a expression through pi3k , through either transcriptional upregulation of cdc25a or enhanced cdc25a mrna stability . further supporting the notion that inhibition of gsk-3 is an important target of npm - alk signalling , treatment of alk+ alcl cell lines with either gsk-3 shrna or a gsk-3 inhibitor could partially rescue the decreased viability associated with alk inhibitor treatment . npm - alk / pi3k / akt signalling also activates the sonic hedgehog ( shh ) pathway in alk+ alcl . shh is a secreted molecule that , when bound to its receptor patched , relieves inhibition of the smoothened co - receptor by patched . shh and gli1 were found to be highly expressed in primary alk+ alcl patient samples , and their expression in cell lines was dependent on npm - alk and pi3k activity . it was argued in this study that pi3k - mediated activation of akt is important for inhibiting gsk-3 in order to prevent gsk-3 from phosphorylating gli1 and targeting the protein for proteasomal degradation . npm - alk was also found to enhance gli1 transcriptional activity , and expression of the gli1 target gene , cyclin d2 . moreover , the inhibition of gli1 in alk+ alcl cell lines , either through sirna - mediated knockdown or treatment of cells with a smoothened inhibitor , reduced viability and arrested cells in the g1 stage of the cell cycle . the phosphorylation of foxo3a by akt results in its binding to 14 - 3 - 3 proteins , which sequesters foxo3a in the cytoplasm where it is unable to promote transcription . foxo3a is phosphorylated in alk+ alcl cell lines and in cells ectopically expressing npm - alk . accordingly npm - alk signalling results in the down - regulation of the pro - apoptotic protein , bim-1 and the cell cycle - inhibitor , p27 , which are transcriptional targets of foxo3a [ 155 , 156 ] . npm - alk / pi3k / akt signalling also maintains low levels of p27 by phosphorylating p27 , and thereby targeting p27 for proteasomal degradation [ 157 , 158 ] . the activation of the pi3k pathway in alk+ alcl is largely dependent on the activity of npm - alk . pi3k complexes with npm - alk in alk+ alcl cell lines [ 146 , 147 , 159 ] and cells isolated from npm - alk transgenic mice . the activation of akt in this lymphoma is dependent on npm - alk and pi3k activity [ 126 , 127 , 160 ] , and akt activity is upregulated in a pi3k - dependent manner by ectopically expressed npm - alk in ba / f3 cells [ 127 , 146 , 153 ] . pten , a lipid phosphatase that dephosphorylates pi3k lipid products [ 144 , 145 ] , is not expressed in some alk+ alcl patient samples , and this may be a contributing factor to akt activation in these patients . it has been over 15 years since the discovery of the npm - alk oncoprotein . in this time we have learned much about the signalling pathways activated by npm - alk in alk+ alcl , and how these pathways contribute to proliferation and survival of this lymphoma . this information has been critical in directing research towards understanding how alk translocations signal and function in other malignancies . for example , stat3 activation has been observed in clathrin heavy chain- ( ctlc-)alk - expressing dlbcl patient samples , and stat3 , erk , and akt are active in eml4-alk - expressing nsclc cell lines [ 163165 ] ; however , the importance of these pathways in nsclc and their regulation by eml4-alk appears to vary amongst nsclc cell lines [ 163165 ] . yet , even if activation of the stat3 , erk , and pi3k / akt pathways is common to malignancies expressing alk fusion proteins , differences almost certainly exist in the genes regulated by these pathways in the individual cancers . some of these differences may be important in the pathogenesis of their respective malignancies . thus , a more thorough characterization of these signalling pathways in other alk fusion protein - expressing malignancies needs to be a priority of future research . while the information gained from elucidating how npm - alk signals in alk+ alcl has been , and will continue to be , beneficial for understanding how other alk fusion proteins signal , it is clear that these fusion proteins are not identical in their signalling capability . in a study by armstrong and colleagues , nih 3t3 cells expressing the npm- , trk - fused gene ( tfg)- , 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase / imp cyclohydrolase ( atic)- , tropomyosin 3 ( tpm3)- , or ctlc - alk fusion proteins at roughly equal levels , differed in their ability to activate stat3 and akt . the proliferation rate , invasiveness , and ability to form tumours in nude mice also differed amongst the cells expressing the different alk fusion proteins . similarly , gene expression profiling demonstrated that , while tumours from alk+ alcl patients expressing npm - alk or tpm3-alk shared many commonly regulated genes , distinctly regulated genes were observed . accordingly , a second focus of future research needs to be a more detailed examination of whether distinctions exist in the signalling pathways or cellular processes regulated by different alk fusion proteins within the same malignancy .

anaplastic lymphoma kinase ( alk ) was first identified in 1994 with the discovery that the gene encoding for this kinase was involved in the t(2;5)(p23;q35 ) chromosomal translocation observed in a subset of anaplastic large cell lymphoma ( alcl ) . the npm - alk fusion protein generated by this translocation is a constitutively active tyrosine kinase , and much research has focused on characterizing the signalling pathways and cellular activities this oncoprotein regulates in alcl . we now know about the existence of nearly 20 distinct alk translocation partners , and the fusion proteins resulting from these translocations play a critical role in the pathogenesis of a variety of cancers including subsets of large b - cell lymphomas , nonsmall cell lung carcinomas , and inflammatory myofibroblastic tumours . moreover , the inhibition of alk has been shown to be an effective treatment strategy in some of these malignancies . in this paper we will highlight malignancies where alk translocations have been identified and discuss why alk fusion proteins are constitutively active tyrosine kinases . finally , using alcl as an example , we will examine three key signalling pathways activated by npm - alk that contribute to proliferation and survival in alcl .

1. The ALK Receptor Tyrosine Kinase 2. The Identification of NPM-ALK and Other ALK Fusion Proteins 3. The Importance of Dimerization/Oligomerization Domains in ALK Fusion Proteins 4. Signalling Pathways Activated by NPM-ALK in ALK+ ALCL 5. The STAT3 Pathway 6. The MEK/ERK Pathway 7. The PI3K/Akt Pathway 8. Conclusions and Future Perspectives

systemic sclerosis ( ssc ) is a complex autoimmune connective tissue disorder with a prevalence of five per 100,000 and an incidence of one per 100,000 . the skin is invariably involved , but commonly affected internal organs include the cardiovascular system , the lungs and the gastrointestinal system . however , uncommon manifestations present clinical dilemmas . this case of ssc describes the unique involvement of the ovaries leading to premature ovarian failure ( pof ) , without the involvement of any other internal organs . a 31-year - old woman , parity one ( full - term , normal vaginal delivery ) , with no abortions presented with inability to conceive and amenorrhoea of 3 years duration . she had raynaud s phenomenon for 16 years , primary hypothyroidism for 5 years ( though euthyroid on treatment with thyroxine 100 g once daily ) , digital ulcers for 4 years and shortening of the terminal digits , decreased mouth opening and skin tightening for 2 years . there was no history of fever , weight loss , anorexia , sleep disturbance , symptoms of the respiratory , the cardiovascular , the gastrointestinal or the nervous system involvement , or the intake of any drugs ( apart from thyroxine ) or substance abuse . she had an expressionless face [ figure 1 ] , with loss of wrinkles , a taut and shiny skin , a reduced oral aperture ( permitting only two fingers ) and a pinched beak - like nose . there was fixed flexion contracture of the fingers of the upper limbs [ figure 2 ] and amputation of the 2 digit of the left lower limb [ figure 3 ] . an expressionless face , a taut and shiny skin , loss of wrinkles , with a reduced oral aperture and a pinched beak - like nose thickened , fixed flexion contracture of the fingers of the upper limbs amputation of the 2 digit of the left lower limb investigations revealed the following : haemoglobin 8.6 g / dl , total leucocyte count ( tlc ) 4.6 10/mm , platelet count 1.01 10/mm and erythrocyte sedimentation rate ( esr ) 75 mm at the end of the 1 hour ; the liver and the kidney functions and blood sugar were normal . electrocardiogram , echocardiography , chest x - ray and pulmonary function test parameters were also normal . serum tsh level was 4.61 iu / ml ( normal 0.345.6 iu / ml ) , and anti - thyroid antibodies were negative . the following serum immunohistochemistry findings were suggestive of ssc : positive ana ( by indirect immunofluorescence , at 1:100 dilution ) , 3 + positivity for scl-70 , anti - dsdna by elisa was 24 iu / ml ( normal value < 25 ) , and p - anca , c - anca and anti - lkm were negative . ultrasonography revealed the presence of normal - sized ovaries ( left 2 cm 1.8 cm 1 cm and right 2.4 cm 2 cm 0.8 cm ) with absent ovarian follicles and normal - sized uterus ( 7.2 cm 5.4 cm 3.6 cm ) , with an endometrial thickness of 3 mm ( with normal being more than 45 mm ) . serum fsh level was 125.6 and 130 iu / l on two separate occasions 1 month apart ( normal 110 iu / l ) , and serum luteinizing hormone ( lh ) level was 56.42 iu / l ( normal 1.6815 a diagnosis of ssc was made on the basis of the characteristic clinical features on history taking and examination and positive serum immunohistochemistry ; secondary amenorrhoea was attributed to pof . fertility issues in the patients with ssc have been debated over decades . it is surmised that the deleterious effects of ssc begin long before conception because of several reasons . the reasons include the psychosocial issues of reduced self - esteem and altered body image arising as a result of skin discolouration and hardening , perioral skin tightening , finger lesions , and digit amputation , as well as because of physical ailments namely vaginal dryness , dyspareunia , arthritic involvement and contractures . in a comprehensive paper published at the turn of the century , it was reported that fertility in the patients with ssc was not significantly different from that in patients with rheumatoid arthritis and the healthy control population , when adjustments were made for possible contributing factors . pof is a cessation of the ovarian function before the age of 40 years , and it may occur due to chromosomal causes ( turner syndrome and fragile x syndrome ) , iatrogenic causes ( radiation , chemotherapy and surgery ) , infections , autoimmune disorders , galactosemia , savage syndrome , cigarette smoking , etc . pof occurs in < 1% of women ; its incidence is about one in 1000 women by the age of 30 years , one in 250 by the age of 35 years and one in 100 by the age of 40 years . depending on various studies the evidence of autoimmunity in these cases is suggested by the presence of ovarian autoantibodies , lymphocytic oophoritis or other autoimmune disorders . the former two conditions are difficult to assess because of the absence of sensitive and specific tests to measure ovarian autoantibodies . hypothyroidism , diabetes mellitus and addison disease are the common autoimmune disorders associated with pof . there are very few reports in which premature menopause has been reported in the patients with ssc . way back in 1983 , by observing the age of menopause in patients with scleroderma , it was reported that the ovaries were not involved in scleroderma . however , in 1995 , among 60 patients with scleroderma , vaginal dryness , ulceration , dyspareunia and many menstrual abnormalities were observed , but premature menopause was seen in only two patients at the ages of 29 and 38 years , respectively . vural et al . reported pof in a 25-year - old woman with localized scleroderma . however , a recent study in 2012 stated that scleroderma does not cause pof unless treated with alkylating agents such as cyclophosphamide . this patient had marked digital ulcerations and also loss of the digits , depicting severe peripheral vascular involvement , which is reflective of a systemic involvement . however , there was no involvement of the visceral organs , that is , the heart or the lungs , in this patient , although the ovaries were involved , which is very rare and unusual . the inflammation and microvascular damage to the ovaries might have resulted in pof in this case , but in the absence of any other visceral organ involvement , it is very difficult to comment on the exact mechanism . the study of pof as a cause of infertility in the patients with ssc is important , because it is irreversible and can save the couple from unnecessary investigations and psychological trauma . moreover , early menopause in the patients with ssc predicts pulmonary hypertension , which is one of the causes of mortality in these patients . additionally , menopause per se has an additive effect on the risk for common co - morbidities such as cardiovascular disease and osteoporosis . pof can be a possible cause of amenorrhoea in the patients with ssc , and it can be a poor predictor of disease progression . further , the link of pof needs to be correlated with other complications of ssc . 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 .

a 31-year - old woman presented with secondary amenorrhoea and inability to conceive , which was of 3 years duration . she had raynaud s phenomenon for 16 years , primary hypothyroidism for 5 years , digital ulcers for 4 years and skin tightening for 2 years . she had an expressionless face , with loss of wrinkles and restriction of the mouth opening along with flexion contractures of the hands and the terminal digit resorptions . investigations showed antinuclear antibodies ( ana ) and anti - scl 70 positivity confirming the presence of systemic sclerosis ( ssc ) . the follicle stimulating hormone ( fsh ) level was elevated , the ovarian follicles were absent , and the endometrial thickness was reduced confirming premature ovarian failure ( pof ) . pof causing infertility and secondary amenorrhoea in ssc is an unusual manifestation ; moreover , pof occured without the involvement of other internal organs .

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other than skin cancers , prostate cancer ( pca ) is the most common cancer in men and the second cause of death after lung cancer . the estimated numbers of new cases of pca and deaths in the united states in 2014 are 233,000 and 29,480 , respectively . in brazil , the number of deaths in 2011 was 13,129 , and the estimated number of new cases for 2014 is 68,800 . screening for pca is accomplished by digital rectal examination ( dre ) and by measuring serum prostate - specific antigen ( psa ) levels . a dre can be uncomfortable and is not welcomed by patients ; however , this type of examination is an important screening and staging tool despite the disadvantages of subjectivity and interpersonal variability among examiners . the examination can aid in the detection of tumors in men with low levels of psa . transrectal ultrasound ( trus)-guided biopsy is the most accepted method for diagnosing pca , which is detected in 30% to 40% of biopsy specimens . this method did not gain popularity until the mid-1980s , when understanding of the anatomy of the prostate for radical prostatectomy and psa measurement stimulated enthusiasm for early detection of pca . with the advent of trus hodge et al . proposed the sextant technique for pca detection , which consists of the collection of six - core biopsy specimens targeted at the base , middle third , and apex regions of the prostate in sagittal line bilaterally . subsequent studies have shown that sextant biopsies yield false - negative results in 30% of cases . the sextant method was modified by the inclusion of more lateral biopsies ( the method of five regions ) ; four fragments ( two on each side ) of the most lateral regions and three from the median line were added , totaling 13 fragments . with this technique the number of false - negative results decreased by 35% . in a subsequent study , presti jr et al . showed the advantages of prostate biopsy techniques involving a larger number of core fragments and including the latero - lateral regions . in our department of urology at the botucatu medical school , the 10-core protocol is standard ; the sextant biopsy protocol , extended to obtain 12 cores during the first biopsy , is used at the brazilian society of urology . the aim of this study was to compare pca detection in prostate biopsy specimens between the 10-core protocol ( protocol of botucatu medical school ) and the 12-core ( used at the brazilian society of urology ) and 16-core ( overall total ) protocols . the number of cores collected during prostate biopsy was also compared with stratified psa levels , stratified prostate volumes , gleason scores , and detection rates of high - grade prostatic intraepithelial neoplasia ( hgpin ) and atypical small acinar proliferation ( asap ) . the current prospective controlled study was conducted from january 2011 to february 2012 at the department of urology , botucatu medical school , sao paulo state university , after approval by the research ethics committee . the criteria for inclusion in the study were as follows : dre results suggestive of neoplasia , elevated psa ( > 4.0 ng / ml in men older than 55 years and > 2.5 ng / ml in men younger than 55 years ) , a psa density > 0.15 ng / ml , and an annual increase in the rate of psa levels > 0.75 ng / ml . carriers of coagulopathies , individuals with urinary tract infections ( whether diagnosed at the time of biopsy or during treatment ) , and individuals who refused to provide informed written consent were excluded from the study . consecutive patients ( n=354 ) were recruited for the study ; however , three of these patients were excluded : two for not consenting to participate in the study ( did not sign the consent form ) and another because they underwent sextant biopsy as a result of an unfavorable medical condition . the patient 's medical records were reviewed , and variables such as age , race , serum total psa ( current and previous ) , free psa , free psa / total psa , and biopsy indication were analyzed . the biopsy was performed on an outpatient basis in a room equipped with all material necessary for emergency intervention . the biopsies were performed by two experienced urologists . on the morning of the procedure , a rectal enema ( 250 ml ) was performed , and antibiotic prophylaxis was achieved with the oral administration of 500-mg ciprofloxacin 2 hours prior to the procedure and again 8 hours afterward . the procedure was performed while the patient was in the left lateral position with the thighs flexed . the procedure was performed by using dornier trus equipment with a 6.5-mhz multiplanar probe , an auto - fire gun , and an 18-gauge needle . initially 10 punctures were performed , yielding core specimens from the following regions of the prostate bilaterally : base , middle third , apex , medial ( transitional zone ) , and latero - lateral . after these specimens were collected , six additional punctures were performed bilaterally in the same specimens in the more lateral regions of the base , middle third , and apex ( fig . a positive diagnosis of pca was compared between the 10-core protocol ( base , middle third , apex , medial [ transitional zone ] and latero - lateral , bilaterally ) and the 12-core ( base , middle third , apex , and more lateral regions of the base , middle third , and apex , bilaterally ) and 16-core ( overall total ) protocols ( fig . tumor detection with the 10- , 12- , and 16-core protocols was correlated with psa levels , prostate volumes , gleason scores , and detection rates of hgpin and asap . data were collected and recorded on an excel spreadsheet and analyzed by using sas 9.2 ( sas institute inc . , cary , nc , usa ) . results for age , psa level , and prostate volume are expressed as means and standard deviations . chi - square and fisher exact tests were used to evaluate differences between the variables ; the significance level was set at 5% . the prevalence of pca diagnosis , with 90% power , was determined by using a one - sided hypothesis test and a significance level of 5% . a total of 351 consecutive patients underwent trus - guided prostate biopsy with 16-core fragments . mean age , total psa level , prostate volume , and race are shown in table 1 . four patients had psa levels > 100 ng / ml ( 137 , 198 , 2,000 , and 2,585 ng / ml)-greater than the mean and standard deviation . most patients ( 68.81% ) had a high psa level , 13.11% had an abnormal dre result , 12.25% had an association between a high psa and the presence of a nodule or an abnormal dre result , and 5.83% had an increased psa velocity . results of the dre performed before prostate biopsy were abnormal in 93 patients ( 26.50% ) and were normal in 258 patients ( 73.50% ) ( t1c ) . examination of the prostate by trus , before the prostate biopsy , detected hypoechoic nodules in 98 of the patients ( 27.92% ) . pca positivity in prostate biopsy specimens , by the number and location of the biopsy cores , is shown in fig . 3 . the cancer detection rate was not significantly different between the three biopsy protocols . pca was detected in 102 patients ( 29.06% ) with the 10-core protocol , in 99 patients ( 28.21% ) with the 12-core protocol , and in 107 patients ( 30.48% ) with the 16-core protocol ( p=0.79 ) . the psa level was stratified as 0 to 2.5 , 2.6 to 4.0 , 4.1 to 10.0 , and > 10.0 ng / ml , and prostate volume was stratified as 20 , 20 to 50 , and > 50 cm . pca positivity in the prostate biopsy specimens in relation to stratified psa levels ( ng / ml ) , stratified prostate volumes ( cm ) , and number of core biopsy specimens is shown in table 2 . elevated psa levels were associated with greater pca positivity , especially when levels were > 10.0 ng / ml . in two patients with a psa level < 2.0 ng / ml , pca was detected on biopsy . a comparison of psa levels with the number of core biopsy specimens showed no significant differences , nor did a comparison of prostate volumes with the number of core biopsy specimens . pca detection rates were greatest at prostate volumes between 20 and 50 cm and were lower at prostate volumes > 50 cm . correlations between tumor gleason scores , number of prostate biopsy specimens , and psa values are shown in table 3 . most patients had tumors with a gleason score of 7 followed by a gleason score of 6 , and few patients had a gleason score of 8 or 9 . no significant differences in cancer detection rates with the three biopsy protocols were found in relation to gleason scores and psa values . the detection rates of asap and hgpin in patients with negative biopsy results were stratified according to the three biopsy protocols and psa values ( table 4 ) . no statistically significant differences were found in the detection of asap and hgpin in relation to the number ( 10 , 12 , or 16 ) of cores collected . pca is an insidious neoplasm , and , as with any other malignancy , early detection is important . the introduction of serum psa screening for pca was a major breakthrough in the early diagnosis of the disease , which allows for the detection of subclinical malignancies . the development of treatments such as radical prostatectomy has led to permanent cure in a large number of patients or to an improved life expectancy . trus - guided prostate biopsy has been the standard method for diagnosing pca , but there is no consensus about the exact number of fragments to be collected . initially , hodge et al . proposed the sextant technique ; however , subsequent studies have shown that sextant biopsies yield false - negative results in 30% of cases . eskew et al . , in a study of 119 patients , added five more core specimens to the sextant biopsy , which improved the cancer detection rate by 35% . levine et al . added six more core specimens to the sextant biopsy , which resulted in the detection of an additional 30% of cancer cases . however , naughton et al . , in a prospective randomized study of 244 patients , found that the 6- and 12-core protocols yielded similar cancer positivity : 26% and 27% , respectively ( p=0.9 ) . reported that sextant biopsies failed to detect pca in 20% of 483 patients as compared with the 8- and 10-core protocols collected from lateral regions . presti jr reviewed available studies that analyzed various biopsy protocols and suggested that an initial biopsy should include a minimum of 12 cores ( extended biopsy ) , with special attention to the lateral regions of the prostate . in our study , pca detection rates with the 10- , 12- , and 16-core protocols were 29.06% , 28.21% , and 30.48% , respectively ( p=0.79 ) . no statistically significant differences in pca detection rates were found between these three protocols . the saturation biopsies with more than 20 cores were proposed for patients with multiple previous negative biopsy results . however , it is worth noting that the false - negative rate of saturation biopsy was reported to be similar to that found in patients who underwent traditional initial extended biopsy . jones et al . , in a study of 226 patients , reported that pca positivity rates were 44.6% and 51.7% , respectively ( p>0.9 ) , with a saturation biopsy consisting of 24 cores compared with 10 cores . pepe and aragona compared saturation biopsies ( 24 - 37 cores ) with 18- and 12-core protocols ; the detection rates of pca on initial biopsy were 46.9% , 49% ( p=0.6 ) , and 39.8% ( p=0 3 ) , respectively . however , on second and third rebiopsies , the saturation biopsy had a higher detection rate than did the 18-core biopsy : 22% versus 10.9% ( p=0.003 ) and 6.2% versus 0% , respectively . nomikos et al . , in a retrospective study , compared 24-core and 10-core protocols ; pca detection rates were 34.55% and 39.09% , respectively ( p=0.43 ) . however , the study by ceylan et al . showed a benefit of saturation biopsy . in a group of 1,120 patients , 8- , 10- , 12- , 16- , and 20-core protocols yielded cancer detection rates of 18.3% , 14.8% , 24% , 22.1% , and 30.3% , respectively ( p=0.008 ) . according to the literature reviewed , the sextant biopsy yields false - negative results in 30% of cases and should not be used . saturation biopsy as initial biopsy does not appear to be more effective at diagnosing pca than does extended biopsy . in summary , extended biopsy is indicated for the first biopsy and saturation biopsy may be indicated for rebiopsies . establishing a cutoff value for psa aims to ensure greater diagnostic accuracy . published data show that high - grade tumors may be found in patients with a psa level as low as < 4.0 ng / ml , which led to a reduction in the cutoff value to 2.5 ng / ml in some guidelines , especially for younger men , i.e. , < 60 years . physicians should evaluate psa cutoff values for each patient individually to allow the diagnosis of aggressive tumors without increasing the diagnosis of indolent tumors . the higher the psa level , the greater the pca positivity rate ( especially when > 10 ng / ml ) , which was also evidenced in other studies . in two patients with a psa level < 2.0 ng / ml , pca was detected on biopsy . the indication for biopsy was the presence of prostatic nodules detected on dre , which indicated that this type of examination may reveal tumors in men with a low psa level . in other studies also , no statistically significant differences were found between psa levels by the number of cores . the number of cores required to diagnose pca in relation to the size of the prostate is not yet defined . many studies have shown that the greater the number of cores collected in larger prostates , the greater the pca detection rate . the vienna nomogram was developed to define an appropriate number of prostate biopsy cores to improve the detection of pca based on the age of the patient and the prostate volume . . showed that the detection rate of pca with the vienna nomogram was 36.7% compared with 22% on first biopsy in the control group of eight cores . however , lecuona and heyns , in a prospective controlled clinical trial , suggested that there was no significant advantage to using the vienna nomogram to determine the number of prostate biopsies to be performed compared with the control group of eight fragments . in our study , we found no statistically significant differences in cancer detection rates when we compared prostate volumes with the number of cores collected ( 10 , 12 , and 16 ) . pca positivity in prostates > 50 cm was lower when compared with prostates between 20 and 50 cm . it remains doubtful whether more than 16 core fragments could increase the cancer positivity rate in prostates > 50 cm . the correlation between gleason scores and the biopsy protocol and psa values in other studies also showed no significant differences in tumor detection rates . studied 426 patients , 221 of whom had undergone sextant biopsy and 205 saturation biopsy before radical prostatectomy . the sextant biopsy had a lower concordance rate with gleason scores than did the saturation biopsy when compared with gleason scores with radical prostatectomy . other studies have also shown that the increase in the number of cores collected improves the concordance rate between gleason scores with prostate biopsy and radical prostatectomy . in our study , the correlation of prostate biopsy gleason scores with radical prostatectomy gleason scores was not evaluated . few studies have addressed the influence of increasing the number of fragments in prostate biopsy on the detection rates of hgpin and asap . published a study in which hgpin and asap were detected in 35.7% of cases by sextant biopsy , in 28.6% of cases with 6 additional cores ( total of 12 cores ) , and in 35.7% of cases with 21 cores . epstein and potter also showed no relationship between the number of cores collected during prostate biopsy and the incidence of hgpin and asap . nomikos et al . , in a retrospective study involving patients with a psa level < 10 ng / ml , reported that a 24-core prostate biopsy protocol increased the detection rate of hgpin by 20% ( p=0.0008 ) compared with a 10-core protocol . in our study , no statistically significant difference was found in the detection rates of asap and hgpin in relation to the number of core fragments ( 10 , 12 , or 16 ) collected . the detection rates of asap and hgpin were approximately 6% and 2.5% , respectively , consistent with the literature . in our study the detection rate was low ( 1.8% ) . corroborating the data of pelzer et al . , we found that it did not improve the cancer detection rate , and there is currently no requirement to collect cores from the transitional zone . in our sample of 351 patients , only 1 patient ( 0.28% ) would have remained undiagnosed with pca if cores from the transitional zone were not collected . considering the data presented , we concluded that the protocol with 10 cores on first biopsy is sufficient to obtain a high positivity rate ( 29.06% ) as compared with protocols with more cores . the most important fact is that additional biopsies should capture more lateral regions of the prostate . in the current study , extended biopsies of additional lateral tissue increased the pca detection rate by 20% compared with the sextant biopsy . thus , we observed that the number of lateral biopsies collected is more important than the total number of biopsies collected . however , it is clear from the literature that a greater number of biopsies is required to yield higher detection rates with rebiopsies ( extended or saturation biopsies ) . we also propose a modification to the biopsy protocol used at the botucatu medical school , i.e. , collect 10 cores , do not collect the 2 medial cores , and collect more lateral cores ( base , middle third , apex and two latero - lateral , bilaterally ) . furthermore , we must consider that increasing the number of biopsy specimens collected increases the duration of the procedure and , consequently , the discomfort of patients , especially when analgesia is induced with local anesthetics , which is the most commonly used method . however , the number of biopsy specimens collected is not as important when using intravenous sedation and analgesia , as is performed when it is necessary to obtain a large number of fragments . however , collection of a greater number of fragments could be a risk factor for complications after biopsy . de jesus et al . reported that the collection of more than eight fragments increases the likelihood of infectious complications . cancer positivity with the 10-core protocol was not significantly different from that with the 12- and 16-core protocols , which indicates that the 10-core protocol is acceptable for performing a first biopsy .

purposewe evaluated the utility of 10- , 12- , and 16-core prostate biopsies for detecting prostate cancer ( pca ) and correlated the results with prostate - specific antigen ( psa ) levels , prostate volumes , gleason scores , and detection rates of high - grade prostatic intraepithelial neoplasia ( hgpin ) and atypical small acinar proliferation ( asap).materials and methodsa prospective controlled study was conducted in 354 consecutive patients with various indications for prostate biopsy . sixteen - core biopsy specimens were obtained from 351 patients . the first 10-core biopsy specimens were obtained bilaterally from the base , middle third , apex , medial , and latero - lateral regions . afterward , six additional punctures were performed bilaterally in the areas more lateral to the base , middle third , and apex regions , yielding a total of 16-core biopsy specimens . the detection rate of carcinoma in the initial 10-core specimens was compared with that in the 12- and 16-core specimens.resultsno significant differences in the cancer detection rate were found between the three biopsy protocols . pca was found in 102 patients ( 29.06% ) using the 10-core protocol , in 99 patients ( 28.21% ) using the 12-core protocol , and in 107 patients ( 30.48% ) using the 16-core protocol ( p=0.798 ) . the 10- , 12- , and 16-core protocols were compared with stratified psa levels , stratified prostate volumes , gleason scores , and detection rates of hgpin and asap ; no significant differences were found.conclusionscancer positivity with the 10-core protocol was not significantly different from that with the 12- and 16-core protocols , which indicates that the 10-core protocol is acceptable for performing a first biopsy .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

approximately 217,730 persons were diagnosed with prostate cancer in the united states in 2010 , which makes up 28% of all cancers in men . of the 217,730 persons , 32,250 died , which is the second highest mortality rate of male cancers despite the small number when considering the number of people diagnosed . therefore , risk assessment is essential in prostate cancer in both high - risk and low - risk patients to avoid the potential effects of additional treatment on quality of life . proposed the cancer of the prostate risk assessment ( capra ) score , which is a prognosis - predicting method that uses age , clinical stage , prostate - specific antigen ( psa ) before surgery , biopsy gleason score , and malignancy ratio of the biopsy cores . the accuracy of the capra score has been verified in the united states and europe [ 3 - 6 ] . however , the clinical stage , biopsy gleason score , and malignancy ratio of the biopsy cores , 3 of the variables that define the capra score , can overestimate or underestimate the actual stage or the range of cancer . information on pathologic specimen gleason score ( pgs ) , surgical margin ( sm ) , extracapsular extension ( ece ) , seminal vesicle invasion ( svi ) , and lymph node invasion ( lni ) that can be obtained after prostate resection may help to assess the risk of cancer . accordingly , cooperberg et al . proposed the capra - s score , which uses additional information that can be obtained after surgery and that can predict relapse of cancer accurately and easily . the aim of the present study was therefore to assess the validity of applying the capra - s score , which is an assessment measure of biochemical recurrence ( bcr ) that uses histopathologic data after prostate cancer surgery , to koreans . in 2011 , cooperberg et al . analyzed the data of 3,837 patients who underwent prostatectomy in ucsf cancer of the prostate strategic urologic research endeavor ( capsure , university of california , san francisco , ca , usa ) to investigate the capra - s score and reported that it can be applied to the prediction of bcr after surgery and for determining suitable treatment . on the basis of these findings , 203 prostate cancer patients who underwent radical prostatectomy between february 1997 and november 2010 were observed for longer than 6 months and the capra - s score of 134 patients for whom records for preoperative psa , pgs , sm , svi , ece , and lni were available were calculated . bcr was defined as repetitive measurement of psa 0.2 ng / ml at least 6 months after surgery with at least a 4-week interval . the purpose of the capra - s score is to predict bcr more accurately than with existing pathologic data . the capra - s score was calculated by using psa , sm , svi , pgs , ece , and lni ( table 1 ) . these variables were each verified for bcr by use of the cox proportional hazards regression model ( table 2 ) . for statistical analysis , we used a cox proportional hazards regression model and kaplan - meier survival analysis by use of ibm spss ver . 18.0 ( ibm co. , armonk , ny , usa ) . the 134 patients were divided into groups with a capra - s score of 0 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 and 9 , respectively , ( table 1 ) , and survival analysis was performed for each group ( fig . 16 patients had a capra - s score of 0 , 15 patients had a score of 1 , 13 patients had a score of 2 , 17 patients had a score of 3 , 12 patients had a score of 4 , 13 patients had a score of 5 , 11 patients had a score of 6 , 6 patients had a score of 7 , 11 patients had a score of 8 , and 20 patients had a score of 9 or higher . when the patients were divided according to capra - s score into 10 groups , the 5-year disease - free survival rate was predicted in each group differently . although the groups with scores of 4 and 8 showed different tendencies for 5-year disease - free survival from the other neighboring groups , overall , the 5-year disease - free survival rate decreased with increasing capra - s score ( p=0.03 ) ( fig . sm positivity showed statistical significance with a hazard ratio ( hr ) of 1.663 ( p=0.048 ) , and pgs also showed statistical significance ( hr , 1.603 ; p=0.01 ) . the survival rate was analyzed according to the capra - s score , with a score of 0 - 2 as the low - risk group , a score of 3 - 5 as the intermediate - risk group , and a score of 6 and higher as the high - risk group . the 5-year disease - free survival rate differed significantly between the groups ( p=0.002 ) ( fig . radical prostatectomy is done in more than one third of all patients diagnosed with prostate cancer . some of the patients scheduled for surgery experience bcr , and some of these patients experience clinical recurrence and metastasis , eventually leading to death . variation in the postoperative psa level helps to identify high - risk patients , but there are demerits such as the need for repetitive tests and delayed initiation of some treatments such as radiation therapy and hormonal therapy . also , a number of high - risk patients , in whom only restricted pathological factors were considered , can avoid the morbidity caused by additional treatment because they do not progress after surgery [ 10 - 12 ] recently , several models for predicting biochemical outcomes after prostatectomy have been reported , and the validity of the prediction formula suggested by bauer et al . , the nomogram developed by kattan et al . , and the updated version of stephenson et al . have been verified externally on the basis of standard clinical and pathological variables . the c - index of these models is reported to be in the range of 0.77 to 0.86 , and the possibility of non - progression tends to be exaggerated in high - risk patients . the original capra - s study reported by cooperberg et al . showed relatively accurate distinction with a c - index of 0.77 [ 13 - 15 ] . the c - index in the present study was 0.776 , which indicates that this score can predict the prognosis of prostate cancer in koreans very accurately . the results , however , represent only relative risks , and continuous correction may be required with the use of additional clinical data . two studies have directly compared the pretreatment capra score and the established popular nomogram ; one is a united states study that compared the capra score with the original kattan preoperative nomogram , and the other is a european study that compared the capra score with the updated preoperative nomogram of stephenson et al . [ 2,13 - 15 ] . the accuracy of the capra score was shown to be similar to these nomograms in these two studies , but the capra score showed superiority in calibration and decision curve analysis in the european study [ 2,13 - 15 ] . reported that the postoperative nomogram , which is different from the capra - s score , shows similar distinction when looking at the calculation by c - index , and the capra - s score showed superiority in calibration and decision curve analysis [ 13 - 15 ] . reported that although the capra - s score shows a tendency of relatively many patients converging in the lower score , there was wider patient distribution than the original pretreatment capra score . therefore , the capra - s score is expected to be more useful for understanding the risk of recurrence and the availability of adjuvant therapy . the capra - s score shows more diverse score distribution compared with the capra score , which is the preoperative recurrence prediction score , and the capra - s score can be said to be more useful than the capra score at predicting the recurrence risk or determining whether to perform adjuvant therapy because the clinical staging , biopsy gleason score , and malignancy ratio of the biopsy cores used in the capra score can overestimate or estimate the actual stage or range of cancer [ 2,9,16 - 18 ] . however , it has to be acknowledged that lni , which is expected to have more influence on recurrence , only contributes 0 to 1 points in the capra - s score despite the fact that the original capra - s score is a pathologic record recorded with various subjective criteria by several pathologists . it seems that although the actual lni rate is high , lymph node dissection is done restrictively even in high - risk patients in the united states , where the capra - s score was devised . thus , the score contributed by lni has been limited to minimize the effects of unknown lni [ 19 - 21 ] . therefore , active lymph node dissection as well as giving more contributable points to lni in the capra - s score must be considered for a more accurate capra - s score in koreans . the groups with a capra - s score of 4 and 8 showed different tendencies for the 5-year disease - free survival rate from other neighboring groups . however , it is estimated that this arose as a statistical error owing to the small population number and also owing to the nonequality of score distribution of lni mentioned before . however , a significant difference was shown when the capra - s score divided into low - risk ( 1 - 2 ) , intermediate - risk ( 3 - 5 ) , and high - risk ( 6 ) groups , which is easier for clinical application ( p=0.002 ) ( fig . although bcr does not necessarily correlate with the ultimate mortality of prostate cancer , the capra - s score is expected to be able to predict cancer - specific mortality , and it is also expected to be used to predict cancer - specific and overall mortality through accumulation of more patient data and partial correction and supplementation in the future . application of the capra - s score in koreans is significant in that it becomes easier to apply in practice by objectively charting the prediction of prognosis , which is partially dependent on empirical data . the capra - s score can be easily calculated with data on several variables and can be applied to predicting postoperative recurrence and classifying high - risk patients who need adjuvant therapy . however , the relatively few data compared with the united states .

purposeto evaluate the validity of the university of california san francisco cancer of the prostate risk assessment - s score ( capra - s score ) , a biochemical indicator of recurrent prostate cancer that uses histopathologic data , in korean prostate cancer patients.materials and methodsa total of 203 prostate cancer patients who underwent radical prostatectomy between february 1997 and november 2010 were observed for longer than 6 months . the capra - s score of 134 patients for whom records were available for preoperative prostate - specific antigen ( psa ) , pathologic specimen gleason score , surgical margin , seminal vesicle invasion , extracapsular extension , and lymph node invasion were calculated . biochemical recurrence was defined as repetitive measurement of psa 0.2 ng / ml at least 6 months after surgery with at least a 4-week interval . the cox proportional hazard model and kaplan - meier analysis were used for the statistical testing.resultsthe capra - s scores were divided into nine groups . the 5-year disease - free survival rate was reduced as the capra - s score increased compared with the group with a capra - s score of 0 - 1 . the capra - s score in this study was more sensitive to biochemical recurrence than was the capra score conducted at this institution ( capra - s concordance index , 0.776 ; capra concordance index , 0.728).conclusionsthe capra - s score is judged to be a useful tool for predicting the disease - free survival rate of korean prostate cancer patients and is thought to assist in establishing postoperative management .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

the most common type of acrylic denture failure is debonding or fracture of the denture teeth , accounting for approximately 33% of failures.1 this failure usually occurs in the anterior region of the denture . dentists and patients both prefer chair - side repair using auto - polymerized acrylic resin , because it is a straightforward procedure and takes less chair time than repair in the laboratory . a likely explanation for teeth debonding is weak bond due to contamination of the tooth surfaces with substances such as wax during laboratory processing . another cause is a chemical difference between the tooth and denture base because of different processing methods.2 weakness of the repaired interface leads to recurrent debonding . therefore , the surface treatment method , mechanical or chemical , is important for denture repair . the placement of a diatoric recess ( mechanical ) and the use of a bonding agent ( chemical ) on denture teeth resulted in higher bond strengths compared with no treatment.34 methyl methacrylate ( mma ) is the most frequently used chemical surface treatment agent . mma is effective as an adhesion promoter because of its chemical similarities to denture base . however , surface treatment with mma requires 3 minutes to effectively prime the surface for ultimately reducing adhesive failure,5 thus requiring an excessive time . other solutions have been used as chemical surface treatments ( e.g. , chloroform , methylene chloride ( dichloromethane ) , and 4-methacryloxyethyl trimellitate anhydride ( 4-meta ) ) . although chloroform and methylene chloride have been used , they have been identified as being carcinogenic.6 researchers6 found that two solutions , methyl formate and methyl acetate , were non - toxic and resulted in similar bond strengths to poly(methyl methacrylate ) to those obtained with methylene chloride . another study found that methyl formate , methyl acetate , and their mixture significantly enhanced the flexural strength of heat - cured acrylic denture base resin that had been repaired with self - cured acrylic resin . the scanning electron micrographs in this study demonstrated that the application of these solutions to heat - cured acrylic resin resulted in a 3d honeycomb appearance , whereas specimens treated with methyl methacrylate developed shallow pits.7 in addition , other studies have been conducted for investigating the surface treatment of acrylic denture base and reline resin with methyl formate - methyl acetate ( mf - ma ) . these studies have shown that mf - ma significantly enhanced the bond strength between acrylic denture base and reline resin.89 a study of mf - ma application on acrylic denture teeth revealed that a 15-second application before packing the teeth with heat - cured acrylic denture base resulted in significantly higher micro - tensile bond strength between the teeth and the denture base , compared with the no surface treatment group . moreover , there was no significant difference between the mf - ma and mma groups using a 15-second application on micro - tensile bond strength.10 therefore , mf - ma may be an acceptable alternative for mma because it is less toxic . the acrylic denture tooth structure can also influence the bond strength to the denture.311 the conventional tooth has low wear resistance . therefore , increased wear resistance is useful,12 and cross - linking the polymer matrix can increase wear resistance.13 because of this complex structure , the acrylic has less polymer penetrability than into conventional denture teeth , resulting in lower bond strength . in contrast , another study stated that there was no significant difference in bond strength between conventional and cross - linked denture teeth to auto - polymerized acrylic resin.14 thus , crosslinking is not a major factor in reducing the strength of the joint between teeth and denture base.15 there are various methods for determining the bond strength between denture teeth and denture base , such as the american dental association specification number ( ada 15),16 international organization for standardization for synthetic resin teeth ( iso 3336),17 or the finite element stress analysis technique.16 however , these methods have been criticized concerning their accuracy in determining bond strength . moreover , the lack of uniformity in the tooth - denture base testing methods does not allow bond strength to be investigated in a standardized manner and/or the results to be directly compared.2 past studies using mf - ma solutions have indicated that it is a non - toxic surface treatment agent and improves the bond strength of acrylic resin materials . however , there are no studies of the bond strength when using mf - ma for the repair of denture teeth with auto - polymerized acrylic resin . the objective of this study was to evaluate the effect of surface treatment with mf - ma solution , using various application times and comparing to that of mma , on the tensile bond strength of different acrylic denture teeth repaired with auto - polymerized acrylic resin . three brands of denture teeth were used : yamahachi new ace ( ya ) , major dent ( md ) , and cosmo hxl ( cm ) , ( table 1 ) . seventy maxillary central incisor acrylic denture teeth of each brand were embedded with incisal surface down in auto - polymerized poly(methyl methacrylate ) ( pmma ) and were packed in 20-mm diameter polyethylene pipes . their ridge - lap surfaces were polished with 500- and 1200-grit silicon carbide paper in a polishing machine ( nano2000 , pace technologies , st . chemical solutions were applied to the groups as follows ( table 2 ) : no treatment ; methyl formatemethyl acetate ( mf - ma ) solution at a ratio of 25:75 ( by volume ) ( cu acrylic bond , faculty of dentistry , chulalongkorn university , bangkok , thailand ) for 15 seconds , 30 seconds , 60 seconds , 120 seconds , 180 seconds , and mma for 180 seconds . a polyethylene sheet with a 3-mm diameter hole was placed over the treated surface and a 10-mm diameter acrylic resin ring was placed centrally over the hole in the polyethylene sheet . auto - polymerized acrylic resin ( unifast trad , gc dental products co. , aichi , japan ) was loaded into the ring and compressed with a 1-kg weight . the specimen was placed in a pressure cooker at 2 mpa at 60. after the acrylic resin had set , an acrylic resin rod was attached on the top with cyanoacrylate glue ( super glue , alteco chemical pte ltd . , japan ) to connect the specimen to the tensile testing machine ( fig . the polymerized specimens were stored in deionized water at 37 for 48 2 hours and tested using a universal testing machine ( shimadzu , ez - s , bara scientific co. , ltd . , phra nakhon si , ayutthaya , thailand ) with a 500 n load cell at a crosshead speed of 10 mm / min . the tensile bond strength ( in mpa ) was calculated by dividing the failure force by the bond surface area . the data were statistically analyzed using spss for windows 22 ( ibm corporation , new york , ny , usa ) . the mean and standard deviation ( sd ) for the tensile bond strength of each group was calculated and statistically analyzed using two - way analysis of variance ( anova ) and the post hoc dunnett t3 test at the 95% confidence level . the results ( table 2 ) demonstrated that the tensile bond strengths of the negative control groups ( yac ; mdc ; cmc ) were not significantly different from each other ( p > .05 ) . for each brand , the surface treatment groups had significantly higher tensile bond strengths compared with the negative control group ( p < .05 ) . the tensile bond strengths of the mma 180-second group of each brand were not significantly different from the mf - ma 15-second , 30-second , 60-second , 120-second , and 180-second groups ( p > .05 ) . however , the ya mf - ma 180-second group had a significantly higher tensile bond strength compared with the mma 180-second group ( p < .05 ) . the bond strength of acrylic denture teeth to auto - polymerized acrylic resin can be improved by chemical surface treatment . this improvement begins when the solvents in the surface treatment agent contact the denture teeth , dissolving their surfaces , and causing swelling of the surface layers . subsequently , the monomer of the auto - polymerized acrylic resin material diffuses and penetrates into the interpenetration polymer network ( ipn ) matrix of the denture teeth during polymerization , which is known as the swelling phenomenon.17 this mechanism is affected by application time , polymerization temperature , type of solvent , denture teeth structure , and glass transitional temperature of the denture teeth.18 when used on acrylic denture teeth as a surface treatment , a methyl formate - methyl acetate solution acts by swelling and dissolving their surfaces , and then evaporating . in addition , there are no carbon carbon double bonds ( c = c ) in methyl formate or methyl acetate molecules to polymerize with the monomer in the auto - polymerized acrylic material . thus , it would not obstruct the interlocking of the auto - polymerized resin polymer chains and the denture teeth , and the tensile bond strength would be increased . polymer dissolving and swelling occurs when the polymer 's and solvent 's solubility parameters and polarities are close to each other . the solubility parameter of pmma ( acrylic denture teeth ) is 18.3 mpa , whereas those of mma , mf , and ma are 18.0 , 20.9 , and 19.6 mpa , respectively.19 in addition , mma , mf , and ma molecules have the methyl ester group , which increases their ability to soften pmma.6 the mf , ma , and mf - ma mixture increased the bond strength of repaired acrylic denture base and acrylic denture base relined with rebasing material.78 thus , it is hypothesized that mf - ma mixture would result in higher tensile bond strength between acrylic denture teeth and auto - polymerized acrylic resin compared with using mma liquid . although the results indicated that surface treatments with mf - ma and mma improved the bond strength compared to no treatment , mf - ma was superior to mma for the three brands of denture teeth for chairside repairs due to mf - ma 's reduced application time and lack of tissue irritation . the bond strengths when using mma for 180 s for all brands was not significantly different compared with the mf - ma 15 second , 30 second , 60 second , 120 second , and 180 second groups ( p > .05 ) , and mf - ma treatment resulted in bond strengths similar to mma treatment . the shortest application time of mf - ma treatment that had a higher bond strength compared with the control was 15 seconds , thus is the best time for chairside use for both conventional and cross - linked acrylic denture teeth . according to denture teeth types , the results showed that the bond strengths between cross - linked ( cosmo hxl ) and conventional type ( yamahachi new ace ; major dent ) were not significantly different ( p > .05 ) , the result which agrees with a previous study.15 the reason may be the type of acrylic denture base because the bond repaired with heat - cured acrylic resin was greater than that with auto - polymerized resin.20 the stronger bond is the result of processing temperature , time , and pressure . the polymer chain network size in conventional acrylic denture teeth is larger compared with that in the cross - linked type.21 therefore , there is more space between the polymer chains in conventional acrylic denture teeth compared with the cross - linked type ( crosslink density ) . thus , monomer from the auto - polymerized acrylic resin can diffuse more into the conventional acrylic denture teeth , resulting in increased polymerization . therefore , denture tooth type influences the bond strength for heat - cured acrylic resin , but not for auto - polymerized acrylic . another reason may be that the cross - linked structure of cosmo hxl , an interpenetrating polymer network ( ipn ) consisting of only 10% highly crosslinked if the denture tooth has a true ipn structure , the tensile bond strength between the tooth and auto - polymerized resin will be reduced because ipn denture tooth swells less after surface treatment with chemical agents ( mf - ma , mma ) . a limitation of this study was that only conventional and cross - linked denture teeth were investigated . the ipn denture teeth have an advantage of high wear resistance.13 this type of tooth is appropriate for dentures that occlude natural teeth . however , its bond strength to the denture base is low . the ipn consists of two or more polymer networks , resulting in a more complex structure compared with the cross - linked type . therefore , this type of tooth should also be studied because it will generate useful data for determining the most suitable surface treatment method in each case . within the limitations of the present study , cu acrylic bond ( mf - ma solution ) and mma increased the bond strength of either conventional or crosslinked acrylic denture teeth to auto - polymerized acrylic resin compared to no treatment . the application of mf - ma for 15 second can be an alternative chemical surface treatment for repairing a denture base and rebonding both conventional and cross - linked acrylic denture teeth with auto - polymerized acrylic resin .

purposethis study evaluated the effect of chemical surface treatment using methyl formate - methyl acetate ( mf - ma ) solution on the tensile bond strength between acrylic denture teeth and auto - polymerized acrylic resin.materials and methodsseventy maxillary central incisor acrylic denture teeth for each of three different brands ( yamahachi new ace ; major dent ; cosmo hxl ) were embedded with incisal edge downwards in auto - polymerized resin in polyethylene pipes and ground with silicone carbide paper on their ridge lap surfaces . the teeth of each brand were divided into seven groups ( n=10 ) : no surface treatment ( control group ) , mf - ma solution at a ratio of 25:75 ( v / v ) for 15 seconds , 30 seconds , 60 seconds , 120 seconds , 180 seconds , and mma for 180 seconds . auto - polymerized acrylic resin ( unifast trad ) was applied to the ground surface and polymerized in a pressure cooker . a tensile strength test was performed with a universal testing machine . statistical analysis of the results was performed using two - way analysis of variance ( anova ) and post - hoc dunnett t3 test ( =.05).resultsthe surface treatment groups had significantly higher mean tensile bond strengths compared with the control group ( p<.05 ) when compared within the same brand . among the surface treatment groups of each brand , there were no significantly different tensile bond strengths between the mf - ma groups and the mma 180 second group ( p>.05 ) , except for the yamahachi new ace mf - ma 180-second group ( p<.05).conclusion15-second mf - ma solution can be an alternative chemical surface treatment for repairing a denture base and rebonding acrylic denture teeth with auto - polymerized acrylic resin , for both conventional and cross - linked teeth .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

the endoplasmic reticulum ( er ) serves as a way station for the transit of secreted proteins , and specifically as a depot devoted to the folding and maturation of proteins and to the assembly of macromolecular protein complexes . before the protein passenger can exit the station , however , it is subject to a ' search ' by the quality - control manager of the er . the quality - control manager , sensing trouble , can retain undesirable passengers in the er , and in certain cases banish them from the station . undesirable passengers - aberrant polypeptides that can not fold properly or mature - are sent back to the cytoplasm from which they arrived , and there they are degraded by the proteasome . this brutal treatment clears the er of polypeptides that have the potential to aggregate , and prevents potentially toxic molecules from being secreted . the process by which aberrant , secreted polypeptides are degraded by the cytoplasmic proteasome is known as er - associated degradation , or erad . the mechanism of erad has been uncovered in recent years through the analysis of erad - defective yeast mutants , through the development of in vitro assays in which erad can be measured , and through analyses of the fates of misfolded proteins in the secretory pathway . many disease - causing mutations in secreted polypeptides escape erad , and in some cases the over - zealous actions of the quality - control manager can promote disease - for example , in cystic fibrosis . in addition , viruses may co - opt erad to destroy plasma - membrane - targeted host proteins and to elude the immune system , for example in infection by hiv or cytomegalovirus . bacterial toxins travel in reverse through the secretory pathway and finally exit from the er , like erad substrates , to the cytoplasm . foul conditions may increase the concentration of misfolded proteins in the er and overwhelm the capacity of this way station . unlike rail stations , though , overwhelmed eukaryotic cells simply increase the capacity of the er to house the greater number of misfolded proteins , and synthesize molecular chaperones to help solubilize the denatured polypeptides . this unfolded protein response ( upr ) , a response to increased concentrations of misfolded polypeptides in the er , is initiated by an er - resident transmembrane protein , ire1p , thought to sense the presence of aberrant proteins in the er lumen . the cytoplasmic domain of this molecule , which has homology to both kinases and an rnase , then cleaves an intron from the primary transcript for the hac1p transcription factor . ligation of the mrna , which allows for subsequent translation , requires the trna ligase , rlg1p . once made , the transcription factor transits into the nucleus and initiates the synthesis of genes containing a 5 ' ' unfolded protein response element ' and other upr target genes ( figure 1 ) . although the identities of a few of the genes up - regulated by the upr have been known for several years ( for example , er lumenal molecular chaperones ) , the full spectrum of proteins induced directly or indirectly by the upr has been ill - defined . a recent paper in cell from the walter and weissman laboratories rectifies this problem , and provides fascinating details on the breadth of the upr and the connection between the upr and erad . to establish the complete profile of genes induced in response to unfolded proteins , yeast cells ( saccharomyces cerevisiae ) were incubated with compounds known to compromise polypeptide folding in the er : dithiothreitol ( dtt ) , a reducing agent , and tunicamycin , an inhibitor of n - linked glycosylation . yeast genomic oligonucleotide arrays were screened using biotinylated crna probes in collaboration with researchers from affymetrix , inc . the criterion for identifying upr - target genes was that their transcription should not be stimulated in ire1 or hac1 mutant strains , but in wild - type cells transcription should be induced to levels similar to that of a group of seven previously identified upr targets . one of these target genes , kar2 , encodes the lumenal hsp70 molecular chaperone , bip , which is known to play a vital role in protein translocation into the er , protein folding in the er , and erad . thus , travers et al . chose genes as upr targets only if the level of their over - expression correlated to the canonical set at least as well as that observed for bip . some information on the function of the corresponding proteins is available for 208 of these , and no information is available for the other 173 . one of the previously uncharacterized genes uncovered from the screen , per100 , is homologous to a gene required for protein translocation in a related yeast , yarrowia lipolytica , and a per100 mutant was shown by travers et al . to exhibit erad defects . clearly , continued analysis of the 173 previously uncharacterized orfs will yield many exciting discoveries . about one half ( 103 ) of the previously characterized genes identified by travers et al . play roles in protein translocation , glycosylation , vesicular transport from the er , protein targeting to the vacuole ( which can also dispose of aberrant proteins in yeast ) , cell wall biosynthesis , and erad . in a related work , a screen to isolate yeast mutants that can not survive in the absence of the upr was performed , and the synthetic lethal mutants isolated from this screen uncovered genes similarly required for protein translocation , protein folding and glycosylation , and erad ( d. ng , personal communication ) . it is not difficult to imagine why these processes may be induced when the secretory way station is under siege . third , if the upr , or the concentration of misfolded proteins , was elevated to very high levels by the introduction of dtt , tunicamycin , or an over - expression system for an erad substrate , erad was reduced . taken together , these results indicate that the upr helps reduce the mass of unfolded proteins in the er via erad , but when this mass raises above a threshold level the efficiency of erad is compromised . fourth , deletion of genes that are not essential for viability but are required for erad led to a modest ( around twofold ) induction of the upr . and fifth , strains lacking one of these erad genes and ire1 were nonviable at elevated temperatures , indicating that cells survive either a certain level of aberrant proteins in the er or an inability to respond to unfolded proteins , but they succumb when the two are combined . collectively , the results of travers et al . support a model in which the upr and erad cooperate to eliminate misfolded proteins from the er , and provide insights into the nature of this collective and compensatory action . half of the upr target genes that have previously been characterized encode proteins generally associated with secretory pathway functions . these proteins are required to maintain the specialized environments of the er and beyond , an environment necessary for protein folding and quality control , vesicle trafficking , vacuolar protein sorting , and cell wall biogenesis . furthermore , the other half of the characterized upr target genes encode proteins with functions in unidentified processes . so , more surprises are likely to emerge from studies of the upr , studies that may reveal divergent intra - cellular signaling pathways linked to several , as yet unidentified , physiological responses . unfolded proteins in the endoplasmic reticulum activate both er - associated degradation ( erad ) and the unfolded protein response ( upr ) . unfolded proteins are banished to the cytosol , by sec61p - mediated retro - translocation , for degradation by the proteasome . unfolded proteins also activate the er transmembrane kinase / nuclease , ire1p , which in conjunction with the trna ligase , rlg1p , splices the hac1 primary transcript . mrna passes to the cytoplasm for translation , and the newly synthesized transcription factor , hac1p , enters the nucleus where it affects transcription of the upr target genes . translation of the target gene mrnas provides proteins for secretory pathway functions and other cellular processes .

using dna microarrays , 381 genes have been found to be induced in response to unfolded proteins . the identity of the previously characterized 208 of these , and further experiments , have revealed new details on the scope of the unfolded protein response and its connection to the degradation of proteins at the endoplasmic reticulum .

None ER-associated protein degradation The unfolded protein response A protein profile of the UPR Links between the UPR and ERAD

several studies have shown that elevated blood pressure ( bp ) is a major risk factor for cardiovascular morbidity and mortality . this relationship is strong and continuous in a range of patient populations and age groups.15 most of the major guidelines for the treatment of hypertension recommend that individuals with a bp 140/90 mmhg should be regarded as hypertensive , and treated in order to keep bp below this threshold . systolic bp and diastolic bp ( sbp and dbp , respectively ) targets generally must be lower in patients at high cardiovascular risk and in those with diabetes or renal disease.6,7 nearly 40 years ago , data from the framingham heart study showed that sbp was more closely associated with the risk of cardiovascular disease than dbp . although dbp was shown to be a more useful predictor of cardiovascular risk in hypertensive patients younger than 45 years , for the majority of hypertensive patients , the ability of sbp to predict ischemic cardiovascular disease was not improved by addition of dbp data . the superior predictive ability of sbp was more recently confirmed by the prospective studies collaboration , a meta - analysis of 61 prospective observational studies that recorded bp and cause - specific mortality.5 in this study , lewington et al found that sbp at baseline was more informative than dbp as a predictor of stroke and mortality from ischemic heart disease . moreover the study concluded that , in middle - aged individuals , prolonged reductions in usual sbp of only 2 mmhg would lead to substantial reductions in the incidence of death secondary to stroke ( a 7% reduction ) and other vascular causes ( 10%).5,11 these data are supported by those of stamler et al who found that sbp had a stronger association with cardiovascular risk than dbp in middle - aged and elderly individuals . at every level of dbp in this population , a higher sbp value was associated with greater cardiovascular risk and lower life expectancy.12 recently , benetos et al aimed to determine whether the high cardiovascular mortality rate in treated hypertensive patients was due to hypertension or to the presence of associated risk factors and/or diseases . using cardiovascular mortality data from treated hypertensive patients ( n = 8893 ) and from untreated age- and gender - matched normotensive and hypertensive controls ( n = 25,880 ) enrolled in the investigations prventives et cliniques cohort , benetos et al observed that the two - fold increase in cardiovascular and coronary mortality found in treated hypertensive patients persisted after adjustment for cardiovascular risk factors . adjustment for sbp was necessary to make the mortality rates similar in the two populations . subsequent inclusion of dbp in the model did not modify the between - group risk ratio . these results suggest that the increased cardiovascular mortality in treated hypertensive patients is mainly due to uncontrolled sbp levels.13 moreover , the incidence of most adverse cardiovascular events appears to follow a circadian pattern , reaching a peak in the morning shortly after waking and arising . it has been suggested that , during the post - awakening hours , the phases of hemodynamic , hematologic , and humoral cycles synchronize , thus creating an environment that predisposes to atherosclerotic plaque rupture and thrombosis in susceptible individuals . the increased sbp and dbp in the morning may act as a trigger for cardiovascular events , including myocardial infarction and stroke . the clinical implication of these observations is that antihypertensive therapy should provide bp control over the entire interval between doses.14 there is strong evidence that sustain the high variability of bp over 24 hours.15 furthermore , data from cross - sectional studies16 suggest that target organ damage is greater in hypertensive persons with high bp variability . the daytime sbp variability is a strong predictor of early carotid atherosclerosis progression and is useful to define the risk - benefit ratio of therapeutic approaches . a major impact of circadian bp patterns on the development of early carotid atherosclerosis has been demonstrated.17,18 until a few years ago , the endothelium was considered to be just a cellular barrier between circulating blood and the arterial muscle wall , and was thought to be involved only in the processes of transport , metabolism , and coagulation . however , it has now been demonstrated that most of the pathophysiologic mechanisms that lead to atherosclerosis are located in the endothelium.19 the physiologic vascular action of the endothelium is modulated by a series of molecules controlling vasodilation and , to a minor extent , vasoconstriction.20 there is mounting evidence to suggest that hypertension is associated with endothelial abnormalities that are not likely to be related to high bp values , and more to hypertension per se.21 accordingly , endothelial dysfunction is not only improved by reduction of bp values , but may also be improved by pharmacologic treatment for hypertension.22 the mean feature of endothelium - mediated vasodilatation in hypertensive patients is the reduced availability of nitric oxide ( no ) . in patients with essential hypertension , activation of endothelial cells induces production of cyclo - oxygenase - dependent factors , such as free radicals , which are responsible for the impaired bioavailability of no . when cyclo - oxygenase activity is blocked , the bioavailability of no is immediately restored . in hypertensive patients , vitamin c infusion is able to improve acetylcholine ( ach)-induced vasodilatation.23 no bioavailability is usually decreased either by decreased formation or by enhanced removal . the presence of classic cardiovascular risk factors is associated with enhanced generation of radical oxygen species . superoxide anions ( o ) play a pivotal role by reacting with no , resulting in the formation of peroxynitrite ( onoo ) , and hence decreasing bioavailability of no.24 studies with simultaneous measurement of relaxation and release of no clearly show that endothelial cell signal transduction is altered in hypertension.25 moreover , in studies with healthy human subjects , several agonists ( including ach and bradykinin ) have been found to induce vasodilation when injected directly into the brachial or coronary circulation.2628 this vasodilation appears mainly regulated by specific no synthase inhibitors , such as nv - monomethyl - larginine ( l - nmma ) . in patients with essential hypertension , the response to endothelium - dependent agonists ( mainly ach or bradykinin ) is blunted in different vascular regions when compared with healthy controls . this diminished relation response to ach or bradykinin is , moreover , resistant to l - nmma , which suggests the presence of compromised no availability caused mainly by oxidative stress.2932 some authors suggest that chronic inflammation may play a significant role in hypertension . a persistent low - grade inflammatory state could be associated with increased levels of cytokine plasma concentration . by impairing the capacity of the endothelium to generate vasodilating factors , particularly no , elevated cytokines may lead to the development of endothelial dysfunction , chronically impaired vasodilation , and hypertension.33 other studies suggest that endothelial dysfunction may be reversible . physical exercise , calcium channel blockers ( ccbs ) , angiotensin - converting enzyme inhibitors ( aceis ) , and angiotensin receptor antagonists ( arbs ) are effective in improving flow - stimulated endothelium - dependent vasodilation in patients with hypertension and diabetes mellitus.34,35 the arb irbesartan shows high efficacy in lowering bp , to a degree comparable with aceis and superior to other arbs , including losartan and valsartan . moreover , irbesartan has been shown to be effective in both early- and late - stage diabetic nephropathy.36 irbesartan has furthermore demonstrated considerable cost savings over standard therapy , including beta - blockers , diuretics , and nondihydropyridine ccbs in all stages of kidney disease . efficacy data from the irbesartan diabetic nephropathy trial and reduction of endpoints with the angiotensin ii antagonist losartan study37 has also demonstrated better cost savings with irbesartan than losartan in late - stage renal disease . while both irbesartan and losartan are registered for the treatment of late - stage diabetic nephropathy , irbesartan is also registered for early - stage diabetic nephropathy in the european union . some investigators claim there is a genuine value in using irbesartan instead of other arbs in the treatment of hypertension,38 and there is strong evidence of a positive impact from irbesartan in the treatment of hypertensive patients with endothelium - dependent vasodilation.39 this effect is partially shared with other arbs , although the specific activity of irbesartan has been shown in experimental studies.40 several studies have shown that elevated blood pressure ( bp ) is a major risk factor for cardiovascular morbidity and mortality . this relationship is strong and continuous in a range of patient populations and age groups.15 most of the major guidelines for the treatment of hypertension recommend that individuals with a bp 140/90 mmhg should be regarded as hypertensive , and treated in order to keep bp below this threshold . systolic bp and diastolic bp ( sbp and dbp , respectively ) targets generally must be lower in patients at high cardiovascular risk and in those with diabetes or renal disease.6,7 nearly 40 years ago , data from the framingham heart study showed that sbp was more closely associated with the risk of cardiovascular disease than dbp . although dbp was shown to be a more useful predictor of cardiovascular risk in hypertensive patients younger than 45 years , for the majority of hypertensive patients , the ability of sbp to predict ischemic cardiovascular disease was not improved by addition of dbp data . the superior predictive ability of sbp was more recently confirmed by the prospective studies collaboration , a meta - analysis of 61 prospective observational studies that recorded bp and cause - specific mortality.5 in this study , lewington et al found that sbp at baseline was more informative than dbp as a predictor of stroke and mortality from ischemic heart disease . moreover the study concluded that , in middle - aged individuals , prolonged reductions in usual sbp of only 2 mmhg would lead to substantial reductions in the incidence of death secondary to stroke ( a 7% reduction ) and other vascular causes ( 10%).5,11 these data are supported by those of stamler et al who found that sbp had a stronger association with cardiovascular risk than dbp in middle - aged and elderly individuals . at every level of dbp in this population , a higher sbp value was associated with greater cardiovascular risk and lower life expectancy.12 recently , benetos et al aimed to determine whether the high cardiovascular mortality rate in treated hypertensive patients was due to hypertension or to the presence of associated risk factors and/or diseases . using cardiovascular mortality data from treated hypertensive patients ( n = 8893 ) and from untreated age- and gender - matched normotensive and hypertensive controls ( n = 25,880 ) enrolled in the investigations prventives et cliniques cohort , benetos et al observed that the two - fold increase in cardiovascular and coronary mortality found in treated hypertensive patients persisted after adjustment for cardiovascular risk factors . adjustment for sbp was necessary to make the mortality rates similar in the two populations . subsequent inclusion of dbp in the model did not modify the between - group risk ratio . these results suggest that the increased cardiovascular mortality in treated hypertensive patients is mainly due to uncontrolled sbp levels.13 moreover , the incidence of most adverse cardiovascular events appears to follow a circadian pattern , reaching a peak in the morning shortly after waking and arising . it has been suggested that , during the post - awakening hours , the phases of hemodynamic , hematologic , and humoral cycles synchronize , thus creating an environment that predisposes to atherosclerotic plaque rupture and thrombosis in susceptible individuals . the increased sbp and dbp in the morning may act as a trigger for cardiovascular events , including myocardial infarction and stroke . the clinical implication of these observations is that antihypertensive therapy should provide bp control over the entire interval between doses.14 there is strong evidence that sustain the high variability of bp over 24 hours.15 furthermore , data from cross - sectional studies16 suggest that target organ damage is greater in hypertensive persons with high bp variability . the daytime sbp variability is a strong predictor of early carotid atherosclerosis progression and is useful to define the risk - benefit ratio of therapeutic approaches . a major impact of circadian bp patterns on the development of early carotid atherosclerosis has been demonstrated.17,18 until a few years ago , the endothelium was considered to be just a cellular barrier between circulating blood and the arterial muscle wall , and was thought to be involved only in the processes of transport , metabolism , and coagulation . however , it has now been demonstrated that most of the pathophysiologic mechanisms that lead to atherosclerosis are located in the endothelium.19 the physiologic vascular action of the endothelium is modulated by a series of molecules controlling vasodilation and , to a minor extent , vasoconstriction.20 there is mounting evidence to suggest that hypertension is associated with endothelial abnormalities that are not likely to be related to high bp values , and more to hypertension per se.21 accordingly , endothelial dysfunction is not only improved by reduction of bp values , but may also be improved by pharmacologic treatment for hypertension.22 the mean feature of endothelium - mediated vasodilatation in hypertensive patients is the reduced availability of nitric oxide ( no ) . in patients with essential hypertension , activation of endothelial cells induces production of cyclo - oxygenase - dependent factors , such as free radicals , which are responsible for the impaired bioavailability of no . when cyclo - oxygenase activity is blocked , the bioavailability of no is immediately restored . in hypertensive patients , vitamin c infusion is able to improve acetylcholine ( ach)-induced vasodilatation.23 no bioavailability is usually decreased either by decreased formation or by enhanced removal . the presence of classic cardiovascular risk factors is associated with enhanced generation of radical oxygen species . superoxide anions ( o ) play a pivotal role by reacting with no , resulting in the formation of peroxynitrite ( onoo ) , and hence decreasing bioavailability of no.24 studies with simultaneous measurement of relaxation and release of no clearly show that endothelial cell signal transduction is altered in hypertension.25 moreover , in studies with healthy human subjects , several agonists ( including ach and bradykinin ) have been found to induce vasodilation when injected directly into the brachial or coronary circulation.2628 this vasodilation appears mainly regulated by specific no synthase inhibitors , such as nv - monomethyl - larginine ( l - nmma ) . in patients with essential hypertension , the response to endothelium - dependent agonists ( mainly ach or bradykinin ) is blunted in different vascular regions when compared with healthy controls . this diminished relation response to ach or bradykinin is , moreover , resistant to l - nmma , which suggests the presence of compromised no availability caused mainly by oxidative stress.2932 some authors suggest that chronic inflammation may play a significant role in hypertension . a persistent low - grade inflammatory state could be associated with increased levels of cytokine plasma concentration . by impairing the capacity of the endothelium to generate vasodilating factors , particularly no , elevated cytokines may lead to the development of endothelial dysfunction , chronically impaired vasodilation , and hypertension.33 other studies suggest that endothelial dysfunction may be reversible . physical exercise , calcium channel blockers ( ccbs ) , angiotensin - converting enzyme inhibitors ( aceis ) , and angiotensin receptor antagonists ( arbs ) are effective in improving flow - stimulated endothelium - dependent vasodilation in patients with hypertension and diabetes mellitus.34,35 the arb irbesartan shows high efficacy in lowering bp , to a degree comparable with aceis and superior to other arbs , including losartan and valsartan . moreover , irbesartan has been shown to be effective in both early- and late - stage diabetic nephropathy.36 irbesartan has furthermore demonstrated considerable cost savings over standard therapy , including beta - blockers , diuretics , and nondihydropyridine ccbs in all stages of kidney disease . efficacy data from the irbesartan diabetic nephropathy trial and reduction of endpoints with the angiotensin ii antagonist losartan study37 has also demonstrated better cost savings with irbesartan than losartan in late - stage renal disease . while both irbesartan and losartan are registered for the treatment of late - stage diabetic nephropathy , irbesartan is also registered for early - stage diabetic nephropathy in the european union . some investigators claim there is a genuine value in using irbesartan instead of other arbs in the treatment of hypertension,38 and there is strong evidence of a positive impact from irbesartan in the treatment of hypertensive patients with endothelium - dependent vasodilation.39 this effect is partially shared with other arbs , although the specific activity of irbesartan has been shown in experimental studies.40 irbesartan is nearly completely absorbed following oral administration , with an average absolute bioavailability of 60%80% . about 25% of an administered radioactive dose is excreted in the urine and the remainder is eliminated in feces.41 several determinants of drug disposition , such as gastric acidity , gastric motility , glomerular filtration rate , plasma albumin , and renal and hepatic blood flow are altered in elderly subjects.42 it is possible that one or more factors may contribute to the observed significant increases in the cmax and auc of irbesartan . results obtained in renally impaired patients indicated that renal impairment had no clinically important effects on the pharmacokinetics of irbesartan.43 food does not affect the bioavailability of irbesartan , so it can be administered without regard to meals.44 although there is an effect of age on the pharmacokinetics of irbesartan , based on the safety and efficacy profile , no adjustment in irbesartan dosage is necessary with respect to age or gender.45 irbesartan is a selective antagonist for the angiotensin ii type 1 ( at1 ) receptor . the irbesartan concentration needed to reduce specific binding of 125-angiotensin ii to rat adrenal cortical microsomes by 50% is 0.9 nmol / l . the effects of arbs on hypertension are well known from the literature , and the efficacy of irbesartan in lowering bp is evident both in experimental and clinical studies.47 all irbesartan regimens significantly reduced mean 24-hour ambulatory bp values and were well tolerated . administration of irbesartan 150 mg once a day provides significant reduction of bp for 24 hours , equivalent to that obtained with the same total daily dose divided into two doses.48 the antihypertensive effect of irbesartan on both sbp and dbp appears increased when administered in combination with diuretics . in a study by mugellini et al , reduction of dbp and sbp with irbesartan - hydrochlorothiazide was comparable with that obtained with manidipine - delapril ( 26.4/20.2 mmhg and 27.6/21.8 mmhg , respectively ) and better than that of the respective monotherapies ( 16.3/11.3 mmhg with delapril and 15.2/11.7 mmhg with irbesartan ) in hypertensive patients with type 2 diabetes mellitus.49 recently , an uncontrolled , multicenter study has confirmed the efficacy of the combination of irbesartan and hydrochlorothiazide.50 the aim of this large study was to compare the antihypertensive efficacy of valsartan 80 mg versus irbesartan 150 mg when combined with hydrochlorothiazide 12.5 mg . untreated or uncontrolled hypertensive adults ( n = 800 ) were enrolled by primary care physicians . after a five - week , open - label , lead - in phase in which all patients received 12.5 mg hydrochlorothiazide once daily , subjects whose bp remained uncontrolled ( n = 464 ) were randomized to valsartan - hydrochlorothiazide ( 80/12.5 mg ) or irbesartan - hydrochlorothiazide ( 150/12.5 mg ) . after eight weeks , the irbesartan - hydrochlorothiazide combination produced greater reductions in average sbp and dbp measured by home bp monitoring than did valsartan - hydrochlorothiazide ( sbp 13.0 versus 10.6 mmhg , p = 0.0094 ; dbp 9.5 versus 7.4 mmhg , p = 0.0007 , respectively ) . the overall drug safety was similar between the two treatment groups.51 an irbesartan - hydrochlorothiazide fixed - dose combination has been approved for clinical use , and its efficacy and safety has recently been evaluated in a study of 96 hypertensive diabetic patients randomized to 12 months of double - blind treatment with doxazosin 4 mg / day or irbesartan 300 mg / day.52 at the end of the study , sbp and dbp were significantly ( p < 0.01 ) reduced from 152 to 140 mmhg and from 97 to 87 mmhg , respectively , with doxazosin . sbp and dbp were reduced from 150 to 134 mmhg and from 94 to 83 mmhg , respectively , with irbesartan ( p < 0.01 ) . irbesartan had significantly better antihypertensive efficacy than doxazosin ( p < 0.05).53 in patients with increased dbp , irbesartan shows comparable efficacy to that of amlodipine . in a study of non - african - american patients with a seated dbp of 95100 mmhg , irbesartan 150 mg / day did not show any significant difference in dbp - lowering effect compared with amlodipine 5 mg / day.54 in a recent study by fogari et al , 94 hypertensive patients were randomized to valsartan 160 mg + amlodipine 5 mg or irbesartan 300 mg + hydrochlorothiazide 12.5 mg for 24 weeks after a four - week placebo period . both combinations significantly reduced clinical seated and lying bp values , with no difference between treatments . bp changes from the lying to standing position were significantly greater in the irbesartan - hydrochlorothiazide group ( 17.2/9.1 mmhg ) than in the valsartan - amlodipine group ( 10.1/1.9 mmhg , p < 0.05 for sbp and p < 0.01 for dbp versus irbesartan - hydrochlorothiazide ) . both combinations were similarly effective in reducing ambulatory and clinical bp in very elderly hypertensive subjects.55 compared with aceis , irbesartan has a similar effect on bp reduction , with fewer adverse events recorded for irbesartan . in a double - blind , randomized study , an irbesartan - based antihypertensive regimen reduced sbp / dbp by 40/30 mmhg after 12 weeks in patients with severe hypertension . this reduction was at least equivalent to that of a regimen using enalapril up to 40 mg . the irbesartan - based regimen had a better tolerability profile with fewer adverse events ( 55% versus 64% ) and significantly less cough ( 2.5% versus 13.1% , p = 0.007).56 these results have been confirmed in a larger clinical trial comparing irbesartan and enalapril . two hundred and thirty - eight patients were randomized to treatment , and the study was completed by 111 patients in the irbesartan group ( dose titrated to 300 mg / day in 72.0% of patients ) and 115 patients in the enalapril group ( dose titrated to 20 mg / day in 76.5% of patients ) . bp reductions were similar in the two groups , both as measured in the clinic ( dbp 12.7 8.8 mmhg for irbesartan versus 12.4 7.4 mmhg for enalapril ; sbp 19.0 14.1 mmhg versus 17.5 14.0 mmhg , respectively ) and by 24-hour ambulatory bp monitoring ( dbp 9.4 8.5 mmhg versus 8.8 8.5 mmhg ; sbp 14.7 14.7 mmhg versus 12.6 13.1 mmhg ) . the overall incidence of adverse events ( 40.0% for irbesartan , 51.2% for enalapril ) was not statistically different between the treatment groups , although the incidence of adverse events , probably related to antihypertensive treatment , was significantly higher with enalapril than with irbesartan ( 24.6% versus 9.2% , respectively , p = 0.026 ) , and were essentially accounted for by a higher incidence of cough ( 8.1% versus 0.9% , respectively).57 compared with other arbs , irbesartan shows equal or greater efficacy in reducing both sbp and dbp . in a study by mancia et al , irbesartan was more effective than valsartan in reducing dbp and sbp at trough levels and in providing greater overall 24-hour bp lowering . in another study , after a three - week , single - blind , placebo lead - in period , 426 subjects were randomized to receive either irbesartan 150 mg or valsartan 80 mg for eight weeks . at the end of the study , irbesartan provided significantly greater reductions than valsartan for mean change from baseline in diastolic ambulatory bp at trough ( 6.73 versus 4.84 mmhg , respectively , p = 0.035 ) and in mean systolic ambulatory bp at trough ( 11.62 versus 7.5 mmhg , respectively , p < 0.01 ) . similar results were obtained for mean 24-hour diastolic ambulatory bp ( 6.38 versus 4.82 mmhg , respectively , p = 0.023 ) and systolic ambulatory bp ( 10.24 versus 7.76 mmhg , p < 0.01 ) . irbesartan also produced significantly greater reductions than valsartan for office - measured seated dbp ( 10.46 versus 7.28 mmhg , respectively , p < 0.01 ) and sbp ( 16.23 versus 9.96 mmhg , respectively , p < 0.01 ) and for self - measured morning dbp ( 6.28 versus 3.75 mmhg , respectively , p < 0.01 ) and sbp ( 10.21 versus 6.97 mmhg , respectively , p < 0.01).58 recent findings suggest that at1 receptor blockade improves superoxide production and no bioavailability more than do other classes of antihypertensive agents . in a study by brosnan et al , in vivo irbesartan , amlodipine , and hydrochlorothiazide - hydralazine produced similar reductions in bp values , but irbesartan caused a greater reduction in superoxide and p22phox in carotid arteries . four - hour in vitro exposure to irbesartan decreased superoxide levels in the aorta and increased no bioavailability in carotid arteries . neither 30-minute incubation with irbesartan nor four hours with amlodipine or hydrochlorothiazide - hydralazine altered superoxide levels . reduced expression of components of nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase may contribute to these effects.59 moreover , a protective effect of at1 receptor blockers on endothelial function during postprandial hypertriglyceridemia has been reported in healthy subjects.60 in 2005 , ceriello et al demonstrated , for the first time , that irbesartan , like statins , is able to ameliorate the damage done to endothelial function by postprandial hyperglycemia and hypertriglyceridemia , oxidative stress , and inflammation in diabetic patients . these results appeared not to be linked to the bp - lowering effect and were obtained after short - term treatment . in this study , combination treatment of atorvastatin and irbesartan showed a more powerful effect on vascular damage compared with monotherapy using irbesartan or atorvastatin.61 pharmacodynamic data suggest that irbesartan at a dose of 300 mg provides more effective and persistent at1 blockade than losartan 100 mg . in a short - term ( eight week ) study by kassler - taub et al , after a placebo lead - in , 567 patients were randomized to once - daily therapy with placebo , losartan 100 mg , irbesartan 150 mg , or irbesartan 300 mg . at the end of the study , reductions from baseline in seated dbp and seated sbp with irbesartan 300 mg were greater than those obtained with losartan 100 mg by 3.0 and 5.1 mmhg , respectively ( p < 0.01 for both comparisons ) . larger reductions were also demonstrated at weeks 1 and 4 ( p < 0.01 and p = 0.017 , respectively , for dbp and sbp ) of the study , and the antihypertensive effects of the treatments were not significantly different.62 the renoprotective effect of irbesartan has been tested in a study by rossing et al . in this study , 52 hypertensive patients with type 2 diabetes and microalbuminuria already on antihypertensive treatment were included . after a two - month wash - out of other drugs , patients were randomized to receive irbesartan 300 , 600 , and 900 mg once daily . after a 10-week treatment period , ultrahigh doses of irbesartan were well tolerated and provided additional renoprotection independent of changes in bp values and glomerular filtration rate , compared with irbesartan 300 mg.63 administration of irbesartan twice daily has been evaluated in a study by polnia et al who investigated whether blockage of angiotensin ii receptors by irbesartan could reverse the nondipper circadian rhythm of bp to a dipper pattern in 12 black salt - sensitive hypertensive patients on a high sodium diet ( 300 mmol per day ) after a two - week placebo administration , followed by two weeks on irbesartan 150 mg / day , two weeks on placebo , and two weeks on irbesartan 300 mg / day . on the last day of placebo , and of the irbesartan 150 mg / day and irbesartan 300 mg / day treatments , 24-hour bp and urinary 24-hour excretion of sodium and potassium were measured . compared with placebo , irbe - sartan significantly increased serum potassium and plasma renin activity and reduced fractional excretion of potassium and plasma aldosterone levels in a dose - dependent manner , without significant changes in body weight . the authors concluded that irbesartan can reverse the nondipper bp profile in salt - sensitive hypertensive patients on a high - salt diet , restoring the nocturnal bp decline by a predominantly dose - dependent reduction of night time bp . evidence has been accumulating to suggest that ambulatory bp values are more strongly correlated with markers of cardiovascular disease than are clinic bp values.64 the main studies that showed a significant effect of irbe - sartan in reducing microalbuminuria and decreasing the risk of diabetic nephropathy were irbesartan microalbuminuria type 2 diabetes in hypertensive patients ( irma ii)65 and irbesartan in diabetic nephropathy trial ( idnt).66 these are two long - term ( 23 year ) trials showing that the irbesartan is effective in reducing the progression of renal disease in patients with type 2 diabetes and high bp . in each trial , the standard of care for diabetes mellitus was maintained . using conventional antihypertensive therapy , including diuretics , beta - blockers , and ccbs ( but not aceis or other arbs ) , bp control was similar in the placebo- and arb - treated groups . the irma ii study ( table 1 ) evaluated the renoprotective effect of irbesartan , in hypertensive patients with type 2 diabetes and microalbuminuria.65 this multinational , randomized , double - blind , placebo - controlled study enrolled a total of 590 hypertensive patients with type 2 diabetes and persistent microalbuminuria who received irbesartan at a dose of either 150 mg daily or 300 mg daily , or placebo . the primary efficacy measure was the time from the baseline visit to the first detection of overt nephropathy , defined by a urinary albumin excretion rate in an overnight specimen > 200 g / min and at least 30% higher than the baseline rate on at least two consecutive visits . at the end of the study , nephropathy developed in 30 patients in the placebo group , compared with 19 patients in the 150 mg irbesartan group and 10 patients in the 300 mg irbesartan group . the unadjusted hazard ratio ( hr ) for diabetic nephropathy was 0.61 ( p = 0.08 ) in the 150 mg group and 0.30 ( p < 0.001 ) in the 300 mg group . the average bp during the course of the entire study was 144/83 mmhg in the placebo group , 143/83 mmhg in the 150 mg irbesartan group , and 141/83 mmhg in the 300 mg irbesartan group ( p = 0.004 for the comparison of sbp between the placebo group and the combined irbesartan groups ) . after adjustment for bp achieved during the study and the baseline level of microalbuminuria , the hr for diabetic nephropathy was 0.56 in the 150 mg group ( p = 0.05 ) and 0.32 in the 300 mg group ( p < 0.001 ) . serious adverse events were less frequent among the patients treated with irbesartan ( p = 0.02 ) . irbesartan reduced the level of urinary albumin excretion throughout the study . in the irbesartan 150 mg group , urinary albumin excretion decreased by 24% and , in the 300 mg group , decreased by 38% , whereas the placebo group had a 2% decrease ( p < 0.001 for the comparison between placebo and combined irbesartan groups ) . there was a significantly smaller reduction in the level of albuminuria in the 150 mg group than in the 300 mg group ( p < 0.001 ) . during this 24-month study , overt nephropathy developed in 30 patients in the placebo group , compared with 19 patients in the 150 mg group ( p = 0.08 ) and 10 patients in the 300 mg group ( p < 0.001 ) . the unadjusted hr for diabetic nephropathy was 0.61 ( p = 0.08 ) in the 150 mg group and 0.30 ( p < 0.001 ) in the 300 mg group . after adjustment for the baseline level of microalbuminuria and the bp achieved during the study , the hr for diabetic nephropathy was 0.56 in the 150 mg group ( p = 0.05 ) and 0.32 in the 300 mg group ( p < 0.001 ) . at the three - month visit , the decrease in creatinine clearance for the placebo group and the 300 mg irbesartan group separated and continued to diverge . the decline in creatinine clearance during the initial three - month period was greater than the sustained decline from three months to 24 months . the initial declines were 0.9 , 1.0 , and 1.9 in the placebo , and irbesartan 150 mg and 300 mg groups , respectively , compared with declines of 0.1 , 0.2 , and 0.2 between months 3 and 24 . urinary albumin decreased by 24% in the 150 mg group , 38% in the 300 mg group , and 2% in the placebo group ( p < 0.001 for the comparison between placebo and the combined irbesartan groups ) . there was a significantly smaller reduction in the level of albuminuria in the 150 mg group than in the 300 mg group ( p < 0.001 ) . in the other relevant large study , the idnt66 compared the renoprotective effect of irbesartan and amlodipine in patients with type 2 diabetes and microalbuminuria ( table 2 ) , beyond the effect of these two drugs on bp reduction . it was a prospective , randomized , double - blind , placebo - controlled study including 1175 patients from 210 clinical centers . patients were randomized to receive irbesartan 300 mg / day , amlodipine 10 mg / day , or placebo . groups were compared according to time to the primary composite endpoint of a doubling of baseline serum creatinine concentration , development of end - stage renal disease , and death from any cause . the mean follow - up duration was 2.6 years . at the end of the study , treatment with irbesartan was associated with a 20% and 23% reduction in risk of the primary composite endpoint compared with placebo and amlodipine , respectively ( p = 0.006 ) . the relative risk of doubling of the serum creatinine concentration was 33% and 37% lower in the irbesartan group compared with the placebo and amlodipine groups ( p = 0.003 and p < 0.001 , respectively ) . treatment with irbesartan was associated with a significantly lower risk of end - stage renal disease ( 23% ) compared with that observed in the other groups ( p = 0.07 for both comparisons ) . the increase in serum creatinine levels was slower in the irbesartan group compared with the placebo ( 24% ) and amlodipine ( 21% ) groups ( p = 0.008 and p = 0.002 , respectively ) . no significant differences between groups were observed in the rate of death from any cause or in the cardiovascular composite endpoint . although the irma ii and idnt studies provided strong support for the beneficial effect of irbesartan in renoprotection , both studies included patients with hypertension who had been previously treated with antihypertensive drugs . in a short - term study , irbesartan was administered to newly diagnosed drug - naive patients ; the effects of irbesartan 150 mg bid on microalbuminuria were observed in normotensive type 2 diabetic patients , suggesting that the renoprotective action of irbesartan is probably due to a direct action on renal hemodynamics and glomerular morphology.67 more recently , studies have demonstrated a precise role for irbesartan and statins in regulation of vascular dysfunction . in the endothelial protection , at1 blockade and cholesterol - dependent oxidative stress ( epas ) trial , the investigators showed that statin and at1 blocker therapy independently and in combination improved both the antiatherosclerotic endothelial expression quotient and endothelial function . in this study , 60 patients with stable coronary artery disease undergoing elective coronary artery bypass grafting surgery were randomized four weeks before surgery to no arbs and/or statins ( controls ) , pravastatin 40 mg / day , irbesartan 150 mg / day , or pravastatin in combination with irbesartan at the same dosages . the primary endpoint was a priori therapy - dependent regulation of the antiatherosclerotic endothelial expression calculated marker of endothelial regulation . at the end of the study , association of irbesartan and pravastatin significantly improved endothelial expression of antiatherosclerotic and proatherothrombotic genes and endothelial function in arteries in these patients with coronary artery disease . moreover , preoperative therapy with irbesartan and pravastatin and their combination improved endothelial function in internal mammary artery rings.68 recently , many studies have focussed on molecular markers of the inflammatory mechanisms involved in atherogenesis , and particular attention has been given to inflammatory molecules which interact with the endothelium , such as tumor necrosis factor- , vascular cell adhesion molecule , and superoxides . in a study by navalkar et al , irbesartan significantly improved levels of inflammatory molecules in 33 normotensive patients with coronary artery disease , and the maximal suppression of inflammatory markers was observed after 12 weeks.69 in another study , treatment with irbesartan was effective in reducing the pro - oxidative environment seen in patients with coronary artery disease . lag time for ldl oxidation increased 32% at 12 weeks , suggesting that increased resistance of ldl modification during serum and lipid peroxidation decreased by 36% with irbesartan compared with placebo . in addition , superoxide levels and monocyte - binding capacity were also significantly reduced in coronary artery disease patients receiving irbesartan.70 the effects of arbs on hypertension are well known from the literature , and the efficacy of irbesartan in lowering bp is evident both in experimental and clinical studies.47 all irbesartan regimens significantly reduced mean 24-hour ambulatory bp values and were well tolerated . administration of irbesartan 150 mg once a day provides significant reduction of bp for 24 hours , equivalent to that obtained with the same total daily dose divided into two doses.48 the antihypertensive effect of irbesartan on both sbp and dbp appears increased when administered in combination with diuretics . in a study by mugellini et al , reduction of dbp and sbp with irbesartan - hydrochlorothiazide was comparable with that obtained with manidipine - delapril ( 26.4/20.2 mmhg and 27.6/21.8 mmhg , respectively ) and better than that of the respective monotherapies ( 16.3/11.3 mmhg with delapril and 15.2/11.7 mmhg with irbesartan ) in hypertensive patients with type 2 diabetes mellitus.49 recently , an uncontrolled , multicenter study has confirmed the efficacy of the combination of irbesartan and hydrochlorothiazide.50 the aim of this large study was to compare the antihypertensive efficacy of valsartan 80 mg versus irbesartan 150 mg when combined with hydrochlorothiazide 12.5 mg . untreated or uncontrolled hypertensive adults ( n = 800 ) were enrolled by primary care physicians . after a five - week , open - label , lead - in phase in which all patients received 12.5 mg hydrochlorothiazide once daily , subjects whose bp remained uncontrolled ( n = 464 ) were randomized to valsartan - hydrochlorothiazide ( 80/12.5 mg ) or irbesartan - hydrochlorothiazide ( 150/12.5 mg ) . after eight weeks , the irbesartan - hydrochlorothiazide combination produced greater reductions in average sbp and dbp measured by home bp monitoring than did valsartan - hydrochlorothiazide ( sbp 13.0 versus 10.6 mmhg , p = 0.0094 ; dbp 9.5 versus 7.4 mmhg , p = 0.0007 , respectively ) . the overall drug safety was similar between the two treatment groups.51 an irbesartan - hydrochlorothiazide fixed - dose combination has been approved for clinical use , and its efficacy and safety has recently been evaluated in a study of 96 hypertensive diabetic patients randomized to 12 months of double - blind treatment with doxazosin 4 mg / day or irbesartan 300 mg / day.52 at the end of the study , sbp and dbp were significantly ( p < 0.01 ) reduced from 152 to 140 mmhg and from 97 to 87 mmhg , respectively , with doxazosin . sbp and dbp were reduced from 150 to 134 mmhg and from 94 to 83 mmhg , respectively , with irbesartan ( p < 0.01 ) . irbesartan had significantly better antihypertensive efficacy than doxazosin ( p < 0.05).53 in patients with increased dbp , irbesartan shows comparable efficacy to that of amlodipine . in a study of non - african - american patients with a seated dbp of 95100 mmhg , irbesartan 150 mg / day did not show any significant difference in dbp - lowering effect compared with amlodipine 5 mg / day.54 in a recent study by fogari et al , 94 hypertensive patients were randomized to valsartan 160 mg + amlodipine 5 mg or irbesartan 300 mg + hydrochlorothiazide 12.5 mg for 24 weeks after a four - week placebo period . both combinations significantly reduced clinical seated and lying bp values , with no difference between treatments . bp changes from the lying to standing position were significantly greater in the irbesartan - hydrochlorothiazide group ( 17.2/9.1 mmhg ) than in the valsartan - amlodipine group ( 10.1/1.9 mmhg , p < 0.05 for sbp and p < 0.01 for dbp versus irbesartan - hydrochlorothiazide ) . both combinations were similarly effective in reducing ambulatory and clinical bp in very elderly hypertensive subjects.55 compared with aceis , irbesartan has a similar effect on bp reduction , with fewer adverse events recorded for irbesartan . in a double - blind , randomized study , an irbesartan - based antihypertensive regimen reduced sbp / dbp by 40/30 mmhg after 12 weeks in patients with severe hypertension . this reduction was at least equivalent to that of a regimen using enalapril up to 40 mg . the irbesartan - based regimen had a better tolerability profile with fewer adverse events ( 55% versus 64% ) and significantly less cough ( 2.5% versus 13.1% , p = 0.007).56 these results have been confirmed in a larger clinical trial comparing irbesartan and enalapril . two hundred and thirty - eight patients were randomized to treatment , and the study was completed by 111 patients in the irbesartan group ( dose titrated to 300 mg / day in 72.0% of patients ) and 115 patients in the enalapril group ( dose titrated to 20 mg / day in 76.5% of patients ) . bp reductions were similar in the two groups , both as measured in the clinic ( dbp 12.7 8.8 mmhg for irbesartan versus 12.4 7.4 mmhg for enalapril ; sbp 19.0 14.1 mmhg versus 17.5 14.0 mmhg , respectively ) and by 24-hour ambulatory bp monitoring ( dbp 9.4 8.5 mmhg versus 8.8 8.5 mmhg ; sbp 14.7 14.7 mmhg versus 12.6 13.1 mmhg ) . the overall incidence of adverse events ( 40.0% for irbesartan , 51.2% for enalapril ) was not statistically different between the treatment groups , although the incidence of adverse events , probably related to antihypertensive treatment , was significantly higher with enalapril than with irbesartan ( 24.6% versus 9.2% , respectively , p = 0.026 ) , and were essentially accounted for by a higher incidence of cough ( 8.1% versus 0.9% , respectively).57 compared with other arbs , irbesartan shows equal or greater efficacy in reducing both sbp and dbp . in a study by mancia et al , irbesartan was more effective than valsartan in reducing dbp and sbp at trough levels and in providing greater overall 24-hour bp lowering . in another study , after a three - week , single - blind , placebo lead - in period , 426 subjects were randomized to receive either irbesartan 150 mg or valsartan 80 mg for eight weeks . at the end of the study , irbesartan provided significantly greater reductions than valsartan for mean change from baseline in diastolic ambulatory bp at trough ( 6.73 versus 4.84 mmhg , respectively , p = 0.035 ) and in mean systolic ambulatory bp at trough ( 11.62 versus 7.5 mmhg , respectively , p < 0.01 ) . similar results were obtained for mean 24-hour diastolic ambulatory bp ( 6.38 versus 4.82 mmhg , respectively , p = 0.023 ) and systolic ambulatory bp ( 10.24 versus 7.76 mmhg , p < 0.01 ) . irbesartan also produced significantly greater reductions than valsartan for office - measured seated dbp ( 10.46 versus 7.28 mmhg , respectively , p < 0.01 ) and sbp ( 16.23 versus 9.96 mmhg , respectively , p < 0.01 ) and for self - measured morning dbp ( 6.28 versus 3.75 mmhg , respectively , p < 0.01 ) and sbp ( 10.21 versus 6.97 mmhg , respectively , p < 0.01).58 recent findings suggest that at1 receptor blockade improves superoxide production and no bioavailability more than do other classes of antihypertensive agents . in a study by brosnan et al , in vivo irbesartan , amlodipine , and hydrochlorothiazide - hydralazine produced similar reductions in bp values , but irbesartan caused a greater reduction in superoxide and p22phox in carotid arteries . four - hour in vitro exposure to irbesartan decreased superoxide levels in the aorta and increased no bioavailability in carotid arteries . neither 30-minute incubation with irbesartan nor four hours with amlodipine or hydrochlorothiazide - hydralazine altered superoxide levels . reduced expression of components of nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase may contribute to these effects.59 moreover , a protective effect of at1 receptor blockers on endothelial function during postprandial hypertriglyceridemia has been reported in healthy subjects.60 in 2005 , ceriello et al demonstrated , for the first time , that irbesartan , like statins , is able to ameliorate the damage done to endothelial function by postprandial hyperglycemia and hypertriglyceridemia , oxidative stress , and inflammation in diabetic patients . these results appeared not to be linked to the bp - lowering effect and were obtained after short - term treatment . in this study , combination treatment of atorvastatin and irbesartan showed a more powerful effect on vascular damage compared with monotherapy using irbesartan or atorvastatin.61 pharmacodynamic data suggest that irbesartan at a dose of 300 mg provides more effective and persistent at1 blockade than losartan 100 mg . in a short - term ( eight week ) study by kassler - taub et al , after a placebo lead - in , 567 patients were randomized to once - daily therapy with placebo , losartan 100 mg , irbesartan 150 mg , or irbesartan 300 mg . at the end of the study , reductions from baseline in seated dbp and seated sbp with irbesartan 300 mg were greater than those obtained with losartan 100 mg by 3.0 and 5.1 mmhg , respectively ( p < 0.01 for both comparisons ) . larger reductions were also demonstrated at weeks 1 and 4 ( p < 0.01 and p = 0.017 , respectively , for dbp and sbp ) of the study , and the antihypertensive effects of the treatments were not significantly different.62 the renoprotective effect of irbesartan has been tested in a study by rossing et al . in this study , 52 hypertensive patients with type 2 diabetes and microalbuminuria already on antihypertensive treatment were included . after a two - month wash - out of other drugs , patients were randomized to receive irbesartan 300 , 600 , and 900 mg once daily . after a 10-week treatment period , ultrahigh doses of irbesartan were well tolerated and provided additional renoprotection independent of changes in bp values and glomerular filtration rate , compared with irbesartan 300 mg.63 administration of irbesartan twice daily has been evaluated in a study by polnia et al who investigated whether blockage of angiotensin ii receptors by irbesartan could reverse the nondipper circadian rhythm of bp to a dipper pattern in 12 black salt - sensitive hypertensive patients on a high sodium diet ( 300 mmol per day ) after a two - week placebo administration , followed by two weeks on irbesartan 150 mg / day , two weeks on placebo , and two weeks on irbesartan 300 mg / day . on the last day of placebo , and of the irbesartan 150 mg / day and irbesartan 300 mg / day treatments , 24-hour bp and urinary 24-hour excretion of sodium and potassium were measured . compared with placebo , irbe - sartan significantly increased serum potassium and plasma renin activity and reduced fractional excretion of potassium and plasma aldosterone levels in a dose - dependent manner , without significant changes in body weight . the authors concluded that irbesartan can reverse the nondipper bp profile in salt - sensitive hypertensive patients on a high - salt diet , restoring the nocturnal bp decline by a predominantly dose - dependent reduction of night time bp . evidence has been accumulating to suggest that ambulatory bp values are more strongly correlated with markers of cardiovascular disease than are clinic bp values.64 the main studies that showed a significant effect of irbe - sartan in reducing microalbuminuria and decreasing the risk of diabetic nephropathy were irbesartan microalbuminuria type 2 diabetes in hypertensive patients ( irma ii)65 and irbesartan in diabetic nephropathy trial ( idnt).66 these are two long - term ( 23 year ) trials showing that the irbesartan is effective in reducing the progression of renal disease in patients with type 2 diabetes and high bp . in each trial , the standard of care for diabetes mellitus was maintained . using conventional antihypertensive therapy , including diuretics , beta - blockers , and ccbs ( but not aceis or other arbs ) , bp control was similar in the placebo- and arb - treated groups . the irma ii study ( table 1 ) evaluated the renoprotective effect of irbesartan , in hypertensive patients with type 2 diabetes and microalbuminuria.65 this multinational , randomized , double - blind , placebo - controlled study enrolled a total of 590 hypertensive patients with type 2 diabetes and persistent microalbuminuria who received irbesartan at a dose of either 150 mg daily or 300 mg daily , or placebo . the primary efficacy measure was the time from the baseline visit to the first detection of overt nephropathy , defined by a urinary albumin excretion rate in an overnight specimen > 200 g / min and at least 30% higher than the baseline rate on at least two consecutive visits . at the end of the study , nephropathy developed in 30 patients in the placebo group , compared with 19 patients in the 150 mg irbesartan group and 10 patients in the 300 mg irbesartan group . the unadjusted hazard ratio ( hr ) for diabetic nephropathy was 0.61 ( p = 0.08 ) in the 150 mg group and 0.30 ( p < 0.001 ) in the 300 mg group . the average bp during the course of the entire study was 144/83 mmhg in the placebo group , 143/83 mmhg in the 150 mg irbesartan group , and 141/83 mmhg in the 300 mg irbesartan group ( p = 0.004 for the comparison of sbp between the placebo group and the combined irbesartan groups ) . after adjustment for bp achieved during the study and the baseline level of microalbuminuria , the hr for diabetic nephropathy was 0.56 in the 150 mg group ( p = 0.05 ) and 0.32 in the 300 mg group ( p < 0.001 ) . serious adverse events were less frequent among the patients treated with irbesartan ( p = 0.02 ) . irbesartan reduced the level of urinary albumin excretion throughout the study . in the irbesartan 150 mg group , urinary albumin excretion decreased by 24% and , in the 300 mg group , decreased by 38% , whereas the placebo group had a 2% decrease ( p < 0.001 for the comparison between placebo and combined irbesartan groups ) . there was a significantly smaller reduction in the level of albuminuria in the 150 mg group than in the 300 mg group ( p < 0.001 ) . during this 24-month study , overt nephropathy developed in 30 patients in the placebo group , compared with 19 patients in the 150 mg group ( p = 0.08 ) and 10 patients in the 300 mg group ( p < 0.001 ) . the unadjusted hr for diabetic nephropathy was 0.61 ( p = 0.08 ) in the 150 mg group and 0.30 ( p < 0.001 ) in the 300 mg group . after adjustment for the baseline level of microalbuminuria and the bp achieved during the study , the hr for diabetic nephropathy was 0.56 in the 150 mg group ( p = 0.05 ) and 0.32 in the 300 mg group ( p < 0.001 ) . at the three - month visit , the decrease in creatinine clearance for the placebo group and the 300 mg irbesartan group separated and continued to diverge . the decline in creatinine clearance during the initial three - month period was greater than the sustained decline from three months to 24 months . the initial declines were 0.9 , 1.0 , and 1.9 in the placebo , and irbesartan 150 mg and 300 mg groups , respectively , compared with declines of 0.1 , 0.2 , and 0.2 between months 3 and 24 . urinary albumin decreased by 24% in the 150 mg group , 38% in the 300 mg group , and 2% in the placebo group ( p < 0.001 for the comparison between placebo and the combined irbesartan groups ) . there was a significantly smaller reduction in the level of albuminuria in the 150 mg group than in the 300 mg group ( p < 0.001 ) . in the other relevant large study , the idnt66 compared the renoprotective effect of irbesartan and amlodipine in patients with type 2 diabetes and microalbuminuria ( table 2 ) , beyond the effect of these two drugs on bp reduction . it was a prospective , randomized , double - blind , placebo - controlled study including 1175 patients from 210 clinical centers . patients were randomized to receive irbesartan 300 mg / day , amlodipine 10 mg / day , or placebo . groups were compared according to time to the primary composite endpoint of a doubling of baseline serum creatinine concentration , development of end - stage renal disease , and death from any cause . the mean follow - up duration was 2.6 years . at the end of the study , treatment with irbesartan was associated with a 20% and 23% reduction in risk of the primary composite endpoint compared with placebo and amlodipine , respectively ( p = 0.006 ) . the relative risk of doubling of the serum creatinine concentration was 33% and 37% lower in the irbesartan group compared with the placebo and amlodipine groups ( p = 0.003 and p < 0.001 , respectively ) . treatment with irbesartan was associated with a significantly lower risk of end - stage renal disease ( 23% ) compared with that observed in the other groups ( p = 0.07 for both comparisons ) . the increase in serum creatinine levels was slower in the irbesartan group compared with the placebo ( 24% ) and amlodipine ( 21% ) groups ( p = 0.008 and p = 0.002 , respectively ) . no significant differences between groups were observed in the rate of death from any cause or in the cardiovascular composite endpoint . although the irma ii and idnt studies provided strong support for the beneficial effect of irbesartan in renoprotection , both studies included patients with hypertension who had been previously treated with antihypertensive drugs . in a short - term study , irbesartan was administered to newly diagnosed drug - naive patients ; the effects of irbesartan 150 mg bid on microalbuminuria were observed in normotensive type 2 diabetic patients , suggesting that the renoprotective action of irbesartan is probably due to a direct action on renal hemodynamics and glomerular morphology.67 more recently , studies have demonstrated a precise role for irbesartan and statins in regulation of vascular dysfunction . in the endothelial protection , at1 blockade and cholesterol - dependent oxidative stress ( epas ) trial , the investigators showed that statin and at1 blocker therapy independently and in combination improved both the antiatherosclerotic endothelial expression quotient and endothelial function . in this study , 60 patients with stable coronary artery disease undergoing elective coronary artery bypass grafting surgery were randomized four weeks before surgery to no arbs and/or statins ( controls ) , pravastatin 40 mg / day , irbesartan 150 mg / day , or pravastatin in combination with irbesartan at the same dosages . the primary endpoint was a priori therapy - dependent regulation of the antiatherosclerotic endothelial expression calculated marker of endothelial regulation . at the end of the study , association of irbesartan and pravastatin significantly improved endothelial expression of antiatherosclerotic and proatherothrombotic genes and endothelial function in arteries in these patients with coronary artery disease . moreover , preoperative therapy with irbesartan and pravastatin and their combination improved endothelial function in internal mammary artery rings.68 recently , many studies have focussed on molecular markers of the inflammatory mechanisms involved in atherogenesis , and particular attention has been given to inflammatory molecules which interact with the endothelium , such as tumor necrosis factor- , vascular cell adhesion molecule , and superoxides . in a study by navalkar et al , irbesartan significantly improved levels of inflammatory molecules in 33 normotensive patients with coronary artery disease , and the maximal suppression of inflammatory markers was observed after 12 weeks.69 in another study , treatment with irbesartan was effective in reducing the pro - oxidative environment seen in patients with coronary artery disease . lag time for ldl oxidation increased 32% at 12 weeks , suggesting that increased resistance of ldl modification during serum and lipid peroxidation decreased by 36% with irbesartan compared with placebo . in addition , superoxide levels and monocyte - binding capacity were also significantly reduced in coronary artery disease patients receiving irbesartan.70 irbesartan is an angiotensin ii receptor type 1 antagonist which has demonstrated significant efficacy in reducing bp in patients with high cardiovascular risk . in comparative trials , irbesartan seems significantly more effective than other arbs in the treatment of mild to moderate essential hypertension , and is as effective as enalapril or atenolol . many studies also show an additive antihypertensive effect when hydrochlorothiazide is added to irbesartan monotherapy . in hypertensive patients , the presence of an abnormal circadian bp rhythm as a result of lack of nocturnal bp decrease ( nondippers ) has been reported to be associated with a higher risk of cardiovascular end organ damage and a poorer prognosis for cardiovascular events compared with dippers ( who show a normal nocturnal bp decrease ) . irbesartan is able to modulate circadian bp rhythms and thereby significantly modifies the risk of organ damage due to hypertension and endothelial dysfunction , such as increased intima media thickness and left ventricular hypertrophy . irbesartan also induces statistically significant regression of left ventricular mass in patients with hypertension and left ventricular hypertrophy , and preliminary evidence suggests it has beneficial hemodynamic effects in patients with heart failure . moreover , irbesartan counteracts a number of other defects that lead to high cardiovascular risk , in particular insulin resistance and endothelial dysfunction . the so - called ancillary effects of irbesartan , which are partially shared by other arbs , have been highlighted in recent studies . particular interest has arisen about the effects of irbesartan on vascular dysfunction , which represents one of the major mechanisms that lead to atherosclerosis , and metabolic and cardiovascular diseases . a beneficial effect of irbesartan on glomerular filtration and microalbuminuria has been established in large clinical trials , such as idnt and irma ii . many data suggest a relationship between bp and endothelial dysfunction , so the role of irbesartan in modulation of vasodilatation and cytokines disorders could be explained in further experimental and clinical studies . furthermore , the connection between insulin resistance , hypertension , and endothelial dysfunction , as well as the effects of arbs on these mechanisms , has not been yet completely explained . in conclusion irbesartan is useful for clinicians for treatment of hypertension and prevention of organ damage , and appears well tolerated also at high dosage .

irbesartan , an angiotensin ii type 1 receptor antagonist , is approved as monotherapy , or in combination with other drugs , for the treatment of hypertension in many countries worldwide . data in the literature suggest that irbesartan is effective for reducing blood pressure over a 24-hour period with once - daily administration , and slows the progression of renal disease in patients with hypertension and type 2 diabetes . furthermore , irbesartan shows a good safety and tolerability profile , compared with angiotensin ii inhibitors and other angiotensin ii type 1 receptor antagonists . thus , irbesartan appears to be a useful treatment option for patients with hypertension , including those with type 2 diabetes and nephropathy . irbesartan has an inhibitory effect on the pressor response to angiotensin ii and improves arterial stiffness , vascular endothelial dysfunction , and inflammation in hypertensive patients . there has been considerable interest recently in the renoprotective effect of irbesartan , which appears to be independent of reductions in blood pressure . in particular , mounting data suggests that irbesartan improves endothelial function , oxidative stress , and inflammation in the kidneys . recent studies have highlighted a possible role for irbesartan in improving coronary artery inflammation and vascular dysfunction . in this review we summarize and comment on the most important data available with regard to antihypertensive effect , endothelial function improvement , and cardiovascular risk reduction with irbesartan .

Introduction Hypertension and cardiovascular risk Endothelial physiology and dysfunction Pharmacology Efficacy studies of irbesartan Effects on blood pressure Effects on vascular endothelium Conclusions

a major constraint to sustainable aquaculture of penaeid prawns is the current industry reliance on wild - caught broodstock . dependence on wild - caught broodstock stems , for the most part , from reproductive dysfunction in captive - reared females . the precise physiological factors inhibiting maturation and spawning in captive - reared females remains unknown , although the inability of these animals to undergo final ovarian maturation has been well documented . currently , eyestalk ablation , which involves the surgical removal of one or both eyestalks , remains the only method widely utilised by industry to induce ovarian maturation in both wild - caught and captive - reared females . a better understanding of the hormonal regulation of prawn reproduction would enable the development of methods for manipulation of reproduction to the exclusion of eyestalk ablation . accordingly , endocrine control of prawn reproduction is an area that has received considerable attention , with emphasis placed on the roles of cephalothorax and eyestalk neuropeptides and hormones , the sesquiterpene methyl farnesoate , and to a lesser extent , the biogenic amines and vertebrate - type steroid hormones . the neurosecretory system of the eyestalk is known to consist of a cluster of peptidergic neurons located in the medulla terminalis x - organ ( mtxo ) and their globular axonic terminals that constitute the sinus gland ( sg ) , a neurohemal organ that releases peptide hormones into the hemolymph 1 . the effect of eyestalk ablation has been attributed to a reduction in the level of the gonad - inhibiting hormone ( gih ) normally present in the neurosecretory cells of the eyestalk . however , the procedure presumably also produces animals with other hormonal imbalances and consequential detrimental effects including high mortality . the cephalothorax contains target organs involved in reproduction including the hepatopancreas , the mandibular organ and the y - organ . one critical process in ovarian maturation that requires further study is the mtxo - sg regulation of vitellogenesis . vitellogenesis involves the production of vitellogenin - the female - specific high density lipoprotein and precursor to the lipo - glycolcarotenopoprotein vitellin ( the major egg yolk protein ) . vitellogenesis also requires the accumulation of vitellogenin / vitellin and other proteins , carbohydrates , lipids , vitamins and minerals by the oocytes 2 . in crustaceans , vitellogenesis is generally understood to be negatively regulated by the gih , which is produced and released from neurosecretory sites within the eyestalk . the mtxo - sg also secretes a variety of neuropeptides additional to gih including crustacean hyperglycaemic hormone ( chh ) , moult - inhibiting hormone ( mih ) , mandibular organ - inhibiting hormone , and the chromatophorotropins : pigment dispersing hormone and red pigment concentrating hormone . the vast majority of neuropeptides signal through cell - surface guanosine - protein coupled receptors ( gpcrs ) . these neuropeptide - receptor systems control a variety of physiological functions , for instance the numerous involuntary movements of internal ducts that are precisely timed for transporting reproductive materials 3 . meanwhile , arrestins are important components for desensitization of gpcr cascades that mediate neurotransmission 3 . it is unclear whether neuropeptide hormone levels in the eyestalk are regulated primarily at the transcriptional or translational level , or via levels of secretion of stored products to the hemolymph , or whether their involvement in these processes is modulated chiefly via various mechanisms at the target tissue . several studies on a variety of crustaceans have examined transcript and neuropeptide levels during reproduction and moulting cycles . in the crayfish homarus americanus , gih mrna in the xo is at low levels at immature stage compared with pre - vitellogenic and vitellogenic stages 4 . chh - a levels are highest at pre - vitellogenic stage , while chh - b levels are highest at pre - vitellogenic stage and mature stage . meanwhile , neuropeptide levels in the sg are not different in total gih levels , and chh levels are highest at the pre - vitellogenic stage . hemolymph gih levels are at their lowest during the vitellogenic stage compared with other stages , while hemolymph chh levels are highest at mature stage . together , these results provide strong evidence to suggest that chh - a and -b may initiate vitellogenesis , while gih prevents onset of vitellogenesis . further investigation into the biological activities of these neuropeptides at the target tissue ( y - organ ) via bioassays at the tissue , receptor and cellular levels , determined that intracellular signalling mechanisms may provide the most important level of moult control . in support of this , the level of an mih mrna appears not to change significantly in the eyestalk of marsupenaeus japonicus during the moult - cycle 5 . oligonucleotide microarrays have proven to be an excellent tool for studying gene expression in a holistic manner . in this study , ovarian maturation stage - specific differential gene expression was performed on the p. monodon cephalothorax and eyestalk ( es ) by utilising custom oligonucleotide microarrays in both wild - caught and captive - reared animals . here we report the identification of genes that are differentially expressed during ovarian maturation and between wild - caught and captive - reared animals . wild - caught adult female p. monodon ( 116.75 17.99 g ) were obtained from commercial hatchery suppliers in innisfail , queensland and air freighted to the queensland department of primary industries and fisheries ( qdpif ) , bribie island aquaculture research centre ( biarc ) , woorim , queensland . captive - reared adult female p. monodon ( 114.5 19.6 g ) were obtained from the australian institute of marine science ( aims ) , townsville , queensland , following grow - out ( pond ) culture at biarc . animals were stocked in 5 tonne tanks with flow - through seawater heated to 26c and acclimated for 7 days whilst fed fresh diet ( squid and mussels ) twice daily . all animals were moult staged according to extent of epidermal retraction . for wild - caught animals , tissue samples were collected at various ovarian maturation stages based on in vivo observation of maturing ovaries , as described by duronslet et al . , ( 1975 ) 6 , for subsequent classification by histological analysis of developing oocytes as follows : whole ovaries , cephalothorax and ess , ( containing the mtxo - sg complex ) were collected from un - ablated inter - moult females ( immature ovaries ) euthanized in saline ice slurry , snap frozen in liquid nitrogen and stored at -80c until processing . additionally , seventy inter - moult females were unilaterally eyestalk ablated to induce ovarian maturation , eye- and carapace - tagged and maintained for a further 7 days as above . animals were sampled at 2 h and 24 h post ablation ( immature ovaries ) and further sampled during this period with tissues of interest collected as outlined above ( including remaining eyestalk ) from random animals representing each of 4 in vivo ovarian maturation stages : immature , early maturing , late maturing and mature . gonadosomatic index ( gsi ) was also calculated ( ovarian weight expressed as a percentage of total body weight ) for all samples . small pieces ( 100 mg ) of the middle ovarian lobes from specimens at selected ovarian maturation stages were fixed in 4% paraformaldehyde in phosphate - buffered saline ( pbs ) solution ( ph 7.2 ) overnight , washed with pbs at room temperature , dehydrated in ethanol series , embedded in paraffin , sectioned ( 6 m ) and stained with either haematoxylin and eosin for detection of acidophilic and basophilic substances , periodic acid schiff ( pas ) for detection of carbohydrates ( glycoproteins ) or luxol blue for detection of phospholipids , or alternatively frozen and cut with cryostat and stained with oil red o for detection of simple lipids , as described by bell & lightner ( 1988 ) 7 . ovarian stages used in the present study were characterized largely as determined by tan fermin & pudadera ( 1989 ) 8 with some modifications as follows : previtellogenic ( p ) stage - the ovary contains only oogonia and basophilic previtellogenic oocytes at chromatin nucleolus and perinucleolus stage ( gsi 1.7 - 2.9 ) ; vitellogenic ( v ) stage - in addition to the presence of oogonia and basophilic previtellogenic oocytes , the ovary contains yolk accumulating oocytes , the ooplasm of which is full of eosinophilic ( acidophilic ) yolk substances and also stains positive to pas and luxol blue indicating presence of glycoproteins and phospholipids respectively . large globules in the ooplasm are also notable which stain positive with oil red o indicating simple lipids ( gsi 3.5 - 6.5 ) ; round cortical rod ( r ) stage - staining affinities of oocytes are similar to those described for v stage ovaries with the addition of the appearance of round cortical rods ( crs ) developing radially at the peripheral cortex of those oocytes containing yolk substances ( gsi 7.7 - 10.5 ) ; elongated cortical rod ( e ) stage - staining affinities of oocytes are similar to those described for r stage ovaries except crs are elongated and extended towards the nucleus ( gsi 6.0 - 14.0 ) . total rna was isolated from small pieces ( 100 mg ) of the middle ovarian lobes , whole cephalothoraxes and whole eyestalks ( initially ground under liquid nitrogen using mortar and pestle ) from prawns of interest , using trizol reagent as recommended by the manufacturer ( invitrogen life technologies , carlsbad , ca , usa ) . the samples were used for synthesis of complementary dna ( cdna ) , creation of cdna libraries and for construction and screening of microarrays . concentration and purity of the rna were determined using a spectrophotometer ( genequant pro , ge healthcare uk ltd . , rna quality was assessed for all samples by visualisation on denaturing formaldehyde rna gels ( protocol recommended by qiagen , valencia , ca , usa ) using ethidium bromide staining . three p. monodon cdna libraries were created in our laboratory from total rna isolated from ovary , eyestalk ( containing x - organ / sinus gland complex ) and whole cephalothorax ( containing target organs including hepatopancreas , y - organ and mandibular organ ) collected from wild - caught animals . briefly , reverse transcription of total rna ( pooled in equal part from three animals from each ovarian maturation stage ) was conducted using the smart iv polymerase chain reaction ( pcr ) cdna synthesis kit ( clonetech laboratories , inc . first strand cdna synthesis was conducted using deoxyribonucleotide triphosphate ( dntp ) mix , smart iv oligonucleotide and cds iii/3 ' pcr primer with powerscript reverse transcriptase . after determining optimal number of cycles in order to reduce the likelihood of producing redundant cdna libraries , single stranded cdna served as template for pcr based cdna amplification using dntp mix , 5 ' pcr primer , cds iii/3 ' pcr primer with advantage ii polymerase . complementary dna was then purified using the qiaquick pcr purification kit ( qiagen , valencia , ca , usa ) . to ensure efficient ligation into pgem - t easy vector ( promega , madison , wi , usa ) a standard a - tailing procedure was conducted . complementary dna size fractionation was then conducted to separate larger cdna species using chroma spin columns ( clontech laboratories , inc . ligations were then conducted and subsequent transformation into xl10 gold ultracompetent cells ( stratagene , la jolla , ca , usa ) according to the manufacturer 's protocol . sequences were edited ( removal of poor sequence , vector and poly - a sequence ) and then assembled into contiguous over - lapping sequence alignments ( contigs ) using sequencher ( gene codes corporation , ann arbor , mi , usa ) . the sequences were annotated with the name of the highest basic local alignment search tool ( blast ) 9 score from an analysis of genbank entries by the blastx and blastn procedures . further putative functional annotation was assigned to each of the sequences by conducting a blastx similarity search using the goanna annotation tool ( http://agbase.msstate.edu/goanna.html ) . a total of 601 sequences were selected from the three cdna libraries created for this study together with an additional 387 sequences obtained from p. monodon expressed sequence tag ( est ) collections created from ovary , hepatopancrease and eyestalk sourced from publicly available databases ( genbank http://www.ncbi.nlm.nih.gov ) for inclusion as probes on the microarrays . these included key transcripts encoding known eyestalk neuropeptides including several crustacean hyperglycemic hormone ( chh ) family members from p. monodon where available and from other penaeids where not , as follows : p. monodon chh-1 ( ay346378.1 ) -2 ( af104931.1 ) , litopenaeus vannamei moult - inhibiting hormone ( mih ) ( s73824.1 ) , p. monodon sinus gland peptide ( pmsgp)-i ( af104386.1 ) -ii ( af104387.1 ) -iii ( af104388.1 ) -iv ( af104389.1 ) -v ( af104390.1 ) , l. vannamei pigment - dispersing hormone ( pdh)-1 ( y11723.1 ) -2 ( y11722.1 ) and marsupenaeus japonicus pdh-3 ( ab247562.1 ) . oligonucleotide microarrays were produced by combimatrix corporation ( mukilteo , usa ) using the customarray 4 2k platform which contains four identical , independent 2240-feature microarrays on each slide . between two and five oligonucleotide probes were designed and incorporated on each microarray for each of the selected transcripts . ovarian rna samples from nine wild - caught animals representing six ovarian maturation stages ( p , 2 , 24 , v , r , e ) were used in microarray hybridisations . similarly , rna samples from three captive - reared animals representing four maturation stages ( p , 24 , v , e ) were used in microarray hybridisations ( fig . for captive - reared animals , samples from each ovarian maturation stage from all three animals were pooled enabling one hybridisation for each stage . importantly , as the four stages for captive - reared animals were ( 1 ) pre - ablation pre - vitellogenic ( p ) , ( 2 ) post - ablation pre - vitellogenic ( 2 for 2 h post ablation , 24 for 24 h post ablation ) , ( 3 ) post - ablation vitellogenic ( v ) , ( 4 ) post - ablation vitellogenic with cortical rods ( e ) , this arrangement allowed for 2 samples of captive - reared pre - vitellogenic and 2 samples of captive - reared vitellogenic , thereby enabling t - tests between samples , while also allowing analysis across the whole 4 stages via cluster analysis . all hybridisations were single channel hybridisations conducted using equal amounts of rna pooled from each individual . reverse transcription of total rna , rna amplification by in vitro transcription and amplified antisense rna ( arna ) labelling were conducted using the rna ampulse amplification and labelling kit ( kreatech biotechnologies , amsterdam , netherlands ) according to manufacturer 's instructions . briefly , reverse transcription reactions were performed using t7 oligo(dt ) primer with arrayscript reverse transcriptase . second strand synthesis was then performed and cdna purified using cdna filter cartridges and used as template for arna synthesis in in vitro transcription reactions . arna was then purified using arna filter cartridges and yield and quality of arna assessed by measuring the absorbance at 260 nm and 280 nm using a spectrophotometer ( genequant pro , ge healthcare uk ltd . , buckinghamshire , england ) . 1 g of non - modified arna was labelled non - enzymatically using the universal linkage system ( uls ) technology coupled with cy5 fluorochrome . unbound uls label was removed using included kreapure columns and arna fragmentation conducted using rna fragmentation reagents ( applied biosystems / ambion , austin , tx , usa ) according to manufacturer 's instructions . briefly , slides were assembled with supplied hybridisation caps in hybridisation clamps and re - hydrated prior to use by adding nuclease - free water and incubating at 65c for 10 min . pre - hybridisation steps ( 30 min ) and hybridisations ( 16 h ) were conducted in 30l volumes using hybridisation solution containing 6 saline - sodium phosphate - edta ( sspe ) , 0.05% tween-20 , 20 mm edta , 5denhardt 's solution , 100ng/l denatured salmon sperm dna , 0.05% sds , and 6sspe , 0.05% tween-20 , 20 mm edta , 25% deionized formamide , 100 ng/l denatured salmon sperm dna , 0.04% sds and 1 g of target arna respectively , with microarrays loaded onto a rotisserie in a hybridisation oven and incubated at 45c . microarrays were then washed according to manufacturer 's instructions and scanned wet using supplied imaging solution with lifterslips ( thermo fisher scientific inc . , , microarrays were stripped for subsequent re - hybridisation up to three times using the combimatrix customarray stripping kit ( combimatrix corporation , mukilteo , usa ) according to manufacturer 's instructions . microarray slides were scanned using a fluorescent microarray scanner ( genepix 4000b , mds analytical technologies , toronto , canada ) . microarray imager ( combimatrix corporation , mukilteo , usa ) was then used to create spot intensity reports , generate gene i d mapping files and assign gene identification . final intensity reports were then used for further data analysis using data mining software acuity 4.0 ( mds analytical technologies , toronto , canada ) . data were pre - processed and normalised by applying background and floor correction , combining replicates ( means ) and global means normalisation . genes displaying significant differential expression were identified via two - sample students t - tests . a total of 1152 clones were sequenced from the three cdna libraries constructed from cephalothorax , eyestalk and ovary ( fig . a description of the sequence content of the cephalothorax and eyestalk libraries when the sequences were edited and assembled is given in table 1(ovary is described elsewhere ) . percentage representations from the hierarchical gene ontology ( go ) mapping : biological process is displayed in fig . 1b . transcripts encoding proteins involved in metabolism ( including lipid , carbohydrate , chitin and nucleic acid metabolism ) featured highly in the cephalothorax library . in the cephalothorax library , further abundant transcripts included those involved in transport ( such as oxygen , iron and lipid transport ) as well as transcripts involved in signal transduction and protein processing . in eyestalk , transcripts known to be involved in sensory / photo perception and signalling were abundant ( 21% ) , as were transcripts encoding proteins involved in dna assembly / repair and transport ( both 10% ) . we examined ovarian maturation stage - specific differential gene expression in the cephalothorax of wild - caught animals . gene expression profiles in the ovary among wild - caught and captive - reared animals throughout ovarian maturation are reported elsewhere . wild - caught cephalothorax - stage - specific profiles : hierarchical cluster analysis was implemented . 2 ) , in accordance with gap statistic analysis revealed two major clusters of genes with stage - specific expression in the wild - caught cephalothorax data set . 2a ) contains 68 genes displaying elevated expression levels at early ( pre - vitellogenic ) stages . these include thrombospondin , shrimp ovarian peritrophin variants 1 , 2 and 3 , metallothionein , cp14 - a calcified cuticle protein , profilin , glycerol-3-phosphate transporter and 40 genes with no significant blast homology , therefore are presented as novel , some of which displaying very high expression levels . cluster 2 contains 145 genes generally displaying elevated expression levels at later ( vitellogenic ) stages . to provide clear presentation of this large cluster , it is represented in figure 2 , and described here , as its sub - clusters : 2a , 2b and 2c . this contains 87 genes displaying elevated expression levels at later ( vitellogenic ) stages with sub - clusters of genes ( within cluster 2a ) displaying either sustained elevated expression at later stages or displaying elevated expression at one or more of the later stages . genes include : ( 1 ) vitellogenin which displays sustained elevated expression at later stages , ( 2 ) scpx / scp2 - the sterol carrier protein-2/3-oxoacyl - coa thiolase displaying elevated expression at later stages with a peak at r stage , ( 3 ) hemocyanin displaying comparatively high expression at all stages with a peak at v stage , ( 4 ) a chitinase ( ensangp00000021035 ) displaying elevated expression at later stages with a peak at r stage , ( 5 ) the high density lipoprotein/1,3-beta - d - glucan - binding protein precursor up - regulated at later stages , ( 6 ) a kazal - type proteinase inhibitor up - regulated at later stages , ( 7 ) ferritin up - regulated at v stage , and ( 8) 25 novel genes , some displaying very high expression levels . 2c ) contains 20 further genes generally displaying elevated expression at later stages with most genes , however , also displaying an expression peak at 24 h post - ablation . 2d ) contains 38 further genes generally displaying elevated expression at later stages , however , also displaying reduced expression levels at 24 h post - ablation and again at r stage . genes include a fatty acid binding protein and 15 novel genes , some displaying very high expression levels . transcripts encoding the various ribosomal proteins are exclusive to cluster 2 ( elevated expression at later stages ) . cluster 2 also contains a greater number of transcripts encoding translational elongation factors compared with remaining clusters . wild - caught cephalothorax - differentially expressed genes : genes displaying significant differential expression between collective early ( pre - vitellogenic ) stages and collective late ( vitellogenic ) stages in the wild - caught cephalothorax were identified via two - sample students t - tests ( p<0.05 ) . an equal number of transcripts were identified as up - regulated at either pre - vitellogenic or vitellogenic stages . eyestalk . to identify transcripts in the eyestalk with potential involvement in neuropeptide control of ovarian maturation , we examined ovarian maturation stage - specific differential gene expression in the eyestalks of both wild - caught and captive - reared animals . to identify possible physiological differences between wild - caught and captive - reared animals , we also examined differences in gene expression in the eyestalks between the two sources . wild - caught eyestalk - stage - specific profiles : to identify groups of genes displaying stage - specific expression profiles in the eyestalk of wild - caught animals , hierarchical cluster analysis was implemented . 3 ) revealed six major clusters of genes with stage - specific expression in the wild - caught data set . 3a ) contains 60 transcripts generally displaying elevated expression at pre - vitellogenic stages : pre - es ablation ( p ) , 2 hours post - es ablation ( 2 ) and 24 hours post - es ablation ( 24 ) compared with vitellogenic stages . however , some transcripts in cluster 1 displayed elevated expression at p and 2 stages and also at several vitellogenic stages . transcripts include those encoding ferritin , myosin , tetraspanin , cyclophilin , rhodopsin , opsin , arrestin and several cuticle proteins . this cluster also contains 21 novel transcripts ( no significant homology to previously characterised genes ( blastx e - value > 1 10 ) . 3c ) contains 34 transcripts generally displaying elevated expression at 24 hours post - es ablation stage . 3d ) contains 54 transcripts generally displaying reduced expression at 24 hours post - es ablation stage , with some transcripts displaying elevated expression at vitellogenic stages . transcripts include those encoding metallothionein , manganese superoxide dismutase , architectural transcription factor - p8 and several mitochondrial enzymes . compared with other clusters , transcripts encoding ribosomal proteins and translation elongation factors are significantly more abundant in cluster iv . this cluster also contains 21 novel transcripts , some of which displayed very high expression levels . 3e ) contains 22 transcripts generally displaying elevated expression at vitellogenic ( v ) stage and at various other vitellogenic stages , including 10 novel transcripts , some of which displayed very high expression levels . 3f ) contains 34 further transcripts generally displaying elevated expression at various vitellogenic stages with highest expression at round cortical rod ( r ) stage . wild - caught eyestalk - differentially expressed genes : genes displaying significant differential expression between ovarian maturation stages ( in either wild - caught or captive - reared eyestalk samples ) and between wild - caught and captive - reared eyestalk samples were identified via two - sample students t - tests ( p<0.05 ) . of the 55 transcripts identified as significantly differentially expressed during ovarian maturation in the eyestalk in either wild - caught or captive - reared animals , 24 transcripts displayed significant blast homology . of the 24 transcripts identified as significantly differentially expressed in the eyestalk between wild - caught and captive - reared animals , 12 transcripts displayed significant blast homology . the majority of transcripts ( 21 ) identified as significantly differentially expressed between wild - caught and captive - reared animals displayed higher expression in captive - reared animals . a total of 1152 clones were sequenced from the three cdna libraries constructed from cephalothorax , eyestalk and ovary ( fig . a description of the sequence content of the cephalothorax and eyestalk libraries when the sequences were edited and assembled is given in table 1(ovary is described elsewhere ) . percentage representations from the hierarchical gene ontology ( go ) mapping : biological process is displayed in fig . 1b . transcripts encoding proteins involved in metabolism ( including lipid , carbohydrate , chitin and nucleic acid metabolism ) featured highly in the cephalothorax library . in the cephalothorax library , further abundant transcripts included those involved in transport ( such as oxygen , iron and lipid transport ) as well as transcripts involved in signal transduction and protein processing . in eyestalk , transcripts known to be involved in sensory / photo perception and signalling were abundant ( 21% ) , as were transcripts encoding proteins involved in dna assembly / repair and transport ( both 10% ) . we examined ovarian maturation stage - specific differential gene expression in the cephalothorax of wild - caught animals . gene expression profiles in the ovary among wild - caught and captive - reared animals throughout ovarian maturation are reported elsewhere . wild - caught cephalothorax - stage - specific profiles : hierarchical cluster analysis was implemented . 2 ) , in accordance with gap statistic analysis revealed two major clusters of genes with stage - specific expression in the wild - caught cephalothorax data set . 2a ) contains 68 genes displaying elevated expression levels at early ( pre - vitellogenic ) stages . these include thrombospondin , shrimp ovarian peritrophin variants 1 , 2 and 3 , metallothionein , cp14 - a calcified cuticle protein , profilin , glycerol-3-phosphate transporter and 40 genes with no significant blast homology , therefore are presented as novel , some of which displaying very high expression levels . cluster 2 contains 145 genes generally displaying elevated expression levels at later ( vitellogenic ) stages . to provide clear presentation of this large cluster , it is represented in figure 2 , and described here , as its sub - clusters : 2a , 2b and 2c this contains 87 genes displaying elevated expression levels at later ( vitellogenic ) stages with sub - clusters of genes ( within cluster 2a ) displaying either sustained elevated expression at later stages or displaying elevated expression at one or more of the later stages . genes include : ( 1 ) vitellogenin which displays sustained elevated expression at later stages , ( 2 ) scpx / scp2 - the sterol carrier protein-2/3-oxoacyl - coa thiolase displaying elevated expression at later stages with a peak at r stage , ( 3 ) hemocyanin displaying comparatively high expression at all stages with a peak at v stage , ( 4 ) a chitinase ( ensangp00000021035 ) displaying elevated expression at later stages with a peak at r stage , ( 5 ) the high density lipoprotein/1,3-beta - d - glucan - binding protein precursor up - regulated at later stages , ( 6 ) a kazal - type proteinase inhibitor up - regulated at later stages , ( 7 ) ferritin up - regulated at v stage , and ( 8) 25 novel genes , some displaying very high expression levels . 2c ) contains 20 further genes generally displaying elevated expression at later stages with most genes , however , also displaying an expression peak at 24 h post - ablation . genes include the vitelline membrane outer layer protein ( vmo1 ) . cluster 2c ( fig . 2d ) contains 38 further genes generally displaying elevated expression at later stages , however , also displaying reduced expression levels at 24 h post - ablation and again at r stage . genes include a fatty acid binding protein and 15 novel genes , some displaying very high expression levels . transcripts encoding the various ribosomal proteins are exclusive to cluster 2 ( elevated expression at later stages ) . cluster 2 also contains a greater number of transcripts encoding translational elongation factors compared with remaining clusters . wild - caught cephalothorax - differentially expressed genes : genes displaying significant differential expression between collective early ( pre - vitellogenic ) stages and collective late ( vitellogenic ) stages in the wild - caught cephalothorax were identified via two - sample students t - tests ( p<0.05 ) . an equal number of transcripts were identified as up - regulated at either pre - vitellogenic or vitellogenic stages . eyestalk . to identify transcripts in the eyestalk with potential involvement in neuropeptide control of ovarian maturation , we examined ovarian maturation stage - specific differential gene expression in the eyestalks of both wild - caught and captive - reared animals . to identify possible physiological differences between wild - caught and captive - reared animals , we also examined differences in gene expression in the eyestalks between the two sources . wild - caught eyestalk - stage - specific profiles : to identify groups of genes displaying stage - specific expression profiles in the eyestalk of wild - caught animals , hierarchical cluster analysis was implemented . 3 ) revealed six major clusters of genes with stage - specific expression in the wild - caught data set . 3a ) contains 60 transcripts generally displaying elevated expression at pre - vitellogenic stages : pre - es ablation ( p ) , 2 hours post - es ablation ( 2 ) and 24 hours post - es ablation ( 24 ) compared with vitellogenic stages . however , some transcripts in cluster 1 displayed elevated expression at p and 2 stages and also at several vitellogenic stages . transcripts include those encoding ferritin , myosin , tetraspanin , cyclophilin , rhodopsin , opsin , arrestin and several cuticle proteins . this cluster also contains 21 novel transcripts ( no significant homology to previously characterised genes ( blastx e - value > 1 10 ) . 3c ) contains 34 transcripts generally displaying elevated expression at 24 hours post - es ablation stage . 3d ) contains 54 transcripts generally displaying reduced expression at 24 hours post - es ablation stage , with some transcripts displaying elevated expression at vitellogenic stages . transcripts include those encoding metallothionein , manganese superoxide dismutase , architectural transcription factor - p8 and several mitochondrial enzymes . compared with other clusters , transcripts encoding ribosomal proteins and translation elongation factors are significantly more abundant in cluster iv . this cluster also contains 21 novel transcripts , some of which displayed very high expression levels . 3e ) contains 22 transcripts generally displaying elevated expression at vitellogenic ( v ) stage and at various other vitellogenic stages , including 10 novel transcripts , some of which displayed very high expression levels . 3f ) contains 34 further transcripts generally displaying elevated expression at various vitellogenic stages with highest expression at round cortical rod ( r ) stage . wild - caught eyestalk - differentially expressed genes : genes displaying significant differential expression between ovarian maturation stages ( in either wild - caught or captive - reared eyestalk samples ) and between wild - caught and captive - reared eyestalk samples were identified via two - sample students t - tests ( p<0.05 ) . of the 55 transcripts identified as significantly differentially expressed during ovarian maturation in the eyestalk in either wild - caught or captive - reared animals , 24 transcripts displayed significant blast homology . of the 24 transcripts identified as significantly differentially expressed in the eyestalk between wild - caught and captive - reared animals , 12 transcripts displayed significant blast homology . the majority of transcripts ( 21 ) identified as significantly differentially expressed between wild - caught and captive - reared animals displayed higher expression in captive - reared animals . we have utilised custom oligonucleotide microarrays to assess differential gene expression in specific stages of ovarian maturation , in the cephalothorax and eyestalk of wild - caught and captive - reared black tiger prawns p. monodon . in an effort to understand the cause of reproductive dysfunction in captive - reared animals , we have identified differences in gene expression between wild - caught and captive - reared animals . here , we discuss those genes whose expression we found to be significantly altered between these two groups and are potentially relevant to oocyte maturation . throughout this study , the term key transcripts is used to refer to transcripts which display significant homology ( through blast ) to genes previously identified as playing key roles in the process of ovarian maturation and other related metabolic processes . we thought it appropriate to focus on transcripts that displayed significant blast homology to genes / proteins previously identified as involved in relevant biological processes . differences between wild - caught and captive - reared animals , and between animals with immature vs mature / maturing ovaries . for some transcripts , which displayed significant blast homology , we could identify no plausible link with the biology in question , and conversely , some transcripts that displayed interesting expression profiles were novel , as they displayed no blast homology and hence form the focus of further studies . clusters identified in the wild - caught cephalothorax data set contain a range of transcripts displaying elevated expression profiles at either collective pre - vitellogenic or vitellogenic stages . the identity of many of these transcripts reflects the dramatic shift in dynamics of energetic and metabolic processes towards mobilisation and biosynthesis of materials for ovarian maturation in tissues other than the ovary , including the hepatopancreas . whereas , clusters identified in the wild - caught and captive - reared eyestalk data sets contain a range of transcripts identified as either expressed at similar levels at all maturation stages or displaying more transient expression profiles . transcripts displaying blast homology to known genes / proteins while also displaying statistically significant differential expression ( via two - sample student 's t tests ( p<0.05 ) ) between maturation stages , groups of stages , and animal source are discussed individually below . we found that 1,3--d - glucan - binding high - density lipoprotein ( pmgbp - hdl ) transcripts were up - regulated at vitellogenic compared with pre - vitellogenic stages in the wild - caught cephalothorax . gbp - hdl has a bi - functional role in penaeids as a non - sex - specific hdl involved in lipid transport and as a pattern recognition protein ( prp ) central to innate immunity 10 . the gbp - hdl identified in litopenaeus gbp - hdl transcripts have also been demonstrated to be predominantly expressed in the hepatopancreas by using rt - pcr 11 . although most of the literature relevant to gbp - hdl gene expression concerns its role as a prp , gbp - hdl gene expression has also been demonstrated to be influenced by starvation and lipid composition of diets in the hepatopancreas of juvenile l. vannamei 12 . using density ultracentrifugation , it was demonstrated that the hdl fraction of ovarian homogenates in p. semisulcatus , comprising vt and gbp - hdl , is increased in mature compared with immature ovaries 13 . up - regulation of gbp - hdl gene expression in the cephalothorax at vitellogenic stages observed in this study provides further evidence of its involvement in delivery of lipid to developing oocytes . a transcript with significant homology to the sterol carrier gene 2/3-oxoacyl - coa thiolase ( pmscp2/scpx ) was isolated in the whole cephalothorax cdna library . whilst not identified as statistically significantly differentially expressed in t - tests ( p>0.05 ) , the transcript displayed elevated expression levels at vitellogenic compared with pre - vitellogenic stages in the wild - caught cephalothorax . conceptual protein domain analysis using the pfam database confirmed that the incomplete p. monodon clone identified in this study contains the sterol - binding domain of scp2 . scp2/scpx is a fusion gene in many , but not all species , which encodes both the scp2 and the scpx with variations in their regulation occurring at the transcriptional and translational level [ reviewed by 14 ] . no complete nucleotide sequence exists yet for a crustacean and therefore the gene structure and mode of its regulation are currently unknown . in vertebrates , the scp2 has many proposed roles in lipid metabolism and intracellular trafficking of cholesterol and other lipids , while the scpx is involved in oxidation of branched - chain fatty acids fas 14 . in vertebrates , the scp2 has a range of ligand partners including fatty acids ( fas ) , fatty acyl coas , sterols and phospholipids 14 , and therefore resolution of its full range of physiological functions is ongoing . scp2 transcripts were detected in the ovary , gut , head and body in aedes aegypti 15 . based only on an est identified in l. vannamei 16 the hepatopancreas remains so far the only site of scp2 transcription identified in crustaceans . up - regulation of scp2/scpx transcripts during vitellogenesis in this study is consistent with its possible involvement in processes associated with the mobilisation and metabolism of sterols towards both the accumulation of sterol in the ovaries and the production of reproductive steroid hormones . given the magnitude of the accumulation of lipids in the ovary during vitellogenesis , and the multitude of ligand partners demonstrated for scp2/scpx , it is possible that scp2/scpx may also serve other key roles during ovarian maturation . a transcript encoding a kazal - type serine proteinase inhibitor ( spi ) was identified as more highly expressed at vitellogenic compared with pre - vitellogenic stages in the wild - caught cephalothorax . spis are widely distributed in all multicellular organisms and play vital roles regulating many biological processes by limiting the level and extent of proteinase activity 17 . kazal - type spis have been previously identified in the hepatopancreas and hemocytes 18 and of p. monodon . recombinant spi exhibited strong inhibitory activity against subtilisin in a tight - binding inhibition assay , suggesting a possible role as a defence component , and also against elastase , for which the inhibitory function is not known 18 . the elevated expression of kazal - type spi transcripts at vitellogenic stages in the wild - caught cephalothorax in this study suggests a possible role for these proteins in regulating physiological processes associated with ovarian maturation . also , a manganese - superoxide dismutase ( mn - sod ) transcript encoding mn - sod was identified as more highly expressed at vitellogenic compared with pre - vitellogenic stages in the wild - caught cephalothorax . sods are ubiquitous enzymes which provide the most important line of antioxidant defence systems against reactive oxygen species 19 . clair et al . , ( 1993 ) 20 also provide evidence of the importance of mn - sods ability to neutralise a cellular hyperoxidant state in processes of cellular differentiation . the elevated expression of transcripts at vitellogenic stages in the wild - caught cephalothorax in this study may be a reflection of the greater metabolic demand during ovarian maturation . a total of 28 transcripts displayed higher expression levels at pre - vitellogenic stages compared with vitellogenic stages in the wild - caught cephalothorax . of these transcripts , only six transcripts had been previously annotated , as identified through blast homology . these include several transcripts encoding tsp and sop isoforms , a transcript encoding cp14 - a calcified cuticle protein and a transcript encoding profilin ( chickadee ) . , 21 have identified transcripts encoding a number of cuticular proteins displaying moult cycle stage - specific differential expression in the crab portunis pelagicus . they also have roles in cellular processes such as membrane trafficking , small - gtpase signalling and nuclear activities 22 , 23 . the significance of the up - regulation of profilin transcripts at pre - vitellogenic stages in the wild - caught cephalothorax remains to be determined . for this study , we chose to isolate rna from whole intact eyestalks rather than dissect the mtxo - sg complex , given that its diffuse nature and proximity to retinal tissue increased the likelihood of irregular dissections . as such , transcripts detected in this study are likely to be expressed in other tissues besides the mtxo - sg including muscle , epidermis and tissues associated with sensory / photo perception . key transcripts encoding known eyestalk neuropeptide hormones were not detected in wild - caught or captive - reared eyestalk samples at any ovarian maturation stage , suggesting low levels of transcript . however , several known and novel transcripts exhibited expression profiles suggestive of involvement in mtxo - sg function . transcripts encoding an arrestin family member were up - regulated at pre - vitellogenic stages [ pre - es ablation ( p ) stage and 2 hours post - es ablation ( 2 ) stage ] compared with later stages in the wild - caught es . although arrestins remain largely uncharacterised in crustaceans , it is well established that they form a family of proteins important for regulating the activity of gpcrs , with the different arrestins affecting the activity of their target gpcrs in different ways 24 . gpcrs form the largest family of cell - surface receptors , involved in numerous processes including hormonal system regulation , but also sensory / photo perception 25 , and include receptors for biogenic amines . in particular , the biogenic amines dopamine and serotonin have been demonstrated to modulate release of various eyestalk neuropeptides . for example , dopamine has been shown to stimulate release of chh from the eyestalk in the crab , carcinus maenas 26 . serotonin ( 5-hydroxytryptamine , 5-ht ) injection has been demonstrated to induce ovarian maturation in penaeids 27 , 28 and enhance release of chh in the crayfish , procambarus clarkia 29 . ongvarrasopone et al . , ( 2006 ) 30 have cloned a putative serotonin receptor from the ovary of p. monodon and further determined its expression in all tissues examined including the es . it is possible that elevated arrestin levels at p and 2 stages in the wild - caught eyestalk indicate changes in gpcr related signalling activity at these stages , potentially associated with regulation of eyestalk neuropeptide release . these observations highlight the possibility that further functional characterisation of the various hormone receptors in the eyestalk , including their modes of regulation and probable coupling to excitation - release mechanisms in the neurosecretory cells of the eyestalk , may offer additional opportunities for manipulation of eyestalk neuropeptide levels . in addition , the results pertaining to arrestin and gpcr highlight further possibilities for manipulation of neuroendocrine functions . a transcript encoding p8 , an architectural transcription factor , was up - regulated at collective vitellogenic stages compared with collective pre - vitellogenic stages in the captive - reared but not the wild - caught es , and also at pre - es ablation ( p ) stage compared with 24 hours post - es ablation ( 24 ) stage in the wild - caught es . the p8 is a nuclear phosphoprotein related to the high mobility group class of proteins which function as architectural transcription factors , possessing considerable flexibility in regulating the expression of large numbers of genes 31 . up - regulation at vitellogenic stages compared with pre - vitellogenic stages in the captive - reared eyestalk suggests p8s possible involvement in initiating expression of large numbers of genes in the eyestalk at vitellogenic stages , while also suggesting possible differences in transcriptional activity in the eyestalk between the two sources . up - regulation in the wild - caught eyestalk at p stage compared with 24 stage also suggests possible reduced transcriptional activity in the eyestalk 24 hours post - es ablation . examination of the promoter regions of genes expressed in the eyestalk during ovarian maturation may further determine the extent of p8s involvement in these processes . up - regulation of ribosomal proteins and translation elongation factors at collective vitellogenic stages compared with collective pre - vitellogenic stages in both the wild - caught and the captive - reared eyestalk , and also at several vitellogenic stages compared with p stage in the wild - caught eyestalk , suggests increased protein biosynthesis in the eyestalk at vitellogenic stages . the significance of further transcripts displaying differential expression during ovarian maturation , which include transcripts encoding cell division , signalling and apoptosis proteins ( 14 - 3 - 3 like protein ) , er protein translocation apparatus membrane proteins ( sec 61 gamma subunit ) , actin - based motility proteins ( myosin ) , as well as 26 novel transcripts , remains to be determined . this is also the case for further transcripts displaying differential expression between wild - caught and captive - reared eyestalk samples . incidental to this study , transcripts encoding opsin and rhodopsin , the gpcrs found in the photoreceptor cells of the retina 32 were up - regulated at both pre - vitellogenic and vitellogenic stages in the eyestalk of captive - reared compared with wild - caught animals and at pre - es ablation stage compared with 24 h post - es ablation stage in the eyestalk of wild - caught animals . also , transcripts encoding two cuticle proteins were up - regulated at collective pre - vitellogenic stages compared with collective vitellogenic stages in the wild - caught es . commonality of these cuticular protein transcripts lies in their localization within the cuticle ( established by andersen , 1999 33 ) , however , they display significant sequence variation and possess different cuticle protein domains . differential expression of these cuticular transcripts , and indeed several other transcripts identified in this study , is likely modulated by moult - cycle stage rather than ovarian maturation stage . kuballa et al . , ( 2007 ) 21 , demonstrated that transcripts encoding various cuticular proteins displayed moult - cycle stage specific differential expression in whole p. pelagicus , where differential expression occurred both between transcripts containing different domains and between transcripts containing the same domain . a transcript encoding dd9b - a further putative cuticular protein , identified as up - regulated in epithelial cells in post - moult metapenaeus japonicus 34 , was also up - regulated at collective pre - vitellogenic stages compared with collective vitellogenic stages in the eyestalk of wild - caught animals in this study . this result could be attributed to low relative expression levels of these transcripts . in our laboratory , we have previously determined the inability to detect eyestalk neuropeptides when hybridising target tissue derived from whole animals to cdna microarrays 21 . we had , however , anticipated that by targeting the eyestalk specifically , detectability of such rare transcripts would be improved . in this study we also utilised oligonucleotide arrays ( synthesised 35 - 40 mers ) rather than cdna arrays . several studies have demonstrated that the various oligonucleotide array platforms perform at least as well as cdna arrays in detecting differential expression ( eg . , 35 ) . moreover , wang et al . , ( 2003 ) 36 demonstrate that oligonucleotide probes and pcr amplicons perform equally well at detecting two - fold changes in rare transcript levels . oligonucleotide arrays also have , in general , some advantages over cdna arrays including higher uniformity of spots , higher specificity and more uniform melting temperatures . amplification of signals on microarrays can be achieved using two basic methods : nucleic acid amplification prior to hybridisation and on - chip signal amplification post - hybridisation . both methods potentially enable improved detection of rare transcripts . however , when using the former method , arrays are saturated at a lower target concentration as a result , while the latter method remains limited at the lower end of the dynamic range by the amount of signal detectable above background . a further explanation for the lack of neuropeptide transcripts identified in this study might be lack of adequate specificity / conservation of these sequences between p. monodon and the other species from which some heterologous sequences were obtained . in summary , numerous transcripts displayed microarray based expression profiles of interest in the cephalothorax and eyestalk among wild - caught and captive - reared animals during ovarian maturation . in the cephalothorax , key transcripts include those encoding the 1,3--d - glucan - binding high - density lipoprotein and the sterol carrier gene 2/3-oxoacyl - coa thiolase , both of which were up - regulated at vitellogenic stages indicative of their involvement in accumulation of lipids toward vitellogenesis . in the eyestalk , up - regulation of arrestin transcripts at pre - vitellogenic stages indicates probable modulation of gpcr activity at these stages , potentially associated with biogenic amine mediated release of reproductive neuropeptides . this observation highlights the prospects that improved understanding of hormone receptor regulation and function in the cephalothorax and eyestalk may , in time , provide opportunities for alternative methods of manipulation of neuropeptide levels . additionally , transcripts encoding the architectural transcription factor p8 were up - regulated at vitellogenic stages , suggestive of its involvement in initiating expression of large numbers of genes at vitellogenic stages .

in crustaceans , a range of physiological processes involved in ovarian maturation occurs in organs of the cephalothorax including the hepatopancrease , mandibular and y - organ . additionally , reproduction is regulated by neuropeptide hormones and other proteins released from secretory sites within the eyestalk . reproductive dysfunction in captive - reared prawns , penaeus monodon , is believed to be due to deficiencies in these factors . in this study , we investigated the expression of gene transcripts in the cephalothorax and eyestalk from wild - caught and captive - reared animals throughout ovarian maturation using custom oligonucleotide microarray screening . we have isolated numerous transcripts that appear to be differentially expressed throughout ovarian maturation and between wild - caught and captive - reared animals . in the cephalothorax , differentially expressed genes included the 1,3--d - glucan - binding high - density lipoprotein , 2/3-oxoacyl - coa thiolase and vitellogenin . in the eyestalk , these include gene transcripts that encode a protein that modulates g - protein coupled receptor activity and another that encodes an architectural transcription factor . each may regulate the expression of reproductive neuropeptides , such as the crustacean hyperglycaemic hormone and molt - inhibiting hormone . we could not identify differentially expressed transcripts encoding known reproductive neuropeptides in the eyestalk of either wild - caught or captive - reared prawns at any ovarian maturation stage , however , this result may be attributed to low relative expression levels of these transcripts . in summary , this study provides a foundation for the study of target genes involved in regulating penaeid reproduction .

Introduction Materials and Methods Results Overview of P. monodon cephalothorax and eyestalk EST collection Microarray based differential gene expression Discussion

animals : the research was conducted in accordance with the declaration of helsinki and was approved by the animal experiments committee of riken ( approved i d no . h24 - 2 - 206 ) . the heterozygous rolling nagoya ( prod - rol/+ ) strain was provided by the riken bioresource center ( tsukuba , japan ) , with support from the national bioresource project of the ministry of education , culture , sports , science and technology , japan . c57bl/6j ( b6 ) mice were purchased from charles river japan ( yokohama , japan ) . the homozygous rolling nagoya ( prod - rol / rol ) and wild - type rolling nagoya ( prod - s / s ) strains were bred by mating with prod - rol/+ mice . the mice were allowed ad libitum access to water and food pellets ( crf-1 ; oriental yeast , tokyo , japan ) and kept under conditions of controlled room temperature ( 23 1c ) and humidity ( 55 5% ) with a 12:12-hr light dark cycle ( lights on from 08:00 to 20:00 ) . reverse transcription - polymerase chain reaction ( rt - pcr ) : total rnas from the brains of prod - s / s(male , female : n=10 , 10 ) , prod - rol / rol ( male , female : n=10 , 10 ) and b6 ( male , female : n=10 , 10 ) mice were isolated using trizol reagent ( invitrogen , burlington , canada ) . first - strand complementary dna ( cdna ) was synthesized using the superscript first - strand synthesis system ( invitrogen ) . the following pcr primer sets were used for amplification of ednrb ( genbank i d : nm_007904.4 ) : forward , 5-gctagtgtgttttcagaggcttg-3 , and reverse , 5-gactaagaatctaagtgcttccc-3. the rt - pcr products were sequenced using an abi prism 3730 ( applied biosystems , foster city , ca , u.s.a . ) . genomic structure analysis : to distinguish normal alleles from those with insertion of a retroposon - like element in intron 1 of the ednrb gene ( genbank i d : ab242436.1 ) , the pcr products were amplified with genomic dna from the tails prod - s / s ( male , female : n=10 , 10 ) , prod - rol / rol ( male , female : n=10 , 10 ) and b6 ( male , female : n=10 , 10 ) mice , and allele - specific primers designed according to a previous report . the pcr products were sequenced and examined for the presence or absence of the insertion . real - time quantitative rt - pcr ( real - time qrt - pcr ) : the levels of ednrb and cacna1a mrna were measured using applied biosystems taqman gene expression assays ( ednrb , assay i d : mm01224433_m1 ; cacna1a , assay i d : mm00432190_m1 ) . all data were normalized relative to 18s ribosomal rna ( assay i d : hs99999901_s1 , applied biosystems taqman gene expression assay ) . the pcr conditions were 94c for 10 min , followed by 40 cycles at 95c for 15 sec and 60c for 1 min . all male samples ( prod - s / s , prod - rol / rol and b6 : n = each 10 ) were analyzed in duplicate , and the threshold cycle ( ct ) value was calculated to determine the relative levels of expression . in situ hybridization : the male gastrointestinal tract ( prod - s / s , prod - rol / rol and b6 : n = each 5 ) was dissected after perfusion with saline followed by 4% paraformaldehyde , fixed with tissue fixative ( gonostaff , co. , ltd . , tokyo , japan ) , embedded in paraffin and cut into section at a thickness of 6 m for in situ hybridization . the probes for the cav2.1 1 subunit cdna fragment ( positions 60686748 ) were labeled with a digoxigenin rna labeling kit ( roche diagnostics , mannheim , germany ) . color reactions were performed with nbt / bcip solution ( sigma - aldrich , st . louis , mo , u.s.a . ) overnight and then washed with phosphate - buffered saline ( pbs ) . the sections were counterstained with kernechtrot stain solution ( mutoh pure chemicals , tokyo , japan ) and mounted with cc / mount ( diagnostic biosystems inc . , microsatellite genotyping : to examine the origin of the ednrb allele from genomic dna obtained from the tails of prod - s / s ( male , female : n=10 , 10 ) , prod - rol / rol ( male , female : n=10 , 10 ) and b6 ( male , female : n=10 , 10 ) mice , microsatellite markers ( d14mit30 , d14mit193 , d14mit93 , d14mit94 , d14mit170 , d14mit42 and d14mit267 ) were typed by pcr amplification using the mouse microsatellite data base of japan ( mmdbj , http://www.shigen.nig.ac.jp/mouse/mmdbj/ ) and the mouse genome informatics ( mgi , http://www.informatics.jax.org/ ) . statistical analysis : the data are presented as the means standard error of the mean ( sem ) . statistical analyses were conducted using excel statistics 2006 ( ssri , tokyo , japan ) . the data were analyzed using dunnett s test between groups where appropriate . in all analyses gross observation of prod strain : prod - s / s and prod - rol / rol mice had irregular white spots on the agouti coat ( fig . 1afig . ( a ) representative photographs showing white spots on agouti coat of prod strain and ataxia of prod - rol / rol mice are presented . ( b ) representative photographs of the open abdomen of prod - s / s and prod - rol / rol mice are shown . ) . prod - rol / rol mice showed poor motor coordination and stiffness of the hindlimbs . neither prod - s / s nor prod - rol / rol mice showed the megacolon phenotype ( fig . ( a ) representative photographs showing white spots on agouti coat of prod strain and ataxia of prod - rol / rol mice are presented . ( b ) representative photographs of the open abdomen of prod - s / s and prod - rol / rol mice are shown . sequence analysis of the ednrb gene coding region : two c to t nucleotide substitutions were found at nucleotides 159 and 1032 in the coding region of the ednrb gene in prod - s / s mice ( fig . ( a ) exons are shown as white boxes , and the retroposon - like region is shown as a black box . white arrows represent the primers used to amplify the normal ( wt ) and mutant ( mu ) alleles by pcr as previously reported . black arrows represent the primers used to examine mrna expression using real - time qrt - pcr . ( c ) the expression levels of ednrb mrna in the colon determined by real - time qrt - pcr . the ednrb mrna expression level for each strain was calculated relative to that in b6 mice . * p<0.05 compared to the appropriate control ( dunnett s test ) . ) . however , the amino sequence of ednrb in prod - s / s mice was not different from the sequence of the b6 strain in the database . these silent nucleotide substitutions in the ednrb gene were detected in s / s mice and jf1 mice as a piebald mutation . we found nucleotide substitutions in the ednrb gene ( genbank i d : u32329.1 ) in both male and female prod - s / s and prod - rol / rol mice ( data not shown ) . ( a ) exons are shown as white boxes , and the retroposon - like region is shown as a black box . white arrows represent the primers used to amplify the normal ( wt ) and mutant ( mu ) alleles by pcr as previously reported . black arrows represent the primers used to examine mrna expression using real - time qrt - pcr . ( c ) the expression levels of ednrb mrna in the colon determined by real - time qrt - pcr . the ednrb mrna expression level for each strain structure analysis of intron 1 of the ednrb gene : intron 1 has the 5.5-kb retroposon - like element in s / s mice and jf1 mice . the f and r 2a ) and amplify a 225-base pair ( bp ) fragment as the wild type . the r - mu primer corresponds to a retroposon - like element in the s / s mutant ( fig . 2a ) , and the f and r - mu primers amplify a 318-bp fragment containing part of intron 1 of the ednrb gene and the retroposon - like element of the s / s mutant type . although a 225-bp fragment was amplified from b6 mice , prod - s / s mice and prod - rol / rol mice showed a 318-bp fragment ( fig . sequence analyses of the pcr products showed that the retroposon - like element was inserted in intron 1 ( data not shown ) . we found the retroposon - like element in both male and female prod - s / s and prod - rol / rol mice ( data not shown ) . ednrb gene expression levels in the colon : real - time qrt - pcr analysis was performed to determine the levels of ednrb mrna expression in enteric neurons of prod - s / s , prod - rol / rol and b6 mice ( fig . the relative expression level of ednrb was not significantly different between prod - s / s and prod - rol / rol mice , both of which showed about 25% lower levels of ednrb expression in the colon compared to control b6 mice . cacna1a gene expression patterns in the colon : to examine whether mutation of the ednrb gene affects the cacna1a expression pattern in the colon , we used in situ hybridization and real - time qrt - pcr analyses . the results of in situ hybridization analysis indicated similar patterns of cacna1a mrna expression in the colon of prod - s / s , prod - rol / rol and b6 mice ( fig 3.expression patterns of cacna1a mrna in the colon of prod - s / s , prod - rol / rol and b6 mice . ( a ) representative photographs indicating the localization of cacna1a mrna in the colon are shown . ( b ) the expression levels of cacna1a mrna in the intestine determined using real - time qrt - pcr analysis are shown . the cacna1a mrna expression level for each strain was calculated relative to that in b6 mice . * p<0.05 compared to the appropriate control ( dunnett s test ) . ) . using the antisense probe , no signals were seen in the colon using the sense probe in any of these strains ( data not shown ) . real - time qrt - pcr analysis was performed to determine the levels of cacna1a mrna expression in enteric neurons in the three mouse strains ( fig . the relative levels of cacna1a mrna were not significantly different between prod - s / s and prod - rol / rol mice , but they were both significantly lower than that in the colon of b6 mice . expression patterns of cacna1a mrna in the colon of prod - s / s , prod - rol / rol and b6 mice . ( a ) representative photographs indicating the localization of cacna1a mrna in the colon are shown . ( b ) the expression levels of cacna1a mrna in the intestine determined using real - time qrt - pcr analysis are shown . the cacna1a mrna expression level for each strain was calculated relative to that in b6 mice . * analysis of microsatellite loci adjacent to the ednrb locus : to examine the origin of the mutant ednrb gene in prod - s / s and prod - rol / rol mice , we typed microsatellite loci on chromosome 14 . ( a ) linkage map of mouse chromosome 14 , including the ednrb and microsatellite loci used in this study , is presented . ( b ) genotyping results of the microsatellite makers are shown .. as shown in fig . 4b , fragments were amplified from prod - s / s , prod - rol / rol and b6 mice . according to mmdbj and mgi , the different sizes between jf1 and b6 strains are produced in d14mit30 ( jf1 , b6 : size=116 bp , 154 bp ) , d14mit193 ( jf1 , b6 : size=134 bp , 119 bp ) , d14mit93 ( jf1 , b6 : size=189 bp , 147 bp ) , d14mit94 ( jf1 , b6 : size=108 bp , 104 bp ) , d14mit170 ( jf1 , b6 : size=163 bp , 146 bp ) , d14mit42 ( jf1 , b6 : size=142 bp , 152 bp ) and d14mit267 ( jf1 , b6 : size=97 bp , 114 bp ) loci . amplified fragments of the same length were obtained in both male and female prod - s / s and prod - rol / rol mice ( data not shown ) . ( a ) linkage map of mouse chromosome 14 , including the ednrb and microsatellite loci used in this study , is presented . the results of the present study indicated that the rolling nagoya strain prod - rol / rol , which exhibits white spots on the agouti coat , has two mutations in the ednrb gene , i.e. , two c to t nucleotide substitutions at positions 159 and 1032 in the coding region without amino acid substitutions and a retroposon - like element insertion into intron 1 . these mutations have been reported to be piebald spotting ( s ) mutations [ 4 , 6 , 9 , 20 ] . in addition to the coat pigment defect , piebald lethal ( s / s ) mice , which carry a severe mutation at the s locus and lack the full - length transcript of the ednrb gene , show the megacolon phenotype . the s / s , s / s , s / s and s/+ mice , in which the relative ratios of ednrb expression are about 0%:12.5%:25%:50% , show a graded coat color phenotype in the extent of white spotting and have a white coat on>95% , 4050% , ~20% and 0% of the body surface area , respectively . the megacolon phenotype occurs in s / s mice , but not in s / s , s / s or s/+ mice . these results indicate that the two neural crest - derived cell lineages require different minimal threshold levels of ednrb gene expression . more than 50% of the wild - type level of ednrb density is likely needed for normal development of epidermal melanocytes , whereas at least 12.5% of the wild - type level is required for a functional enteric nervous system . in the present study , prod - rol / rol and prod - s / s mice showed approximately 25% lower levels of ednrb gene expression compared with b6 mice that have an intact ednrb gene . the white - spotted area on the body surface was detected in prod- s / s and prod- rol / rol mice without a megacolon . regarding the origin of the laboratory mouse , while most regions of the chromosomes and mitochondrial dna are of mus musculus domesticus origin , the y chromosome and some particular regions of the autosomes are of m. m. musculus or m. m. molossinus origin . the c to t nucleotide substitutions at position 1032 in the coding region of the ednrb gene are unique to m. m. molossinus origin . jf1 is thought to be derived from the japanese wild mouse via m. m. molossinus . jf1 mice show irregular black spots on the white coat body without a megacolon and have the piebald s / s mutation . the region between d14mit93 and d14mit42 loci was identical to that of the m. m. molossinus strain . these results indicate that the sequence around the s locus is derived from m. m. molossinus . in the present study , microsatellite genotyping indicated that the alleles of the d14mit193 , d14mit93 , d14mit94 , d14mit170 and d14mit42 loci were identical to those of the jf1 strain , but that d14mit30 and d14mitw267 loci were identical to those of the b6 strain . jf1 strain was derived from fancy mice purchased at a market in denmark in 1987 and established as inbred strain in 1993 . prod strain was derived from the descendants of ( siii x c57bl/6 ) f1 and discovered in 1969 . although the genetic background of the siii strain has not been reported , our results indicated that at least the region of chromosome 14 between the d14mit193 and d14mit42 loci of the jf1 and prod strains was derived from the japanese fancy piebald mouse via m. m. molossinus . the results of this study indicate that prod - rol / rol is a double - mutant strain with an amino acid change at r1262 g in the cacna1a gene on chromosome 8 causing cav2.1 channel dysfunction and the classic piebald mutation in the ednrb gene on chromosome 14 causing the coat pigment defect . expression analyses indicated that the mutation in the ednrb gene did not affect the expression patterns of cav2.11 in the colon of mice with the prod genetic background . previous immunohistochemical experiments indicated that jf1 mice showed a lower density of enteric neurons in the colon compared to b6 mice . in the present study , the level of cav2.11 expression in the prod strain was lower than that in the b6 strain , suggesting lower enteric neuronal density due to the classic piebald mutation . acetylcholine release at the peripheral neuron muscular junctions in the colon depends on calcium channels , including cav2.1 channels . to understand the functions of cav2.1 in the rolling nagoya mouse strain , establishing a strain with an intact ednrb gene will be important

abstract ataxic rolling nagoya ( prod - rol / rol ) mice , which carry a mutation in the 1 subunit of the cav2.1 channel ( cacna1a ) gene , were discovered in 1969 . they show white spots on agouti coat and have a mutation in the piebald spotting ( s ) locus . however , mutation analysis of the s locus encoding the endothelin receptor type b ( ednrb ) gene in prod - rol / rol mice had not been performed . here , we examined the genomic and mrna sequences of the ednrb gene in prod - rol / rol and wild - type rolling nagoya ( prod - s / s ) and studied the expression patterns of ednrb and cacna1a genes in these mice in comparison with c57bl/6j mice . polymerase chain reaction analyses revealed two silent nucleotide substitutions in the coding region and insertion of a retroposon - like element in intron 1 of the ednrb gene . expression analyses demonstrated similar localizations and levels of ednrb and cacna1a expression in the colon between prod - rol / rol and prod - s / s mice , but the expression levels of both genes were diminished compared with c57bl/6j mice . microsatellite genotyping showed that at least particular regions of chromosome 14 proximal to the ednrb locus of the prod strain were derived from japanese fancy piebald mice . these results indicated that prod - rol / rol mice have two mutant genes , ednrb and cacna1a . as no prod strain had an intact ednrb gene , using congenic rolling mice would better serve to examine rolling nagoya - type cav2.1 channel dysfunctions .

MATERIALS AND METHODS RESULTS DISCUSSION

recurrent headaches are prevalent in children and adolescents . up to 89% of these individuals identify stress as a trigger . yoga offers a complementary and alternative method for this population who often lacks effective relaxation tools for stress relief . this presentation describes the yoga group offered as part of the integrative headache clinic ( ihc ) at children 's hospital colorado . one of the objectives is to determine the effect of the yoga group for children and adolescents with recurrent headaches on pain and relaxation scores . over the course of 18 months ( january 2011 to june 2012 ) , a retrospective chart review was conducted of 40 children and adolescents with an average age of 15 years , 62.5% of whom were female . during the first hour of clinic , every individual takes part in a 50-minute group yoga session consisting of 10 asanas that promote relaxation and pain relief . before and after yoga group , participants rate their level of pain and relaxation on a scale from 0 to 10 ( with 0 being no pain or relaxation and 10 being maximum pain or relaxation ) . 3.0 ) and after yoga pain score was 3.4 ( /-3.1 ) ( p=.0672 ) , representing a decrease in pain . 2.3 ) and after yoga relaxation score was 7.5 ( /-1.9 ) ( p<.0001 ) , representing a statistically significant increase in relaxation . yoga may offer an adjunct to common pharmaceutical options for headache management for children and adolescents .

focus areas : integrative approaches to care , supporting behavioral change , alleviating painobjective : recurrent headaches are prevalent in children and adolescents . up to 89% of these individuals identify stress as a trigger . yoga offers a complementary and alternative method for this population who often lacks effective relaxation tools for stress relief . this presentation describes the yoga group offered as part of the integrative headache clinic ( ihc ) at children 's hospital colorado . one of the objectives is to determine the effect of the yoga group for children and adolescents with recurrent headaches on pain and relaxation scores.description:over the course of 18 months ( january 2011 to june 2012 ) , a retrospective chart review was conducted of 40 children and adolescents with an average age of 15 years , 62.5% of whom were female . during the first hour of clinic , every individual takes part in a 50-minute group yoga session consisting of 10 asanas that promote relaxation and pain relief . before and after yoga group , participants rate their level of pain and relaxation on a scale from 0 to 10 ( with 0 being no pain or relaxation and 10 being maximum pain or relaxation ) . the average before yoga pain score was 3.7 ( /- 3.0 ) and after yoga pain score was 3.4 ( /-3.1 ) ( p=.0672 ) , representing a decrease in pain . the average before yoga relaxation score was 5.2 ( /- 2.3 ) and after yoga relaxation score was 7.5 ( /-1.9 ) ( p<.0001 ) , representing a statistically significant increase in relaxation.results:group yoga is effective in promoting relaxation in children and adolescents with recurrent headache . yoga may offer an adjunct to common pharmaceutical options for headache management for children and adolescents .

Objective: Description: Results:

hemimegalencephaly ( hme ) is an uncommon anomaly of the brain characterized by one enlarged and dysplastic cerebral hemisphere with different degree of tissue impairment . it may be an isolated anomaly or associated with syndromes such as neurofibromatosis type i , tuberous sclerosis , epidermal nevus syndrome , proteus syndrome , unilateral hypomelanosis of ito and klippel - trenaunay syndrome . it is generally assumed that hme results due to abnormal neuronal and glial proliferation or apoptosis . macroscopically , one hemisphere is enlarged and there is usually cortical dysgenesis , white - matter hypertrophy , and a dilated and dysmorphic lateral ventricle . the microscopic features include polymicrogyria ( pmg ) , heterotopic grey matter , cortical dyslamination , cystic changes , blurring of the gray - white junction , and an increase in the number of both neurons and astrocytes . the clinical triad of hmeg is typically : ( a ) intractable partial seizures from the neonatal period or early infancy , ( b ) hemiparesis and ( c ) developmental delay . the developmental outcome may be more positive if seizures are well controlled from an early age . seizures are usually resistant to drugs and control may only be achieved by surgery such as anatomical hemispherectomy . the eeg of hme is characterized by three main patterns of abnormal findings : triphasic complexes of large amplitude ; unilateral , rhythmic alpha - like activity ; and asymmetrical suppression - bursts characterized by alpha - like activity . the first pattern is associated with the most severe prognosis and the last one with a good prognosis . mri gives the best high - resolution structural image of the brain , and has whole brain coverage allowing symmetry and volume comparisons . a ten - year - old boy , previously diagnosed with infantile spasms at the age of 6 weeks , presented to the pediatric neurology room with an episode of disinhibited behavior in family environment with coprolalia suggestive for frontal disinhibition for several days . he was born after an uneventful pregnancy , except of a trauma of the mother who fell down the stairs with sacral impact . the birth weight was 3400 g , apgar score = 9 and there were no signs of perinatal distress . from the age of 6 weeks he began to have epileptic episodes with infantile spasms , with the appearance in cluster , on awakening or before falling asleep , the eeg findings show asymmetrical suppression - bursts characterized by alpha - like activity , which is the reason why a neurometabolic disease was suspected , like nonketotic hyperglycemia . he started the treatment with phenobarbital at a dose of up to 9 mg / kg / day , with no control of the seizures . at the age of 4 months , the seizures recurred with focal character : onset with head and eyes deviation to the left , eyelid myoclonus , tonic contracture in extension of the limbs lasting 13 minutes and up to 7 seizures / day . however , the seizures persisted until now , at the age of 10 , regardless of the combination therapy associated with anxiety , hyperkinetic behavior , consciousness partially preserved , 1020 seconds duration , up to 20 seizures / day . eeg recordings showed an aspect of electric status for the right occipital derivations , with a tendency to spread to the contra - lateral hemisphere . in evolution , the neurological status of the patient consists of a left pyramidal syndrome associated with left eye divergent strabismus , horizontal nystagmus on left lateral gaze , central facial palsy of the left side . the psychological status of the patient evolved with moderate motor impairment , especially of the hand with intermittent neglection of the left upper limb . also he presented autistic - like features : avoiding look in lateral angle , anxiety and hyperventilation episodes in unknown environment and disinhibited behavior in family environment with coprolalia , all of these suggestive for frontal disinhibition . during his last admission a brain mri was performed and it showed cortical dysplasia and mild enlargement of the right occipital lobe ( image a , e , i ) , white matter markedly increased in volume and isointense to cortical gray matter , consistent with graymatter heterotopia ( image b , d , f , g and h ) . the occipital gyri were presenting overfolding resembling polymicrogyria ( imagea , b and c ) . there were no brain tumors and no enhancement of the dysplastic structure ( image d , e and f ) also the ventricles were asymmetric and dysmorphic due to hypoplasia of the corpus callosum ( image j and k ) . because of the enlarged hemisphere the midline can be displaced ( image d , e , f and k ) . all these mri findings supported isolated hemimegalencephaly with polymicrogyria of the right occipital lobe diagnose correlated with patient clinical history and evolution of his neurological and psychological status . this case shows peculiarities in terms of evolution of eeg : from asymmetrical suppression - bursts characterized by alpha - like activity to focal hypssarhythmia and focal electric status , and also the evolution of behavior with frontal disinhibition . also this patient has been diagnosed in a very late phase of evolution of this disease . during diagnosis it had nt been included in any syndrome because it presented only few symptoms and the kariotype was nt performed . the poor financial and educational status of this patient s family did not allow an early diagnosis and unfortunately it has a very unfavourable outcome . intractable epilepsy is a common diagnosis in our country because of the lack of experience of radiologists in diagnosing cortical dysplasia with polimicrogirya on mri . hemimegalencephaly on mri consists of cortical gray matter almost uniformly abnormal , areas of increased thickness of the cortical gray matter ( gm ) , abnormal gyral patterns , increased signal intensity in the subcortical white matter ( wm ) on t2-weighted images , blurring of the gmwm transition , atrophy or hemispheric hypertrophy , demyelination . during development of the brain , white matter is generally markedly increased in volume , and often contains tissue isointense to cortical gray matter , consistent with graymatter heterotopia . this patient presented almost all of these mri findings supporting the hemimegalencephaly with polymicrogyria diagnose . the white - matter signal change may be consistent with either dysmyelination or advanced myelination . the ipsilateral ventricle is usually enlarged and dysmorphic , often with extension of the posterior horn of the lateral ventricle across the midline . this patient did not have enlarged ventricles but asymmetric ( image j and k ) . there may be enlargement of the ipsilateral cerebellar hemisphere and brain stem , an appearance which was named . this pathology does not affect the whole hemisphere ; in general , the posterior regions ( parietal , occipital , and posterior aspect of the temporal lobe ) are more frequently involved , compared with frontal or anterior aspect of the temporal lobes . about brain size , the characteristic hme might be compromised by atrophy related with a long history of epilepsy which is also the case here , with occipital lobe involvement that seems to have an only mild enlargement compared to the contra - lateral one . however , any intractable epilepsy should be completely investigated during infancy especially using the mri features for the correct diagnose of hemimegalencephaly with polymicrogyria and only after that the treatment started which consists of surgical resection of the affected area with fully recovery . in this particular case , it is kind of late for surgical treatment , but is the only way to treat the seizure . anyhow , the recovery will not be complete because this patient s brain is fully developed and because the affected occipital lobe with all of the neuronal connections after surgical resection should be replaced somehow . however , it is very important to recognize the abnormal tissue organization to make the correct diagnosis . in children , brain mri became the gold standard diagnostic tool , with brain enlargement and wm changes . different degree of changes in mri t1 and t2 signal intensity reveal the wm abnormalities and are the most important and constant sign in hme . it is a real challenge because those signal abnormalities are related to gliosis and various degrees of destroyed tissue .

hemimegalencephaly on magnetic resonance imaging scan ( mri ) consists of cortical gray matter almost uniformly abnormal , areas of increased thickness of the cortical gray matter ( gm ) , abnormal gyral patterns , blurring of the grey - white matter transition , atrophy or hemispheric hypertrophy , demyelination , gliosis . we present a case of ten - year - old boy with a history of infantile spasms and developmental delay who presented to the pediatric neurology room with an episode of disinhibited behavior in family environment . an mri was performed and isolated hemimegalencephaly with polymicrogyria of the right occipital lobe was diagnosed .

Introduction Case report Disscusion

the enteric nervous system ( ens ) is composed of the cell bodies and fibers that are localized between the muscle layers throughout the entire gastrointestinal tract . the ens integrates the motility , secretion , blood flow and immune response of the digestive tract , independent of the extrinsic autonomic inputs . the function of the ens is to coordinate the complex interactions of the enteric networks , which consist of sensory , inter , motor and secremotor neurons . the myenteric plexus is positioned between the outer longitudinal and inner circular muscle layers throughout the digestive tract . since the myenteric plexus modulates the contraction and relaxation of the smooth muscles , this plexus is an endogenous source for motor innervation to the muscular layers and secremotor innervation to the mucosa . similar to other nervous systems , the functions of the myenteric plexus are mediated by various neurochemical substances . indeed , substance p ( sp ) was isolated from the gut and can be detected in a dense nerve fiber network within the myenteric plexus . sp - containing nerve fibers are an intrinsic contractor of the longitudinal muscle layer . in addition , calcitonin gene - related peptide ( cgrp ) , which was originally identified as a splicing product of the alternative rna processing of the calcitonin gene in the rat brain , affects a variety of biological activities in the ens , such as release of gastrointestinal hormone , co - ordination of gastrointestinal motility , excitation of myenteric neurons and vasodilatation . on the other hand , vitamin - d dependent calcium - binding proteins ( cabps ) were first isolated from the small intestine of the chicken , thus they were presumed to play a primary role in the absorption of dietary calcium . thereafter , it was discovered that cabps transfer calcium across the membranes and regulate free intracellular calcium . thus , cabps act as " buffer / transport " proteins in various cell types . among them , calbindin d-28k ( cb ) and calretinin ( cr ) are observed in ens neurons characterized by a distinct morphology and specific electrophysiological properties . the ruminant stomach serves as a large fermentation chamber , and the motility patterns therein maintain vigorous mixing of the ingested food . neuronal regulation is therefore crucial for the regulation and coordination of region - specific motility in the ruminant stomach . as the exogenous autonomic nervous system is known to play a role in controlling stomach movement , it has been implied that the endogenous ens may also be involved in regulating the local forestomach functions . however , little data are available to elucidate the neurochemical properties of the ens in ruminants . thus , we investigated the localization of cb , cr , cgrp and sp immunoreactivity in the myenteric plexus of the goat stomach to characterize the ens in ruminants . twelve goats ( capra hircus , 10 - 16 months , 15 - 20 kg b.w . ) were used in this study . all experiments were carried out in accordance with the nih guide for the care and use of laboratory animals . the animals were anesthetized with ketamine - xylazine mixture and perfused via the common carotid artery with 3 l of 0.9% normal saline followed by 8 l of 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.4 ) . the stomach was immediately removed and dissected . the stomach contents were washed by rinsing in ice - cold 0.1 m phosphate buffer . the tissues used for cryostat sectioning were stretched and pinned flat on pieces of balsa wood . the tissues were then fixed in 0.1 m phosphate buffer containing 4% paraformaldehyde for 12 h at 4 with the mucosal surface facing up . the fixed tissues were washed in 0.1 m phosphate buffer and cryoprotected in 0.1 m phosphate buffer containing 30% sucrose . serial sections of 12 m were cut using cryostat ( reichert - jung , germany ) and mounted on gelatin - coated slides . the sections were pre - incubated in phosphate - buffered saline ( pbs ) containing 10% normal goat serum ( santa cruz , usa ) for 30 min to reduce nonspecific background staining . the primary antibodies were diluted in 0.1 m pbs containing 0.3% triton x-100 and 2% normal goat serum . the tissues were incubated for 48 h at 4 in the solution containing primary antibodies . the following antisera were used at the indicated concentrations : mouse anti - cb ( 1 : 1,000 ; swant , switzerland ) , rabbit anti - cr ( 1 : 1,000 ; chemicon , usa ) , rabbit anti - sp ( 1 : 2,000 ; peninsula , usa ) and rabbit anti - cgrp ( 1 : 2,000 ; peninsula , usa ) . after the specimens were incubated with the primary antibodies , they were washed three times for 10 min with pbs and incubated for 2 h in buffer solution containing affinity - purified secondary anti - rabbit or -mouse antibodies conjugated to indocarbocyanine ( cy3 ) . finally , the specimens were washed in pbs and coverslipped with a solution of pbs containing 80% glycerol . the slide preparations were observed under an olympus bx51 microscope ( olympus , japan ) attached to an imt2000 digital camera ( imtechnology , korea ) with the appropriate filters ( green filter ; exciter filter 513~565 nm ; beam filter 580 nm ; barrier filter 590 nm ) . the images of immunoreactivity in the myenteric plexus were captured using adobe photoshop version 6.0 software via imt2000 . twelve goats ( capra hircus , 10 - 16 months , 15 - 20 kg b.w . ) were used in this study . all experiments were carried out in accordance with the nih guide for the care and use of laboratory animals . the animals were anesthetized with ketamine - xylazine mixture and perfused via the common carotid artery with 3 l of 0.9% normal saline followed by 8 l of 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.4 ) . the stomach was immediately removed and dissected . the stomach contents were washed by rinsing in ice - cold 0.1 m phosphate buffer . the tissues used for cryostat sectioning were stretched and pinned flat on pieces of balsa wood . the tissues were then fixed in 0.1 m phosphate buffer containing 4% paraformaldehyde for 12 h at 4 with the mucosal surface facing up . the fixed tissues were washed in 0.1 m phosphate buffer and cryoprotected in 0.1 m phosphate buffer containing 30% sucrose . serial sections of 12 m were cut using cryostat ( reichert - jung , germany ) and mounted on gelatin - coated slides . the sections were pre - incubated in phosphate - buffered saline ( pbs ) containing 10% normal goat serum ( santa cruz , usa ) for 30 min to reduce nonspecific background staining . the primary antibodies were diluted in 0.1 m pbs containing 0.3% triton x-100 and 2% normal goat serum . the tissues were incubated for 48 h at 4 in the solution containing primary antibodies . the following antisera were used at the indicated concentrations : mouse anti - cb ( 1 : 1,000 ; swant , switzerland ) , rabbit anti - cr ( 1 : 1,000 ; chemicon , usa ) , rabbit anti - sp ( 1 : 2,000 ; peninsula , usa ) and rabbit anti - cgrp ( 1 : 2,000 ; peninsula , usa ) . after the specimens were incubated with the primary antibodies , they were washed three times for 10 min with pbs and incubated for 2 h in buffer solution containing affinity - purified secondary anti - rabbit or -mouse antibodies conjugated to indocarbocyanine ( cy3 ) . finally , the specimens were washed in pbs and coverslipped with a solution of pbs containing 80% glycerol . the slide preparations were observed under an olympus bx51 microscope ( olympus , japan ) attached to an imt2000 digital camera ( imtechnology , korea ) with the appropriate filters ( green filter ; exciter filter 513~565 nm ; beam filter 580 nm ; barrier filter 590 nm ) . the images of immunoreactivity in the myenteric plexus were captured using adobe photoshop version 6.0 software via imt2000 . in the present study , cb , cr , sp , and cgrp immunoreactivities were observed in the nerve cell bodies or fibers of the stomach myenteric plexuses of the korean native goat . cb immunoreactivity was found in the nerve cell bodies and fibers of the rumen , reticulum and abomasum ; however , only cb - immunoreactive ( cb - ir ) nerve fibers were observed in the omasum ( fig . cb - ir neurons were oval - shaped with generally smooth margins and were classified as dogiel type ii cells based on dogiel 's classification for enteric neurons . the average number of cb - ir cell bodies in the myenteric plexus was 7.8 cells / ganglion . cr - immunoreactive ( cr - ir ) fibers were found in all subregions of the stomach ; in particular , varicosities of cr - ir fibers were dominantly observed in the reticulum ( fig . sp - immunoreactive ( sp - ir ) neurons and fibers were observed in all stomachs , except in the omasum . cgrp - immunoreactive ( cgrp - ir ) fibers were found in all subregions of the stomach , and varicosities of cgrp - ir fibers were prominently observed in the myenteric plexus of goat stomach , compared to cb- , cr- , and sp - ir fibers ( fig . in the present study , we observed cb- , cr- , cgrp- and sp - ir myenteric neurons within the goat stomach . it is known that there are several histologically distinct types of intrinsic neurons in the ens . these include excitatory and inhibitory motor neurons to the muscle , vasomotor neurons , secremotor neurons , interneurons and sensory neurons . many investigators have tried to relate these physiological functions to individual cytoarchitecturally defined enteric neurons . although the ens can function independently of the cns , the latter has an important role in the coordination of the diverse functions of the ens . the ens is well connected to the central autonomic neural network in the cns through both motor and sensory pathways of the sympathetic and the parasympathetic nervous system . the parasympathetic motor pathways consist of the vagus and sacral nerves that control motor and secremotor functions . these neurons are located in the smooth muscle layer and are sensitive to mechanical distention of the gut . in the goat stomach , the cb - ir neurons observed in the myenteric plexus were identified as morphological dogiel type ii cells featuring smooth perikarya with long multiple processes . cb - ir cells are known to have the electrophysiological characteristics of after - hyperpolarization ( ah ) , and may be considered to be intrinsic sensory neurons . moreover , the cb - ir dogiel type ii neurons located in the myenteric plexus of the guinea - pig ileum are known to be cholinergic and to activate the intrinsic primary afferent neurons . in the guinea - pig gi tract , the somata of cb - containing primary afferent neurons in the intestine of several species are located within the myenteric plexus and project to the epithelium . based on these previous studies , cb - ir neurons in the myenteric neurons of goat stomach may also be sensory neurons that project to the epithelium . these findings are similar to another study detailing the profiles of in the equine stomach . therefore , our findings indicate that the localization / functionality of cb in myenteric plexus of the goat may be similar to that of other animals . in the present study , cr - immunoreactivity , cr is known to be present in the myenteric neurons , and related with sensory function of circular muscle layer and villi . moreover , cr - ir neurons are identified as small dogiel type i neurons and sensory intrinsic neurons . however , cr - ir was only found in the myenteric nerve fibers of goat stomach in this study . these data show that there are differences in the distributions and functions of cr in goat stomach compared to that of other animals . in a variety of species , sp has been shown to be the major excitatory neurotransmitter of motor neurons in the gastrointestinal tract . however , a significant difference in the density of sp has been reported in the various regions of the digestive tract ; sp - ir neurons were found in the submucosal and myenteric plexuses of small animals , while sp - ir neurons were rarely observed in large animals . indeed , in the horse , sp - immunoreactivity was not observed in the submucosal plexus , and was only weakly detected in the myenteric plexus . in the pig this discrepancy might be related to the differential ganglionic organization between small and large animals . in the present study , since sp affects the contraction of the smooth muscle of the stomach , this peptide stimulates gastric motility and increases the intragastric pressure . cgrp is a ' marker peptide ' of the dogiel type ii cell population in the ganglionated plexuses of the porcine small intestine and has a sensory function . however , we did not observe cgrp - ir myenteric neurons in the goat stomach . although the function of cgrp - ir neurons in the goat stomach is unclear , our findings indicate that there is species - specific localization of cgrp - ir cells in the myenteric plexus . similar to cb , the distribution of cgrp - ir fibers indicate that cgrp may be involved in the sensory functions of the stomach , which initiate mixing of the ingesta , regurgitation of the forestomach ingesta , and signal satiety . in conclusion , the immunohistochemical localization of cb , cr , cgrp and sp in the myenteric plexus of the goat stomach displayed species - specific patterns . these findings suggest that these substances may be directly and/or indirectly related to the gastric functions of the goat stomach .

to understand the neurochemical properties of the gastric myenteric plexus of ruminants , the expression patterns of calbindin d-28k ( cb ) , calretinin ( cr ) , substance p ( sp ) and calcitonin gene - related peptide ( cgrp ) were explored in the korean native goat . in gastric myenteric plexus , cb and sp immunoreactivity were observed in round- or oval - shaped neurons . cr and cgrp immunoreactivity were detected only in the nerve fibers . this immunohistochemical localization of cb , cr , cgrp and sp in the myenteric plexus of the goat stomach exhibited species - specific patterns . these findings suggest that these substances may be directly or indirectly related to the gastric functions of the goat stomach .

Introduction Materials and Methods Animal and Tissue Preparation Immunohistochemistry Results Discussion

in contrast to colon cancer , transmural ( t3 and t4 ) and node - positive rectal cancer has a propensity for local recurrence . based on a significant morbidity of local - regional failure and evidence from prospective randomized trials demonstrating improvement in disease - free survival time and local - regional recurrences , the national cancer institute sponsored consensus conference convened in 1990 and recommended that postoperative chemoradiotherapy should be administered in all patients with stages ii and iii rectal cancers . despite these recommendations , adoption of these guidelines into clinical practice has been , at best , uneven [ 36 ] . there is little debate that chemoradiotherapy reduces local - regional failures and improves survival in stages ii and iii rectal cancer . the investigators demonstrated improved survival and local control rate in patients receiving a short - course high - dose preoperative radiotherapy . the german rectal study group randomly assigned patients with clinical stages ii and iii rectal cancer to either preoperative or postoperative chemoradiotherapy , and they provided convincing evidence that preoperative chemoradiotherapy significantly improves the local recurrence rate than postoperative chemoradiotherapy . however , no difference in overall survival rate at 11 years of follow - up was observed despite being adequately powered to address this question . based on this compelling high level evidence , the national comprehensive cancer network ( nccn ) recommends radiotherapy ( ideally preoperative ) for most patients with stage ii / iii rectal cancers . we have previously demonstrated that significant numbers of patients undergoing curative surgical resection of stage ii / iii rectal cancer do not receive radiotherapy [ 3 , 6 , 11 ] . it is imperative that patterns of care for patients in whom guideline recommended radiotherapy is not delivered be understood . omission of treatment in at - risk patients is associated with outcome inequality [ 12 , 13 ] . this study sought to examine , in a large population - based cancer registry , adherence to national cancer institute consensus guidelines for the use of adjuvant radiotherapy in resected stage ii / iii rectal cancer . this study utilized data obtained between 1998 and 2007 from the surveillance epidemiology and end results-(seer- ) 17 database . the seer-17 program is the largest population - based us cancer registry that collects information on incidence , prevalence , and survival rate from specific geographic areas representing 28% of the us population . seer routinely collects surgical and radiation data but lacks any information on chemotherapy or site of first recurrence . all patients were restaged using current american joint committee on cancer criteria . in this study , we included patients with either stage ii or stage iii rectal cancer , those who had undergone curative surgical resection of the primary tumor ( site - specific surgery codes 30 , 40 , 50 , 60 , 70 , and 80 ) , and those who were documented to have either received or not received pelvic radiation . patients with seer historic stage a disease with nonregional metastatic disease at the time of diagnosis ( m1 ) , or those who had missing treatment information ( i.e. , delivery of radiotherapy was not documented ) were excluded . the following variables were examined : age , race , gender , year of diagnosis , delivery of radiotherapy , and county - level census data ( median family income , percent families below poverty , percent high school education , percent unemployed , and percent white - collar occupation ) . , which is employed by others to define area - level socioeconomic status ( ses ) in large databases . three domains were defined using the following county - level data : education ( percent of high school graduates ) , income ( median income and percent below poverty ) , and employment ( percent unemployed and percent white - collar occupation ) . each factor was divided into one of 5 quintiles , with one reflecting the lowest socioeconomic quintile . after the quintiles were clearly defined , an overall ses index was derived by adding each factor ; these scores were again divided into quintiles to derive a score from 1 to 5 , with one reflecting the lowest ses and five the highest ses . data was extracted utilizing seerstat 7.0.5 using the case - listing function and exported to the sas platform jmp ( sas institute inc . , cary , nc , usa ) for analysis , and student 's t - tests were utilized where appropriate . table 1 summarizes the demographics of the study patient population stratified by whether or not they received radiation . a majority of patients had stage iii disease ( 54.9% ) , were aged > 60 years ( 64.3% ) , white ( 83% ) , and males ( 58.8% ) . young patients were far more likely to receive radiotherapy than older patients ( p < 0.0001 ) . the percentage of patients who received adjuvant radiotherapy decreased with each successive decade of life . other factors associated with the utilization of adjuvant radiotherapy include patients with stage iii disease ( stage iii , 70.6% versus stage ii , 64.3% ; p < 0.0001 ) and gender ( male , 70.3% versus female , 64.1% ; p < 0.0001 ) . in addition , we observed that patients residing in lower ses index counties were less likely to receive adjuvant therapy than individuals who resided in higher ses index counties . however , we were unable to define any difference in the delivery of radiotherapy between different racial groups . in order to better understand factors associated with delivery of adjuvant radiotherapy patients greater than 80 years old were significantly less likely to be treated with adjuvant radiotherapy than any other age group ( table 2 ) . comparing patients 50 to those 80 , the likelihood of adjuvant radiotherapy was 10 times greater ( odds ratio 10.3 and p value 0.001 ) . the odds of being treated decrease progressively with increased age . patients with stage iii rectal cancer were significantly more likely to be treated with radiotherapy than those with stage ii disease ( odds ratio 1.21 and p value < 0.0001 ) . men were more likely to be treated than women ( odds ratio 1.18 and p value < 0.0001 ) . the chance of receiving radiotherapy was approximately 10% greater for patients residing in the highest ses level counties when compared to the lowest . we also noted a trend over time for an increase in use of radiotherapy , odds ratio of 1.41 for treatment in 2007 when compared to 1998 , p = 0.001 . the optimal management of a patient with rectal cancer involves a multidisciplinary team whose members include surgeons with expertise in total mesorectal excision ( tme ) , radiation oncologists , medical oncologists , and ancillary support staff . despite dramatic improvements in local control and survival with the introduction of neoadjuvant and adjuvant radiotherapy regimens , not all patients are treated with radiotherapy [ 1 , 7 , 9 ] . utilizing a large population - based cancer registry , we report a failure to deliver radiotherapy in one - third of patients with stage ii / iii rectal cancer . older age , stage ii , area - level socioeconomic deprivation , and female gender we found that there was a linear decrease in the use of radiotherapy with increasing age . patients less than 50 years old were treated with radiotherapy in 84.4% of cases , whereas only a third of patients greater than 80 years old were treated . patients less than 50 were 10 times more likely to be treated with radiotherapy when compared to those older than 80 ( table 2 ) . ageism ( reflected by undertreatment of older patients ) is well documented for colorectal cancer . such treatment differences are not explained by comorbidities or poor tolerance of treatment regimens [ 1517 ] . older patients generally derive a similar benefit from adjuvant therapy and can tolerate such regimens [ 1720 ] . despite this , older patients with colorectal cancer are less likely to be treated with adjuvant therapy [ 16 , 17 , 2124 ] . omission of adjuvant radiotherapy in older , medically fit rectal cancer patients may compromise not only local recurrent but survival as well . men were treated in 70% of cases , whereas women received radiation in only 64% of cases . on logistic regression , this difference persisted , with nearly a 20% increase in treatment for men compared to women . this phenomenon of omission of adjuvant therapy for women with colorectal cancer has been documented in both the united states and the netherlands [ 21 , 25 ] . since there is no reason to expect that women would not derive a similar treatment benefit , the reason of this difference is unclear . lower socioeconomic status is associated with treatment and outcome disparities for patients with colorectal cancer [ 26 , 27 ] . we found area - level socioeconomic deprivation , as reflected in a validated socioeconomic index , to be associated with decreased use of adjuvant therapy . these findings are similar to other investigators who have noted a slower integration of advances in medical treatment of colorectal cancer for patients residing in counties with lower ses . this slow adaptation of medical innovation has resulted in increasing outcome inequality in regions with area - level ses deprivation . the lower treatment rates for stage ii cancer reported here may , in part , be appropriate . there may be little benefit to radiotherapy for patients with t3n0 rectal cancer resected using total mesorectal excision with negative radial margins if there is minimal invasion of the mesorectum . these factors are not documented in the seer registry ; as a result , it is impossible to determine whether deviation from standard radiotherapy was rational . although seer data is not robust enough to make definitive conclusions regarding the effects of radiotherapy on survival , the potential impact of optimal local therapy on overall survival rate should not be overlooked . educational initiatives and/or centralization designed to improve the quality of care for rectal cancer patients suggest that optimal local therapy can impact not only local recurrence rates but also overall survival rate . the university of erlangen implemented a strategy to improve the quality of surgery in rectal cancer by routine use of standardized tme surgery . the 5-year local recurrence rate after implementation decreased from 39.4% to 9.1% and survival rate improved from 50% to 71% . quality initiatives in other studies have found that systematic education regarding implementation of and adherence to optimal surgical and adjuvant therapy guidelines significantly improves survival rates [ 5 , 3133 ] . educational programs in the netherlands , designed to increase the use of tme and preoperative radiotherapy , were associated with a 9% improvement in 5-year survival rates . jullumstro found that decreased adherence to guidelines in norwegian patients was associated with an increase in local recurrence and mortality . women with node - positive breast cancer are generally considered to have systemic disease , but they derive survival benefit from postmastectomy radiation [ 35 , 36 ] . a large body of evidence now supports the use of preoperative chemoradiotherapy in patients with stage ii / iii disease along with tme . in this report , utilizing a large population - based database , we demonstrate that pelvic radiotherapy is omitted after presumably curative surgery in one - third of cases . undertreatment is associated with increasing age , female gender , lower ses , and stage ii disease . although limitations in the seer registry ( i.e. , lack of data on chemotherapy and comorbidities data ) precluded a definitive survival analysis , it is likely that interventions that improve compliance with treatment guidelines will improve survival in groups at - risk for undertreatment .

high - level evidence supports adjuvant radiotherapy for rectal cancer . we examined the influence of sociodemographic factors on patterns of adjuvant radiotherapy for resected stage ii / iii rectal cancer . methods . patients undergoing surgical resection for stage ii / iii rectal cancer were identified in seer registry . results . a total of 21,683 patients were identified . majority of patients were male ( 58.8% ) , white ( 83% ) , and with stage iii ( 54.9% ) and received radiotherapy ( 66% ) . on univariate analysis , male gender , stage iii , younger age , year of diagnosis , and higher socioeconomic status ( ses ) were associated with radiotherapy . radiotherapy was delivered in 84.4% of patients < 50 ; however , only 32.8% of those are > 80 years . logistic regression demonstrated a significant increase in the use of radiotherapy in younger patients who are < 50 ( or , 10.3 ) , with stage iii ( or , 1.21 ) , males ( or , 1.18 ) , and with higher ses . conclusions . there is a failure to conform to standard adjuvant radiotherapy in one - third of patients , and this is associated with older age , stage ii , area - level of socioeconomic deprivation , and female sex .

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

aortopulmonary window ( apw ) is a rare entity in congenital heart disease with a prevalence of 0.2%0.4% of all the cardiac lesions . we present an infant with a distal apw who underwent surgical repair of the same and stressed the importance of intraoperative transesophageal echocardiography ( tee ) as an invaluable tool that aids in confirming and assessing the adequacy of surgical repair . a 4-month - old boy , 4.5 kg , presented to our hospital with a history of recurrent upper and lower respiratory infections , feeding difficulties and excessive forehead sweating , progressive tachypnea , and obvious chest retraction at rest and a soft systolic heart murmur . his cardiothoracic ratio was near 65% in chest x - ray [ figure 1 ] . chest x - ray revealing cardiomegaly with dilated pulmonary artery and congested lung fields transthoracic echocardiography showed increased the pulmonary venous return to the left atrium , dilated all four chambers , large distal apw ( 15 mm 11 mm ) , intact interventricular septum . cardiac catheterization was deferred as the defect was quite evident on two - dimensional echo with an increased pulmonary venous return to the left atrium and the infant being 4 months in age with chest x - ray showing plethoric lung fields and cardiomegaly , surgical repair was planned . mid - esophageal view at 86 with probe turned to the right revealing a patent foramen ovale shunting left to right upper esophageal view at 0 revealing the aortopulmonary window extending till the bifurcation of the pulmonary artery upper esophageal view with a transesophageal echocardiography demonstrating the aortopulmonary window measuring 11.2 mm color doppler ( left ) showing turbulent flow in pulmonary artery across the aortopulmonary window the child was subjected to a surgical repair . intraoperative snapshot of the aortopulmonary window from head end cardiopulmonary bypass ( cpb ) was instituted after occluding pulmonary arteries with moderate hypothermia using two venous cannulas and a single arterial cannula positioned in the aorta distal to the defect . upper esophageal view at 24 postrepair showing the gore - tex patch closure of the defect the child had an uneventful postoperative course in the hospital with regression of pulmonary hypertension . an apw is a communication between the pulmonary artery ( pa ) and the ascending aorta in the presence of two separate semilunar valves . mori et al . classified the apw as proximal , distal , and total depending on the extent of the lesion . richardson classified apw as simple defects between the ascending aorta and pulmonary trunk ( type i ) , defects extending distally till the bifurcation of pa ( type ii ) , and anomalous origin of the right main pa from the ascending aorta with no other aortopulmonary communication ( type iii ) . complex apw is associated with ventricular septal defect ( vsd ) , interrupted aortic arch , transposition of the great artery , tetralogy of fallot , or coronary artery anomalies . there can be artifactual dropouts with transthoracic echo in the lateral wall of the aorta with normal or aberrant coronaries in the aortic root area which does not happen with a transesophageal echo that can be superior in imaging the defect . there have been reports regarding the role of intraoperative transesophageal echo in complex apw . cardiac catheterization and cineangiography with retrograde aortography are done in infants 6 months of age for evaluating the presence of irreversible pulmonary vascular disease and in case of complex apw for delineating the exact morphology . closure of apw is indicated in all patients , and it should be performed as soon as possible after diagnosis . successful repair of an apw was reported from several years ago , and many authors have reported their results . the spectrum of the surgical techniques which evolved with time include simple ligation , division and suturing without cpb , division and suturing with cpb , trans - pa closure using cpb , trans - window closure ( anterior sandwich patch closure ) , transaortic direct closure ; to transaortic patch closure of the defect ; using cpb and arresting the heart . transaortic direct closure of an apw was first reported by wright et al . in 1968 . in the year 1969 , deverall et al . we had used a gore - tex patch to close the defect in our patient by transaortic route , and the pfo was closed . as the transaortic repair using cpb allows accurate visualization of coronaries arteries and right pa reconstruction using a dacron patch without distorting either the aorta or the pa , it is the recommended approach for most patients with an apw . the possible occurrence of pulmonary hypertensive crisis should be kept in mind while managing these children in the postoperative period . intraoperative tee aids in confirming the diagnosis and is an invaluable tool in detecting additional intracardiac defects which may have been missed by a transthoracic echocardiography . the severe pulmonary hypertension usually associated with apw may mask underlying small shunts in the heart which become evident once we close the apw . tee is more sensitive in picking up the defects in the prebypass period , as well as postbypass , when it mandates addressing the additional shunts immediately . in addition , tee in postbypass period confirms the adequacy of repair and detects any residual shunt across the window , direction of pfo shunting indicates the severity of pulmonary arterial hypertension , valvular competence of the semilunar valves , the left ventricular function which can have an afterload mismatch when shunt across into the low resistance pulmonary circulation is suddenly removed , and the delineation of coronaries postrepair in type 1 proximal apw . tee guides us in the postoperative plan of an apw repair patient , be it simple or complex , the pulmonary hypertension management and left ventricular afterload reduction strategies . postoperative mortality depends on the age of the patient at operation , status of preoperative pulmonary vascular disease , and presence of associated intracardiac defects . as the natural history of apw is similar to that of a large vsd or pda , early recognition is necessary for a successful surgical result before the onset of irreversible pulmonary vascular disease in both simple and complex apw .

aortopulmonary window ( apw ) is a relatively rare cardiac lesion representing approximately 0.2%0.4% of all cardiac malformation . it is a cardiac abnormality that results from abnormal communication between the proximal aorta and the main pulmonary artery in the presence of two normally separated aortic and pulmonary valves . in the past , the diagnostic and surgical approach to apw was almost always preceded by cardiac catheterization . with recent advances in noninvasive approach and techniques of two - dimensional echocardiography diagnosis of the defect and associated anomalies are facilitated without a cath study . we report a 4-month - old infant with a distal apw who was referred to our center for surgical repair . we emphasize the usage of transesophageal echocardiography as a valuable intraoperative tool which not only confirms the preoperative diagnosis but also helps in assessing the surgical repair of an apw .

INTRODUCTION CASE REPORT DISCUSSION Financial support and sponsorship Conflicts of interest

it presents as a painless and slowly growing mass and also as exophthalmoses like in orbital masses . this study aims to identify the clinical course , surgical treatment and histopathologic properties of pleomorphic adenoma diagnosed in a patient who had painless exophthalmia with an intraorbital mass . a 62-year - old male patient referred to our clinic due to painless exophthalmia of his right eye for the last 2 years . on neurological examination of the patient , there was no abnormality except for the up - gaze restriction and severe exophthalmia . cranial magnetic resonance imaging ( mri ) revealed an isointense mass on the superolateral part of right orbita in the coronal intersections and it was contrasted homogenously ( fig . 2a and b ) . the existing score of our patient according to rose and wright was + 8 points histological examination of the surgical specimen covered the epithelial and stromal components that formed the glandular structures . figure 1:(a and b ) computerized tomography revealed a homogenously contrasted intraorbital mass in the right eye . figure 2:(a and b ) cranial mri revealed a homogenously contrasted mass on the superolateral part of the right orbita . figure 3:(a and b ) postoperative computerized tomography revealed the replacement of the eye . figure 4:(a ) four times h - e biphasic tumor composed of epithelial and stromal components . ( b ) ten times h - e fibromyxoid stroma and epithelium that form glandular structures . ( a and b ) computerized tomography revealed a homogenously contrasted intraorbital mass in the right eye . ( a and b ) cranial mri revealed a homogenously contrasted mass on the superolateral part of the right orbita . ( a ) four times h - e biphasic tumor composed of epithelial and stromal components . ( b ) ten times h - e fibromyxoid stroma and epithelium that form glandular structures . although the orbital tumors originate primarily from vascular , muscle , cartilage , neural tissues , lacrimal glands and lymphoid structures , they can also originate from the surrounding structures and ( low possibility of ) metastasis . epithelial tumors account for 30% of the lacrimal gland lesions , while 12% of epithelial tumors are pleomorphic adenoma . the term pleomorphic ( benign mixed tumor ) was first defined and used by willis . malignant transformation can occur in 1020% of pleomorphic adenomas of the lacrimal gland [ 5 , 6 ] . seventy - five percent of pleomorphic adenomas transform into pleomorphic adenocarcinoma , while the rest transform into cystic carcinoma . pleomorphic adenoma must be considered for the patients whose clinical course lasts for 12 months . malignancy must be considered for cases with pain and progredien symptoms . in his study , wright reported that 39 of 40 cases diagnosed with primary malignant lacrimal gland tumors had a painful mass . in addition to pain , neoplasm of the lacrimal gland , which destroys the bones , can be interpreted as malignancy . while pleomorphic adenomas do not destroy the bones , malignant neoplasm does [ 9 , 10 ] . although pleomorphic adenomas constitute 49% of all orbital tumors , their morbidity is very high . displacement of the mass without its capsule and malignant transformations following the incisional biopsies prior to the diagnosis of pleomorphic adenoma increase the morbidity . to this end , rose and wright developed a scoring system to evaluate the lacrimal gland masses [ 6 , 9 ] ( table 1 ) . according to this scoring system , if the value obtained from this table is below + 2 , the value may indicate carcinoma and thus incisional biopsy can be performed . the values + 3 and higher may indicate pleomorphic adenoma and thus , incisional biopsy must not be performed . the success of the treatment depends on the displacement of the mass with its capsule . it can also lead to malignant transformation of pleomorphic adenoma . the existing score of our patient according to rose and wright was + 8 points . table 1:management of lacrimal gland masses according to rose and wrightclinicalscore1 + 1duration of acute symptoms<10 months>10 monthspersistent painpresentabsentsensory losspresentabsentradiological1 + 1well - defined round or oval massabsentpresentmoulding of mass to globe or along the lateral orbital wallpresentabsenttumor calcificationpresentabsentinvasion of bonepresentabsentduration of symptoms in relation to tumor sizeshort symptomslong symptoms management of lacrimal gland masses according to rose and wright pleomorphic adenoma of the lacrimal gland must be considered in the presence of long - term painless exophthalmoses . for the patients suspected to have pleomorphic adenoma , incisional biopsy should not be performed in order to prevent the likelihood of a relapse to occur . the displacement of the mass with its capsule can extend the patients ' lives and improve their quality of life .

we present a case of a 62-year - old male patient with pleomorphic adenoma and painless solid mass in his right eye . computerized tomography demonstrated a mass with a diameter of 2.5 cm located in the right lacrimal gland . the mass was removed completely by combined orbitofrontal craniotomy through a transcranial approach . histopathologic examination revealed pleomorphic adenoma of the lacrimal gland . orbital tumors originate primarily from vascular , muscle , cartilage , neural tissues , lacrimal glands and lymphoid structures . five percent of all intraorbital masses originate from the lacrimal gland . pleomorphic adenoma presents as a painless and slowly growing mass and also as exophthalmoses . pleomorphic adenoma has a high morbidity . morbidity increases due to the total displacement of the tumor without its capsule and incisional biopsy for the purpose of diagnosis . the success of the treatment depends on the removal of the tumor with its capsule .

INTRODUCTION CASE REPORT DISCUSSION

anaplastic astrocytomas are progressive brain tumors with a tendency to infiltrate the surrounding tissue . recurrence is very common , with recurrent tumors being extremely refractory to existing therapies . case presentation : a 33-year - old woman presented with a history of an unprovoked fall , followed by seizures . an mri scan revealed a mass in the fronto - temporo - parietal region of the brain , suggesting a primary tumor . follow - up mri scans indicated the presence of a residual , rapidly progressing tumor . a 6-week course of fractionated radiation and concurrent chemotherapy with temodar ( temozolomide capsules ) did not stop tumor progression . intervention : due to the failure of conventional therapies in preventing rapid disease progression , the patient volunteered to undergo a 28-day course of sequentially programmed magnetic field ( spmf ) therapy . immediate post - therapy mri scan showed a cessation of tumor growth , and follow - up imaging at 6 , 12 , 24 and 36 months revealed a gradual but steady decrease in the size of the tumor . the patient reported an alleviation of clinical symptoms and a subjective improvement in the quality of life at 6 , 12 , 24 and 36 months following spmf therapy . the remarkable improvement of this patient suggests that spmf therapy may be a valuable option for anaplastic astrocytoma , especially in recurrent and rapidly progressing tumors . anaplastic astrocytomas are pathologically classified by who as grade iii tumors that demonstrate pleomorphism , increased mitosis and vascular proliferation , with a characteristic absence of necrosis seen in glioblastomas . the currently accepted treatment modality for such tumors is surgical debulking , followed by external beam radiation and adjuvant chemotherapy . however , due to the nature of the tumor , a complete surgical resection is almost impossible . the prognosis is highly variable and dependent on the age of the patient , symptom duration , karnofsky performance status , extent of resection , dose of radiation and several other factors . also , recurrence is extremely common , with subsequent tumors being highly refractory to further therapy . anaplastic astrocytomas , in particular recurrent tumors , clearly have an unfavorable prognosis , affirming the need to explore new therapeutic modalities . sequentially programmed magnetic field ( spmf ) therapy is based on the principle that exposure to sequentially programmed electromagnetic fields can suppress tumor growth . the magnetic fields are delivered by a spmf machine that consists of multiple magnetic field generators arranged in a circular fashion around the gantry . the fields from these generators are focused on the centre of the affected tissue with the aid of laser guides . the spmf machine generates sequential pulsed magnetic fields of < 30 milli - tesla in the range of 10 - 1,000 hz . the field strength that the machine delivers is within the international commission for non - ionizing radiation protection 's safety norms . a study assessing the spmf therapy for terminally ill cancer patients showed appreciable pain relief and improvement in karnofsky performance scores in 80% of the cases . the failure of conventional modalities of treatment in preventing rapid tumor progression substantiated the use of spmf therapy in the current study subject . a 33-year - old woman presented at the emory adventist hospital , atlanta , ga . , usa , in october 2005 with a history of an unprovoked fall , followed by seizures a few hours later . a ct scan of the brain revealed a large mass in the right fronto - temporo - parietal region involving the basal ganglia , with features suggestive of a primary tumor . the patient underwent a right craniotomy with debulking of the tumor at the wellstar cobb hospital , atlanta , ga . , usa . an aggressive parieto - frontal resection was done , and tumor tissue , roughly measuring 4 5 5 cm , was removed . histopathological examination of the specimen revealed an astrocytoma with increased cellularity and the presence of mitotic figures , but without any evidence of necrosis or endothelial hyperplasia . the histological features were consistent with the diagnosis of an anaplastic astrocytoma ( who grade iii ) . a post - surgical mri scan revealed a persistent nodular enhancement within the right temporal lobe posterior to the resection cavity , suggesting residual disease in this location . she was administered 34 fractions of 180 centigray ( cgy ) over a 7-week period , and prescribed 105 mg temodar ( temozolomide capsules ) daily for 45 days . this was followed by a 5-day course of high - dose temodar ( 300 mg ) one month after the completion of the initial cycle . monthly follow - up investigations indicated the presence of a progressive tumor with increasing enhancement and edema . pet scan showed a hypometabolic lesion but magnetic resonance spectroscopy showed that the lesion was progressing and the patient was advised to undergo a second surgery for decompression . the patient refused surgery and opted for spmf therapy at the institute of aerospace medicine ( iam ) , indian air force , bangalore , india . at the time of admission in june 2006 , the patient complained of tingling in the lower half of the left side of the face and left extremities , with some loss of recent memory . an mri taken before starting the spmf therapy showed a mass in the right fronto - tempero - parietal region measuring 3.82 2.3 2.32 cm ( antero - posterior x transverse x supero - inferior ) . based on the mri findings , the fields from the magnetic field generators were adjusted to focus on the centre of the tumor . the therapy was given for 1 hour , daily , usually at a fixed time , for 28 consecutive days . an mri scan taken on completion of the therapy showed a marginal reduction in mass ( 3.42 2.19 2.19 cm ) , along with some reduction in the thickness of the peripherally enhancing component . the patient had subjective signs of recovery , with an improvement in memory and a complete alleviation of the tingling sensation in the lower half of the left side of the face and left extremities . the mri scans performed 6 months after spmf therapy showed a reduction in the size of the lesion ( 2 2 1.3 cm ) , as well as a corresponding decrease in the thickness of the peripherally enhancing component and perilesional edema . follow - up mri scans at 12 , 24 and 36 months after the spmf therapy demonstrated a progressive decrease in the lesion size ( fig . pet scans at 12 , 24 and 36 months confirmed that the tumor had become metabolically inactive . the radiological responses were mirrored by similar improvements in subjective assessment . the functional assessment for cancer therapy general ( fact - g ) scores improved dramatically at 6 months post - therapy and were maintained at 12 and 24 months ( table 1 ) . corresponding changes were also seen with the karnofsky performance status , with scores of 70 , 80 and 100 during pre - spmf , immediate post - spmf and 6 months post - spmf therapy periods , respectively . a score of 100 was maintained at 12 , 24 and 36 months post - therapy . several studies have demonstrated the efficacy of magnetic fields in preventing neoplastic progression in cancer cell lines and animal models . prostate cancer cell lines exposed to 60 hz sinusoidal magnetic field have demonstrated increased apoptosis and cell cycle arrest ; the effect being mediated by the generation of reactive oxygen species . gastric adenocarcinoma cell lines treated with magnetic fields have shown a significantly higher incidence of apoptosis in comparison to untreated cells . exposure of mice mammary adenocarcinoma cells to pulsating magnetic fields was reported to cause a reduction in vascularization and an increase in tumor necrosis . similarly , mice with breast cancer xenografts exposed to pulsed magnetic fields were observed to have less lung metastasis and slower growth of tumors compared to sister controls . static magnetic fields , produced by nuclear magnetic resonance spectroscopy , induced apoptosis in tumor cells of hematopoietic origin due to an increase in cytosolic calcium . such a response was not seen in normal mononuclear white blood cells exposed to the same magnetic fields . studies have also demonstrated that electromagnetic fields cause anti - tumor activity and a significant increase in apoptosis in tumors of treated animals , together with reduction in immunoreactive p53 expression . the cells can divide only a defined number of times and then perish ( hayflick limit ) . with each mitotic cycle , they lose one molecule in the cytoplasm , such that the daughter cell contains one molecule less than the mother cell . microtubules and centrioles have their endogenous electromagnetic fields and can condense this energy required for transport of ions and organelles and duplication of centrioles during mitosis [ 14 , 15 ] . in cancer , these electromagnetic fields are aberrant , which can be normalized by exposure of these cancer cells to spmf therapy . it is a well - known fact that the cell membrane potential of cancer cells is about 15 mv to 30 mv . exposure of cancer cells to spmf will normalize this potential , thereby halting the process of cell proliferation . a cascade of effects follows normalization of cell membrane potential , such as an increased influx of calcium , potassium and magnesium ions and efflux of sodium and water out of cells , and reduction in intracellular acidity [ 16 , 17 ] . the frequencies and field strength used in spmf therapy are well within the specified norms of the international commission for non - ionizing radiation protection . the previous studies conducted at the iam have shown that spmf therapy is a safe treatment modality [ 6 , 7 ] . the efficacy of spmf therapy in this patient with a rapidly progressing anaplastic astrocytoma demonstrates the enormous potential of this new treatment modality . considering the poor prognosis of anaplastic astrocytoma grade iii with standard modalities of treatment , spmf holds promise to improve overall survival and quality of life non - invasively .

backgroundanaplastic astrocytomas are progressive brain tumors with a tendency to infiltrate the surrounding tissue . recurrence is very common , with recurrent tumors being extremely refractory to existing therapies . case presentation : a 33-year - old woman presented with a history of an unprovoked fall , followed by seizures . an mri scan revealed a mass in the fronto - temporo - parietal region of the brain , suggesting a primary tumor . she underwent craniotomy and surgical debulking of the tumor . the histology of the tumor tissue revealed an anaplastic astrocytoma . follow - up mri scans indicated the presence of a residual , rapidly progressing tumor . a 6-week course of fractionated radiation and concurrent chemotherapy with temodar ( temozolomide capsules ) did not stop tumor progression . intervention : due to the failure of conventional therapies in preventing rapid disease progression , the patient volunteered to undergo a 28-day course of sequentially programmed magnetic field ( spmf ) therapy.resultsimmediate post - therapy mri scan showed a cessation of tumor growth , and follow - up imaging at 6 , 12 , 24 and 36 months revealed a gradual but steady decrease in the size of the tumor . the patient reported an alleviation of clinical symptoms and a subjective improvement in the quality of life at 6 , 12 , 24 and 36 months following spmf therapy.conclusionthe remarkable improvement of this patient suggests that spmf therapy may be a valuable option for anaplastic astrocytoma , especially in recurrent and rapidly progressing tumors .

Background Results Conclusion Introduction Case Presentation Discussion Conclusion

angiodysplasia is the second most common cause of gi bleeding in elderly patients with end - stage renal disease ( esrd ) . these lesions respectively account for about 20 and 30% of upper and lower gi bleeding and approximately one - half of recurrent upper gi bleeding . a double - balloon endoscope ( dbe ) is a newly developed device that can be inserted more deeply into the small intestine via either an oral or anal approach . it can support endoscopic observation and treatment for the entire small intestine while obviating general anaesthesia . after both endoscope itself and overtube reach the duodenum , the balloon fitted to the tip of overtube is firstly inflated to fix the tube to the intestine . then the endoscope is inserted further through the overtube in place . when the endoscope is inserted as far as possible thereafter , the balloon of the overtube is deflated , and the overtube can reach the tip of the endoscope with the use of the endoscope itself like a guidewire . when the distal end of the overtube reaches the end of the endoscope , the balloon on the overtube is inflated to fix a distal side to the intestine . the above - described sequence is repeated until the endoscope arrives at the target portion of intestine . we present herein those cases as well as several considerations about newly developed techniques that enable us to observe the small intestine . the patient was a 70-year - old male who started to receive haemodialysis because of diabetic nephropathy in 1992 . although he had undergone both upper and lower gastroenteral endoscopy repeatedly , the bleeding point was not determined . scintigraphy performed to detect gi haemorrhage revealed an accumulation of activity on the right side of the common iliac artery , which suggested small intestinal bleeding . moreover , his blood type of ab and d negative was difficult to match for blood transfusion . he was referred to our hospital in june 2006 for more extensive examination and treatment . he underwent dbe , which showed red spots at the middle portion of the jejunum . we treated this lesion using argon plasma coagulation ( apc ) and clipping ( figure 1-a , b ) . thereafter , the same type of lesion appeared four times in different areas of the intestine . however , we treated these lesions successfully using the same method ( figure 1-c ) . the patient was a 58-year - old male who started to receive haemodialysis for a condition of unknown aetiology in 2006 . he had liver cirrhosis and received radiofrequency abrasion therapy for treatment of hepatocellular carcinoma . after he developed a sudden decrease in hb as well as haematochezia , scintigraphy performed to detect gi haemorrhage revealed an accumulation of activity on the upper jejunum . he underwent capsule endoscopy ( ce ) , which showed diffuse antral vascular ectasia ( dave ) and angiodysplasia in the small intestine ( figure 1-d ) . the patient was a 74-year - old male who started to receive haemodialysis for a condition of unknown aetiology in 1989 . he was referred to our hospital in april 2008 for more extensive examination and treatment . he underwent ce , which showed angiodysplasia at the upper side of the jejunum ( figure 1-e ) . immediately after the ce procedure he developed an inflammatory reaction of unknown aetiology . we postponed the scheduled dbe until his general status was resolved . during the follow - up , his haematochezia stopped . we continued to monitor the patient closely without therapy because the active bleeding had ceased . the patient was a 59-year - old male who started to receive haemodialysis because of diabetic nephropathy in 2005 . he had liver cirrhosis caused by hepatitis c virus infection , which was treated with interferon . he received upper and lower endoscopy at an outpatient clinic , but the bleeding point was not detected . he was referred to our hospital in may 2008 for more extensive examination and treatment . although vascular ectasia was detected at the upper jejunum using ce , we were unable to identify the source of the active bleeding point . although he underwent dbe , we were unable to identify the source of bleeding ( figure 1-f ) . during the follow - up , although his hb had been as low as 7.1 g / dl at the outpatient clinic , his haemoglobin concentration recovered to 9.3 g / dl at admission . the patient was a 70-year - old male who started to receive haemodialysis because of diabetic nephropathy in 1992 . although he had undergone both upper and lower gastroenteral endoscopy repeatedly , the bleeding point was not determined . scintigraphy performed to detect gi haemorrhage revealed an accumulation of activity on the right side of the common iliac artery , which suggested small intestinal bleeding . moreover , his blood type of ab and d negative was difficult to match for blood transfusion . he was referred to our hospital in june 2006 for more extensive examination and treatment . he underwent dbe , which showed red spots at the middle portion of the jejunum . we treated this lesion using argon plasma coagulation ( apc ) and clipping ( figure 1-a , b ) . thereafter , the same type of lesion appeared four times in different areas of the intestine . however , we treated these lesions successfully using the same method ( figure 1-c ) . the patient was a 58-year - old male who started to receive haemodialysis for a condition of unknown aetiology in 2006 . he had liver cirrhosis and received radiofrequency abrasion therapy for treatment of hepatocellular carcinoma . after he developed a sudden decrease in hb as well as haematochezia , scintigraphy performed to detect gi haemorrhage revealed an accumulation of activity on the upper jejunum . he underwent capsule endoscopy ( ce ) , which showed diffuse antral vascular ectasia ( dave ) and angiodysplasia in the small intestine ( figure 1-d ) . the patient was a 74-year - old male who started to receive haemodialysis for a condition of unknown aetiology in 1989 . he was referred to our hospital in april 2008 for more extensive examination and treatment . he underwent ce , which showed angiodysplasia at the upper side of the jejunum ( figure 1-e ) . immediately after the ce procedure he developed an inflammatory reaction of unknown aetiology . we postponed the scheduled dbe until his general status was resolved . during the follow - up , his haematochezia stopped . we continued to monitor the patient closely without therapy because the active bleeding had ceased . the patient was a 59-year - old male who started to receive haemodialysis because of diabetic nephropathy in 2005 . he had liver cirrhosis caused by hepatitis c virus infection , which was treated with interferon . he received upper and lower endoscopy at an outpatient clinic , but the bleeding point was not detected . he was referred to our hospital in may 2008 for more extensive examination and treatment . although vascular ectasia was detected at the upper jejunum using ce , we were unable to identify the source of the active bleeding point . although he underwent dbe , we were unable to identify the source of bleeding ( figure 1-f ) . during the follow - up , although his hb had been as low as 7.1 g / dl at the outpatient clinic , his haemoglobin concentration recovered to 9.3 g / dl at admission . both platelet dysfunction and quantitative abnormality of the von willebrand factor have been considered as important predisposing factors for haemorrhagic diathesis in such patients . zuckerman et al . reported a 39% occurrence of clotting abnormalities in patients with chronic renal failure . acute gi haemorrhage in patients with chronic renal failure is a severe complication causing higher mortality and morbidity rates than in cases without chronic renal disease . recurrent bleeding is more frequent in patients with chronic renal failure than in those without chronic renal failure . consequently , early recognition of the exact bleeding site and prompt treatment are important for management of such patients . haemorrhage at the small intestine is less common than bleeding in the upper gi tract or colon , but its underlying cause is more difficult to diagnose . regarding haemorrhage of the small intestine , assessment of the exact localization and the cause of bleeding have been difficult both clinically and endoscopically . dbe , a newly developed endoscopic visualization technique of the small intestine , was developed by yamamoto et al . in 2001 . a typical dbe comprises a 200 cm long endoscope , a 145 cm long semi - elastic external tube and two latex balloons to fix the endoscope inside the small intestine better and enable easier injection . the endoscope can be inserted via either an oral or anal route . the dbe method is applied to cases with pathological conditions of the small intestine , to identify a bleeding source , or to detect obstructive and neoplastic changes in the small intestine . ce is a new diagnostic method that enables non - invasive assessment of lesions of the small intestine mucosa . the capsule contains a miniature digital video camera , which can take pictures with very little light , and a data transmission apparatus . after the capsule is swallowed by the patient , it passively moves along the alimentary tract through its peristaltic movements . ce is applied to cases with chronic bleeding from the alimentary tract and chronic iron - deficiency anaemia , conditions that suggest the presence of a tumour in the small intestine . ce , however , is contraindicated in patients with cardiac pacemakers because the method uses radiofrequency waves to transmit data through the abdominal wall . we first adopted ce for chronic anaemia in case 3 because this method is more non - invasive than dbe . we performed dbe because of deteriorative effects on a pacemaker in case 1 , and because of active bleeding in case 2 . newly available imaging techniques can replace prior approaches to small bowel pathology . because of its ability to take biopsies and to carry out therapeutic interventions , dbe is the most efficacious among newly developed techniques such as ce and radiological diagnosis with respect to minimizing laparotomy and intraoperative enteroscopy . those features bring great benefit to esrd patients because the patients often experience repeated bleeding episodes within the small intestine . additionally , this new system is useful as a therapeutic tool for performing non - surgical haemostasis for bleeding in the small intestine . moreover , the combination of ce and dbe is a promising elective approach for esrd patients with intestinal haemorrhage . case 1 : a bleeding point could be observed by double - balloon endoscopy ( a ) . argon plasma coagulation technique was applied to the lesion ( b ) . clipping under observation by double - balloon endoscopy was also added to the same lesion ( c ) . ( d ) case 2 : diffuse antral vascular ectasia ( dave ) lesion was observed with use of conventional upper gastric fibrescopy . ( f ) case 4 : intestinal erosion was observed by double - balloon endoscopy .

gastrointestinal ( gi ) bleeding is a common and troublesome complication of end - stage renal disease ( esrd ) . patients often have various lesions in the small bowel and in either upper or lower gi tracts . recently developed double - balloon endoscopy ( dbe ) enables observation of the entire small intestine through a combination of anterograde and retrograde approaches . moreover , dbe is useful not only for diagnosis of small intestinal lesions ; it provides a mode of treating the disease . this article presents patients with several small intestinal diseases from our facility . their bleeding sources were identified using dbe . we also report two representative cases of angiodysplasia that had been diagnosed and treated successfully using dbe . one case particularly underscored the usefulness of the combination of capsule endoscopy ( ce ) and dbe as an electively diagnostic approach for patients with gi bleeding . small intestinal bleeding is often observable repeatedly in a single patient , as described for case 1 . in such circumstances , dbe can treat the lesions successfully without surgical procedures . in this report , esrd patients , in whom comorbid conditions made it difficult to perform surgical procedures , receive great benefit from dbe .

Background Case reports Case 1 (A.T.) Case 2 (S.M.) Case 3 (M.K.) Case 4 (I.Y.) Discussion Conclusion

avaliar a prevalncia de sintomas respiratrios como motivo para procura de atendimento de emergncia por pacientes adultos e peditricos , descrevendo as principais sndromes clnicas diagnosticadas e o desfecho dos pacientes . entre novembro de 2008 e novembro de 2009 , o nmero total de atendimentos foi revisado diariamente . durante o perodo do estudo , houve 37.059 admisses na emergncia , das quais 11.953 ( 32,3% ) foram motivadas por sintomas respiratrios . a prevalncia de atendimentos por sintomas respiratrios foi 28,7% e 38,9% nos adultos e crianas , respectivamente . as taxas de internao hospitalar e de mortalidade nos adultos foram 21,2% e 2,7% , respectivamente , comparadas com 11,9% e 0,3% , respectivamente , nas crianas . nos adultos , quanto maior o tempo entre o incio dos sintomas e a visita emergncia , maiores foram a necessidade de hospitalizao ( p < 0,0001 ) , o tempo de hospitalizao ( p < 0,0001 ) e a mortalidade ( p = 0,028 ) . encontramos uma prevalncia elevada de atendimentos por sintomas respiratrios entre os pacientes adultos e peditricos . futuros estudos epidemiolgicos podero colaborar para a melhor elucidao dos fatores de risco para a presena de sintomas respiratrios nesses pacientes . the prevalence of respiratory symptoms in a given population is an indirect indicator of acute and chronic respiratory diseases , an indicator that is quite reliable from an epidemiological standpoint . respiratory diseases are a leading cause of death among adults and children , accounting for 12% of all deaths worldwide . in low- and middle - income countries , upper respiratory tract infections ( urtis ) in addition to exerting enormous pressure on health services , respiratory diseases are common causes of school and work absenteeism . in brazil , chronic and acute respiratory diseases collectively rank second among the leading causes of hospitalization within the brazilian unified health care system , accounting for approximately 14% of all hospitalizations within the system . in southern brazil , respiratory diseases are the leading cause of hospitalization ( accounting for 17% of all hospital admissions ) . respiratory complaints are the motive for a large proportion of outpatient visits , emergency room visits , and hospitalizations . a study conducted in a developing country showed that 10% of all emergency room visits were due to respiratory diseases . in a two - year study conducted in the city of gama , brazil , 641,905 emergency room visits were evaluated , 37,642 ( 5.8% ) of which were found to be due to respiratory complaints . studies on the pattern of emergency room visits by individuals with respiratory symptoms are important , because they could inform decisions regarding the organization and planning of health services in order to meet this demand . the objective of the present study was to evaluate the prevalence of respiratory symptoms as the motive for emergency room visits by adult and pediatric patients at a tertiary care hospital , describing the major clinical syndromes diagnosed and the patient outcomes . this was a cross - sectional study conducted in the emergency room of the hospital de clnicas de porto alegre ( hcpa ) , located in the city of porto alegre , southern brazil . the hcpa is a general tertiary care university hospital with 750 beds and approximately 30,000 admissions per year . the city of porto alegre has 1,409,939 inhabitants , and the surrounding metropolitan area includes 31 cities , the total population of the greater metropolitan area being 3,979,561 . the research ethics committee of the hcpa granted the researchers access to patient medical records . children and adults who presented to the emergency room with at least one respiratory symptom were included in the study . during the study period ( between november of 2008 and november of 2009 ) , we reviewed the total number of visits per day to the pediatric and adult sections of the emergency room of the hcpa . the electronic medical records were reviewed , and the characteristics of the patients treated for respiratory symptoms that were recorded included gender , age , race , smoking history , comorbidities , respiratory symptoms , and familial history of flu symptoms . the respiratory symptoms considered were rhinorrhea , nasal congestion , odynophagia , dysphonia , cough , breathlessness , wheezing , chest pain , and fever . clinical findings on admission ( axillary temperature , hr , rr , pulmonary auscultation findings , and spo2-measured by digital pulse oximetry ) were also recorded , as were the major clinical syndromes diagnosed and the patient outcomes ( icu admission , ward admission , discharge from the emergency room , and death ) . data were entered into a microsoft excel 2007 database , after which they were processed and analyzed with the statistical package for the social sciences , version 18.0 ( spss inc . , we performed a descriptive analysis of the overall prevalence of visits by patients with respiratory symptoms to the pediatric and adult sectors of the emergency room ( for patients < 14 and 14 years of age , respectively ) . we stratified the time from symptom onset to emergency room visit ( as < 7 days , 7 - 21 days , and > 21 days ) , as well as evaluating the major respiratory symptoms , the major clinical diagnoses , the length of the emergency room visit , the rate of admission to the hospital , the rate of icu admission , the rate of discharge from the emergency room , and the mortality rate . during the 12-month study period , there were 37,059 emergency room visits ( 24,189 by adults and 12,870 by children ) , of which 11,953 ( 32.3% ) were motivated by respiratory symptoms . the monthly variation in the number of emergency room visits due to respiratory symptoms is shown in figures 1 and 2 . there was an increase in the number of such visits in april , may , and june . the prevalence of visits due to respiratory symptoms was 28.7% ( 6,942/24,189 ) among adult patients and 38.9% ( 5,011/12,870 ) among pediatric patients . the most common symptoms were cough ( in 73.4% ) , fever ( in 56.1% ) , breathlessness ( in 40.9% ) , chest pain ( in 24.5% ) , and rhinorrhea ( 20.9% ) . the median duration of symptoms prior to admission was 3 days ( interquartile range : 1 - 6 days ) . a total of 2,205 patients ( 18.5% ) admitted to the emergency room required hospitalization . the demographic and clinical characteristics of the study population ( adults and children ) are shown in table 1 . figure 1monthly variation in the number of visits to the adult sector of the hospital de clnicas de porto alegre emergency room ( for patients 14 years of age ) motivated by respiratory symptoms , between november of 2008 and november of 2009 . figure 2monthly variation in the number of visits to the pediatric sector of the hospital de clnicas de porto alegre emergency room ( for patients < 14 years of age ) motivated by respiratory symptoms , between november of 2008 and november of 2009 . characteristicadultschildren(n = 6,942)(n = 5,011 ) age , years 50.0 ( 30.0 - 55.0)2.0 ( 0.8 - 5.0 ) male gender3,125 ( 45.0)2,795 ( 55.8 ) white5,941 ( 85.6)4,007 ( 80.0 ) > 8 years of schooling3,129 ( 51.2)- place of residence porto alegre4,815 ( 69.4)3,635 ( 72.5 ) greater metropolitan area of porto alegre1,851 ( 26.7)1,247 ( 24.9 ) interior of the state of rio grande do sul276 ( 3.9)129 ( 2.6 ) symptom upon arrival at the emergency room cough4,553 ( 65.6)4,214 ( 84.1 ) breathlessness3,521 ( 50.7)1,363 ( 27.2 ) chest pain2,697 ( 38.9)229 ( 4.6 ) rhinorrhea676 ( 9.7)1.821 ( 36.3 ) nasal congestion263 ( 3.8)673 ( 13.4 ) odynophagia897 ( 12.9)472 ( 9.4 ) dysphonia102 ( 1.5)60 ( 1.2 ) wheezing264 ( 3.8)760 ( 15.2 ) fever2,986 ( 43.0)3,718 ( 74.2 ) hemoptysis186 ( 2.7)4 ( 0.1 ) duration of symptoms prior to arrival at the emergency room , days3.0 ( 1.0 - 7.0 ) 2.0 ( 1.0 - 4.0 ) comorbidity systemic arterial hypertension1,730 ( 24.9)0 asthma603 ( 8.7)777 ( 15.5 ) copd730 ( 10.5)0 ischemic heart disease567 ( 8.2)0 heart failure554 ( 8.0)0 diabetes mellitus900 ( 13.0)6 ( 0.1 ) hiv infection400 ( 5.8)32 ( 0.6 ) cancer632 ( 9.1)48 ( 1.0 ) chronic renal failure231 ( 3.3)0 smoking status nonsmoker143 ( 2.1)0 former smoker712 ( 10.3)0 current smoker611 ( 8.8)0avalues expressed as n ( % ) , except where otherwise indicated . bvalue expressed as mean sdcvalues expressed as median ( interquartile range ) values expressed as n ( % ) , except where otherwise indicated . value expressed as mean sd values expressed as median ( interquartile range ) table 2 shows the characteristics of the adult patients and the analysis of these characteristics on the basis of the time from symptom onset to emergency room visit . because data on the time from symptom onset to emergency room visit were not available for all patients , the analysis included only 4,434 patients . of those 4,434 patients , 939 ( 21.2% ) were admitted to the hospital and 120 ( 2.7% ) died . the median ( interquartile range ) length of emergency room visit and the median ( interquartile range ) total length of hospital stay were 0.5 day ( 0.5 - 1.0 day ) and 0.5 day ( 0.5 - 3.0 days ) , respectively . the patients who had a 6-day or shorter history of respiratory symptoms ( < 7 group ) were younger than those who had a 7- to 21-day history ( 7 - 21 group ) and those who had a 21-day or longer history ( > 21 group ; 44.9 20.4 years vs. 49.4 19.7 years and 49.9 17.6 years ; p < 0.0001 ) . a longer time from symptom onset to emergency room visit correlated with a lower level of education of the patients ( p < 0.0001 ) . when the proportion of patients who were residents of the city of porto alegre was higher , the time from symptom onset to emergency room visit was shorter ( p = 0.012 ) . rhinorrhea , odynophagia , and fever were most common in the patients in the < 7 group , whereas cough and breathlessness were most common in those in the 7 - 21 group . the number of patients with asthma was highest in the < 7 group ( p = 0.002 ) , and the number of patients with hiv infection was highest in the > 21 group ( p < 0.0001 ) . among the patients who underwent chest x - ray , normal results were more common in those in the < 7 group ( p < 0.0001 ) , and a consolidation pattern was more common in those in the 7 - 21 group ( p = 0.002 ) . pleural effusion and diffuse infiltrates were most common in the > 21 group ( p = 0.008 and p = 0.003 , respectively ) . the most common diagnoses in the < 7 group were influenza ( in 18.2% ) , pneumonia ( in 12.6% ) , and urti ( in 12.2% ) . in the 7 - 21 group , the most common diagnoses were pneumonia ( in 17.8% ) , urti ( in 10.7% ) , and copd exacerbation ( in 9.6% ) ; whereas the most common diagnoses in the > 21 group were pneumonia ( in 9.1% ) , copd exacerbation ( in 5.2% ) , and sinusitis ( in 4.3% ) . the length of emergency room visit was shortest among the patients in the < 7 group ( p < 0.0001 ) . a longer time from symptom onset to emergency room visit correlated with a greater need for hospitalization ( p < 0.0001 ) , longer hospital stays ( p < 0.0001 ) , and higher mortality ( p = 0.028 ) . table 2characteristics of the adult patients and analysis based on the time from onset of respiratory symptoms to emergency room visit . characteristictotaltime from symptom onset to emergency room visitp < 7 days7 - 21 days > 21 days(n = 4,434)(n = 3,024)(n = 1,179)(n = 230 ) age , years 46.4 20.244.9 20.4 * * * 49.4 19.7 * 49.9 17.6 * * < 0.0001 male gender1,961 ( 44.2)1,355 ( 44.8)500 ( 42.4)106 ( 46.1)0.317 white3,791 ( 85.5)2,600 ( 86.0)598 ( 84.6)193 ( 83.9)0.438 > 8 years of schooling2,103 ( 53.5)1,506 ( 55.8 ) * * * 498 ( 48.4 ) * * * * 99 ( 48.1 ) * * * * * < 0.0001 place of residence porto alegre3,110 ( 70.1)2,162 ( 71.5 ) * * * 799 ( 67.8 ) * * * 149 ( 64.8 ) * * * * * 0.012 greater metropolitan area of porto alegre1,174 ( 26.5)777 ( 25.7)329 ( 27.9)68 ( 29.6)0.189 interior of the state of rio grande do sul150 ( 3.4)86 ( 2.8 ) * * * 51 ( 4.3 ) * * * 13 ( 5.7 ) * * * * * 0.009 symptom upon arrival at the emergency room cough3,132 ( 70.6)2,069 ( 68.4 ) * * * 906 ( 76.8 ) * * * * 157 ( 68.3 ) * * * * * < 0.0001 expectoration1,946 ( 28.0)795 ( 26.3 ) * 445 ( 37.7 ) * * * * 60 ( 26.1 ) * * * < 0.0001 breathlessness2,071 ( 46.7)1,335 ( 44,1 ) * * * 621 ( 52.7 ) * * * * 115 ( 50.0 ) * * * * * < 0 .. 001 chest pain1,841 ( 41.5)1,249 ( 41.3)495 ( 42.0)97 ( 42.2)0.900 rhinorrhea538 ( 12.1)422 ( 14.0 ) * * * 102 ( 8.7 ) * * * * 14 ( 6.1 ) * * * * * < 0.0001 nasal congestion203 ( 4.6)148 ( 4.9)49 ( 4.2)6 ( 2.6)0.201 odynophagia683 ( 15.4)547 ( 18.1 ) * * * 124 ( 10.5 ) * * * * 12 ( 5.2 ) * * * * * < 0.0001 dysphonia75 ( 1.7)49 ( 1.6)25 ( 2.1)1 ( 0.4)0.167 wheezing166 ( 3.7)113 ( 3.7)47 ( 4.0)6 ( 2.6)0.602 fever2,240 ( 50.5)1,645 ( 54.4 ) * * * 518 ( 43.9 ) * * * * 77 ( 33.5 ) * * * * * < 0.0001 hemoptysis118 ( 2.7)75 ( 2.5)35 ( 3.0)8 ( 3.5)0.494 comorbidity systemic arterial hypertension1,071 ( 24.2)698 ( 23.1 ) * * * 316 ( 26.8 ) * * * * 57 ( 24.8 ) * * * * * 0.039 asthma389 ( 8.8)291 ( 9.6 ) * * * 90 ( 7.6 ) * * * * 8 ( 3.5 ) * * * * * 0.002 copd381 ( 8.6)249 ( 8.2 ) * * * 122 ( 10.3 ) * * * * 10 ( 4.3 ) * * * * * 0.006 ischemic heart disease357 ( 8.1)261 ( 8.6)83 ( 7.0)13 ( 5.7)0.092 heart failure310 ( 7.0)212 ( 7.0)90 ( 7.6)8 ( 3.5)0.078 diabetes mellitus542 ( 12.2)376 ( 12.4)136 ( 11.5)30 ( 13.0)0.675 hiv infection278 ( 6.3)150 ( 5.0 ) * * * 95 ( 8.1 ) * * * * 33 ( 14.3 ) * * * * * < 0.0001 cancer302 ( 6.8)202 ( 6.7)88 ( 7.5)12 ( 5.2)0.407 chronic renal failure133 ( 3.0)95 ( 3.1)32 ( 2.7)6 ( 2.6)0.720 smoking status nonsmoker107 ( 45.0)64 ( 43.5)32 ( 49.2)11 ( 42.3)0.714 former smoker452 ( 81.7)299 ( 88.5)124 ( 87.9)29 ( 76.3)0.112 current smoker405 ( 87.4)248 ( 81.0)128 ( 85.3)29 ( 72.5)0.160 radiological pattern normal881 ( 38.7)593 ( 41.8 ) * * * 241 ( 34.2 ) * * * * 47 ( 30.9 ) * * * * * < 0.0001 consolidation498 ( 21.9)298 ( 21.0 ) * * * 180 ( 25.5 ) * * * * 20 ( 13.2 ) * * * * * 0.002 diffuse infiltrate114 ( 5.0)57 ( 4.0 ) * * * 42 ( 6.0 ) * * * * 15 ( 9.9 ) * * * * * 0.003 pleural effusion176 ( 7.7)91 ( 6.4 ) * * * 68 ( 9.7 ) * * * * 17 ( 11.2 ) * * * * * 0.008 primary diagnosis urti503 ( 11.3)368 ( 12.2 ) * * * 126 ( 10.7 ) * * * * 9 ( 3.9 ) * * * * * 0.001 pneumonia612 ( 13.8)381 ( 12.6 ) * * * 210 ( 17.8 ) * * * * 21 ( 9.1 ) * * * * * < 0.0001 influenza614 ( 13.8)550 ( 18.2 ) * * * 64 ( 5.4 ) * * * * 0 ( 0 ) * * * * * < 0.0001 asthma204 ( 4.6)156 ( 5.2 ) * * * 43 ( 3.6 ) * 5 ( 2.2)**0.022 copd331 ( 7.5)206 ( 6.8 ) * * * 113 ( 9.6 ) * * * * 12 ( 5.2 ) * * * * * 0.004 sinusitis227 ( 5.1)134 ( 4.4 ) * * * 83 ( 7.0 ) * * * * 10 ( 4.3 ) * * * * * 0.002 pulmonary tuberculosis42 ( 0.9)13 ( 0.4 ) * * * 13 ( 1.1 ) * 16 ( 7.0 ) * * < 0.0001 length of emergency room visit , days 0.5 ( 0.5 - 1)0.5 ( 0.5 - 1 ) * * * 0.5 ( 0.5 - 2 ) * 0.5 ( 0.5 - 2 ) * * < 0.0001 need for hospitalization939 ( 21.2)560 ( 18.5 ) * * * 303 ( 25.7 ) * * * * 76 ( 33.0 ) * * * * * < 0.0001 need for icu admission119 ( 2.7)70 ( 2.3)39 ( 3.3)10 ( 4.3)0.056 length of hospital stay , days 0.5 ( 0.5 - 3.0)7 ( 3 - 14 ) * * * 10 ( 6 - 19 ) * * * * 14 ( 6 - 24 ) * * * * * < 0.0001 death120 ( 2.7)70 ( 2.3 ) * * * 39 ( 3.3 ) * * * * 11 ( 4.8 ) * * * * * 0.028urti : upper respiratory tract infectionavalues expressed as n ( % ) , except where otherwise indicated.bvalue expressed as mean sdcvalues expressed as median ( interquartile range).*comparison between the < 7 days and 7 - 21 days.**comparison between the < 7 days and > 21 days.***comparison between the 7 - 21 days and > 21 days . : upper respiratory tract infection values expressed as n ( % ) , except where otherwise indicated . table 3 shows the characteristics of the pediatric patients and the analysis of those characteristics on the basis of the time from symptom onset to emergency room visit . because , as was true for adults , data on the time from symptom onset to emergency room visit were not available for the entire group of pediatric patients , the analysis included only 3,860 patients . of those 3,860 patients , 458 ( 11.9% ) were admitted to the hospital and 12 ( 0.3% ) died . the median length of the emergency room visit and median length of hospital stay were equal : 0.5 days ( range , 0.5 - 0.5 days ) . the pediatric patients in the > 21 group were younger than were those in the < 7 and 7 - 21 groups ( p = 0.023 ) . fever and odynophagia were most common in the patients in the < 7 group ( p < 0.0001 and p = 0.008 , respectively ) . in all three of those groups , the most common diagnoses were influenza ( p < 0.0001 ) , pneumonia ( p = 0.014 ) , and sinusitis ( p = 0.002 ) . among the pediatric patients , the type of symptom(s ) presented did not correlate significantly with the length of emergency room visit , length of hospital stay , or mortality . table 3characteristics of the pediatric patients and analysis based on the time from onset of respiratory symptoms to emergency room visit.characteristictotaltime from symptom onset to emergency room visitp < 7 days7 - 21 days > 21 days(n = 3,860)(n = 3,242)(n = 574)(n = 44 ) age , years 2.0 ( 0.8 - 5.0)2.0 ( 0.8 - 5.0 ) * * 1.0 ( 0.7 - 5.0 ) * * * 0.9 ( 0.5 - 3.0 ) * * * * * 0.023 male gender2,116 ( 54.8)1,771 ( 54.6)322 ( 56.1)23 ( 52.3)0.762 white3,085 ( 79.9)2,602 ( 80.3)447 ( 77.9)36 ( 81.8)0.401 place of residence porto alegre2,806 ( 72.7)2,352 ( 72.5)420 ( 73.2)34 ( 77.3)0.754 greater metropolitan area of porto alegre963 ( 24.9)815 ( 25.1)138 ( 24.0)10 ( 22.7)0.806 interior of the state of rio grande do sul91 ( 2.4)75 ( 2.3)16 ( 2.8)0 ( 0)0.461 symptom upon arrival at the emergency room cough3,311 ( 85.8)2,765 ( 85.3)507 ( 88.3)39 ( 88.6)0.136 breathlessness988 ( 25.6)837 ( 25.8)140 ( 24.4)11 ( 25.0)0.767 chest pain181 ( 4.7)151 ( 4.7)30 ( 5.2)0 ( 0)0.280 rhinorrhea1,459 ( 37.8)1,213 ( 37.4)233 ( 40.6)13 ( 29.5)0.184 nasal congestion544 ( 14.1)442 ( 13.6)95 ( 16.6)7 ( 15.9)0.170 odynophagia374 ( 9.7)334 ( 10.3 ) * * * 39 ( 6.8 ) * * * * 1 ( 2.3 ) * * * * * 0.008 dysphonia47 ( 1.2)37 ( 1.1)9 ( 1.6)1 ( 2.3)0.563 wheezing575 ( 14.9)488 ( 15.1)77 ( 13.4)10 ( 22.7)0.203 fever2,985 ( 77,3)2,554 ( 78.8 ) * * * 406 ( 70.7 ) * * * * 25 ( 56.8 ) * * * * * < 0.0001 comorbidity asthma587 ( 15.2)506 ( 15.6)74 ( 12.9)7 ( 15.9)0.246 hiv infection25 ( 0.6)21 ( 0.6)4 ( 0.7)0 ( 0)0.857 cancer35 ( 0.9)30 ( 0.9)5 ( 0.9)0 ( 0)0.809 history of bronchiolitis 117 ( 3.0)98 ( 3.0)18 ( 3.1)1 ( 2.3)0.947 radiological pattern normal651 ( 47.7)539 ( 48.8)98 ( 43.8)14 ( 60.9)0.210 consolidation300 ( 22.0)232 ( 20.8 ) * * * 64 ( 28.4 ) * * * * 4 ( 17.4 ) * * * * * 0.035 diffuse infiltrate137 ( 10.0)115 ( 10.3)19 ( 8.4)3 ( 13.0)0.623 primary diagnosis urti980 ( 25.4)816 ( 25.2)155 ( 27.0)9 ( 20.5)0.487 pneumonia381 ( 9.9)301 ( 9.3 ) * * * 76 ( 13.2 ) * * * * 4 ( 9.1 ) * * * * * 0.014 influenza356 ( 9.2)326 ( 10.1 ) * * * 28 ( 4.9 ) * * * * 2 ( 4.5 ) * * * * * < 0.0001 asthma486 ( 12.6)426 ( 13.1)56 ( 9.8)4 ( 9.1)0.062 bronchiolitis366 ( 9.5)303 ( 9.3)57 ( 9.9)6 ( 13.6)0.580 sinusitis63 ( 1.6)43 ( 1.3 ) * * * 18 ( 3.1 ) * * * * 2 ( 4.5 ) * * * * * 0.002 length of emergency room visit , day 0.5 ( 0.5 - 0.5)0.5 ( 0.5 - 0.5)0.5 ( 0.5 - 0.5)0.5 ( 0.5 - 0.5)0.182 need for hospitalization458 ( 11.9)376 ( 11.6)73 ( 12.1)9 ( 20.5)0.156 need for icu admission27 ( 0.7)22 ( 0.7)5 ( 0.9)0 ( 0)0.751 length of hospital stay , days 0.5 ( 0.5 - 0.5)6 ( 3 - 11)5 ( 3 - 10)11 ( 4.5 - 20.5)0.179 mortality rate12 ( 0.3)9 ( 0.3)3 ( 0.5)0 ( 0)0.582urti : upper respiratory tract infection.avalues expressed as n ( % ) , except where otherwise indicated . bvalue expressed as mean sd.cvalues expressed as median ( interquartile range).*comparison between the < 7 days and 7 - 21 days * * comparison between the < 7 days and > 21 days***comparison between the 7 - 21 days and > 21 days . : comparison between the < 7 days and 7 - 21 days comparison between the < 7 days and > 21 days comparison between the 7 - 21 days and > 21 days . in this cross - sectional study , we have described the characteristics of adult and pediatric patients presenting to the emergency room of a tertiary care hospital with respiratory symptoms . in one year , the prevalence of emergency room visits due to respiratory symptoms was 28.7% and 38.9% among adult and pediatric patients , respectively . in adults , the rates of hospitalization and mortality were 21.2% and 2.7% , respectively , compared with 11.9% and 0.3% , respectively , in children . in a study conducted at various emergency rooms in a city in italy , only 5% of all visits by adults were motivated by respiratory symptoms . the same percentage was found by another group of authors , who also reported that 47% of patients presenting to primary care did so because of respiratory symptoms . at 77 basic health care facilities in algeria , the presence of respiratory symptoms was the motive for seeking medical attention in up to 31.6% of the cases . in our sample , the number of patients who sought treatment because of respiratory symptoms might have been overestimated because of the influenza a ( h1n1 ) pandemic , which occurred during the study period . this also explains the high prevalence of diagnoses of influenza and urti in patients treated at the emergency room of the tertiary care hospital . likewise , upper respiratory tract symptoms , such as rhinorrhea and odynophagia , as well as fever , were more common in the adult patients presenting to the emergency room with a 6-day history of respiratory symptoms , perhaps out of concern regarding the possibility of having a ( h1n1 ) influenza . the adult patients who had a 6-day history of respiratory symptoms were younger than were the other patients . it is known that elderly patients with pneumonia have lower body temperature and that it can take them longer to seek medical attention , increasing the likelihood of delayed diagnosis and delayed treatment . in fact , the higher number of patients diagnosed with pneumonia in the 7 - 21 and > 21 groups corroborates this finding . a higher level of education was found in the group of adult patients who presented to the emergency room earlier ( < 7 group ) . studies have demonstrated that individuals with a higher level of education have a healthier behavior , have more information about their health , and clearly understand the instructions provided at discharge from the emergency room . in general , those with a higher level of education tend to make better health - related decisions . therefore , considering the context of the influenza a ( h1n1 ) epidemic , it is possible that these patients sought medical attention earlier for fear of this pathology . in the adult patients , we found that a longer time from symptom onset to emergency room visit correlated with a greater need for hospitalization , longer hospital stays , and higher mortality . respiratory symptoms are associated with various diseases and are considered significant predictors of mortality . among respiratory symptoms , in fact , in our study , we found that breathlessness was more common in the 7 - 21 and > 21 groups than in the < 7 group , probably indicating greater severity and chronicity of symptoms in the first two groups . we included only patients presenting to the emergency room , and those probably represented cases that were more severe than those treated in primary care . in addition , the diagnoses were syndromic ( i.e. , the etiologic agent was not identified ) . however , despite these limitations , the study achieved its objective of showing the characteristics of patients with respiratory symptoms and is relevant for the evaluation of these patients in the context of emergency care . in conclusion , we have described the epidemiological profile of adult and pediatric patients presenting to an emergency room with respiratory symptoms . we found a high prevalence of respiratory symptoms as the motive for emergency room visits by adult and pediatric patients ( in 28.7% and 38.0% , respectively ) . in addition , we found that a longer time from symptom onset to emergency room visit correlated with a greater need for hospitalization , longer hospital stays , and higher mortality among adults . further longitudinal epidemiological studies are needed in order to clarify the risk factors for severe respiratory symptoms .

objective : to evaluate the prevalence of respiratory symptoms as the motive for emergency room visits by adult and pediatric patients , describing the major clinical syndromes diagnosed and the outcomes of the patients . methods : a cross - sectional study conducted in the emergency room of a tertiary care university hospital . between november of 2008 and november of 2009 , we reviewed the total number of emergency room visits per day . children and adults who presented with at least one respiratory symptom were included in the study . the electronic medical records were reviewed , and the major characteristics of the patients were recorded . results : during the study period , there were 37,059 emergency room visits , of which 11,953 ( 32.3% ) were motivated by respiratory symptoms . the prevalence of emergency room visits due to respiratory symptoms was 28.7% and 38.9% among adults and children , respectively . in adults , the rates of hospitalization and mortality were 21.2% and 2.7% , respectively , compared with 11.9% and 0.3% , respectively , in children . among the adults , the time from symptom onset to emergency room visit correlated positively with the need for hospitalization ( p < 0.0001 ) , the length of the hospital stay ( p < 0.0001 ) , and the mortality rate ( p = 0.028 ) . conclusions : we found a high prevalence of respiratory symptoms as the motive for emergency room visits by adult and pediatric patients . our results could inform decisions regarding the planning of prevention measures . further epidemiological studies are needed in order to clarify the risk factors for severe respiratory symptoms .

OBJETIVO: MTODOS: RESULTADOS: CONCLUSES: Introduction Methods Results Discussion

the quest for important medical knowledge the kind that clarifies understanding of or that leads to successful interventions for important clinical problems has always required departure from accepted norms of medical practice . innovation in medicine ( i.e. trying out new or revised clinical practices to improve health outcomes for individual patients ) remains a central feature of medical practice and varies from minor adjustments to ' radical new procedures ' . it is doubtful that any thoughtful physician or surgeon has not at some time tailored a patient 's treatment to fit specific circumstances , or adjusted a standard dose of a medication in an attempt to maximize the patient 's benefit . when thomas percival suggested , in 1803 , that some of these departures from accepted practice were sufficiently bold to warrant seeking the opinion of medical colleagues before conducting them in patients , he ushered in the age of peer review in medicine . as recently as the early 1960s , peer review in research was viewed by many investigators and research administrators with the same suspicion that percival undoubtedly encountered in the 1800s . however , in the wake of the thalidomide tragedy of the late 1950s and early 1960s , revelations in 1963 of researchers injecting elderly indigent patients with live cancer cells at the brooklyn jewish chronic diseases hospital , and in the same year an ethically dubious and unsuccessful chimpanzee to human kidney transplant at tulane university , the specter of lost public trust in the research enterprise forced two critical issues onto the agenda . first , how should the risks associated with medical research be dealt with , and , second , how should the burgeoning research enterprise be governed ? in 1965 , the us national institutes of health ( nih ) director dr james shannon championed a policy whereby the nih would fund research in human subjects only if , ' the judgment of the investigator is subject to prior review by his [ sic ] institutional associates ' . this policy formalized the practice of institutionally based research ethics review , a form of ethics peer review that has for better or worse become the norm throughout the world . the vast majority of research ethics review takes the form of protocol review alone , conducted in advance of the research . the goal of these reviews is to determine the ethical acceptability of the proposed research . when this has been done , authorization may be given to the investigators to proceed with their proposed research . investigators are required to conduct the research according to the approved protocol and to give regular status reports ( at least annually ) in order for approval to be extended . however , research ethics scandals have shown that the limited scope of research ethics review can provide sufficient opportunity for those few investigators who choose to take shortcuts , proceed carelessly , and even occasionally engage in willfully unethical conduct to avoid the scrutiny of their peers . such deviations from the approved protocol can have grave consequences , both in terms of the safety of research participants and in public trust in the research enterprise . the lack of effective oversight and monitoring in clinical research makes it similar in many respects to clinical practice , especially that by independent , individual physicians . the similar levels of latitude and independence of action raise the question of whether oversight of clinical research may be sufficiently served , post hoc , through litigation by injured parties a familiar mode of oversight in clinical practice . since shannon 's institution of peer review at the nih , the process of ethics approval for research studies by institutional research ethics boards ( rebs ; also known as institutional review boards or research ethics committees ) , including formal informed consent procedures as well as annual status reports , have been widely accepted as a sufficient institutional response to ethical challenges in research . according to mcdonald , ' ( t)he reb process ( and with it the focus on the research proposal and the consent form ) has become the reification of the sum total of responsibilities and accountabilities for researchers , research institutions , research sponsors , and research regulators . in effect , this rationalizes the avoidance of major responsibilities that arise before , after and on the peripheries of the reb review process . ' the current state of oversight and monitoring of clinical research make them the achilles ' heel of research ethics , the flaw that fatally weakens the rest of the operation . although these practices have long been recognized as essential features of sound research ethics , they are seldom exercised in ways that fulfill their motivating goals , which are ' to ensure that research is conducted as planned , that research subjects comprehend the information presented to them in the consent process , and that the potential benefits and risks of study participation remain acceptable ' . in part , this reflects the fact that , despite important improvement in awareness and interest in research ethics within the research community , the review and oversight of research are still viewed by many investigators as an intrusion on their professional discretion and as obstacles to research , rather than integral and complex challenges for enhancing research conduct and governance . as such , research ethics review , oversight and monitoring remain among the small handful of essential research related activities that are conducted on an almost entirely voluntary basis . institutions either lack the resources or the motivation ( or both ) to ensure that these activities occur and are done well . research monitoring and oversight encompass four types of activity : annual review of continuing research , monitoring of informed consent , monitoring of adherence to approved protocols and monitoring the integrity of data . these activities are intended as means of quality assurance in research , and as means of establishing expectations of rigorous and ethical conduct in research . however , there are very few empirical research data to demonstrate how well these practices fulfill these functions , and the few epidemiological studies of monitoring and continuing review suggest much room for improvement . furthermore , it is also taken on faith that when these activities do occur they contribute to the higher order aims of protecting human participants and promoting ethical conduct in research , but similar deficits in empirical evidence make these claims increasingly suspect and raise a deeper question about what we are trying to achieve through research ethics review , monitoring and oversight practices , and how . according to the canadian tri - council policy statement : ethical conduct for research involving humans , ' ( p)rinciples of accountability require that , regardless of the review strategy , the reb continue to be responsible for the ethics of all research involving human subjects that is carried out within the institution . ' in interviews with reb members at several major canadian universities , however , mcdonald found them to be apprehensive about monitoring . this seems a reasonable response to the ambiguous message of the tri - council policy statement , which , in a subsequent section , states that ' ( b)eyond scrutinizing reports , the reb itself should not normally carry out the continuing ethics review , except in specific cases where the reb believes that it is best suited to intervene ' . so , the committees most responsible for research ethics continue to be given few , if any , resources and little helpful guidance on these matters . our own hospital , st . michael 's hospital , has taken the unusual step of implementing a monitoring programme with a full - time research ethics monitor . through judicious use of skill , diplomacy and diligence , and with strong support from the hospital 's research administration , we are forging what we believe is a fruitful new direction in research monitoring and oversight . we are working to create a climate and culture within the institution that support investigators with the myriad technical requirements of research , particularly the canadian and us regulatory requirements for clinical trials , but also to provide them with meaningful opportunities to discuss and examine the complex ethical and regulatory issues that arise in the conduct of their research . we provide investigators with detailed information about research responsibilities through regularly scheduled rounds and a variety of other educational activities , such as courses in the international conference on harmonisation 's good clinical practice guidelines . we conduct internal quality assurance audits of ongoing trials , initiated either by random selection , investigator request , or in response to specific events or concerns . we have also monitored informed consent discussions between research staff and prospective research participants , and we are currently planning to survey research participants themselves in order to gain a better understanding of their perspective . these activities not only engage investigators with the issues , but they also provide the reb with a real and meaningful set of mechanisms to help it fulfill its obligations for monitoring and oversight . the reb members receive feedback about the actual conduct of the research that they approved , and this constitutes an important learning loop for their review of future proposals . the institution is also exercising due diligence by gathering more details about the conduct of its research , especially for investigator - initiated drug studies , in which the institution must assume sponsor responsibilities . our researchers have responded very favourably to acknowledgement and support for the high standards of their conduct , and to recommendations for improvement , when these are warranted . given the many ethical challenges inherent in critical care research , it would be tempting to propose a separate and distinct approach to their monitoring and oversight . although perplexing challenges related to monitoring and oversight in critical care research will invariably arise [ 13 - 15 ] , such as informed consent from a substitute decision maker for those patients whose capacity changes over time , similar challenges are equally likely to arise in other clinical research fields . we believe that our institutionally based system of research ethics review and responsibility requires greater engagement and participation of researchers and research administrators so that they can help to compensate for the limitations of the ethics review process , rather than ignoring or taking advantage of them . we need better professional ownership of these issues by investigators and clinical departments , and institutional pride in affording these issues the time , resources and intellectual commitment that they deserve . the appropriate role for critical care researchers and administrators in this respect is to help provide the necessary leadership to move beyond the limits of current research ethics review practices and toward a greater recognition of the importance of monitoring and oversight for the performance of ethical and high quality clinical research .

institutionally based research ethics review is a form of peer review that has for better or worse become the norm throughout the world . the vast majority of research ethics review takes the form of protocol review alone , conducted in advance of the research . although oversight and monitoring in clinical research have long been recognized as essential features of sound research ethics , they are seldom exercised in ways that fulfill their motivating goals : to ensure that research is conducted as planned ; that research participants comprehend the information presented to them in the consent process ; and that the potential benefits and risks of study participation remain acceptable . annual review of continuing research , monitoring informed consent , monitoring adherence to approved protocols and monitoring integrity of research data comprise the main types of monitoring and oversight activity . we believe that our institutionally based systems of research ethics review and responsibility require greater engagement and participation from researchers and research administrators . the appropriate role of critical care researchers and research administrators is to provide leadership to move toward a greater recognition of the importance of monitoring and oversight for ethical and high quality clinical research .

Introduction Challenges in oversight and monitoring Oversight and monitoring practices Conclusion Competing interests Abbreviations

complaints are upper abdominal discomfort and pain , poor appetite and a mass in the abdomen which , in many cases , expressed by the patient himself . physical findings may include hepatomegaly , a mass palpated on the surface of the liver or other organs and abdominal distention in chronic patients ( 2 ) . herein , we evaluated different emergency presentations of hydatid cyst disease and their treatment under emergency conditions . we studied pre - operative laboratory findings of patients in emergency room who had hydatid disease , to identify any parameters to predict any hydatid cyst - biliary system communication . in this retrospective study approved by a research ethics committee ( ankara numune trining and research hospital ethics committee/20.01.2016/e-16 - 733 ) , we retrospectively reviewed the files of patients who underwent surgery due to hydatid cysts between march 2010 and march 2014 in numune research and training hospital , which is a high volume reference hospital in an endemic region . all liver hydatid cyst patients who admitted emergency room and underwent laparotomy accordingly are included in the study . patients with history of trauma , or recent hydatid cyst surgery ( in last six months ) are excluded . patients diagnosed with liver hydatid disease only , during emergency diagnostic laparotomy are also included in the study . we collected information of demographics , cyst sizes , presence of perforation , type of the hydatid cysts ( 3 ) , imaging modalities , preferred treatment methods , complications , time between hospital arrival and diagnosis , time between hospital arrival and surgery and outcome of the disease . intraoperative diagnosis of biliary communication of hydatid cysts and postoperative biliary leakage were regarded as confirmed presence of biliary system involvement . only the patients whose cysts are proven unrelated with biliary system are grouped as biliary system not involved group . patients hydatid cysts confirmed or suspected to have a biliary system communication were grouped as biliary involved group . groups were compared regarding initial laboratory findings of the patients ( the blood tests made upon arrival in the emergency room ) ; using mann - whitney u test . dissection of hospital database revealed 243 patients underwent surgery due to liver hydatid disease between mar 2010 and mar 2014 . only twelve of these patients ( 3 females , 9 males ) underwent surgery following their admission to the emergency surgery department due to peritoneal irritation with acute abdomen findings and/or abdominal sepsis is included in this study ( 4.93% ) . the median age was 42 yr ( 1884 yr ) . only in three patients , disease was somehow communicated with the biliary system . in six cases , cyst rupture into the biliary system was identified in operation room , and t - tube drainage was performed in four of them , while choledochoduodenostomy was preferred in two . biliary leakage was identified postoperatively in two cases and one particular case had only a bilioma due to prior hydatid cyst surgery . biliary system involved group had significantly higher pre - operative gamma glutamine transferase ( ggt ) and alkaline phosphatase ( alp ) levels ( p=0.036 ) . in this group , median ggt level was 190 u / l and median alp level was 249 u / l , which are around , two - times the upper limits . no significant difference was noted regarding other pre - operative laboratory findings ( table 1 ) . n : number of patients , alt : alanine aminotransferase , alp : alkaline phosphatase , ggt : gamma glutamyl transferase detailed information about demographics , preferred treatment method and outcome of each patient is summarized in table 2 . table 3 gives detailed information about the complaints of the patients on admittance , imaging modalites used , gharbi types of the cysts , cyst diameters , and the elapsed time between admittance and diagnosis / treatment . demographic features , preferred treatment methods and disease outcome f : female , m : male , hc : hydatid cyst , ercp : endoscopic retrograde cholangiopancreatography patients complaints on admission , diagnostic methods , cyst characteristics and elapsed time between admission and diagnosis / treatment hc : hydatid cyst , xr : x - ray , usg : ultrasonography , ct : computerized tomography , mrcp : magnetic resonance cholangiopancreatography , ctgpd : compurerized tomography guided percutaneous drainage , ercp : endoscopic retrograde cholangiopancreatography , dpl : diagnostic peritoneal lavage in two of twelve cases , hydatid disease was diagnosed during laparotomies initially performed for exploration purposes because of peritoneal irritation . he had a known history of subtotal gastrectomy due to peptic ulcers , in addition to hydatid cyst surgery and was on follow - up . emergerncy endoscopic retrograde cholangiopancreatography ( ercp ) could not be performed because of a duodenal diverticulum . patient s health status did not improve . during this period of decision - making , emergency laparotomy revealed an infected bilioma , probably due to prior hydatid cyst surgery and t - tube drainage was performed . he was referred to the interventional radiology unit due to liver abscess and a percutaneous drainage catheter was placed in it . on the follow - up , meanwhile , the patient s vital signs worsened and he was moved to the surgical intensive care unit . laparotomy revealed a superinfected hydatid cyst , which has a relation with the biliary system as well . standard cystotomy , drainage , omentoplasty was performed and t - tube drainage was added . unfortunately , biliary leakage was detected on the postoperative third day and sphincterotomy with ercp was performed . type 4 hydatid cyst was detected in the liver of case 11 , and its relation to the choledoch was unclear . an unlucky iatrogenic choledoch injury has occurred during dissection of biliary structures from hydatic cyst wall . he had a history of subtotal gastrectomy with billroth ii gastrojejunostomy , due to gastric ulcer . patient was deceased due to hemodynamic instability and respiratory failure , in the intensive care unit , on the day of surgery . out of twelve patients , imaging modalities used , cyst diameters , elapsed time before diagnosis and surgery are also presented in table 2 . cyst rupture into the peritoneal cavity or the biliary system is also rare ; however , these complications may cause severe complications such as anaphylaxis and may even threaten life ( 46 ) . as we observed in our sample , abdominal pain is the common cause for emergency room admittance in complicated hydatid cyst cases . nonetheless , the emergency clinicians may not consider hydatid disease as the primary cause of abdominal pain , reasonably due to rarity ; even when the patient has a history of treated hydatid disease . totally , 66.7% of 15 confirmed intrabiliary rupture of hydatid cysts could have been suggested by ultrasonography , preoperatively ( 7 ) . emergency physicians are likely to get indeterminate reports from the radiologists , therefore , they need to rely more on the patient s physical examination and laboratory findings in such cases . twenty - four patients with confirmed cyst - biliary communication out of a total of 116 hydatid cyst patients were studied and pre - operative history of vomiting , preoperative elevated alp , total bilirubin levels and cyst sizes greater than 10.5 cm were related with intrabiliary rupture ( 8) . moreover , greater cyst size , elevated eosinophil count , alp , ggt , alanine aminotransferase , aspartate aminotransferase and bilirubin levels are associated with biliary leakage after hydatid cyst surgery ( 9 ) . nevertheless , none of these reports evaluated predictive potential of preoperative findings . several cases of spontaneous hydatid cyst ruptures are reported , and these_ruptures may cause peritoneal irritation and even subsequent anaphlylaxis ( 4 , 10 ) . in turkey , rate of intraperitoneal rupture has been reported to be 7.8 ( 11 ) . however , in our database , no patients with intraperitoneal rupture of hydatid cyst could be found in a four yr period . on the other hand , we saw that twelve out of 243 liver hydatid cyst surgeries in our hospital were performed in emergency conditions because of acute abdomen and/or abdominal sepsis ; and in nine of them biliary system was somehow involved . in our study , preoperative ggt and alp levels were higher in the patients with confirmed or suspected biliary system involvement . although , this study was conducted in a high volume hospital in an endemic region , sample size was not adequate . as the sample size is tiny , we used mann whitney u test to see if we would be able to demonstrate any differences between groups . it is not surprising to fail to demonstrate any difference in such analyses with low power . however , our analyses succeeded in demonstrating higher ggt and alp levels in biliary system involved group . due to the limitations of our sample size , it is not possible to draw any definite conclusions . however , this result is compatible with previous studies on electively managed hydatid cyst patients . t - tube drainage is a safe method for treating intrabiliary ruptures of hydatid cysts ( 12 , 13 ) . in our study group , all emergency room patients who underwent t - tube drainage recovered perfectly . we think that t - tube drainage is a safe and relatively minimalistic surgical option in patients with hydatid cyst - biliary system communication , even in emergency conditions there is very limited information about surgical emergencies of the patients who have hydatid cyst due to the limited sample sizes . we think that preoperatively elevated alp and ggt levels may be considered as a warning sign for biliary involvement of the hydatid cyst in these patients .

background : vast majority of complaints and physical examination findings of hydatid disease are common in emergency room patients . different emergency presentations of hydatid cyst disease and their treatment are evaluated . we studied preoperative laboratory findings of these patients to identify any parameters to predict hydatid cyst - biliary system communication.methods:we reviewed the files of patients who underwent emergency surgery due to liver hydatid cysts and related conditions between march 2010 and march 2014 in ankara numune research and training hospital , turkey , retrospectively . patients were grouped , regarding to the presence of biliary system involvement.results:twelve patients ( 9 males , 3 females ) were included . we identified two groups . biliary system involved group ( n=9 ) had significantly higher pre - operative gamma glutamine transferase and alkaline phosphatase levels ( p=0.036 ) . no significant difference was noted regarding other pre - operative laboratory findings . mortality rate was 17%.conclusion : medical literature lacks sufficient information about hydatid disease related non - traumatic emergency surgeries . preoperative elevated gamma glutamyl transferase and alkaline phosphatase levels may be questioned as a warning about cyst - biliary communication in hydatid cyst patients with abdominal pain in the emergency room . future studies with larger sample sizes are needed . in addition , prolongation of the time before diagnosis in these patients may result in life threatening complications .

Introduction Materials and Methods Results Discussion Conclusion

drug - induced phospholipidosis ( pld ) is a lipid storage disorder characterized by the intralysosomal accumulation of polar lipids , membranous lamellar inclusions , and drug . common primary tissue targets are alveolar macrophages , hepatic kupffer cells , bile duct epithelia , and peripheral blood mononuclear cells , although numerous other cellular targets have been reported . most have basic and hydrophobic physicochemical properties ( amphiphilic cations ) that enable binding with phospholipids , crossing of biological membranes , and lysomotropism ( partitioning of drug into lysosomes once it is protonated at acidic intralysosomal ph ) . lysomotropic properties and drug accumulation potential are underscored by studies employing lysosomal modulators which have a demonstrated ability to abolish the ph gradient from cytoplasm to lysosome , preclude accumulation of drug in the lysosome ( depot effect ) , and/or rescue cells in vitro [ 46 ] . there is a consensus view that pld itself is largely an adaptive rather than an adverse response to xenobiotics as a number of ex vivo studies have failed to consistently reveal specific functional detriment in responding phospholipidotic tissues [ 2 , 79 ] . yet there is suggestive evidence that phospholipogenic compounds are associated with concurrent toxicities preclinically and clinically . these toxicities include liver necrosis ( amiodarone ) , motor neuropathies ( imipramine , amitriptyline ) , proximal tubule kidney injury ( gentamicin ) , pulmonary alveolitis that can progresses to fibrosis ( amiodarone ) , pneumonitis ( fluoxetine ) , peripheral neuropathies ( suramin , amiodarone , chloroquine , perhexiline ) , and corneal opacity ( chloroquine , amiodarone ) [ 1015 ] . despite this potential association , to date , there have been no comprehensive published reviews of pharmaceutical industry data that enumerate these risks or that probe the statistical connection between phospholipogenic compounds and concurrent toxicities . understanding whether such a connection exists would help define the relative importance of thorough pathological characterization of phospholipidosis and concurrent toxicities in early drug discovery and assist in reducing attrition in drug development . to investigate the link between phospholipogenic compound administration and the incidence of preclinical histological signals , phospholipogenic and nonphospholipogenic compounds were identified in an internal astrazeneca in vivo toxicology report database and their histological results annotated . we herein report on a robust , unbiased analysis revealing that in vivo phospholipogenic compounds are statistically more likely to demonstrate non - pld histologic changes in all organs investigated . this observation is even more compelling when one considers that , in these studies , the nominal dose ( or exposures ) for nonphospholipogenic compounds typically exceeded that for phospholipogenic compounds . an internal astrazeneca database containing repeat - dose in vivo toxicology reports was searched to identify phospholipogenic compounds using the common pld indicator terms phospholipidosis , histiocytosis , foamy macrophages , lamellar lysosomes , and myeloid bodies . to identify reports on nonphospholipogenic compounds , species , study duration , histological findings , nominal doses , and plasma exposures were the key data extracted . we focused on nine responding organs : adrenal gland ; bone marrow ; kidney ; liver ; lung ; lymph node ; reproductive organs ; spleen ; thymus . ( while we have observed nervous and muscle tissue phospholipidosis in our studies , reports were not archived in time , or search terms were inadequate for the purpose of performing an unbiased analysis . ) reports were individually read to verify the presence or absence of indicator terms , and both pld and all non - pld histologic findings were recorded and annotated . although pld often presents as vacuolation by light microscopy , due to its common occurrence , by itself , this finding was not considered sufficient for pld classification . one or more of the following was considered an additional confirming measure for membership in the phospholipogenic dataset : a positive result in an in vitro pld assay , a positive prediction from our in - house in silico pld model , a pathologist interpretation / description of pld in the report ( pld had to be considered by the pathologist a familiar and salient finding and not that of background ) , or an electron microscopic finding of pld , the latter being the gold standard for detecting lysosomal phospholipid - laden myeloid inclusions indicative of pld . one or more of the following was considered an additional confirming measure of a compound having a nonphospholipogenic status : a negative result in an in vitro pld assay or a negative result in our in silico pld predictive model . nominal dose ( mol / kg ) , aucmhr and cmax ( m ) plasma exposure data for the two compound classes were extracted from the study reports if available ( table 1 ) . if a study had four dose groups , the second lowest dose was designated as the low dose . auc exposure information for at least two doses was available for 77% of the phospholipogenic reports and 86% of the negative 's reports ; cmax exposure information for at least 2 doses was available for 73% of the phospholipogenic compounds and 79% of the nonphospholipogenic compounds . exposures were not adjusted for plasma protein binding as complete measurements were not available for a majority of the studies . statistically significant differences between the two datasets for organ histological findings were assessed using chi square analysis at a 95% confidence level . statistically significant differences are indicated by chi square values greater than 3.84 in table 2 . in vivo data extracted from an internal in vivo toxicology report archive resulted in compilation of forty - six phospholipogenic compounds and sixty - two nonphospholipogenic compounds . to obtain the forty - six phospholipogenic compound set , fifty - three compounds initially identified as phospholipogenics based on verbal descriptors in the study reports were tested in our in vitro pld assay , found to be positive , and selected for further analysis . the majority of these compounds exhibited in vitro ec50 values less than 50 m ( mean 44 m , sd 66 ; ranging from 2.4 to 273 m ) . for comparison , the phospholipogenics amiodarone , chloroquine , and citalopram yield 4.8 , 12 , and 38 m ec50 values in this assay , demonstrating that many positives selected for analysis were intrinsically weaker in their phospholipidosis inducing potential than these standard reference drugs . clustering based on 2-dimensional molecular fingerprints resulted in seventy - five sets of compounds , with the largest cluster containing eight compounds . physical and computed molecular properties also revealed this diversity , with molecular weights ( mw ) ranging from 139 to 696 ( mean 434 , sd 115 ) , polar surface area ( psa ) between 20.5 and 194.9 ( mean 85.9 , sd 33.4 ) , and clogp between 2.53 and 8.95 ( mean 3.0 , sd 2.0 ) . the number of hydrogen bond donors ranged from 0 to 4 ( mean 1.7 , sd 1.2 ) and hydrogen bond acceptors from 2 to 14 ( mean 6.9 , sd 2.4 ) . at ph 7 , the phospholipogenic compounds were predicted to contain 37 bases , 4 neutrals , 3 zwitterions , and 1 acid , while the nonphospholipogenic compounds contain 9 bases , 34 neutrals , 1 zwitterion , and 18 acids . the mw for the phospholipogenic compounds ranged between 256 and 645 ( mean 476 , sd 85 ) and 139 and 695 ( mean 404 , sd 124 ) for the negative compounds . clogp for the phospholipogenic compounds range between 0.67 and 8.95 ( mean 3.8 , sd 1.6 ) and for the negative compounds between 2.53 and 7.46 ( mean 2.4 , sd 2.0 ) . psa for the phospholipogenics ranged between 30.6 and 138.1 ( mean 76.4 , sd 25 ) , while , for the nonphospholipogenic compounds it ranges between 20.5 and 194.9 ( mean 92.8 , sd 37.2 ) . the number of hydrogen bond donors ranges between 0 and 4 ( mean 1.5 , sd 1.1 ) and the number of hydrogen bond acceptors between 3 and 12 ( mean 6.9 , sd 1.8 ) for the phospholipogenic compounds and between 0 and 4 ( mean 1.8 , sd 1.2 ) and 2 and 14 ( mean 6.9 , sd 2.7 ) , respectively , for the negatives . aside from the obvious difference in the ionization characteristics of the two sets , these simple physical chemical parameters alone do not appear to effectively distinguish between phospholipogenic and nonphospholipogenic compounds . mouse , rat , and dog are the three species represented in the compound datasets . study durations ranged from five days to two years , with the majority of repeat dose studies having been conducted for 1 to 4 weeks duration . there were on average 4.8 ( 4.6 sd ) and 3.2 ( 2.3 sd ) reports per compound in the phospholipogenic and nonphospholipogenic datasets , respectively . where there were multiple study reports for a compound tested in the same species , reports were counted as a single report to minimize weighting bias . there were some notable differences in the distribution of study species and duration for both compound classes . there were 29% ( of the total ) ( or 1.6-fold ) more rat study reports available in the phospholipogenic dataset than the negatives dataset , whereas there were 28% ( of the total ) ( or 2.8-fold ) more dog study reports available in the negative dataset than the phospholipogenic dataset . the two - week and four - week groupings had the greatest variation in study duration distribution . there were 15% ( of the total ) ( or 1.7-fold ) more 2-week reports available for the phospholpogenic dataset than for the negatives ; there were 26% ( of total ) ( or 1.7-fold ) more reports available for the negative dataset than for the phospholpogenics . except for mouse where aucmhr and cmax ( m ) exposure data were similar between datasets at high nominal doses , nominal dose ( mol / kg ) , aucmhr , and cmax ( m ) values for the negative compounds were on average higher for the other two species ( table 1 ) . multiorgan pld was more likely to occur when predicted percent free compound ( free of protein binding ) was greater than ~10% , whereas pld more often occurred in only one to two organs when the predicted percent free was less than 10% . lung , lymph node , and spleen presented with the highest incidence , bone marrow , and thymus the lowest . in contrast , the rank order of non - pld histologic findings incidence for this dataset differed from that of organ pld ranking ( figure 5 ) . liver and thymus exhibited the highest incidences of non - pld findings , lung , and bone marrow the lowest . to ascertain which dataset was associated with more histologic findings , frequencies of non - pld histologic findings were compared . phospholipogenic compounds consistently exhibited more non - pld histologic changes for all organs ( figure 6 ) , and among these the liver exhibited the highest percentage of histologic findings for both datasets . compared to nonphospholipogenic compounds , phospholipogenic compounds yielded a statistically significant higher percentage of liver necrosis findings ( table 2 ) . for those phospholipogenic compounds in this subset where exposure data were available ( i.e. , 11 of 22 compounds that caused necrosis ) , top plasma exposures did not on average exceed those phospholipogenics that did not cause necrosis ( data not shown ) . a statistically significant , higher frequency of lymphocytolysis / lymphoid depletion in lymphoid organs increased apoptosis in spleen , and alveolitis / pneumonitis in lung were observed with this subset of compounds . finally , statistically significant higher levels of no histologic findings in the liver , lung , thymus , lymph nodes , and spleen were reported for the nonphospholipogenic compounds . the purpose of this study was to test whether pharmaceutically relevant phospholipogenic compounds were associated with a higher frequency of histologic changes in repeat dose in vivo preclinical toxicology studies than that for nonphospholipogenic compounds . for the comparison , forty - six phospholipogenic compounds were identified ; sixty - two negative compounds were identified . phospholipogenic administrations were found to yield proportionally more non - pld histologic changes in general in all organs examined and for select histological findings in liver , lung , thymus , lymph node , and spleen but not for kidney and adrenal glands . phospholipogenic compounds exhibited statistically significant increases in lung alveolitis / pneumonitis and in true toxicities relating to liver necrosis . not surprisingly , phospholipogenic compounds demonstrating no liver findings tended to exhibit fewer histological findings across the 9 organs overall ( data not shown ) . increased incidences of lymphocytolysis and/or lymphoid depletion in three lymphoid tissues ( thymus , lymph nodes , and spleen ) were also observed in the phospholipogenic dataset . lymphocytolysis / lymphoid depletion is often attributed to high - dose - mediated stress responses ; but they may in some instances represent a true toxicological response . without other hematologic correlates it is not possible to shed light on this matter , and for this reason labeled these results conservatively simply as excess histological findings . finally , it also appears from the study duration analysis that two - week study designs and the rat model are typically sufficient to capture phospholipidotic signals in vivo . although concurrent toxicities were not specified , it is of interest that a recent report indicates that a 24 h duration may be sufficient to detect / predict phospholipidosis accurately by employing a small biomarker set in toxicogenomic evaluation . several additional comparisons were made for the phospholipogenic dataset , particularly regarding physical - chemical properties and toxicokinetic behavior . consistent with the well - described cationic amphiphilic nature of phospholipogenic compounds , the phospholipogenic dataset was comprised mostly of amphiphilic bases and the negative dataset contained primarily neutrals and acids with only a few bases . plasma exposures tended to be lower in animals dosed with phospholipogenic compounds . for the dog and mouse , this pharmacokinetic behavior can be explained in part by the selection of lower phospholipogenic compound nominal doses in the study designs but not entirely since relative exposures are still lower if dose - normalized ( data not shown ) . nominal doses for rat were on average higher than that of the nonphospholipogenic dataset , but plasma exposures in the phospholipogenic dataset were considerably lower . there is an acknowledged general tendency for bases , and particularly lipophilic bases , to have higher volumes of distribution , although it has recently been demonstrated that this property alone does not differentiate phospholipogenics from nonphospholipogenics . finally , it should be noted that not only were the average doses and exposures of the nonphospholipogenic compounds higher , but , as can be seen in figure 2 , the average duration of their studies is somewhat longer as well . all of these factors should bias the number of nonphospholipidosis histological findings profile towards the higher dose , higher exposure , longer study duration group , but , as demonstrated in this study , we see the opposite . this thorough and systematic analysis of a large preclinical toxicology dataset containing both confirmed phospholipogenics and nonphospholipogenic compounds indicates that phospholipogenic administration is likely to be associated with more and various histological findings , including liver toxicities , than do nonphospholipogenic administrations . the data , while compelling , are correlative and can not be used to prove causality between phospholipidosis and toxicities . it should also be noted that speculation on functional compromise can not be made and that histologic findings identified in this exercise were not equal in severity . findings ranged from relatively benign to severe , and we did not apply risk assessment analysis where it may be found that compounds could proceed normally through the drug development process , taking concurrent toxicities , tissues affected , severity , and safety margins ( therapeutic window ) into account . even with these caveats , it is clear that phospholipogenic compounds tend to produce more histological findings and may require additional monitoring and risk assessment , thereby increasing the overall burden on a drug development project .

while phospholipidosis is thought to be an adaptive response to chemical challenge , many phospholipogenic compounds are known to display adverse effects in preclinical species and humans . to investigate the link between phospholipogenic administration and incidence of preclinical histological signals , an internal astrazeneca in vivo toxicology report database was searched to identify phospholipogenic and nonphospholipogenic compounds . the datasets assembled comprised 46 phospholipogenic and 62 nonphospholipogenic compounds . the phospholipogenic potential of these compounds was confirmed by a pathologist 's interpretation and was supported by well - validated in silico and in vitro models . the phospholipogenic dataset contained 37 bases , 4 neutral compounds , 3 zwitterions , and 1 acid , whereas the nonphospholipogenic dataset contained 9 bases , 34 neutrals , 1 zwitterion , and 18 acids . histologic findings were tracked for adrenal gland ; bone marrow ; kidney ; liver ; lung ; lymph node ; spleen ; thymus ; and reproductive organs . on average , plasma exposures were higher in animals dosed with the nonphospholipogenics . phospholipogenics yielded proportionally more histologic changes ( exclusive of phospholipidosis itself ) in all organs . statistically significant higher frequencies of liver necrosis , alveolitis / pneumonitis , as well as lymphocytolysis in the thymus , lymph nodes , and spleen occurred in response to phospholipogenics compared to that for nonphospholipogenics .

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

dramatic advances in imaging technology , especially for small animal imaging , have been an important driving force in establishing the field of molecular and genomic imaging . micro - ct system for small animal imaging has been studied since 1990s [ 27 ] and has played a critical role in the evolution of molecular imaging [ 8 , 9].it can obtain high - resolution anatomic information and can be combined with other modalities [ 10 , 11 ] , such as nuclear imaging [ 12 , 13 ] and optical imaging . a prototype cone - beam micro - ct system for small animal imaging has been developed by our group . in this system , we use a microfocus x - ray source and a complementary metal oxide semiconductor ( cmos ) based flat - panel detector . fdk method is adopted for 3d reconstruction . in order to accelerate ct reconstruction speed , we develop reconstruction software using graphics processing unit ( gpu ) hardware . the aim for building such a system is to develop a dual modality system integrated with a micro - ct scanner and a bioluminescence tomography ( blt ) system . however , the spatial resolution of blt is about several millimeters or submillimeters , so the anatomic structure with less than 100 micron is enough . moreover , main organs , such as lung , bone , kidney , and liver , should be discriminated in ct image . in this paper , the prototype micro - ct system is introduced , including overview of the system , geometric misalignment calibration , gpu - based image reconstruction and post - processing software . system performances have been evaluated in terms of spatial resolution , image uniformity and noise , and low contrast resolution . furthermore , some mouse images with and without contrast agent are presented to show the overall performance of the system . the prototype micro - ct system consists of a microfocus x - ray source , a three - dimensional ( x - y translation and rotation ) programmable stage with mouse holder and a flat - panel x - ray detector . all of them are mounted on an optical bench in a shielded laboratory and controlled by a host computer . a schematic diagram of the system is shown in figure 1 . the source to detector distance ( sdd ) and source to object distance ( sod ) are variable to change the magnification ratio , cone - beam angle , and field of view ( fov ) of the system . in most cases , the sdd is set to 498 mm and the magnification ratio m is set to 1.3 , where m = sdd / sod . the x - ray source ( ultrabright , oxford instruments , usa ) has a microfocus continuously adjustable from 13 m to 40 m and the power density has been rated to 2.5 w/m . the target voltage of the x - ray tube is 20 to 90 kvp , with maximum output power 80 w. the x - ray flat panel detector ( c7942ca-02 , hamamatsu , japan ) , based on cmos technology with a column csi scintillator plate , has a 120 mm 120 mm photodiode area with 50 m pixel size . the image data are transferred to the host computer by a frame grabber card ( imaq pci-1424 , national instruments , usa ) in the system . the three - dimensional programmable stage consists of two motorized translation stages ( psa200 - 11 , zolix instruments , china ) and one motorized rotation stage ( rak-100 , zolix instruments , china ) . the resolution of the rotation stage is up to 0.00125. the magnification ratio can be changed by adjusting the position of the object ( mouse or rat ) using the motorized translation stages , which will significantly affect the spatial resolution and the fov . the fov is usually large enough to acquire the whole body imaging of a normal size mouse in a single scan with a small magnification ratio . several methods [ 1719 ] have been proposed to evaluate and calibrate the geometric misalignment . in our system , the method proposed by yang is used . as is stated in , the rotation axis is defined as the z axis of the system . the axis which passes through the cone vertex ( x - ray tube focal spot ) and perpendicular to the z axis the axis perpendicular to the x - z plane is defined as the y axis . there are seven parameters to describe the system geometry : source to detector distance ( sdd ) , source to object distance ( sod ) , u0 , horizontal location of the intersection of the x axis and detector plane , v0 , vertical location of the intersection of the x axis and detector plane , , in - plane rotation angle , the rotation angle of the detector plane along x axis , , out - of - plane rotation angle , the rotation angle of the detector plane along the axis of v = v0 , , out - of - plane rotation angle , the rotation angle of the detector plane along the axis of u = u0 , the two out - of - plane rotation angles , and , are quite difficult to determine with reasonable accuracy and have only a small influence on image quality , so and could be assumed to zero during geometric calibration , and then five other parameters need to be estimated . therefore , the method with multiple projection images acquired from rotating point - like objects ( metal ball bearings ) are used to estimate these five parameters . during experiments , it is found that the horizontal location parameter u0 has a great influence on image quality . though yang 's method could achieve high accuracy , there still exists a small residual detector horizontal offset sometimes , which may be caused by the projection noise in the geometric evaluation . we denote the residual horizontal offset as u , which is the difference between the actual and estimated value of u0 after yang 's geometric calibration . if the residual offset value is large , it will degrade the reconstruction image , especially when high resolution is desired . in the developed system , a wire phantom is utilized to assess the geo - calibration result , and a simple method is proposed to evaluate the horizontal offset through the wire phantom reconstruction image . considering a wire phantom with the wire diameter significantly smaller than the system spatial resolution , cross - sectional view of the phantom reconstruction image can be regarded as the point spread function ( psf ) of the system . through 1d psf , the profile of the psf image , one can access geometric calibration effect quantitively . if there is no detector residual horizontal offset , the wire in the cross - sectional view will be a point . while if a small horizontal offset exists after geometric calibration , psf will be spreaded and its intensity will become smaller . camparing the peak value of psf with different geometry calibration , we can determine which one leads to a better calibration result . if the residual horizontal offset is large enough , a ring will appear in the cross - sectional view instead of a point , and the radius of the ring is related to the horizontal offset value . we have proposed a simple method to assess the horizontal offset , which is derived as follows . let s , o and od be the focal spot of the x - ray source , the center of the rotational stage and the center of the flat panel detector , respectively . we use p to denote a point in the tungsten wire near horizontal mid - plane of the cone beam , where the cone angle can be neglected . the distance between the wire and the rotational axis is l. figure 2 shows that the rotational stage turns an angle ( 0 < 2 ) , and the projection of the point p will be along the radial sp or sn which makes an angle with the center ray so . owing to the effect of the horizontal offset u , back - projection will start from n and along the radial ns which makes an angle with sp . the distance from point p to the line ns is r. figure 3 illuminates that the tail portions of the line are cancelled and a ring with radius r around the point p is formed when the neighboring views are closely placed . as u is very small , the following relationships can be derived : ( 1)usdd,(2)=arctan(l sin sod+l cos ),(3)=2,(4)r=rsin ,(5)ur=sddsod+l cos . substituting ( 3 ) and ( 4 ) into ( 5 ) , we have ( 6)ur = sdd(sod+l cos )sin(/2 ) . as the wire is near the phantom center , the value l will be very small , only a few millimeters , when the phantom is put in the center of the rotational stage , thus l sod . u is significantly smaller than sdd , u sdd , then ( 6 ) can be simplified to ( 7)ur = sddsod = m , where m is the magnification ratio of the system . by ( 7 ) , the horizontal offset can be evaluated and the ring radius value can be measured from the wire reconstruction image . reconstruction image with smaller voxel though the offset value could be calculated , yet the offset direction could not be determined . so the offset needs to be calibrated in both left and right to confirm the correct direction . it should be noted that the above analysis is an ideal case . in real experiments , the ring may be not very clear because of the effects of noise and wire size . so we have to average many cross - sectional slices to identify the ring clearly . in addition , when the horizontal offset is small , the ring may not appear in the image . in this condition , if one wants to dertermine whether the calibration result is good or not , a small horizontal offset ( about a pixel ) should be added artificially . if the evaluted value is equal to the added one , it indicates that the former geometric calibration is good . due to dark imaging offset , pixel gain and defective pixels , raw data from x - ray detector show spatial variation , therefore it is necessary to perform data preprocessing before reconstruction , including dark current subtraction , flat field division , and defective pixels interpolation . after data pre - processing , fdk method is adopted for 3d reconstruction in the developed system . because an exact reconstruction is impossible for circular cone - beam scans due to data insufficiency [ 20 , 21 ] , fdk is an approximate algorithm and the reconstruction images using fdk algorithm have artifacts such as low - intensity drop when the cone angle is large . however , our experiments and other literatures [ 22 , 23 ] have suggested that the reconstruction images are acceptable when the cone angle is relatively small ( about less than 10 ) . it is well known that 3d ct image reconstruction is computationally demanding , so a software using gpu hardware is developed to improve the reconstruction speed . it takes about 5.2 seconds to reconstruct a 512 cubed volume from 360 views of the size of 512 512 on a 2.66 ghz dual - core intel pc with 2 gb ram hosting a nvidia geforce 8800gtx card . large data reconstruction may need tens of or hundreds of seconds , because data reading from hard disk to ram costs lots of time . we use 3d medical image processing and analyzing software 3dmed ( v. 2.2.0 ) , developed by the medcal image group , institute of automation , chinese academy of sciences ( http://www.3dmed.net/ , http://www.mitk.net/ ) , to display and post - process the 3d micro - ct data . it is a free software and combines the function of medical image segmentation , registration and visualization . furthermore , it supports the creation of plug - ins to incorporate new processing algorithms . in order to evaluate the system characteristics , the reconstruction images are assessed in terms of modulated transfer function ( mtf ) , uniformity and noise level , and low contrast resolution . phantoms and mouse scanning protocols are listed detailedly in table 1 . a wire phantom ( micro - ct wire phantom , qrm , germany ) , as shown in figure 4 , is utilized to measure the psf and mtf of the micro - ct system in our study . the cylinder phantom contains two tungsten wires in solid material aligned parallel to the phantom axis of rotation . one of the wires is slightly off center , the other 12 mm away from the center in order to allow estimating image quality in the periphery . the two tungsten wires in the phantom are designed to parallel to the phantom axis . when the cylinder phantom is put on the rotation stage perpendicularly , we can judge whether the wire slopes or not by the sagittal and coronal view of the reconstruction image . if there is no slope observed from the sagittal view and coronal view , we assume that the wire is perpendicular to the horizontal plane . the wire phantom is scanned according to the above protocols to acqure the system modulation transfer function . the reconstruction image of the wire can be regarded as the psf of the system . calculating the modulus of the fourier transform of the psf and normalizing to 1 at spatial frequency at zero different magnification ratios , 1.3 and 2.36 , are set to observe geometric position effect on spatial resolution . as there are two wires in the phantom , one could evaluate the spatial resolution both near the rotation axis and off away from the axis in a single scan measurement . ramp function and cosine window function ( hamming window ) are used respectively as the filter kernel during 3d reconstruction to study the influence of different filter kernels . imaging uniformity and noise of the reconstruction image are investigated with a cylindrical phantom filled with distilled water ( micro - ct water phantom , qrm , germany ) , as shown in figure 5 . the radial profile of the reconstruction image is plotted to illuminate the uniformity qualitatively . a quantitative assessment of the signal variation , from center to periphery , is performed by calculating the mean values and standard deviations in five regions of interest ( rois ) , one in the center of phantom and four in the periphery , and the average differences in signal intensity values between the central and the perihperal regions are calculated . five different exposure intensities are used to determine the relationship between exposure and image noise . the tube is set at 50 kvp with the tube current varying from 0.4 ma to 1.2 ma , which corresponds to exposure from 0.24 mas to 0.72 mas for a single projection . beam hardening correction is implemented to reduce artifacts caused by polychromatic x - ray beam . the total measured noise can be considered as a quadrature summation of photon noise and system noise , then one can obtain ( 8)m2=ae+s2 , where m is the measured noise variance , m is standard deviation . also , e is the exposure for a single view in mas , and a and s are constant terms . in our system , if we want to distinguish the low - contrast objects from their background with false positive and false negative rate 5% , the means of the two distributions should be separated by 3.29 m . for 50% false - positive and false - negative rate , 0.83 m separation are needed . for in vivo small animal imaging , to reduce the motion blurring due to breathing or heart beating , the systems with high framing rate or respiratory gating has been studied [ 25 , 28 , 29 ] . none of these techniques has been implemented in our system and great efforts have been made to reduce motion effects under the current condition . in addition , as the purpose of the system is to provide anatomic structure for blt , which resolution is about several millimetres or submillimetres , so some slight motion artifacts are acceptable . the overall performance of the micro - ct system is assessed by mouse scanning . the mouse was anesthetized with pentobarbital sodium and no contrast agent was used during scanning . the cross - sectional view , coronal view , sagittal view , and the bone structure of the mouse are illuminated in the following section . in order to enhance soft tissues contrast resolution , after fasting for 24 hours , a 20 g balb / c mouse is slowly injected fenestra lc ( art , montreal , canada ) at a dose of 15 ml / kg over a period of 3060 seconds via the lateral tail vein . it is anesthetized with urethane and scanned by our micro - ct system at 3.5 hours postinjection . the wire phantom shown in figure 4 is used to assess the geometric calibration results . it is reconstructed with ram - lak filter and the voxel size is 0.01 0.01 0.05 mm . however , 100 slices near the horizontal mid - plane are averaged to reduce the noise effect . figure 6(a ) shows that a ring exists in the cross - sectional view of the wire phantom , which arises from a small horizontal offset after geometric calibration . in figure 6(a ) , the radius of the ring is mesured about 0.035 mm . the magnification ratio is 1.3 . according to ( 7 ) , the horizontal offset can be determined , that is , 0.045 mm , about 0.9 pixel . because the misalignment direction is unknown , the calibration have to be implemented in left and right to determine the correct direction . figure 6(c ) gives an excellent geometric calibration results , where a point appears in the image instead of a ring . in order to assess the geometric results quantitively , normalized 1d psfs are ploted in figure 7 , which are corresponding to figures 6(a ) and 6(c ) . due to horizontal geometric offset it should be noted that when the horizontal offset is not large , the forked peak will not appear but the peak value will be still lower than the excellent geometric calibration result . campared the psf peak values obtained with different horizontal offsets , the optimal geometric configration can be determined . figure 8 shows the modulation transfer function of the cone - bean micro - ct system . the effects of the magnification ratio and filter kernel are illuminated in the figure clearly . the spatial resolution in the periphery of the phantom is a little lower than that near the center , but not obviously . at a lower magnification ratio ( m = 1.3 ) , the spatial resolution is about 9 lp / mm at 10% of the mtf curve with ramp function kernel and 6.5 lp / mm with cosine window kernel , while it reaches about 14.5 lp / mm with ramp function kernel and 10.5 lp / mm with cosine window kernel at a higher magnification ratio ( m = 2.36 ) . the radial profile of the reconstruction water phantom is plotted in figure 9 to illuminate the uniformity of the image . table 2 gives the mean value and standard deviation of the signals in five different rois with different exposure intensities . signal average values acquired in different exposure intensities share almost the same value , while the standard deviation decreases with the increment of the exposure intensity . for all exposure intensities , the differences of the average signal intensities from center to periphery are all less than one standard deviation of the signals . figure 10 shows the relationship between standard deviation of voxel noise ( in hu ) and the exposure intensity ( in mas ) , which has been fitted to ( 8) and obtains ( 9)m2=883e , where the system noise variance s is neglected because it is significant small . equation ( 9 ) indicates that the voxel noise variance is inversely proportional to the exposure intensity . as shown in figure 10 , the standard derivation of the signal noise is about between 35 hu and 60 hu . take 50 kvp , 1.2 ma for example , the standard derivation is 34.8 hu , which means that 114.5 hu difference is needed to distinguish the low contrast objects from their background with false positive and false negative rate 5% , and 28.9 hu is needed to reach 50% false positive and false negative rate . . figures 11(a ) , 11(b ) , and 11(c ) are the transaxial view , the sagittal view , and the coronal view of the mouse , respectively . figure 11(d ) shows the bone structure of the mouse segmented from the reconstruction data using the software 3dmed . while figure 12 shows excellent liver contrast enhancement which is achieved at 3.5 hours postinjection fenestra lc at the dose of 15 ml / kg . it is possible to distinguish liver from surrounding soft tissues in this image . a prototype cone - beam micro - ct system for small animal imaging is described in this paper . to acquire images with high spatial resolution and low artifacts , data preprocessing and geometry calibration system spatial resolution is investigated in terms of mtf , which reaches about 14.5 lp / mm at a magnification ratio of 2.36 with ram - lak filter . water phantom study shows that the voxel noise variance is inversely proportional to the exposure intensity . low contrast resolution is also studied in the paper . some living mouse imaging results with and without contrast agents are presented to give an overall performance of the system . for micro - ct , relative low soft tissue contrast resolution is a drawback which hinders its widespread . as is measured in water phantom experiments , the standard deviation is about 35 hu for 50 kvp , 1.2 ma , while ct number of human 's main organs , except lung and bone , are mostly in the range from 20 hu to 80 hu . so it is a big challenge to distinguish one from others , especially for the liver . fortunately , exogenous contrast agent overcomes this problem to some extent . with specific micro - ct contrast agent fenestra lc beside contrast agent method , techniques to reduce image noise could also be used in micro - ct to improve contrast resolution . at present , several commercial micro - ct systems have been developed . compared with these systems , one characteristic of our system is reconstruction using gpu hardware , which reaches a high reconstruction speed with relative low cost , and will achieve software upgrade easily . a simple method has been proposed to access geometric calibration results and evaluate the detector horizontal offset utilizing a wire phantom . in addition , as mentioned , the final purpose is to build a blt - ct dual modality system . a prototype micro - ct system will be more conveniently to integrate with blt system .

a prototype cone - beam micro - ct system for small animal imaging has been developed by our group recently , which consists of a microfocus x - ray source , a three - dimensional programmable stage with object holder , and a flat - panel x - ray detector . it has a large field of view ( fov ) , which can acquire the whole body imaging of a normal - size mouse in a single scan which usually takes about several minutes or tens of minutes . fdk method is adopted for 3d reconstruction with graphics processing unit ( gpu ) acceleration . in order to reconstruct images with high spatial resolution and low artifacts , raw data preprocessing and geometry calibration are implemented before reconstruction . a method which utilizes a wire phantom to estimate the residual horizontal offset of the detector is proposed , and 1d point spread function is used to assess the performance of geometric calibration quantitatively . system spatial resolution , image uniformity and noise , and low contrast resolution have been studied . mouse images with and without contrast agent are illuminated in this paper . experimental results show that the system is suitable for small animal imaging and is adequate to provide high - resolution anatomic information for bioluminescence tomography to build a dual modality system .

1. Introduction 2. System Description 3. System Performance Evaluation 4. Results 5. Conclusion

the prevalence of type 2 diabetes mellitus in korea is estimated to be 7.3% ( in those over 20 years of age ) , according to a report by the korea national health and nutrition examination survey ( knhns iii , 2005 ) , an approximately five - fold increase from the value 30 years ago [ 1 - 3 ] . the number of patients with type 2 diabetes is expected to increase dramatically from about 3.5 million in 2010 ( 7.08% of the total population ) to about 5.5 million ( 10.85% ) by 2030 . the obese and overweight population is also increasing steadily in korea ; in addition , korea is becoming one of the aged societies in the world . this increase in societal age has contributed to the dramatic increase in type 2 diabetic patients . furthermore , diabetes mellitus was the fifth leading cause of death in korea in 2008 . therefore , the early detection and prevention of type 2 diabetes are major health concerns for korean people and the government . the diagnosis and appropriate treatment of type 2 diabetes mellitus are very important issues in establishing and implementing high - priority health policies in korea . recently , several hospital - based or community - based cross - sectional and cohort studies undertaken in korea have been published . in 2007 , the committee of the korean diabetes association ( kda ) on the diagnosis and classification of diabetes mellitus analyzed four of these community - based epidemiological studies performed after 1990 , which included 6,234 subjects , to demonstrate the diagnostic equivalence of fasting plasma glucose ( fpg ) value to a 2-hour plasma glucose ( pg ) value range of 140 and 200 mg / dl . from a receiver operating characteristic ( roc ) analysis , an fpg cutoff of 110 mg / dl corresponded to a 2-hour pg value of 200 mg / dl ( sensitivity 76.4% , specificity 92.2% ) . the optimal cutoff point for fpg for the diagnosis of impaired glucose tolerance ( igt ) in korean people was 97 mg / dl ( sensitivity 57.5% , specificity 71.0% ) . another study that compared the fpg criterion for diagnostic screening of diabetes with the 2-hour pg criterion in 1,731 middle - aged korean adults showed that the level of agreement between the two diagnostic criteria was low ( 0.268 ) . an roc analysis determined that an fpg of 100 mg / dl yielded optimal sensitivity and specificity and corresponded to a 2-hour pg result of 200 mg / dl . if these lower cutoff values are applied to the diagnosis of diabetes , the prevalence of diabetes is more than two - fold higher than when the 2-hour pg level of 200 mg / dl is used . this large discrepancy between the fpg and 2-hour pg criteria in the diagnosis of diabetes and igt in koreans is cause for hesitation before changing the diagnostic criteria for diabetes . we also suggest that the fpg level required for a diagnosis of type 2 diabetes mellitus is lower in the korean and asian populations than in western populations . for this reason , oh et al . analyzed the ifg population according to fasting glucose level , as follows : stage 1 ifg ( 100 to 109 mg / dl ) and stage 2 ifg ( 110 to 125 mg / dl ) . for subjects in the stage 2 group , poorer metabolic profiles were detected in terms of total cholesterol , triglyceride , and high blood pressure . moreover , a greater proportion of people in the stage 2 group were diagnosed with diabetes based on the oral glucose tolerance test ( ogtt ) . from these results , we suggest that korean individuals with stage 2 ifg should be treated or screened in a different manner from those with stage 1 ifg . to avoid missing further cases of diabetes , however , in patients with stage 1 ifg , additional risk factors for diabetes should be assessed and an annual glucose test be performed ( table 1 ) . the diagnostic criteria for type 2 diabetes are based on the results of either the fasting or 2-hour 75 g ogtt , according to the observed association between glucose level and the presence of retinopathy . until now , there have been no cross - sectional epidemiological studies of koreans to demonstrate an association between pg level and the development of diabetic retinopathy . the kda has adopted the diagnostic criteria for type 2 diabetes of the american diabetes association ( ada ) ( table 2 ) . moreover , the variable association between fpg and 2-hour pg level between populations must be attributed to different genetic or environmental backgrounds across different ethnic groups . therefore , more large epidemiological studies are required to confirm the suitable cutoff values for the korean population . the ogtt has low repeatability , requires more time and blood samples , and is more expensive than the fpg test . it has not been used to screen for type 2 diabetes mellitus and is not recommended for the diagnosis of type 2 diabetes in routine clinical practice in korea . however , although fpg is a simple and convenient method , a considerable number of type 2 diabetic patients can not be diagnosed based on fpg results alone , as discussed above . several previous studies have shown that the fpg criterion is less sensitive for the detection of diabetes in elderly korean people than the ogtt - based criterion [ 8 - 10 ] . therefore , the ogtt remains a valuable test in the diagnosis of diabetes and the classification of igt , especially in elderly koreans . moreover , ogtt should particularly be considered if the subject has a history of impaired fasting glucose , is a high - risk individual , is pregnant , or to exclude an igt state . in addition to its role in monitoring and targeting glycemic control , hba1c has received attention as a diagnostic criterion for type 2 diabetes since the ada included a hba1c value threshold 6.5% as a factor with which to diagnose diabetes . if we are to consider hba1c level as a diagnostic criterion , priority must be given to standardization of the assay method . a second consensus meeting on the worldwide standardization of the hba1c measurement , which included the ada , the european association for the study of diabetes , the international diabetes federation , the international federation of clinical chemistry and laboratory medicine ( ifcc ) , and the international society for pediatric and adolescent diabetes , stated that the ifcc reference system for hba1c represents the only valid method to standardize the measurement . the ada recommends that hba1c should be measured according to the method certified by the national glycohemoglobin standardization program and standardized or traceable to the diabetes control and complication trial reference assay . because the hba1c assay was not standardized throughout the country , hba1c was not recommended as a diagnostic criterion for diabetes mellitus in korea until 2007 . however , hba1c standardization has been widely performed since 2007 by the korean association of quality assurance for clinical laboratory , the metabolic disorders subcommittee , the korean society of laboratory medicine , and the kda , including a nation - wide survey for the current status of the hba1c test . most commercial laboratories in korea are certified by designated comparison methods , and accuracy - based proficiency tests are underway . based on this background , we included hba1c as a diagnostic criterion and renewed our clinical practice guidelines in 2011 . several studies have been performed in korea to determine the cutoff point for hba1c required to predict an abnormal glucose tolerance status in non - diabetic korean subjects . analyzed 1,482 subjects without diabetes and reported a cutoff point of 5.95% hba1c to predict diabetes , with a sensitivity of 60.8% and specificity of 85.6% . one study that included 392 subjects with risk factors for diabetes showed that the current guideline of fpg 126 mg / dl for diabetes screening detected only 55.7% of diabetic subjects . the optimal cutoff points for hba1c and fpg for the diagnosis of diabetes were 6.1% ( sensitivity 81.8% , specificity 84.9% ) and 110 mg / dl ( sensitivity 85.2% , specificity 88.5% ) , respectively . ku et al . reported a cutoff point for hba1c derived from the data collected from 19,178 subjects without diabetes . when the fpg criterion was used , the cutoff value was 5.9% ( sensitivity 84.6% , specificity 85.9% ) , and when the 2-hour glucose level after ogtt was used , the cutoff value was 6.1% ( sensitivity 86.5% , specificity 85.8% ) . based on these clinical results , we suggest that the hba1c cutoff level should be lower than the ada criterion . however , larger epidemiological studies are required to confirm the suitable cutoff values for the korean population . the ogtt has low repeatability , requires more time and blood samples , and is more expensive than the fpg test . it has not been used to screen for type 2 diabetes mellitus and is not recommended for the diagnosis of type 2 diabetes in routine clinical practice in korea . however , although fpg is a simple and convenient method , a considerable number of type 2 diabetic patients can not be diagnosed based on fpg results alone , as discussed above . several previous studies have shown that the fpg criterion is less sensitive for the detection of diabetes in elderly korean people than the ogtt - based criterion [ 8 - 10 ] . therefore , the ogtt remains a valuable test in the diagnosis of diabetes and the classification of igt , especially in elderly koreans . moreover , ogtt should particularly be considered if the subject has a history of impaired fasting glucose , is a high - risk individual , is pregnant , or to exclude an igt state . in addition to its role in monitoring and targeting glycemic control , hba1c has received attention as a diagnostic criterion for type 2 diabetes since the ada included a hba1c value threshold 6.5% as a factor with which to diagnose diabetes . if we are to consider hba1c level as a diagnostic criterion , priority must be given to standardization of the assay method . a second consensus meeting on the worldwide standardization of the hba1c measurement , which included the ada , the european association for the study of diabetes , the international diabetes federation , the international federation of clinical chemistry and laboratory medicine ( ifcc ) , and the international society for pediatric and adolescent diabetes , stated that the ifcc reference system for hba1c represents the only valid method to standardize the measurement . the ada recommends that hba1c should be measured according to the method certified by the national glycohemoglobin standardization program and standardized or traceable to the diabetes control and complication trial reference assay . because the hba1c assay was not standardized throughout the country , hba1c was not recommended as a diagnostic criterion for diabetes mellitus in korea until 2007 . however , hba1c standardization has been widely performed since 2007 by the korean association of quality assurance for clinical laboratory , the metabolic disorders subcommittee , the korean society of laboratory medicine , and the kda , including a nation - wide survey for the current status of the hba1c test . most commercial laboratories in korea are certified by designated comparison methods , and accuracy - based proficiency tests are underway . based on this background , we included hba1c as a diagnostic criterion and renewed our clinical practice guidelines in 2011 . several studies have been performed in korea to determine the cutoff point for hba1c required to predict an abnormal glucose tolerance status in non - diabetic korean subjects . bae et al . analyzed 1,482 subjects without diabetes and reported a cutoff point of 5.95% hba1c to predict diabetes , with a sensitivity of 60.8% and specificity of 85.6% . one study that included 392 subjects with risk factors for diabetes showed that the current guideline of fpg 126 mg / dl for diabetes screening detected only 55.7% of diabetic subjects . the optimal cutoff points for hba1c and fpg for the diagnosis of diabetes were 6.1% ( sensitivity 81.8% , specificity 84.9% ) and 110 mg / dl ( sensitivity 85.2% , specificity 88.5% ) , respectively . ku et al . reported a cutoff point for hba1c derived from the data collected from 19,178 subjects without diabetes . when the fpg criterion was used , the cutoff value was 5.9% ( sensitivity 84.6% , specificity 85.9% ) , and when the 2-hour glucose level after ogtt was used , the cutoff value was 6.1% ( sensitivity 86.5% , specificity 85.8% ) . based on these clinical results , we suggest that the hba1c cutoff level should be lower than the ada criterion . however , larger epidemiological studies are required to confirm the suitable cutoff values for the korean population . large prospective , randomized controlled clinical trials such as the kumamoto study and the uk prospective diabetes study ( ukpds ) have showed that early intensive glycemic control in type 2 diabetic patients significantly reduced microvascular and macrovascular diabetic complications . based on these results , most of the clinical practice guidelines for glycemic control target of type 2 diabetes since early the 2000s have recommend the achievement of near - normoglycemia to prevent diabetic vascular complications . however , three recently published large rct studies ( accord , advance , vadt ) have demonstrated no significant benefits in cardiovascular outcome with intensive glycemic control in type 2 diabetes . from these findings , we agreed not to overlook the potential risks of very strict glycemic control in specific situations , such as old age with long diabetic duration , combined severe complications , recurrent severe hypoglycemic episodes , or limited life expectancy . however , it should be kept in mind that many meta - analysis consistently provide evidence for the clinical benefits of achieving and maintaining intensive glycemic control to prevent diabetic complications . therefore , a glycemic goal of hba1c 6.5% should be attained , especially in the recently diagnosed , young type 2 diabetic patients without severe complications or hypoglycemia through lifestyle modification and glucose - lowering agents , including insulin ( table 3 , fig . in addition to glycemic control within the target range , the comprehensive management of metabolic risk factors is also clinically important . after diagnosis of type 2 diabetes , lifestyle modification , as well as medical treatment , should be emphasized to prevent cardiovascular events . lifestyle modification could be accomplished by diabetes self - management education , including knowledge , self - care behavior , self monitoring of blood glucose , medical nutrition therapy , exercise , and hypoglycemia . blood pressure and dyslipidemia should be normalized by lifestyle modification and anti - hypertensive or lipid - lowering agents . regular screening for diabetic microvascular or macrovascular complications should be performed ( table 4 ) . the dramatic increase in the prevalence of type 2 diabetes and its accompanying acute or chronic complications are major health concerns in korea . screening of high risk individuals , early detection , and proper management of type 2 diabetes are urgently needed . although more evidence and clinical trials should be undertaken , appropriate clinical practice guidelines characterized in korean people with type 2 diabetes have been developed and updated to provide better glycemic control and favorable clinical outcomes .

as in other countries , type 2 diabetes is major health concern in korea . a dramatic increase in the prevalence of type 2 diabetes and its chronic complications has led to an increase in health costs and economic burdens . early detection of high risk individuals , hidden diabetic patients , and improvement in the quality of care for the disease are the first steps to mitigate the increase in prevalence . the committee of clinical practice guidelines of the korean diabetes association revised and updated the ' 3rd clinical practice guidelines ' at the end of 2010 . in the guidelines , the committee recommended active screening of high risk individuals for early detection and added the hemoglobin a1c level to the diagnostic criteria for type 2 diabetes based on clinical studies performed in korea . furthermore , the committee members emphasized that integrating patient education and self - management is an essential part of care . the drug treatment algorithm based on the degree of hyperglycemia and patient characteristics were also updated .

INTRODUCTION SCREENING OF TYPE 2 DIABETES MELLITUS DIAGNOSIS OF TYPE 2 DIABETES MELLITUS Oral glucose tolerance test (OGTT) Hemoglobin A1c (HbA1c) TARGET OF GLYCEMIC CONTROL COMPREHENSIVE MANAGEMENT OF METABOLIC RISK FACTORS BEYOND GLYCEMIC CONTROL CONCLUSION

listeria monocytogenes ( lm ) is a facultative anaerobic bacterium which can grow and reproduce inside the host 's cells , making it one of the most virulent food - borne pathogens . the genus consists of six species i.e. , listeria monocytogenes , l. ivanovii , l. seeligeri , l. innocua , l. welshimeri and l. grayi , of which only l. monocytogenes is the primary human pathogen although there have been rare reports of illnesses caused by l. seeligeri and l. ivanovii [ 1 - 3 ] . listeria monocytogenes , commonly referred to as listeria , is a pathogen that causes listeriosis , a severe human illness [ 4 , 5 ] . it is unlike most other food - borne pathogens because it can grow and multiply at proper refrigeration temperatures . in addition , listeria is widely distributed in nature , and has been recovered from farm fields , vegetables , animals and other environments such as surfaces of food processing facilities , retail stores and home kitchens and ready - to - eat foods [ 8 - 10 ] . listeria monocytogenes represents a constant challenge for the food industry , health regulatory officials and consumers since it remains one of the most virulent foodborne pathogens for immunodeficient individuals , it has been extensively studied over the past few decades due to its high case / fatality rate ( 20 - 30% ) , its high burden of healthcare costs during chronic episodes of infection and its ability to survive for longer periods under adverse environmental conditions than many other non - spore - forming bacteria . in man , outbreaks usually occur following consumption of unpasteurized milk , contaminated cheeses and other dairy products . reports of outbreaks have also followed ingestion of undercooked meat , poultry as well as coleslaw where it was first recognized as a food - borne zoonosis . it is frequently present in the gut of cattle , poultry and pigs and can be transmitted to ready - to - eat ( rte ) foods as well as raw meat products . listeria species are isolated from a diversity of environmental sources , including decaying vegetation , soil , water , effluents , a large variety of foods , and the faeces of humans and animals . most reported isolations of this species were from abortions , stillbirths , and neonatal septicemias in sheep and cattle [ 16 , 17 ] . packaged raw foods can represent a potential source of contamination when opened at home , and listeriosis is associated with the consumption of such undercooked raw foods . human to human transmission is rare , except in cases of pregnancy where infected mothers transmit the infection via the placenta to the unborn child . transmission in domestic animals can occur by ingestion of contaminated feed and poor quality silage with ph greater than 5.5 , hence the name " silage disease " . there has been a dearth of information on the epidemiology of listeriosis in most african countries , including nigeria with only few reports , when compared to europe and usa . while antibiotic resistance has been reported severally in literature with clinical isolates from human beings , recent evidences however , show that antibiotic resistance traits have entered the microflora of farm animals and the food produced from them . thus , the food microflora is not separated from its human counterpart in cases of antibiotic resistance . this trend has been worsened by prophylactic use of common broad spectrum antibiotics , indiscriminate usage in humans and in animal feed as growth promoters , particularly in developing nations [ 23 , 24 ] . despite these and the increase in the consumption of poultry products coupled with enormous untrained hands in the poultry industry in nigeria and the associated public health implications , there is paucity of information on the prevalence and antibiotic susceptibility profiles of l. monocytogenes among commercial chickens as well as raw processed chicken meat ; hence , this study . the study was carried out in 13 local government areas ( lgas ) known for the presence of high number poultry industries through a pilot survey across three senatorial districts of oyo state , south - western nigeria . the state was chosen as it possesses the majority of poultry industries in the region aside the backyard small scale poultry farming being practised by many . in addition , consumption of chicken and other poultry products is increasingly high in the state . this cross - sectional study involved a total of 71farms randomly selected from 100 available farms with different poultry types ( layers , broilers ) , breeds , management ( deep litter , battery cage ) and biosecurity levels ( high , average , low ) located in the 13 lgas of the state . the purpose of the study as well as the potential benefits was explained to the farm owners and they were told that participation was voluntary . it was also emphasized that declining participation did not have any attached penalty and that participation would not have any negative effects on their farms . the total number of poultry farms sampled was based on random selection of three of every four poultry farms through a transect walk guided by an initial pilot survey conducted . however , four of the selected farms declined participation . at each of the participating farms , cloacal swabs were collected using sterile swabs to scoop about one gram from each randomly selected chicken . 1ml of peptone water was then dispensed into each of the swab containers to moisten the samples in order to prevent the samples from drying up . these were then placed in coolers containing ice packs for transportation to the meat hygiene laboratory of the department of veterinary public health and preventive medicine , university of ibadan , nigeria for processing . isolation was done using a slight modification of the methods described by gibbons et al . and indrawattana et al . peptone water was prepared by dissolving 15 g of the powder in 1000mls distilled water and autoclaved at 121c for 15min . nutrient agar was prepared by dissolving 28 g of the powder in 1000mls of distilled water and autoclaved at 121c for 15min . listeria selective agar ( lsa ) ( brilliancetm ) was prepared by dissolving 33.6 g of the powder base in 1000ml of distilled water , autoclaved for 15min at 121c , cooled to 40c and lsa antibiotics supplements was added . one gram of each sample was homogenized and transferred into a test tube containing sterile and freshly prepared peptone water . this was incubated at 37oc for 18 hours to 24 hours to revive viable but non - culturable cells . thereafter , 100ul ( 0.1ml ) each of the peptone water culture was transferred to a freshly prepared lsa and spread plated . incubation was done at 37c for 36 - 48 hours . following incubation , discrete bacterial colonies counts were transformed to colony forming unit ( cfu ) [ 27 , 28 ] . discrete lm colonies from the lsa plates were then streaked onto freshly prepared lsa plates to obtain pure listeria isolates and the streaked plates were incubated at 37c for 36 - 48 hours . pure listeria monocytogenes isolates were gram stained , then subjected to various morphological and biochemical tests which included catalase , oxidase and sugar fermentation using glucose , mannitol , sucrose , maltose , fructose and lactose . serial dilution of each sample was also done up to the 6-fold dilutions , using freshly - prepared peptone water . 100ul ( 0.1ml ) each of the 4 and 6 dilutions were then spread plated on nutrient agar plates and incubated at 37c for 18 - 24 hours for counting . following incubation , discrete bacterial colonies the sensitivity discs were specifically designed and contained appropriate concentrations of different gram positive antibiotics which include : ciprofoxacin ( 10g / disc ) , norflaxacin ( 10g / disc ) , gentamycin ( 10g / disc ) , streptomycin ( 30g / disc ) . the plates were then incubated at 37c for 18 - 24 hours . following incubation , data were analyzed using spss version 15 . chi - square test was used to test for association between the variables and prevalence of listeria monocytogenes . bacteria counts at the two different dilutions were compared among the sample types using paired t - test . the prevalence of listeria monocytogenes contamination was calculated by dividing number of contaminated samples with the total number of samples collected . a flock was considered contaminated by listeria monocytogenes if at least one sample taken from the poultry house tested positive . the outcome variable " listeria monocytogenes status " was dichotomous ( contaminated ( positive ) versus non - contaminated ( negative ) flock ) . prevalence was calculated based on the 100cfu / unit limit set by the european commission regulation ( ec ) no.2073/2005 on microbiological criteria for foodstuffs . the study was carried out in 13 local government areas ( lgas ) known for the presence of high number poultry industries through a pilot survey across three senatorial districts of oyo state , south - western nigeria . the state was chosen as it possesses the majority of poultry industries in the region aside the backyard small scale poultry farming being practised by many . in addition , consumption of chicken and other poultry products is increasingly high in the state . this cross - sectional study involved a total of 71farms randomly selected from 100 available farms with different poultry types ( layers , broilers ) , breeds , management ( deep litter , battery cage ) and biosecurity levels ( high , average , low ) located in the 13 lgas of the state . the purpose of the study as well as the potential benefits was explained to the farm owners and they were told that participation was voluntary . it was also emphasized that declining participation did not have any attached penalty and that participation would not have any negative effects on their farms . the total number of poultry farms sampled was based on random selection of three of every four poultry farms through a transect walk guided by an initial pilot survey conducted . however , four of the selected farms declined participation . at each of the participating farms , cloacal swabs were collected using sterile swabs to scoop about one gram from each randomly selected chicken . 1ml of peptone water was then dispensed into each of the swab containers to moisten the samples in order to prevent the samples from drying up . these were then placed in coolers containing ice packs for transportation to the meat hygiene laboratory of the department of veterinary public health and preventive medicine , university of ibadan , nigeria for processing . isolation was done using a slight modification of the methods described by gibbons et al . and indrawattana et al . . peptone water was prepared by dissolving 15 g of the powder in 1000mls distilled water and autoclaved at 121c for 15min . nutrient agar was prepared by dissolving 28 g of the powder in 1000mls of distilled water and autoclaved at 121c for 15min . listeria selective agar ( lsa ) ( brilliancetm ) was prepared by dissolving 33.6 g of the powder base in 1000ml of distilled water , autoclaved for 15min at 121c , cooled to 40c and lsa antibiotics supplements was added . one gram of each sample was homogenized and transferred into a test tube containing sterile and freshly prepared peptone water . this was incubated at 37oc for 18 hours to 24 hours to revive viable but non - culturable cells . thereafter , 100ul ( 0.1ml ) each of the peptone water culture was transferred to a freshly prepared lsa and spread plated . incubation was done at 37c for 36 - 48 hours . following incubation , discrete bacterial colonies counts were transformed to colony forming unit ( cfu ) [ 27 , 28 ] . discrete lm colonies from the lsa plates were then streaked onto freshly prepared lsa plates to obtain pure listeria isolates and the streaked plates were incubated at 37c for 36 - 48 hours . pure listeria monocytogenes isolates were gram stained , then subjected to various morphological and biochemical tests which included catalase , oxidase and sugar fermentation using glucose , mannitol , sucrose , maltose , fructose and lactose . serial dilution of each sample was also done up to the 6-fold dilutions , using freshly - prepared peptone water . 100ul ( 0.1ml ) each of the 4 and 6 dilutions were then spread plated on nutrient agar plates and incubated at 37c for 18 - 24 hours for counting . following incubation , discrete bacterial colonies the sensitivity discs were specifically designed and contained appropriate concentrations of different gram positive antibiotics which include : ciprofoxacin ( 10g / disc ) , norflaxacin ( 10g / disc ) , gentamycin ( 10g / disc ) , streptomycin ( 30g / disc ) . the plates were then incubated at 37c for 18 - 24 hours . following incubation , data were analyzed using spss version 15 . chi - square test was used to test for association between the variables and prevalence of listeria monocytogenes . bacteria counts at the two different dilutions were compared among the sample types using paired t - test . the prevalence of listeria monocytogenes contamination was calculated by dividing number of contaminated samples with the total number of samples collected . a flock was considered contaminated by listeria monocytogenes if at least one sample taken from the poultry house tested positive . the outcome variable " listeria monocytogenes status " was dichotomous ( contaminated ( positive ) versus non - contaminated ( negative ) flock ) . prevalence was calculated based on the 100cfu / unit limit set by the european commission regulation ( ec ) no.2073/2005 on microbiological criteria for foodstuffs . of the 450 samples screened in this study , an overall prevalence of lm contamination was found to be 91.8% comprising 95.8% ( 23/24 ) in meat and 91.5% ( 390/426 ) in cloacal swabs . all the flocks sampled had at least one positive sample yielding a flock prevalence of 100.0% . cloacal samples from broilers had significantly higher prevalence ( 98.8% ) than 89.8% from the layers ( tab . listeria monocytogenes prevalence was highest among the leghorn white ( 98.5% ) and least among the isa brown breed ( 85.6% ) . samples from poultry raised on deep litter ( 92.6% ) and those from farms with low biosecurity level ( 93.2% ) also recorded higher lm prevalence . overall , poultry type ( x= 7.13 ; p = 0.008 ) ; breed ( x = 15.25 ; p = 0.000 ) , but not management ( x = 1.09 ; p = 0.297 ) as well as biosecurity level ( x2 = 0.173 ; p = 0.917 ) were significantly associated with the prevalence of lm among the cloacal samples obtained ( tab . i ) . occurrence of listeria monocytogenes contamination based on poultry types , breed , management and biosecurity levels . harco black , anak white , cobb usa table ii shows the comparison of bacteria counts ( log cfu / ml ) obtained at two different dilutions based on sample types . mean bacteria counts obtained at 10 dilution were significantly higher ( p = 0.0001 ) than those obtained at 10 dilution when compared across the sample type . the variations in mean logcfu / ml differences were significant across sample types ( p = 0.0001 ) . total bacterial counts among the different samples taken ( log cfu / ml ) . a 100% resistance to both ampicillin - cloxacillin ( 30 ug ) and cefuroxime ( 20 ug ) antibiotics was demonstrated by the lm isolates tested while the highest sensitivity ( 86.1% ) was obtained with amocillin clavulanate ( 30ug ) ( tab . of the 450 samples screened in this study , an overall prevalence of lm contamination was found to be 91.8% comprising 95.8% ( 23/24 ) in meat and 91.5% ( 390/426 ) in cloacal swabs . all the flocks sampled had at least one positive sample yielding a flock prevalence of 100.0% . cloacal samples from broilers had significantly higher prevalence ( 98.8% ) than 89.8% from the layers ( tab . listeria monocytogenes prevalence was highest among the leghorn white ( 98.5% ) and least among the isa brown breed ( 85.6% ) . samples from poultry raised on deep litter ( 92.6% ) and those from farms with low biosecurity level ( 93.2% ) also recorded higher lm prevalence . overall , poultry type ( x= 7.13 ; p = 0.008 ) ; breed ( x = 15.25 ; p = 0.000 ) , but not management ( x = 1.09 ; p = 0.297 ) as well as biosecurity level ( x2 = 0.173 ; p = 0.917 ) were significantly associated with the prevalence of lm among the cloacal samples obtained ( tab . i ) . occurrence of listeria monocytogenes contamination based on poultry types , breed , management and biosecurity levels . table ii shows the comparison of bacteria counts ( log cfu / ml ) obtained at two different dilutions based on sample types . mean bacteria counts obtained at 10 dilution were significantly higher ( p = 0.0001 ) than those obtained at 10 dilution when compared across the sample type . the variations in mean logcfu / ml differences were significant across sample types ( p = 0.0001 ) . total bacterial counts among the different samples taken ( log cfu / ml ) . a 100% resistance to both ampicillin - cloxacillin ( 30 ug ) and cefuroxime ( 20 ug ) antibiotics was demonstrated by the lm isolates tested while the highest sensitivity ( 86.1% ) was obtained with amocillin clavulanate ( 30ug ) ( tab . the overall high prevalence of 91.8% obtained in this study shows that listeria monocytogenes is a common and constant contaminant of chicken flocks and chicken meat in the study area . this is similar to the findings of gaffa & ayo and chukwu et al . in ready - toeat ( rte ) dairy products ; and nwachukwu et al . , in kunu . our findings further corroborate previous reports that listeria monocytogenes is an important food - borne pathogen and is widely distributed in food , environmental and clinical samples [ 2 , 34 , 35 ] . as observed from our findings , the meat samples had higher incidence of l. monocytogenes ( 95.8% ) when compared to cloacal samples ( 91.5% ) . these higher counts in meat could have resulted from the unhygienic handling practices of meat handlers and processors . as reported , contamination usually arises from unwholesome contacts of meat with excretions from skin , mouth and nose of the meat processors [ 36 , 37 ] . it also suggests likely crosscontamination of raw processed chicken by improperly cleaned and disinfected processing environment and to a lesser degree from the live chicken . this finding concurs with similar findings by cox et al . and kanarat et al . which put processing as a major hazard of crosscontamination . the very high prevalence in raw processed chicken meat samples in this study is similar to the report by gibbons et al . which indicated 90.9% prevalence in raw meat . these findings coupled with poor food handling practices in the study area therefore portends serious health hazards to the public considering possible contamination with other raw food items during food preparation . comparatively , most listeria cases are reported in highincome countries , while cases are much more likely to go unreported in developing countries . most cases of listeriosis are sporadic and have been reported in high - income countries , where incidence is quite low but fatality rate is high . recently , effimia reported a 14.4% prevalence of l. monocytogenes in ready - to - eat food products in greece while wu et al . observed a 20% prevalence in retail foods in china . important outbreaks have also occurred - for example , an outbreak of listeriosis from cantaloupes in colorado , usa , in 2011 resulted in infection of 147 people and 33 deaths , making it the deadliest recorded us foodborne outbreak since the us centers for disease control and prevention ( cdc ) began tracking outbreaks in the 1970s [ 43 - 44 ] . listeriosis often results in admission to intensive - care units , which makes l. monocytogenes the third most costly foodborne pathogen in the usa per case in 2010 , after clostridium botulinum and vibrio vulnificus . ivanek and colleagues estimated that the annual cost of l. monocytogenes in the usa was us$23 billion to 22 billion , and the annual benefit of listeria food safety measures was $ 001 billion to 24 billion . our findings also observed a higher lm prevalence among poultry flocks on deep litter than those in battery cage system . a previous report indicated that lm can survive and multiply in wet litter and thus serves as a source of contamination to the poultry flock . this may also explain the higher lm prevalence recorded among broilers than layers in this study since broilers were in most cases raised on deep litter system . litter should therefore be regularly changed and be protected from moisture . also , it should always be stored in an enclosed location in order to protect it from pests such as wild bird so as to avoid contamination by wild life . similarly , the results of this study also suggest a significant association between the breeds of poultry flocks and lm prevalence , with the isa brown breed showing the least prevalence . this could be as a result of possible varying resistance associated with different breed types . a further research into the genetic variations of breeds of poultry with reference to resistance / susceptibility to disease organism is required . on the other hand , while there was no statistically significant association between lm prevalence and biosecurity levels of the different farms , lm prevalence was highest in farms with low biosecurity level . previous studies have also showed that farms with low biosecurity level have increased risk of lm contamination [ 47 , 48 ] . in addition , most of the listeria monocytogenes isolates obtained in this study showed profound resistance to the majority of the common antibiotics with 100% resistance to ampicillin cloxacillin and cefuroxime . a similar report was previously made by adetunji and ishola and nwachukwu et al . who revealed a profound resistance to ampicillin , which is the drug of choice for treating listeriosis . it was , however in contrast to the report by david and odeyemi who found that broad - spectrum drugs like chloramphenicol and fluoroquinolones were significantly effective against this organism . again , the susceptibility of most of the listeria monocytogenes to gentamicin sulphate in this study is similar to previous reports [ 51 , 52 ] which indicated susceptibility of all the l. monocytogenes obtained to this antimicrobial agent . the susceptibility of most of the l. monocytogenes in this study and previous studies to gentamicin sulphate plausibly suggests that this antimicrobial remains an alternative regimen against the organism . given the multiple resistance shown by the l. monocytogenes to anti - microbial agents , the implication could be that the cost of treatment will be very high when humans are infected with these zoonotic pathogens ; assertions which are in agreement with other reports [ 52 , 53 ] . similarly , the high incidence of listeria monocytogenes in cloacal samples ( 64.8% ) may be attributed to the constant ingestion of listeria - contaminated feed and water . this is similar to findings by schlech et al . and gravani which stated that listeria are mainly found in soil , silage and water . though , the gut of birds is a usual habitat for listeria monocytogenes ; skovgaard and larpent reported a common occurrence of liseria monocytogenes in animal feaces . despite the high prevalence of listeria monocytogenes in this study , most of the chickens showed no sign of infection . , that chickens are faecal carriers of the organism and may contaminate the litter and environment of the poultry house . also , there seemed to be no significant increase in lm counts with total microbial load , as samples with highest lm counts did not necessarily have the highest microbial load , and vice - versa . this could be explained by the fact that lm is very hardy and persists in the environment , resisting most cleaning and disinfectant techniques , unlike many other bacteria which are eliminated by cleaning and disinfecting [ 49 , 57 ] . this study showed a high overall incidence ( 91.8% ) of listeria monocytogenes in poultry flocks and poultry meat in oyo state , nigeria . the higher incidence in meat suggests post - slaughter contamination and portends health hazards to the public through contact between these raw meat and other processed foods . it also shows that poultry flock types and breeds were significant factors associated with lm contamination . in addition , the resistance of listeria monocytogenes isolates to most of the antibiotics in this study is a matter of concern both to the future management of poultry diseases as well as public health . we therefore recommend that farm tofork principles of hygiene should be stepped up particularly among poultry and other food handlers in order to limit contamination with food pathogens . standard operating procedures ( sop ) and hazard analysis and critical control points ( haccp ) should be developed and implemented by poultry regulatory agencies such as poultry association of nigeria ( pan ) and poultry farmers of nigeria ( pfn ) . government should enforce prompt registration and periodic monitoring of all poultry farms and abattoirs in order to institute measures to check the sanitary levels of farms and abattoirs and enforce strict adherence to hygiene standards on a continual basis .

summaryintroduction.food contamination with listeria monocytogenes is on the increase posing threats to public health with growing trends in food products recalls due to suspected listeria contamination.methods.we conducted a cross - sectional study to determine the prevalence and antibiotic susceptibility profiles of listeria monocytogenes ( lm ) among 71 randomly selected poultry farms in oyo state , nigeria . a total of 450 samples comprising cloacal swabs ( 426 ) and randomly selected dressed chicken meat ( 24 ) were cultured for lm isolation using brilliancetm selective listeria agar with antibiotics and microbial load count with nutrient agar . further identification was done using microscopic , biochemical characterization and antibiotic sensitivity tests . data were analysed using bivariate analysis and student t-test.results.an overall prevalence of 91.8% lm contamination was obtained comprising 91.5% ( 390/426 ) in cloacal swabs and 95.8% ( 23/24 ) in meat . the prevalence of lm in cloacal samples was significantly associated with poultry type ( p = 0.008 ) and breed ( p = 0.000 . in addition , all the flocks had at least one positive sample yielding 100% flock prevalence . antibiotic sensitivity test revealed that most of the isolates were resistant to common antibiotics like ampicillin - cloxacillin and cefuroxime.conclusions.the results revealed a high level of contamination with lm in the poultry flock and meat and the observed resistance to most common antibiotics has implications for future disease control as well as public health . there is need to step up routine screening of food animal products for listeria contamination as well as measures towards reducing such contaminations .

Introduction Methods STUDY SITE, DESIGN, POPULATION AND SAMPLING MICROBIOLOGICAL ANALYSIS FOR ASSESSMENT OF THE MICROBIAL LOAD ON SAMPLES SCREENED ANTIBIOTIC SUSCEPTIBILITY TESTING DATA ANALYSIS Results PREVALENCE OF TOTAL BACTERIA COUNT AND ANTIBIOTIC SENSITIVITY TEST Discussion Conclusions

a survey of modern studies of colonial medicine suggests that there is a lack of precision about what colonial medicine is or was , and a lot of overlap exists with two other terms : tropical medicine and imperial medicine . haynes , imperial medicine , 111 ; chakrabarti , medicine and empire , ix xxix . for a wider discussion , see loomba , colonialism / postcolonialism , 17 ; osterhammel , colonialism , 122 . these terms are not interchangeable , even if often used indiscriminately . tropical medicine is seen as a discipline of medical science that developed in the late - nineteenth century to tackle the diseases of hot climates . it grew out of older discourses and experiences of illness in the colonies , flourished in research laboratories in europe and was exported back to the colonies to tackle malaria , sleeping sickness , cholera and nutritional deficiencies . arnold ; chakrabarti , medicine and empire , 14159 . for older applications of the tropics , see cagle , beyond the senegal , in this issue . on the need to define medicine , see ernst , beyond east and west , 510 ; chakrabarti , medicine and empire , xiii xiv ; roy , science , 448 . arguably once exported , it became a form of colonial medicine . colonial medicine has been seen as a tool of empire enabling settlement in the colonies , and tropical medicine as a method of carrying out the imperial project , however that is perceived , but the relationship between them is not clear - cut . the main difference between colonial and imperial medicine seems to be that the former refers to medicine practised in the colonies . as mark harrison notes , colonial medicine was pre - eminently a medicine of place . , on the other hand , refers to health policies imposed by the imperial power and linked to metropolitan policies and institutions . the policies affected the colonies but usually did not originate there , although overseas experience could influence implementation of policy in europe . a good example of this is smallpox vaccination imposed , resisted and negotiated in european countries and european colonies alike from the nineteenth century . bulletin of the history of medicine 83 ( 2009 ) is a special issue on global smallpox vaccination . to a very great extent , haynes , imperial medicine , 111 ; chakrabarti , medicine and empire , ix xxix . for a wider discussion , see loomba , colonialism / postcolonialism , 17 ; osterhammel , colonialism , 122 . arnold ; chakrabarti , medicine and empire , 14159 . for older applications of the tropics , see cagle , beyond the senegal , in this issue . on the need to define medicine , see ernst , beyond east and west , 510 ; chakrabarti , medicine and empire , xiii xiv ; roy , science , 448 . bulletin of the history of medicine 83 ( 2009 ) is a special issue on global smallpox vaccination . it is possible to identify common themes and debates that have emerged over time in studies of colonial or imperial medicine . the first historians of empire in the nineteenth century primarily saw european medicine as a means to keep european troops , sailors and settlers alive in a hostile environment and only later did it become the gift of civilization to the benighted peoples of a disease - ridden world . from the 1920s , however , medicine in the colonies slowly began to be seen in a more critical light as a method of subjugating indigenous peoples with disruptive and often unhealthy consequences ; imperial expansion began to be seen as the cause of disease . the picture shifted again from the 1960s as decolonized nations considered their approaches to disease and malnutrition . as a result , historians of the new fields of colonial and postcolonial medicine focused from the 1980s on themes of oppression and racial tension leading to or resulting from inequalities in healthcare , especially in the context of epidemic or nutritional disease and madness . studies of colonial medicine are often full of dichotomies : centre / periphery , colony / metropole , east / west , western / indigenous . postcolonial studies usually try to deconstruct these dichotomies or at least to show that the relationships were two - way even if still unequal . ; ernst , beyond east and west ; chakrabarti , medicine and empire ; arnold , colonizing the body ; haynes , imperial medicine ; anderson , postcolonial histories of medicine ; western medicine as contested knowledge , ed . cunningham and andrews ; vaughan , curing their ills . marks , what is colonial about colonial medicine ? ; ernst , beyond east and west ; chakrabarti , medicine and empire ; arnold , colonizing the body ; haynes , imperial medicine ; anderson , postcolonial histories of medicine ; western medicine as contested knowledge , ed . cunningham and andrews ; vaughan , curing their ills . the most recent scholarship tries to explore the agency of numerous groups : indigenous healers , indigenous medical practitioners trained in europe or in colonial institutions based on european models , local government officials and both expatriate and native sick people . the historical approaches of subaltern studies and history from below have interacted very well in the field of the history of medicine to recover the stories of previously neglected or marginalized actors . this process mirrors the enlarging scope of medical history in recent years beyond institutions , physicians and technologies to include patients , low - status health - workers , and more diverse practices that affect mind and body such as bathing , waste disposal , drug use and eating . digby , ernst and mukharji ; chakrabarti , medicine and empire ; chakrabarti , materials and medicine ; imperial medicine and indigenous societies , ed . arnold ; attewell , interweaving ; winterbottom , of the china root ; mobilising medicine , ed . cook and walker ; roy , science ; porter , the patient 's view ; subaltern studies , ed . ernst also argued in 2007 for area studies : why not a social history of south asian medicine(s ) rather than a history of colonial medicine in india ? it seems that the former is the approach of the oxford handbook of the history of medicine ( 2013 ) , which includes chapters on medicine in latin america , sub - saharan africa and australasia , rather than general chapters on colonial medicine and tropical disease , which was the approach taken by the companion encyclopedia of the history of medicine ( 1993 ) . the only chapter to mention the word colonial in its title in the new handbook is interestingly mark harrison 's study of south asian medicine . this title suggests that there will continue to be debate about how to study medicine in former colonies . ernst has already warned that national histories of medicine run the risk of repeating the old grand narratives , and that decentring colonialism diminishes the immensity of the abuses that were carried out in its name . ever since michel foucault put pen to paper it has been a truism that medicine is always about power . yet ernst feels that to compare power relations in indian medicine too closely to those in british medicine might excuse atrocities specific to colonialism . digby , ernst and mukharji ; chakrabarti , medicine and empire ; chakrabarti , materials and medicine ; imperial medicine and indigenous societies , ed . arnold ; attewell , interweaving ; winterbottom , of the china root ; mobilising medicine , ed . cook and walker ; roy , science ; porter , the patient 's view ; subaltern studies , ed . other historians will disagree with ernst , especially those working on parts of europe where healthcare relationships are not yet well - studied , such as portugal . it does not seem surprising that timothy walker is one of the most prominent historians working on medicine in the portuguese empire in the english language . he began his career by researching the role of physicians in the portuguese inquisition who sought to eradicate magical healing practices in portugal . he now explores the role of jesuit physicians , indigenous healers and pharmaceutical commodities across the portuguese empire , similarly drawing out tensions between different kinds of healing and different attitudes towards illness . moving from one hegemonic yet contested system in portugal to others in asia walker , doctors , folk medicine and the inquisition ; walker , acquisition and circulation ; walker , medicines trade . for health and hegemony , see arnold , colonizing the body , 24089 . european medicine was not monolithic enforced colonization of spaces , minds and bodies occurred in european indigenous medicine as well as abroad and it would be a mistake to see these as separate phenomena . walker , doctors , folk medicine and the inquisition ; walker , acquisition and circulation ; walker , medicines trade . for health and hegemony , the most recent research on colonial medicine focuses on these complex connections , not just those between colonizing powers and the colonized , but also those between colonies and different empires . too great an emphasis on the british empire is now recognized as a hindrance to understanding other colonial contexts . comparative work on healthcare in many different places seems to be the way forward , with the long - term goal of provincializing europe within its global context , however impossible this might seem and however fraught the term global might be . middle ages just as the term becomes problematic for some modernists . a new journal the medieval globe launched in 2014 with a fine special issue on the black death . see also abu - lughod , before european hegemony ; ruz , medieval historians and the world . for an overview of modern debates , see potter and saha , global history . what is sometimes called the centre or the metropole in colonial or imperial history is only another locality , often one that is peripheral to many people . the sociologist of science bruno latour argues that no place dominates enough to be global and no place is self - contained enough to be local ; for him some places are just more connected than others . global knowledge can therefore be defined as knowledge that passes through multiple localities always changing in nature as it travels . focusing too much on the global in medical history can obscure the specificity of healthcare in particular localities . colonial medicine should therefore continue to mean medicine practised in the colonies , connected to the colonizing power through imperial / global processes and policies , but unique to each locale due to climate , indigenous beliefs and practices , regional geo - politics and other complex factors . this essay will therefore consider colonial medicine in the middle ages as the medicine of places into which certain modern colonial medicine is seen as a tool enabling colonization ; a method of creating others in the colonies and of othering the colony itself ; a method of drawing boundaries between them and us by pathologizing racial difference or by controlling access to or regulating healthcare . medicine is seen as a prism through which to view colonialism in action and as a method of critiquing it since healthcare practices usually accompanied and were influenced by colonial settlement . some of these aspects can be identified in the middle ages . this essay presents some preliminary research that will help to bring medieval colonial medicine into view . for example , chakrabarti , materials and medicine , compares india and the caribbean . chakrabarty , provincializing europe ; loomba , colonialism / postcolonialism , 2556 . a new journal the medieval globe launched in 2014 with a fine special issue on the black death . see also abu - lughod , before european hegemony ; ruz , medieval historians and the world . for an overview of modern debates , see potter and saha , global history . before considering whether there was a colonial medicine in the middle ages , it is important to discuss the concept of medieval colonialism . the idea that there was medieval colonization became attractive to scholars after the publication of robert bartlett 's highly influential the making of europe in 1993 , although the matter had already been debated for decades . by , most medievalists mean the migration of peoples of one ethnicity and/or region to another to become the culturally and politically dominant people in that new region through the imposition of their own laws , and the marginalization of the existing population . abulafia and berend ; muldoon and fernndez armesto , introduction ; jensen and reynolds , european colonial experience ; verlinden , beginnings of modern colonization ; laiou , many faces of medieval colonization . it has been argued by bartlett and his followers that there was colonization of this kind during the period of the crusades to the holy land , especially after the establishment of four latin christian states in syria and palestine ( 10991291 ) , during the norman invasions of england and sicily in the late eleventh century and the anglo - norman / english conquest of ireland and wales during the twelfth and thirteenth centuries , and during the settlement of large areas of central europe and the iberian peninsula over many centuries . all of these forms of expansion fit into at least one of the colony types identified in osterhammel , colonialism , 410 . it is interesting that earlier visigothic , suevi , arab and berber settlements of the iberian peninsula are rarely seen in the same light as the later christian expansion , whereas ancient roman , greek and phoenician settlements often are seen as forms of colonialization . i would like to thank both my anonymous reviewers for raising this point in different ways . it is clear that there is a great deal of myopia involved in the history of expansion and much more comparative cross - cultural and cross - chronological work needs to be done . in more recent years , critics have argued that the concept of medieval colonization relates to the nineteenth - century contexts of the first professional historians rather than to medieval reality . piskorski . nevertheless , there is still a general acceptance that there were aggressive migrations during the middle ages . abulafia and berend ; muldoon and fernndez armesto , introduction ; jensen and reynolds , european colonial experience ; verlinden , beginnings of modern colonization ; laiou , many faces of medieval colonization . all of these forms of expansion fit into at least one of the colony types identified in osterhammel , colonialism , 410 . it is interesting that earlier visigothic , suevi , arab and berber settlements of the iberian peninsula are rarely seen in the same light as the later christian expansion , whereas ancient roman , greek and phoenician settlements often are seen as forms of colonialization . i would like to thank both my anonymous reviewers for raising this point in different ways . it is clear that there is a great deal of myopia involved in the history of expansion and much more comparative cross - cultural and cross - chronological work needs to be done . there has been less acceptance of the idea that there was a medieval colonialism ; that is , colonization as a deliberate centralized policy with specific aims . this is perhaps ironic considering the growing interest in expanding medieval empires , the special thematic strand of the international medieval congress in leeds in 2014 was empire. see also reynolds , empire ; burbank and cooper , empires in world history ; muldoon , empire and order ; hart , comparing empires . for debates about what constituted an empire , see mccleery , from the edge of europe ; morier - genoud and cahen , introduction . and the important influence of postcolonialism on medieval literature . kabir and williams ; holsinger , medieval studies ; decolonizing the middle ages critics of medieval postcolonialism argue that medievalists lack the engagement with temporal and spatial colonialism that their modernist colleagues had to have in order to develop the concept of postcolonialism in the first place . although in the 1970s joshua prawer and robert i. burns argued strongly for the colonialism of the crusader kingdom of jerusalem and the post - conquest kingdom of valencia respectively , their approaches seem to have been abandoned . these approaches were explored in many works but see especially , prawer , latin kingdom ; burns , medieval colonialism . iberianists have preferred to focus on convivencia and reconquista as methods of debating population movements , exploitation of minorities and cultural exchange , although these terms are also hotly debated , especially convivencia . wacks , reconquest colonialism ; soifer , beyond convivencia ; wolf , convivencia in medieval spain ; ray , beyond tolerance ; glick and pi - sunyer , acculturation . thomas madden , historian of venice and the crusades , categorically denied that the crusades were the first form of european colonialism in an interview he gave in 2006 . he argued that europe was not a dominant cultural force in the middle east , the numbers of europeans in the crusader states were tiny , the motivation of crusaders was religious not economic , and maintaining the latin east was a drain on european resources while at the same time the crusader states were independent of any european state . most medievalists believe that colonialism has to mean a mercantilist policy to conquer , settle and exploit overseas territories on a large scale . colonies must remain dependent on the colonizing power and from them the colonizer has to draw great wealth in the form of natural resources and markets for exported goods . this fairly narrow interpretation of colonialism may apply to some european colonies in some periods but it continues to be contested . loomba , colonialism / postcolonialism , 5 , 2243 ; osterhammel , colonialism , 1617 . although it is not possible here to investigate thoroughly the varying natures of colonialism , it seems that there is scope to open up the medieval debate much more . empire. see also reynolds , empire ; burbank and cooper , empires in world history ; muldoon , empire and order ; hart , comparing empires . for debates about what constituted an empire , see mccleery , from the edge of europe ; morier - genoud and cahen , introduction . altschul , future of postcolonial approaches ; altschul , postcolonialism ; postcolonial middle ages , ed . kabir and williams ; holsinger , medieval studies ; decolonizing the middle ages , ed . these approaches were explored in many works but see especially , prawer , latin kingdom ; burns , medieval colonialism . wacks , reconquest colonialism ; soifer , beyond convivencia ; wolf , convivencia in medieval spain ; ray , beyond tolerance ; glick and pi - sunyer , acculturation . loomba , colonialism / postcolonialism , 5 , 2243 ; osterhammel , colonialism , 1617 . prawer thought nearly forty years ago that the crusader states were indeed anomalous colonial societies because of their political independence . yet the extent to which they were culturally , religiously and economically independent of europe is much debated . it is important to distinguish , on the one hand , between the military and religious expeditions sent out from western europe from the late eleventh century , which are strictly what should be known as the crusades , and on the other hand , the latin christian polities established in the holy land , which had settlement needs that could be described as colonialist but were easily subjected to royal authority when rulers of england , france and the german empire came to the holy land . as far as the iberian peninsula is concerned , it is difficult not to see the deliberately conquered and systematically dominated and exploited former muslim territories of the iberian peninsula in a colonialist light , especially from the 1230s1240s after the conquests of seville , crdoba , valencia and faro . fletcher , reconquest and crusade ; o'callaghan , reconquest and crusade ; lay , reconquest kings of portugal . for an alternative view , see linehan , at the spanish frontier . although it might be useful to describe seville as a colonized city in the thirteenth century , it does not seem appropriate to do so in the fifteenth century , any more than london was still a colonized city in the thirteenth century . fletcher , reconquest and crusade ; o'callaghan , reconquest and crusade ; lay , reconquest kings of portugal . for an alternative view , see linehan , at the spanish frontier . we also need to consider catalan and genoese settlements found across the mediterranean , and the key role played by pisa , genoa and venice in the crusades , the crusader states and , in the case of the last two cities , in atlantic expansion . it is not an accident that columbus was genoese or that a venetian wrote the best source for the portuguese exploration of west africa in the fifteenth century . fernndez armesto , before columbus . for the writings of the venetian alvise da cadamosto , see voyages of cadamosto , and cagle , beyond the senegal . abu - lughod , before european hegemony , argues that italians entered the atlantic because plague caused the collapse of their previous economy , but this explanation ignores iberian merchants and the effects of plague on the iberian peninsula . catalan , genoese and venetian policy towards the indigenous populations of menorca , sardinia and crete respectively can certainly look like colonialism . late medieval genoese settlement at caffa in the black sea is similar to the colonial enclaves or island settlements of later centuries . fernndez armesto , before columbus ; laiou , many faces of medieval colonization , 1718 ; lourie , crusade and colonization ; balard , genuensis civitas in extremo europae . in fact , islands and enclaves were amongst the first european , i.e. latin christian , colonies in the mediterranean , the black sea and the atlantic , and amongst the last european colonies around the world ( in order of acquisition : ceuta , goa , maco , the falklands , gibraltar , hong kong ) . european settlement in much of africa , east asia and south asia was always limited in contrast to the white settler colonies of north america , south africa and australasia . not all colonies were financially lucrative all of the time and most were not initially ; some failed altogether . relationships between colonies and the colonizer were often fraught and could be quite weak across the vast distances involved . fernndez armesto , before columbus . for the writings of the venetian alvise da cadamosto , see voyages of cadamosto , and cagle , beyond the senegal . abu - lughod , before european hegemony , argues that italians entered the atlantic because plague caused the collapse of their previous economy , but this explanation ignores iberian merchants and the effects of plague on the iberian peninsula . fernndez armesto , before columbus ; laiou , many faces of medieval colonization , 1718 ; lourie , crusade and colonization ; balard , genuensis civitas in extremo europae . fernndez armesto , before columbus ; gillis , islands of the mind . the narrow approach to colonialism relates to a tendency to see colonialism as a british phenomenon in the interview in 2006 madden compared the crusader states to british north america thereby obscuring other forms of colonialism . some modern colonialist forms were developed over centuries , often through conscious copying of older imperial models , and many had medieval precursors . see loomba , colonialism / postcolonialism , 1103 ; hart , comparing empires , 5376 , for debates about modern colonialism , continuities with medieval colonialisms , and the copying of models . as bartlett says in the concluding lines to the making of europe : see loomba , colonialism / postcolonialism , 1103 ; hart , comparing empires , 5376 , for debates about modern colonialism , continuities with medieval colonialisms , and the copying of models . the european christians who sailed to the coasts of the americas , asia and africa in the fifteenth and sixteenth centuries came from a society that was already a colonizing society . europe , the initiator of one of the world 's major processes of conquest , colonization and cultural transformation , was also the product of one . bartlett , making of europe , 314 . for a similar argument in relation to the colonialist making of medieval france , see symes , middle ages between nationalism and colonialism . in relation to the colonialist making of medieval france , see symes , middle ages between nationalism and colonialism . this argument is of course similar to that of felipe fernndez armesto , who asserts that patterns of island colonialism as practised by genoa and the crown of aragon were replicated in the canary islands and the caribbean . however , fernndez armesto downplays the portuguese role in this process , describing portugal 's expansionist royal family as an impecunious and parvenu dynasty , a portrayal that researchers are still trying to combat . see in contrast , hair , before vasco da gama ; miranda , before the empire ; subrahmanyam , portuguese empire , 3054 . stevens - arroyo , the inter - atlantic paradigm , argues instead for different processes of colonization on either side of the atlantic . see in contrast , hair , before vasco da gama ; miranda , before the empire ; subrahmanyam , portuguese empire , 3054 . stevens - arroyo , the inter - atlantic paradigm , argues instead for different processes of colonization on either side of the atlantic . the negative portrayal may stem from the problem of fitting portuguese expansion into the patterns found in british or spanish colonialism . the portuguese experience started before the traditional date of 1492 on the uninhabited island archipelagoes of madeira , cabo verde , the azores and so tom e prncipe , and in west africa using a negotiated model of settlements and factories similar to that of the genoese . only in morocco , starting with the capture of ceuta in 1415 , and for a brief period a hundred years later , was there an aggressive policy of conquest . the early - sixteenth - century aggression was in fact the viceroy afonso de albuquerque 's ( d. 1515 ) grand strategy for controlling maritime commerce not an attempt at a land empire . he focused on commercial chokepoints such as the straits of malacca and hormuz , and identified goa , which he captured in 1510 , as key to transoceanic routes . disney , history of portugal , ii : 177 , 11927 ; newitt , portuguese overseas expansion , 3697 . isabel dos guimares s refers to the azores , settled from the 1430s , as a premature colonial experiment , and her comment could equally well apply to madeira , officially discovered in c.1420 . . some of these islands must have been visited much earlier as they are depicted on fourteenth - century maps : disney , history of portugal , ii : 84 . , however , does not mean that the islands were not colonies , but that their early settlement causes them to fly under the historiographical radar . the uninhabited nature of most of portuguese islands excluded them from discussions about indigenous rights , in sharp contrast to the canaries and the caribbean islands where contact with native peoples triggered intense debate . in the case of the canaries this happened as early as the mid - fourteenth century in the writings of petrarch ( d. 1374 ) and boccaccio ( d. 1375 ) . abulafia , discovery of mankind , 3348 . outside portugal , few historians show interest in its pre-1498 expansionism . the portuguese sphere of economic interests is not mapped in janet abu - lughod 's influential study of medieval world systems . disney , history of portugal , ii : 177 , 11927 ; newitt , portuguese overseas expansion , 3697 . . some of these islands must have been visited much earlier as they are depicted on fourteenth - century maps : disney , history of portugal , ii : 84 . in fact , much of the iberian peninsula ( catalonia sometimes excepted ) is left out of surveys of european religious and political culture , something that nadia altschul calls the coloniality of knowledge . the portuguese could share the view expressed by dagenais and greer that the spanish middle ages is a product of a double colonization of both territory and of historiography : the story of the middle ages has largely been told from a northern european perspective , a perspective that pushes the iberian middle ages to an exotic , orientalised fringe . yet it is a mistake to see the portuguese princes conquest of ceuta in north africa in 1415 as a fringe activity : that same year their first cousin henry v of england , also the son of a usurper , pursued his own expansionist ambitions at agincourt . the decision to retain ceuta , flying in the face of military logic and at huge cost , surely does not differ so much from english attempts to hang on to calais ( the last vestige of henry v 's ambitions ) until 1558 against all the odds . although the german physician jerome mnzer thought at the end of the fifteenth century that the portuguese king got more honour than profit out of ceuta , this does not mean it was not a colony . mnzer , viaje , 76 ; miranda , before the empire , 78 . mnzer , viaje , 76 ; miranda , before the empire , 78 . similarly the portuguese atlantic islands were subjected to deliberate colonialist policies from the 1420s , involving the mass implantation of european institutions , tax systems , commercial activities , agriculture and slavery ( a mode of production common in mediterranean sugar production ) . none of this means that settlement was straightforward or non - negotiable : the islands had a diverse population of portuguese peasants and artisans , merchants of jewish , flemish , italian and iberian backgrounds , and enslaved peoples from the canary islands and north and west africa . greenfield , ilhas ; vieira , emigrao ; rodrigues , organizao ; catz , christopher columbus . the atlantic islands deserve much closer study by both colonial historians and medieval historians interested in empire and expansion . recognizing a colonial context to atlantic and intra - peninsular expansion does not run counter to the widely accepted crusading , reconquest or chivalric motives for it ; it simply suggests that colonialism was both an economic and an ideological phenomenon that flourished in the name of different ideologies and within a variety of economic systems . for chivalric and crusading arguments , see russell , prince henry ; goodman , chivalry and exploration . for reconquest arguments , see paviot , les portugais et ceuta . for economic arguments , see barata , portugal and the mediterranean trade ; miranda , before the empire . greenfield , ilhas ; vieira , emigrao ; rodrigues , organizao ; catz , christopher columbus . for chivalric and crusading arguments , see russell , prince henry ; goodman , chivalry and exploration . for reconquest arguments , see paviot , les portugais et ceuta . for economic arguments , see barata , portugal and the mediterranean trade ; miranda , before the empire . the foregoing discussion of medieval colonialism explains why there are few studies of medieval colonial medicine . after the present author gave a conference paper on this topic in 2011 , a member of the audience queried whether it was tautological to ask how colonial was medieval colonial medicine . the question implies that medieval medicine was colonial ; perhaps , it would make more sense to ask whether medieval medicine was colonial . yet the title of marks 's essay on which this essay 's title is based has no quotation marks around either of the words colonial . her title is not tautological since it is widely accepted that the modern world is colonial ; the problem was determining what is colonial about modern medicine . the rest of this essay will argue that there was indeed a medieval colonial medicine in territories where there was colonization . taking the zones in which colonization is widely believed to have occurred before 1492 , the crusader states , norman england and sicily , anglo - norman wales and ireland , central europe , the iberian peninsula , italian and catalan enclaves , the atlantic islands and north / west africa , the fact is that until the 1980s there was little interest in healthcare for most of these regions . the grand narrative of medieval medical history focused on institutional and intellectual matters , often interpreted in a positivist way from a nationalist perspective . the daily practice of healthcare tended to be neglected in favour of intellectual developments . even since the 1980s , health encounters between normans and english or syrians and franks are rarely seen in the same way as similar encounters in early - modern india or mexico . although see bartlett , hanged man , for a resuscitation set in thirteenth - century colonial wales . there has been much written about the dissemination of greek , muslim and jewish medicine via the twelfth - century latin translation movement , but this rarely embeds intellectual concerns in social and cultural contexts . most text books refer simply to disembodied centres of translation in southern italy and spain . there is much less focus on the multi - cultural interface in the eastern mediterranean . although see bartlett , hanged man , for a resuscitation set in thirteenth - century colonial wales . the implication in this neglect of the eastern mediterranean is that crusaders were more intent on killing than healing , but this approach seems simplistic . it is worth comparing the interests of crusaders to those of spanish settlers in the new world , where rebecca earle has been able to study in detail the attitudes of often illiterate conquistadores towards food and health . piers mitchell has done a tremendous amount of work to establish crusader medicine as a viable field , but there is still need for further research on healthcare issues in the eastern mediterranean . for example , the reflections of the syrian nobleman usamh ibn munqidh ( d. 1188 ) , the sole manuscript of which survives in the library of el escorial near madrid , seem ripe for postcolonial analysis ; his much - quoted but little understood writings about food and medical practices are rich in hybridity , mimicry , acculturation and ambiguities about the other . my student joanna phillips is completing a doctoral thesis at the university of leeds on representations of health and disease in christian chronicles of the crusades . my student joanna phillips is completing a doctoral thesis at the university of leeds on representations of health and disease in christian chronicles of the crusades . as far as the western mediterranean is concerned there are many detailed studies of medical practice , especially for well - documented catalonia and valencia . yet when these discuss health encounters between muslims , christians and jews they tend to draw on the conventions of convivencia . it is shared learning or peaceful collaborations that feature most in the scholarship rather than the unequal relationships based on shifting needs and pragmatism that one tends to find in colonial studies ; the work that has been done on medicine and religion in the atlantic world and south asia has a much sharper edge to it . for a recent post - medieval approach , david nirenberg 's work on religious violence in castile and aragon suggests this difference might be how medieval research is framed rather any major contextual difference . if convivencia remains a useful term , it no longer refers solely to peaceful interactions . it is time that medical historians also begin to take a more critical view of patient - practitioner relationships , not because they involved people from different faiths but because they may represent the conquered and the conquerors . moving away from formal medicine to a wider range of technicians of the body is more revealing : studies of wet nurses , prostitutes and food providers in the western mediterranean highlight the vulnerabilities of muslims and converts , especially women . winer , conscripting the breast ; mummey and reyerson , whose city is this ? constable , food and meaning . for technicians of the body , see fissell , introduction . see most of the excellent research by luis garca ballester and michael mcvaugh . for a recent post - medieval approach , see hayden and walker , intersecting religioscapes . winer , conscripting the breast ; mummey and reyerson , whose city is this ? constable , food and meaning . for technicians of the body , see fissell , introduction . even within formal medicine , historians working on licensing systems in the iberian peninsula have interpreted new legislation in valencia in 1329 as a method of limiting the practice of medicine by non - christians , just over ninety years after the conquest of the city in 1238 . garca ballester , mcvaugh and rubio vela , medical licensing ; lpez terrada , medical pluralism ; lepicard , medical licensing ; ferragud , expert examination . this is comparable to the ban on the practice of indigenous practitioners without a licence in goa in 1618 , one hundred and eight years after its conquest by the portuguese . , 268 , gives the date of the ban as 1563 , but pearson , , 41618 , explains that this earlier ban does not seem to have had much effect . , 230 ; boxer , some remarks , 296 . in both the newly conquered parts of the iberian peninsula and in post - conquest mexico and goa , healers of all kinds were originally tolerated due to the needs of the community . it is well - known that the medical licensing systems of the new world were based on those of fifteenth - century spain . similarly , king manuel of portugal ( r. 14951521 ) reformed a licensing system that had been in place since at least 1338 . lpez terrada , control ; mccleery , medical licensing ; mendona , reforma ; lanning , royal protomedicato ; miville , medical pluralism ; dutra , practice of medicine ; boxer , some remarks . there could , for example , be more postcolonial scrutiny of how knowledge , manuscripts , medicinal drugs and foodstuffs were circulated , appropriated , ( mis)attributed and consumed north and south of the pyrenees . such research could draw on debates and approaches developed by early - modern historians of science who have looked at the spanish and portuguese atlantic or who follow the trajectories of particular commodities and practices . garca ballester , mcvaugh and rubio vela , medical licensing ; lpez terrada , medical pluralism ; lepicard , medical licensing ; ferragud , expert examination . upanov , conversion , illness and possession , 268 , gives the date of the ban as 1563 , but pearson , portuguese state , 41618 , explains that this earlier ban does not seem to have had much effect . lpez terrada , control ; mccleery , medical licensing ; mendona , reforma ; lanning , royal protomedicato ; miville , medical pluralism ; dutra , practice of medicine ; boxer , some remarks . to provide one example of how fruitful medieval - modern comparisons could be , it is worth exploring the similarities between the scholars who came down from north of the pyrenees from the twelfth century to find manuscripts in the libraries of castile , exploiting the learning of muslim and jewish physicians and translators , and the later bio - prospecting for plants and therapeutic knowledge that went on in the spanish atlantic . historians interested in what happened to the manuscripts of medieval spain after the christian conquest of toledo in 1085 could pay more attention to research on the construction and circulation of knowledge . there is a debate within the history of science about the extent to which indigenous knowledge travelled with traded commodities : some doubt that imperial bio - prospectors were interested in the cultural baggage that came with a drug or foodstuff . it is possible to argue that the flemish botanist charles de lcluse ( carolus clusius ) in his 1567 latin translation of garcia de orta 's colloquies on the simples , drugs and materia medica of india , first published in portuguese in goa in 1563 , stripped out the indian geo - political material that orta had used to contextualize the drugs , diseases and treatments because they did not matter to him . orta , colquios ; upanov , drugs , health , bodies and souls ; xavier and upanov , catholic orientalism , 8596 ; costa and carvalho , between east and west ; costa , geographical expansion ; medicine , trade and empire , ed . harold cook and londa shiebinger contend that not only were early - modern european botanists primarily interested in the utility and profit to be got from drugs , but they struggled to understand alternative ways of ordering and understanding the world and therefore had to fit their new knowledge into existing european frameworks such as humoural theory . cook , matters of exchange ; cook , global economies and local knowledge ; schiebinger , prospecting ( quotation at 128 ) . orta , colquios ; upanov , drugs , health , bodies and souls ; xavier and upanov , catholic orientalism , 8596 ; costa and carvalho , between east and west ; costa , geographical expansion ; medicine , trade and empire , ed . cook , matters of exchange ; cook , global economies and local knowledge ; schiebinger , prospecting ( quotation at 128 ) . in contrast , marcy norton argues in the case of chocolate that both the equipment and the rituals associated with the drink in elite aztec culture travelled with it to spain ; not via european learned texts but through the undocumented daily pragmatism of travellers . similarly , kapil raj , teresa huguet termes , timothy walker and jnia ferreira furtado have argued for much more complex and less flowing methods of dissemination . they also focus more on the continued use of remedies and foods in their original location . raj , relocating modern science ; huguet termes , new world materia medica ; walker , acquisition and circulation ; furtado , tropical empiricism . it is probable that these diverse approaches represent some of the differences between colonial and imperial medicine discussed at the start of this essay ; some historians focus on writings produced as knowledge for immediate use in the colonies , others explore how these writings were produced for knowledge creation elsewhere . raj , early - modern european botanising ; mccleery , from the edge of europe . so to return to medieval iberian medical manuscripts : these should equally be seen as traded commodities that were produced in an indigenous cultural context but commodified for the cathedral schools , monasteries , universities and royal courts of northern europe and italy . the original identities of the non - christian authors were of limited interest ; even the ancient greek medical authority galen became quasi - christian in the late middle ages . the knowledge within these texts was itself a commodity that travelled from one cultural context to another , adapted for new purposes . alongside the texts also travelled the materials they mention ( sugar , figs , raisins , lemons , saffron ) , many of which became desirable foodstuffs among the same northern elites that collected manuscripts . miranda , before the empire ; childs , trade and shipping ; regional cuisines , ed . nevertheless , these texts were also used in iberian towns by muslims , christians and jews , and later became the mainstay of iberian universities as elsewhere ; the foodstuffs were closely linked to muslims within the peninsula but widely consumed nonetheless . marginal learned medical world ; constable , food and meaning . for the consumption of some of these foodstuffs in portugal , for the curricula of the university of lisbon and the approach of its professors to both new and old learning , see farelo , garcia de raj , relocating modern science ; huguet termes , new world materia medica ; walker , acquisition and circulation ; furtado , tropical empiricism . raj , early - modern european botanising ; mccleery , from the edge of europe . miranda , before the empire ; childs , trade and shipping ; regional cuisines , ed . adamson . marginal learned medical world ; constable , food and meaning . for the consumption of some of these foodstuffs in portugal , see rodrigues and s , sugar and spice , in this issue . for the curricula of the university of lisbon and the approach of its professors to both new and old learning , see farelo , garcia de there was considerable respect already in the twelfth century amongst latin scholars for medical and natural philosophical learning written in arabic or hebrew but based on shared graeco - roman theories . muslim and jewish practical knowledge about foods , magic , drugs and treatments circulated as a by - product of a search for aristotle , galen and ptolemy . yet there was a similar respect for ayurvedic medicine in sixteenth - century india , perhaps because it recognized concepts akin to the humours . furthermore , the transferability of pagan authors to latin christendom was partly due to their prior adaption to monotheism within islamic cultures . galenic medicine was largely taught at european universities through authors like the persians ab al al - usayn ibn abd allh ibn sn ( avicenna , d.1037 ) and abu bakr muhammad ibn zakariy al - rz , ( rasis , d. 925 ) . this process is not dissimilar to the way in which a codex of herbal medicine was produced in mid - sixteenth - century mexico ; this text was not pure nahua medical knowledge but a reinvention of it combined with spanish medicine for new purposes . aztec herbal , ed . gates ; viesca trivio , cdice de la cruz - badiano . a very good recent study of the northern europe 's appropriation of iberian medical knowledge is monica green 's fascinating analysis of the transmission of the writings of the late - tenth - century cordoban surgeon ab al - qsim al - zahrw ( albucasis , d. 1013 ) , normally seen as the high point of medieval surgical expertise . first translated into latin by gerard of cremona ( d. 1187 ) in toledo , no latin manuscripts survive until the mid - thirteenth century , as is the case with most of gerard 's work , and most manuscripts thereafter are italian . most owners were practising physicians and surgeons who may have understood the precise clinical detail . amongst these individuals were a surgeon of the aragonese kings , a jewish physician of the catalan town of perelada and a physician of valencia , but their possession of this book was not a neat circular path back home . green , moving from philology to social history ; savage - smith , practice of surgery . the present essay 's premise is that differences between medical encounters in often fraught contexts such as post - conquest andalusia and mexico may be quantitative rather than qualitative . a useful term is that of the contact zone developed by mary louise pratt for eighteenth- and nineteenth - century travel writing and further developed by londa schiebinger as the biocontact zone where scientific encounters occurred . pratt uses the term to refer to the space of colonial encounters , the space in which peoples geographically and historically separated come into contact with each other and establish ongoing relations , usually involving conditions of coercion , radical inequality , and intractable conflict . pratt , imperial eyes , 610 ; schiebinger , prospecting , 125 ; roberts , situating science . pratt , imperial eyes , 610 ; schiebinger , prospecting , 125 ; roberts , situating science . this kind of contact zone is familiar to medievalists , who might associate it with the idea of frontier , as indeed pratt does also , but they need to start seeing cultural frontiers as much more fraught spaces for colonial healthcare encounters . not long after the conquest of 1492 , an unidentified christian minister , whom l.p . harvey argues was likely to have been the new archbishop of granada , hernando de talavera ( d. 1507 ) , received medical care from an eminent imam , ibn al - mawwaq , when his hand began to swell up . in 1501 , the archbishop of toledo , francisco jimnez de cisneros ( d. 1517 ) , was treated by an elderly curandera , possibly a highly respected midwife and religious leader known from other sources as the mora de beda . she was ironically the kind of muslim healer that cisneros tried prematurely to ban in 1498 ; not only did her community still need her but so did he ! as noted earlier , it was decades before anyone seriously attempted to limit indigenous practice in post - conquest valencia and goa . although these granadan examples of medical practice seem to have been conducted according to the ideals of peaceful convivencia , harvey sets them in the context of growing tensions within the muslim community , partly due to cisneros 's harsh approach to conversion , which led to the revolt of the alpujarras in 14991500 , and the burning of all books in arabic in 1501 , except notably for medical texts . in these two cases , the clinical encounter with the other could be interpreted as something done out of necessity by sick foreigners or as a means to communicate with local power brokers . the methods of healing are significant ; the imam behaved like some saints in both muslim and christian traditions ; that is to say , his touch may have caused the illness in the first place as he had previously kissed the christian 's hand . perhaps it was a form of punishment of the unbeliever followed by a carefully timed healing that increased the healer 's authority . the curandera used non - invasive herbal ointments without giving purges , letting blood or other [ internal ] medicines , providing an alternative to the failed treatments of the court doctors . perhaps the point being made here was that gentler methods might enable a stronger body politic without the need for bloodshed . both these cases can usefully be compared to accounts of cross - cultural clinical encounters in sixteenth- to eighteenth - century india , brazil , mexico and west africa , several of which saw conflicts of opinion , divergent practices and considerable therapeutic success , but also worked symbolically to reflect community tensions . see for example , walker , roles and practices ; cagle , botany of colonial medicine ; cagle , dead reckonings ; salazar simarro and owens , cloistered women ; pearson , hindu medical practice ; fields , pestilence ; sweet , domingos lvares ; miville , medical pluralism . it is not just a case of daily healthcare continuing amidst tensions ; the unequal encounters between new political authorities and conquered practitioners signified much deeper dis - ease and pointed towards the need for political healing that with hindsight was lacking in granada . harvey could have made more of his point that ibn al - mawwaq 's intervention is only known from a seventeenth - century biographical dictionary . the story of the curandera is told by juan de vallejo ( d. after 1547 ) , who wrote after cisneros died in 1517 but whose words were only published in an official see for example , walker , roles and practices ; cagle , botany of colonial medicine ; cagle , dead reckonings ; salazar simarro and owens , cloistered women ; pearson , hindu medical practice ; fields , pestilence ; sweet , domingos lvares ; miville , medical pluralism . harvey could have made more of his point that ibn al - mawwaq 's intervention is only known from a seventeenth - century biographical dictionary . the story of the curandera is told by juan de vallejo ( d. after 1547 ) , who wrote after cisneros died in 1517 but whose words were only published in an official the second example of a spanish biocontact zone that should be seen as medieval is the caribbean in the 1490s . diego lvarez chanca ( d. c.1515 ) was a prominent royal physician from seville who accompanied columbus on his second voyage in 1493 , returning as soon as he could the following year . in a letter to the municipal council of seville , chanca used his medical skills of observation to describe the food and environment of the new world in which he found himself , including accounts of illness , poisonous fruit and cannibalism . he played a crucial role in examining the wound of an indigenous ruler said to have been injured in a conflict related to the deaths of the europeans whom columbus had left behind on hispaniola . chanca did not believe that the ruler was injured , thereby suggesting that the local people had lied about what happened . strangely , chanca 's account has not been much studied from a medical point of view , except in relation to the history of disease , or discussed much in the light of colonial discourse , except in relation to cannibalism . cook , born to die , 2839 ; mccleery , from the edge of europe ; gallinari , diego alvarez chanca ; paniagua , doctor chanca . it might be thought that since chanca 's journey occurred after 1492 and took place in the caribbean , his narrative could be accepted as a modern colonialist account without question . although chanca wrote to the councillors of seville , he was also a royal physician . his role on the expedition was therefore similar to that of the catalan physician arnau de vilanova ( d. 1311 ) , who explained to the king of aragon how to provision his army and avoid health problems on a proposed military expedition against muslim almera in 1309 . chanca is also not dissimilar to the aforementioned german physician mnzer , whose account of a journey around the ( for him ) exotic iberian peninsula in the 1490s may have been a form of espionage authorized by the emperor maximilian ( d. 1519 ) in order to investigate iberian exploration . both chanca and mnzer used food and illness as gauges of otherness . mcvaugh , arnau de vilanova 's regimen almarie ; mnzer , viaje , is rich in medical references . in chanca 's case , his narrative ensured that the councillors of seville saw the indigenous peoples of the caribbean as untrustworthy man - eaters and the new world as an unhealthy place to live , based on standard medieval environmental theories and his clinical skills . it is surely the case that the main difference between earlier and later accounts of medicine used as a tool of empire to describe and thereby dis - empower the other is the vastly greater number of accounts that survive from later centuries rather than anything intrinsically different in their method and approach . cook , born to die , 2839 ; mccleery , from the edge of europe ; gallinari , diego alvarez chanca ; paniagua , doctor chanca . mcvaugh , arnau de vilanova 's regimen almarie ; mnzer , viaje , is rich in medical references . the final biocontact zone to be studied here is the island of madeira . on 27 april 1496 , the town councillors of funchal , the main settlement on the island , discussed the case of a man called andre janoes , newly arrived on madeira and probably from genoa , who claimed to be able to cure a disease called boulhas . he said he could cure it within nine or ten days , eighteen to twenty at the most , as long as the sick person kept to the regimen that he recommended . he was supported in his claims by simo gonalves , the hereditary captain of funchal , who said that andre had already cured his female slave . boulhas is similar to the word bubas widely used from the late 1490s to refer to the apparently new disease much later known as syphilis . for the terminology of plague in portugal , casa das bubas attached to the new hospital of all saints in lisbon : regimento , ed . neither boulhas nor bubas were previously used in the minutes of the funchal town council meetings to refer to disease , despite there often being a lot of detail provided . for example , in 1495 the symptoms of a woman who died of pestilence ( pestenencia ) were described as an ingoa ( swelling ) on the leg and a vergam ( pustule ? ) on the stomach . the town authorities were experienced in tackling disease but they took a different approach to boulhas in 1496 . casa das bubas attached to the new hospital of all saints in lisbon : regimento , ed . not , it is highly significant that the first person on the island to be treated for it was an unidentified enslaved woman . the relative attractiveness of the climate and the gradually less intensive agriculture ( moving for a variety of reasons from a sugar to a wine economy ) meant that madeira saw considerable european immigration and needed far fewer slaves , compared to the other portuguese archipelagos further south where conditions seemed much harsher . it has been suggested that most of the enslaved people to be found on madeira by the mid - sixteenth century ( c.3000 people or roughly 15% of the population ) were probably domestic servants or skilled artisans , many of whom were manumitted at the death of their owner . despite this relative ease of absorption into the population , life was not easy for slaves . they were likely to have been most vulnerable to the frequent periods of dearth , bad weather and pestilence experienced by the isolated islanders . carita , histria da madeira ; matos , origem ; greenfield , ilhas ; nepomuceno , histria de madeira ; vieira , escravos . a woman called cumba , described as a moura so probably originally a north african muslim , was treated so harshly by her master , ferno nunes of funchal , that she died of her injuries . he buried her in his house to conceal the death but the body was found and he had to flee , later in 1489 and 1490 seeking royal pardons which document the crime . the fact that a hue and cry was raised when the body was found implies that this was not deemed acceptable behaviour in funchal . the 1491 pardon of joo vaz ousel , a knight of madeira , describes how he was exiled to tangiers for killing his male slave ; therefore the prosecution that followed cumba 's death was not an isolated example . carita , histria da madeira ; matos , origem ; greenfield , ilhas ; nepomuceno , histria de madeira ; vieira , escravos . documents like these suggest that the otherwise unknown slave of simo gonalves who was cured by andre janoes in 1496 was fortunate in that she was likely to have been a domestic servant of the most powerful man on the island and therefore probably on her way towards fuller integration into the community and deserving of a reasonable level of protection . however , it is also the case that the dead slaves referred to above had become the objects of an increasingly bureaucratic state ; their deaths reflect the expansion of legal process , not care for individuals . this situation intensified when duke manuel of beja , the feudal lord of the islands , became king of portugal in 1495 , shifting their administration towards lisbon . costa , ii : i v ; the slaves were elite skilled workers whose deaths represented an economic loss to the local community ( whereas the king overrode these local interests by pardoning the culprits ) . similarly , the anonymous female slave 's medical treatment represented an investment in her person which no doubt was intended to have a positive economic outcome . one should not sentimentalize her care ; her treatment by a previously unknown and untested itinerant healer could be seen as a form of medical experimentation on an expendable patient . the possible venereal nature of the woman 's illness suggests that she may have been sexually vulnerable , a condition that she shared with earlier mediterranean slaves . the royal chief - surgeon master fernando sought a pardon in santarm for his muslim slave mariam because she had been forced to sleep with a christian squire . ii : 278 . in goa in 1512 , two portuguese settlers , one of them a physician master afonso , fought bitterly over who would be able to marry a probably hindu woman who had been enslaved and converted for this purpose . costa , ii : i v ; winer , conscripting the breast . yet that woman of goa was considered a suitable wife , at least in india ; mariam 's honour could be upheld by the king ; and andre janoes was permitted to practise medicine on other islanders , not necessarily slaves . his licence implies that the authorities believed that an enslaved person 's symptoms and cure could be repeated on non - enslaved bodies ; there was no fundamental humoural or physical difference between peoples . medicine here can be seen as a tool of empire in that cases involving sex , health and injury allowed royal authorities to intervene in local politics in order to expand imperial power . there was a complaint about the number of foreigners in funchal later in 1496 , but a foreign healer was welcome as was the portuguese physician master afonso in goa . he failed to get a local wife on that occasion but was not punished because his medical skills were desirable in the precarious new colony . vereaes , i : 56970 ; mccleery , from the edge of europe . medicine can also be seen in these cases as a prism through which to view empire in action . race , power , inequality and disease were as significant in granada , funchal , hispaniola and goa in the fifteenth and early sixteenth century as they were already in west africa , as hugh cagle demonstrates elsewhere in this issue , and would be later in asia and the americas . these cases show too that colonial settlement required pragmatism , flexibility and endurance in order to succeed against the odds . biocontact zones were not just places of colonial encounter ; they were spaces were people lived and died . vereaes , i : 56970 ; mccleery , from the edge of europe . this essay has argued that healthcare encounters between conquerors and the conquered across europe and the middle east as well as in the iberian peninsula and its early colonies should be studied as forms of colonial medicine . the essay endeavoured to break down standard periodizations . it also presented a very much broader picture of medicine than readers might have been used to previously , using it as an umbrella term to cover a wide range of interventions affecting the human body . yet a number of issues remain . throughout the essay it is probable that they are not just professional identities and that chronological differences need to be discussed further . first of all , there is the matter of using medical history to critique colonialism , a major feature of modern colonial and postcolonial scholarship . although the abuse of enslaved people has featured as a topic , it is difficult to critique fifteenth - century expansionism from the perspective of modern civil rights . david abulafia takes a rare apologist stance throughout his major book on cultural encounters in the medieval atlantic . historians of nineteenth- and twentieth - century colonial medicine can see direct relationships between past and present healthcare in the countries they study and can use their research to critique relatively recent policies . historians of the middle ages are able to demonstrate the relevancy of their fields to the modern world and can certainly draw attention to modern abuses of power through their writing . yet relevancy and reflection modern europeans can not be held responsible for acts carried out by fifteenth - century people . on the other hand , taking a colonial approach to medicine might be less problematic for medievalists than doing global history . global history is fast becoming a new grand narrative , one that warwick anderson argues is really postcolonial history in disguise . it does seem to be the case in any time period that documenting disease or daily life or following the bumpy connections between people and commodities on the move is very closely tied to expanding networks of power and trade as is the survival of much of the evidence . the fact that there is so much more evidence for healthcare in later centuries should not be seen as mere chance ; the sheer volume of paperwork that was later produced ( not just survived ) is representative of major differences in the exploiting and archiving of power relations . the explosion of information that occurred during the reign of king manuel of portugal from 1495 , although there were precursors , indicates that the 1490s were a very real watershed for portuguese medical history . although there has been considerable research on the network of portuguese charitable confraternities , the misericrdias , as a tool of imperial control , much more work could be done on the bureaucratization of healthcare . see also s , shaping social space ; s , quando o rico se faz pobre ; abreu , training health professionals . just the tip of the iceberg is touched in mendona , reforma . see also s , shaping social space ; s , quando o rico se faz pobre ; abreu , training health professionals . having said all this , medievalists can learn a great deal from modern colonial and postcolonial scholarship . waltraud ernst argues that medical historians should be able to produce good - quality regional histories of medicine(s ) without resorting to colonial discourses . it might be possible to write a new history of medicine in madeira of this kind . yet studies of post - conquest communities must be wary of official documentation that tends to emphasize continuities . reading the town council minutes of funchal from the late fifteenth century might make one think that the town had always been there . its records are closely comparable to those of oporto in northern portugal and both are crucial sources for portuguese urban history . it is worth remembering , however , that the minutes of oporto 's council meetings date back to the fourteenth century ; funchal 's only survive from 1470 . fifty years prior to that there was no town and the island was deserted of inhabitants . understanding the story of how a diverse population came to the island , built a town , resisted disease , regulated markets , brothels and privies and within thirty years became a major sugar producer benefits from a colonial framework . unlike with cabo verde and so tom further south , this approach rarely happens with madeira or the azores , despite similarities in their initial settlement : an example of how political choices that were made in the 1970s during decolonization have affected medieval studies . similarly , understanding how healthcare worked both practically and symbolically in granada , valencia , jerusalem or wales is worth studying comparatively through a colonial lens even if it is just for a short period after the conquest in order to observe similar processes of inequality , resistance , acculturation and hybridization . as nadia altschul pointed out in the first issue of this journal , medieval postcolonial scholars have long been doing this , with particularly fruitful insights to be gained from study of latin america . it is time that medical historians joined in to gain a better appreciation of the colonial past of medieval and modern europe . serious discussion of the health impact of the early empires of spain and portugal may become a very fruitful way of writing the global history of medieval medicine .

colonial medicine is a thriving field of study in the history of nineteenth- and twentieth - century medicine . medicine can be used as a lens to view colonialism in action and as a way to critique colonialism . this article argues that key debates and ideas from that modern field can fruitfully be applied to the middle ages , especially for the early empires of spain and portugal ( mid - fourteenth to mid - sixteenth centuries ) . the article identifies key modern debates , explores approaches to colonization and colonialism in the middle ages and discusses how medieval and modern medicine and healthcare could be compared using colonial and postcolonial discourses . the article ends with three case studies of healthcare encounters in madeira , granada and hispaniola at the end of the fifteenth century .

Modern answers to the question of what colonial medicine is Medieval colonialism Medieval colonial medicine Three biocontact zones Conclusion

zinc is one of the imperative micronutrients required relatively in small concentrations ( 5100 mg kg ) in tissues for healthy growth and reproduction of plants . zinc deficiency in plants leads to reduced membrane integrity and synthesis of carbohydrates , auxins , nucleotides , cytochromes , and chlorophyll and develops susceptibility to heat stress . excessive use of zinc fertilizers also poses problems to humans causing the impaired absorption of iron and copper . the solubility of zn is highly dependent upon soil ph and moisture and hence arid and semiarid areas of indian agroecosystems are often zinc - deficient . in india , maize is grown in a wide range of environments , extending from extreme semiarid to subhumid and humid regions . it is grown in about 8.26 mha with yield being 19.3 mt ( ministry of agriculture , government of india ) . voluminous literature indicates that zn concentration in the grain is inherently very low , particularly when grown on zn - deficient soils . the major reason for the widespread occurrence of zn deficiency problems in crop plants is attributed to low solubility of zn in soils rather than a low total amount of zn . customary application of inorganic zinc partially caters the plant need as 9699% of applied zn is converted into different insoluble forms depending upon the soil types and physicochemical reactions within 7 days of application . microbes are potential alternate that could cater plant zinc requirement by solubilising the complex zinc in soil . microbes solubilise the metal forms by protons , chelated ligands , and oxidoreductive systems present on the cell surface and membranes [ 57 ] . these bacteria also exhibit other traits beneficial to plants , such as production of phytohormones , antibiotics , siderophores , vitamins , antifungal substances , and hydrogen cyanide . in this study we reported the in vitro zinc solubilisation ability of selected strains and their ability to enhance the growth of zea mays l. bacterial strains used in this study were obtained from the culture bank of central research institute for dryland agriculture , hyderabad , india . five each of pseudomonas spp . were designated as p17 , p21 , p29 , p33 , and p74 and bacillus spp . the cultures were originated from composite and rhizospheric soils of diverse rainfed agroecosystems of india . pseudomonas and bacillus spp . were maintained on king 's b and nutrient agar medium at 4c . all the isolates were inoculated into liquid mineral salts medium ( glit ) specified by saravanan et al . containing dextrose : 10.0 ; ( nh4)2so4 : 1.0 ; kcl : 0.2 ; k2hpo4 : 0.1 ; mgso4 : 0.2 ; ph : 7.0 and insoluble zn compound ( zno and znco3 : 0.1% ; agar : 15.0 g ) and autoclaved at 121c for 20 min . actively growing cultures of each strain were spot - inoculated ( 3 l ) onto the agar and plates were incubated at 28c for 48 h. the clearing zone around colony was recorded . quantitative study of zinc solubilization was studied in 150 ml conical flasks containing 50 ml of liquid mineral salt medium . the broth was inoculated with 10 l of overnight grown bacterial inoculum and incubated for 72 h at 160 rpm in an incubator shaker at 28 2c . after incubation , the culture broth was centrifuged and the concentration of zn in the supernatant was estimated in atomic absorption spectrophotometer ( gbc , australia ) . maize seeds of cultivable variety were surface sterilized with 1% sodium hypochlorite for 5 min and washed five times with sterile distilled water . seeds were treated with talc - based inoculum containing 10 cfug of each strain with 0.5% carboxymethylcellulose ( cmc ) as adhesive . the pot culture experiment was conducted in 10 kg plastic pots ( 20 cm dia ) filled with 9 kg sterile red soil ( presterilized for three consecutive days ) with six replications for each treatment . maize cultivable variety seeds treated with bacterial inoculant were sown and the glasshouse condition was set at 28 2c and 70% humidity . pots were watered once in two days with sterile distilled water until 60 days . the experimental setup consisted of 15 treatments namely , five treatments each of bacillus and pseudomonas strains as seed dresser @ 10 mgkg seed ( t1 to 10 ) , commercially available zinc solubilizing bacteria ( t11 ) , farm yard manure ( fym ) @ 10 kgacre ( t12 ) , seeds primed by soaking overnight in 1.0% znso4 ( t13 ) , positive control @ znso4 @ 10 kgacre ( t14 ) , and uninoculated control ( t15 ) . after 60 days of sowing ( das ) , plants were uprooted from the pots carefully and biometric parameters like root volume , shoot length , leaf area ( measured by li 3100 , lincoln , nebraska , usa leaf area meter ) , and dry mass of plants were recorded as the indicative of plant growth . dried plants were finely ground in a mortar and pestle to amorphous powder and 100 mg was taken in 150 ml conical flask containing 10 ml nitric acid ( hno3 ) and perchloric acid ( hclo4 ) in 9 : 4 ratio . the flasks were placed on a hot plate and digested at 300c until the entire plant material turned colourless . the extract was taken in 100 ml volumetric flask and the volume was made to 100 ml with distilled water . these samples were used for estimation of sodium , potassium , and calcium by flame photometer . similarly , micronutrients such as iron , copper , manganese , zinc , and magnesium were estimated by atomic absorption spectrophotometer . the values presented are the means of two independent experiments each with six replicates performed at different occasions . data obtained from all the experiments were subjected to two - way analysis of variance ( anova ) . mean values between treatments were compared with fisher 's least significant difference ( lsd ) test ( p < 0.05 ) . all the selected strains of pseudomonas and bacillus used could effectively solubilize the insoluble zn compounds used , namely , znco3 and zno , under the assay conditions . the zone of solubilisation was comparatively high in zno amended medium as compared to znco3 . size of the solubilisation zone ranged from 14 to 22 mm in znco3 and from 17 to 33 mm in zno incorporated medium . among the cultures , p29 , p33 , and b40 showed the highest solubilisation zone in znco3 ( 22 mm ) , whereas p17 and b40 showed 31 mm zone in zno amended medium ( table 1 ) . quantitative assay for zinc solubilisation revealed that p29 , p33 , and b40 were able to dissolve 17 , 16 , and 16.8 ppm from znco3 , respectively , in liquid medium ( figure 1 ) and they were consistent with the observations on solid medium . however , p17 which was found to be the leading solubilizer on plate agar did not imitate the result in broth amended with zno though significant fall in ph ( 4.1 ) was noted . instead , p29 showed the highest zn solubilisation of 18 ppm available zn , followed by b40 ( 17 ppm ) ( figure 2 ) . across the treatments , significant reduction of ph was observed in the broth cultures amended with znco3 ( ph 3.96.1 ) and zno ( ph 4.16.4 ) . enhanced the plant growth significantly after 60 das ( table 2 ) . among all the treatments , inoculation of maize with talc - based p29 strain showed increased root volume ( 18.3 cm ) . other growth indicating parameters like total dry mass ( tdm ) and leaf area ( la ) were recorded to be the highest in znso4 treatment with 15.25 g and 1161.3 cm , respectively . however , they are statistically on par with p29 treatment where 12.96 g and 1147.5 cm of tdm and la were recorded , respectively ( figure 1 and table 2 ) . significant concentration of n ( 2.268% ) and k ( 2.0% ) was observed in p29 treatment . the highest p ( 0.28% ) concentration was noted in b40 treated plants as compared to other treatments ( table 3 ) . the highest sodium ( na ) concentration in plants was recorded in zn primed , p29 , p33 , and znso4 treatments which were not significantly different . significant ca ( 0.434% ) concentration in plant tissues was found in znso4 treatment followed by p74 ( 0.354% ) and p29 ( 0.341% ) . na k ratio was higher with zsb ( 0.0047 ) and priming ( 0.004 ) treatment ; p29 ( 0.0033 ) and b118 ( 0.0029 ) also recorded the highest ratio of na k ions . p29 significantly enhanced the concentration of zn content ( 278.8 ppm ) in plant tissue as high as 36 , 32.35 , and 43.11% compared to zn priming , znso4 , and control treatment , respectively ( table 4 ) . the highest mn ( 60 ppm ) concentration was also noted in p29 treatment which is on par with znso4 treatments . interestingly , p21 treated plants showed the highest quantity of fe ( 707 ppm ) which is statistically on par with b40 treatment ( 672.8 ppm ) . mg ( 0.23 ppm ) and cu ( 4.8 ppm ) were significantly found higher in inorganic znso4 plants . solubilisation of zinc can be accomplished by a range of mechanisms , which include excretion of metabolites such as organic acids , proton extrusion , or production of chelating agents [ 12 , 13 ] . in addition , production of inorganic acids such as sulphuric acid , nitric acid , and carbonic acid could also facilitate the solubilisation [ 8 , 14 ] . it is apparent from the zinc solubilization data that the solubilization potential varied with each isolate . organic acid production by microbial isolates has been reported to be a major mechanism of solubilization [ 15 , 16 ] . higher solubilization of the insoluble zinc sources was achieved in 72 h. the zinc solubilizing potential also correlated with the zinc levels that are accumulated by plant leaves . the zinc solubilization in our studies could be due to production of organic acids , like gluconic acids ( especially 2-keto - gluconic acids ) . zinc phosphate solubilization by a strain of pseudomonas fluorescens was investigated by di simine et al . . they identified that gluconic acids produced in culture medium helped in solubilization of zinc salts . in our present study also , the acidic ph shown by all the bacterial isolates gives a clue that the solubilization could be due to production of organic acids and higher the production of the same higher is the available zinc in the culture broth ( figure 3 ) . desai et al . reported that higher availability of zn is directly proportional to acidic ph of the culture broth . however , in some potent strains , ph did not fall drastically suggesting that in those strains other mechanisms may be active and this aspect is being accentuated . the present study clearly demonstrated that inoculation with plant growth promoting rhizobacteria significantly enhanced the growth of maize in all dimensions . in this study application of znso4 alone has increased the total dry mass of plants and leaf area . increased leaf area with p21 and p29 treated plants significantly enhanced the root volume and dry mass of plants , also supported by the studies carried out by richardson , who showed that pgpr inoculation effectively increases surface area of roots and root weight . the variation in enhancement of root volume by these strains may be due to the difference in the quantity of phytohormones produced by each strain . auxin is a class of plant hormones of which indole-3-acetic acid is well studied , which has the capacity to enhance long term responses in plants . pseudomonas strains have increased root and shoot elongation in canola , lettuce , and tomato . the plant growth promoting effects of bioinoculant pgpr strains were clearly demonstrated in many studies [ 23 , 24 ] . the positive effects of pgprs on yield and growth of maize were explained by egamberdiyeva , which may be due to n2 fixation ability , p - solubilizing capacity , and phytohormones production . from the tabulated data in tables 24 it could be observed that no particular treatment with the bacterial strain or znso4 or priming or fym treatment could be able to enhance the growth of plants in all aspects . in case of dry mass and leaf area which were higher with the treatments where no bacteria were applied this minor variation in between bacterial treatments could be due to difference in mechanisms of plant growth promotion exerted by different bacterial strains . untreated plants showed poor growth . in the current study , seed bacterization with zinc solubilizing plant growth promoting bacteria resulted in increased plant height ( root volume and shoot height ) ; leaf area ; and dry mass . similar increases in plant parameters were observed in different crops inoculated with pseudomonas , azospirillum , and azotobacter strains [ 26 , 27 ] . this improved growth by pgpr is due to making the increased availability of nutrients and decreasing metal toxicity . the present study indicated that bacterial inoculation of maize with pseudomonas and bacillus significantly increased the nutrient content of n and p in leaves of maize ( table 3 ) . this higher uptake of essential nutrients compared to uninoculated control plants could be justified from the fact that the unavailable forms of these nutrients were solubilized and made available in the root region by applying pgpr . this fact is further strengthened by studies conducted by murty and ladha who demonstrated that azospirillum inoculation increased phosphate and ammonium uptake in rice plants . even though k concentration was higher in other treatments than control , similar trend was not seen in case of na levels ( table 3 ) . results showed that all bacterial treated plants showed significant differences in fe , cu , mn , and zn content in maize leaves ( table 4 ) although differences between various bacterial strains were insignificant . the enhancement of macro- and micronutrient uptake by plants by inoculation with pgpr may be due to their effect on initiation and development of lateral roots , increased root weight , and nutrient uptake . studies by goldstein and liu showed that phosphate and potash solubilizing bacteria may enhance mineral uptake in plants . this evidence confirmed that the percentage of various nutrients estimated in maize plants was significantly and/or relatively increased in bacterial treated plants ( table 4 ) . on observing the critical zn levels of treatments it can be said that the znso4 treatment recorded lower zn levels compared to other treatments . this can be explained from the fact that the presence of readily available zinc source in soil itself is not sufficient for uptake , but also the mobility of the mineral element is required which can be clearly seen in bacterial treatments there by higher presence of zinc levels in maize plants treated with pgpr . z-score statistical ranking method was followed to identify the promising bacterial isolate among all the strains and p29 strain was marked as the top strain compared to other bacteria for maize growth promotion . our study with pgpr and maize revealed that inoculation with beneficial rhizobacteria is an effective method for enhancing the growth of maize and maintaining the nutrient quality . identified potential plant growth promoting rhizobacteria ( p29 ) could be used as bioinput for improving the plant productivity as a substitute to chemical fertilizers and also to correct the nutrient deficiencies in maize for sustainable agriculture .

zinc ( zn ) is one of the essential micronutrients required for optimum plant growth . substantial quantity of applied inorganic zinc in soil is converted into unavailable form . zinc solubilising bacteria are potential alternates for zinc supplement . among 10 strains screened for zn solubilisation , p29 , p33 , and b40 produced 22.0 mm clear haloes on solid medium amended with znco3 . similarly , p17 and b40 showed 31.0 mm zone in zno incorporated medium . p29 and b40 showed significant release of zn in broth amended with znco3 ( 17 and 16.8 ppm ) and zno ( 18 and 17 ppm ) , respectively . the ph of the broth was almost acidic in all the cases ranging from 3.9 to 6.1 in znco3 and from 4.1 to 6.4 in zno added medium . short term pot culture experiment with maize revealed that seed bacterization with p29 @ 10 gkg1 significantly enhanced total dry mass ( 12.96 g ) and uptake of n ( 2.268% ) , k ( 2.0% ) , mn ( 60 ppm ) , and zn ( 278.8 ppm ) .

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

the recent upsurge of interest to explore the medicinal properties of three - dimensional molecular structures and fragments[14 ] has driven the need for synthetic methodologies that can generate complex scaffolds from acyclic precursors in a single step . an exemplar of such reactions is the cycloisomerisation of 1,6-enynes , which generates carbo- and heterobicyclo[4.1.0]heptene derivatives with up to three stereocentres , thus provides extremely valuable compounds for organic synthesis.[57 ] the reaction was first discovered in 1995 by blum and co - workers by using a catalytic amount of ptcl4 . since then , a number of other -acidic transition - metal catalysts were reported , including rh,[9 , 10 ] ir , pt[1214 ] and au catalysts.[15 , 16 ] the first enantioselective examples were provided by michelet and co - workers in 2009 , by using a gold(i ) complex of the chiral diphosphine ligand l1 ( scheme 1).[17 , 18 ] subsequently , the enantiomeric excess ( ee ) values obtained have been superseded by other transition - metal catalysts ( pt , rh[2022 ] and ir ) , but gold is the only reported system that is catalytically active at room temperature . enantioselective au - catalysed cycloisomerisation of 1,6-enynes [ z = o , n - tosyl ( ts ) ] . the catalytic activity of cationic gold complexes continues to be a subject of considerable intrigue and debate . diphosphine ligands were frequently found to be effective for many asymmetric transformations catalysed by gold . more often than not , silver salts are required to activate [ ( diphosphine)au2cl2 ] precursors towards catalysis . this is thought to generate a cationic species i ( scheme 2 ) with sufficient -lewis acidity for catalysis , but the characterisation of such cationic gold complexes remains elusive ; no x - ray crystal structure has been determined yet . most diphosphine ligands do not form chelate rings with gold(i ) , instead linear geometries that may contain short gold gold distances , indicative of aurophilic interactions , are favoured . thus , a significant gold gold cooperative effect may be important for the catalytic activity . in reactions catalysed by [ aucl(pr3)]/agx systems , chloro - bridged species ii ( scheme 2 ) has been isolated and identified . also , the participation of silver in the catalytic reaction ( presumably via bimetallic complexes ) can not be ruled out.[26 , 27 ] formation of cationic gold complexes . in this paper , we report the results of a preliminary study conducted with different diphosphine we have attempted to correlate the structural parameters of the gold complexes to the observed enantioselectivity and to investigate the role of the silver salt on the outcome of these reactions . the study began with a search in the ccdc database for reported structures of chiral atropisomeric biaryl [ ( diphosphine)au2cl2 ] complexes , from which 14 discreet structures corresponding to 9 compounds can be found ( diphosphine = l1l9 , table 1 ) . the major conformational parameters that vary between the complexes are given in table 1 ; namely , the torsional twist about the central aryl aryl bond ( ) , the au cl bond length and the intramolecular auau separation . selected structural parameters for [ ( diphosphine)au2cl2 ] complexes [ a ] dihedral angle of the biaryl backbone , c2-c1-c1-c2 torsion angle , obtained from crystal structures of the [ ( diphosphine)au2cl2 ] structures . [ b ] this work . additionally , two complexes that contained p - phos ( l10 ) and xylyl - p - phos ( l11 ) were also prepared and their structures were determined by x - ray crystallography ( figures 1 and 2 ) . complex [ ( l10)au2cl2 ] was found to crystallise with two independent molecules ( a and b ) in the asymmetric unit ( figure 1 ) . the compositions of [ ( l10)au2cl2 ] ( figure 1 ) and [ ( l11)au2cl2 ] ( figure 2 ) differ only in the substituents on the phosphorus atoms ; phenyl in [ ( l10)au2cl2 ] and 3,5-dimethylphenyl in [ ( l11)au2cl2 ] , yet all three molecules have noticeably different conformations . structures of the two independent molecules present in the crystals of [ ( l10)au2cl2 ] , a ( top ) and b ( bottom ) . the two independent molecules of [ ( l10)au2cl2 ] have similar twists about the central bond ( 88 and 82 ) , whereas that for [ ( l11)au2cl2 ] is substantially different ( 66 ) . for [ ( l10)au2cl2]-a the orientation of the p au bond relative to its adjacent pyridyl ring is the same in both instances ( 47 ) , whereas in [ ( l10)au2cl2]-b they differ markedly ( 88 and 29 for p1 and p2 , respectively ) . in [ ( l11)au2cl2 ] the twists are again similar to each other , but at approximately 25 they are very different to those seen in [ ( l10)au2cl2]-a . as a consequence , both [ ( l10)au2cl2]-a and [ ( l11)au2cl2 ] have approximate c2 symmetry , whereas [ ( l10)au2cl2]-b does not . the consequence of these differing twists is markedly varied intramolecular au1au2 separations of 3.6701(3 ) , 3.1821(2 ) and 4.7921(4 ) in [ ( l10)au2cl2]-a , [ ( l10)au2cl2]-b and [ ( l11)au2cl2 ] , respectively ( table 1 ) . manifestation of aurophilicity is generally indicated by auau distances that are shorter than the sum of two van der waals radii of 3.7 . for the atropisomeric diphosphine ligands ( meobiphep ( l1l3 ) , binap ( l7l9 ) , segphos ( l4l5 ) and p - phos ) it is expected that the auau distance is dependent upon the degree of rotational freedom between the aromatic rings that defines the axial chirality , which is , in turn , determined by the nature of the biaryl that constitutes the scaffold , as well as the p substituents . with this in mind , a scatter plot of the torsional angle between the two central aryl rings against the auau distance was produced ( figure 3 ) and revealed some interesting trends . scatter plot of torsional angles between axially - chiral aromatic rings ( ) [ ] against the auau distances [ ] for atropisomeric diphosphine au complexes . binap series ( ) , meobiphep series ( ) , segphos series ( ) , p - phos series ( ) . greater conformational flexibility was found in the biaryl series meobiphep , segphos and p - phos , for which it is common to find more than one independent molecule for each complex . nevertheless , the auau distance tends to increase with a greater torsional angle between the two aromatic rings . within the meobiphep series ( l1l3 , , figure 3 ) the introduction of tert - butyl and methoxy substituents on the p - aryl group appear to augment both of these values . the difference between l1 and l3 is particularly notable : the presence of an additional methoxy substituent at the para position appears to lengthen the auau distance quite significantly . the same trend can be observed in the p - phos series ( l12 and l13 , , figure 3 ) , in which the presence of electron - donating xylyl groups increased the auau distance . for the segphos series ( l4 and l5 , , figure 3 ) , substitution of pph2 by pcy2 ( cy = cyclohexyl ) groups also has a dramatic effect on the intermetallic distance . as a result , auau distances of < 3.7 were only observed in four of these biaryl diphosphine ligated complexes , which all contained pph2 substituents . thus , it appeared that intermetallic interactions are discouraged by more - electron - rich p donors . in contrast , auau distances in the gold complexes of the binap series ( l7l9 ) are essentially insensitive to the p substituent ( , figure 3 ) . this is attributed to the higher barrier of rotation for the binaphthyl ring . for this particular series of gold complexes , these observations suggest that aurophilicity is relatively uncommon for these atropisomeric [ ( diphosphine)au2cl2 ] complexes , inhibited by more - electron - rich p - donor groups and by restriction of the conformational flexibility of the aryl , the catalytic activity of selected complexes was tested in the cycloisomerisation of ether substrates 1 a d ( scheme 3 ) . effect of chiral diphosphine ligands and au / ag stoichiometry in the cycloisomerisation of 1,6-enynes ( scheme 3 ) [ a ] reaction conditions : substrate 1 in toluene ( 0.5 m ) , [ au]2 ( 5 mol % ) , [ ag ] ( 5 or 10 mol % ) , rt , 24 h. [ b ] isolated yield . the cyclisation of ether - tethered enyne substrate 1 a was examined initially as the model reaction , performed in toluene at room temperature . the product was somewhat unstable , therefore quick purification by flash chromatography was necessary to achieve good isolated yields . the benchmark for this study was set by reproduction of the result reported by michelet and co - workers by using ligand l1:[17 , 18 ] the product 2 a can be obtained in 87 % yield with 99 % ee ( table 2 , entry 1 ) . guided by the structural analysis ( table 1 ) , two complexes from the binap series ( l7 and l8 ) were tested as catalysts because they have similar auau distances ( > 5.3 ) . in both cases , a modest 46 % ee was obtained , whereas tolyl - substituted l8 gave a much higher 75 % ee ( table 1 , entries 2 and 3 ) . similarly , two ligands from the p - phos series ( l10 and l11 ) were selected for their similar torsional angles to l1 . in these cases , , the more - electron - rich p - xylyl - substituted complex l11 offered significantly better enantioselectivity than l10 ( 38 vs. 60 % ee ; table 1 , entries 4 and 5 ) . correlations between structural parameters obtained from solid - state structures and homogeneous catalytic reactions should always be exercised with caution . nevertheless , these preliminary studies seem to suggest that the enantioselectivity of atropisomeric biaryl diphosphine ligands can be enhanced by using more - electron - rich p substituents . in previous studies , the choice of silver salt / counter - anion often had a profound effect on the catalytic activity and it has been suggested that the participation of silver catalysis can not be discounted in such reactions . in this case , a control reaction showed that the linear substrate 1 a remained unchanged in the presence of agbf4 alone at ambient temperature ( table 2 , entry 7 ) , that is , any background racemic process due to the silver salt can be categorically ruled out . subsequently , by using the l11-ligated complex , the cycloisomerisations of three other substrates 1 b d were performed in the presence of various amounts of silver salt , with the stoichiometric ratio of au / ag either 1:1 or 2:1 ( table 2 , entries 812 ) . in each case , the product yield was lower when silver salt ( 0.5 equiv ) was employed but the level of enantioselectivity remained invariant . this is a significant observation . considering the different cationic complexes that can be generated in each case ( scheme 4 ) , the involvement of a bimetallic au / ag complex as the active catalyst can be ruled out because this can only be generated in a significant quantity when a sub - stoichiometric amount of silver salt is used . furthermore , the result also strongly suggests that each metal centre operates independently as an active catalytic site , that is , the presence of any cooperative effect between the two proximal au metal centres may be discounted . this is commensurate with the previous study , in which greater enantioselectivity was observed with complexes that had larger intermetallic distance / less propensity to form auau interactions for steric ( l1 ) and/or electronic ( l8 , l11 ) reasons . finally , the active catalyst was also generated in situ by pre - mixing the gold chloride complex with the silver additive prior to addition of the substrate , or by introducing an intermediate operation , in which the agcl formed was removed by filtration through a bed of celite before the introduction of the substrate . hence , the involvement of [ l au cl ag ] is also highly unlikely . we have conducted a structural study on a series of [ ( diphosphine)au2cl2 ] complexes . even within the small selection of examples , some trends can be observed within the diaryl diphosphine complexes , for instance an increase in the electron - donating character of the p - ar substituent increases the auau distance . this effect is not observed , however , in the binap series of ligands , which is attributed to the greater barrier to rotation / more - rigid structure . the catalytic activity of selected complexes was tested in the cycloisomerisation of 1,6-enynes ; the incorporation of electron - rich p donors enhanced the stereoselectivity . critically , the enantioselectivity of the process was not affected by using different gold / silver ratios or by removal of the silver by - product . this suggests that each metal centre is likely to operate independently and that silver does not participate in the process . as a service to our authors and readers , this journal provides supporting information supplied by the authors . such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors

x - ray crystal structures of two [ ( diphosphine)au2cl2 ] complexes ( in which diphosphine = p - phos and xylyl - p - phos ; p - phos=[2,2,6,6-tetramethoxy-4,4-bis(diphenylphosphino)-3,3-bipyridine ] ) were determined and compared to the reported structures of similar atropisomeric gold complexes . correlations between the auau distances and torsional angles for the biaryl series of ligands ( meobiphep , segphos , and p - phos ; biphep=2,2-bis(diphenylphosphino)-1,1-biphenyl , segphos=[(4,4-bi-1,3-benzodioxole)-5,5-diyl]bis[diphenylphosphine ] ) can be made ; these measurements appear to be very dependent upon the phosphorous substituent . conversely , the same effect was not observed for ligands based on the binaphthyl ( binap ) series . the catalytic activity of these complexes was subsequently assessed in the enantioselective cycloisomerisation of 1,6-enynes and revealed an over - riding electronic effect : more - electron - rich phosphines promote greater enantioselectivity . the possibility of silver acting as a ( co-)catalyst was ruled out in these reactions .

Introduction Results and Discussion Conclusion Supporting Information

clinical , case -control , and epidemiologic studies show an association between late - life depression and persistent cognitive deficits , and between history of depression and subsequent dementia , especially ad . studies of late - life depression generally find significant cognitive impairment , concurrent with affective symptoms , ( eg , refs 2,3 ) that is mediated almost , entirely by slowed information processing or working memory deficits . the cognitive deficits accompanying late - life depression often persist following treatment and remission of affective symptoms . one year after good treatment response for a major depressive episode , significant impairment was found in 23% of subjects who had been deemed cognitively intact while depressed . two recent meta - analyses found that a history of depression approximately doubles an individual 's risk of subsequent dementia in general and ad in particular . yet , many large individual studies have found no such relationship , giving rise to varying conclusions regarding the temporal and directional association between depression and mild cognitive impairment ( mci ) and/or dementia . that is , it is not clear whether prior depression is a true etiologic risk factor for dementia or rather represents a prodromal clinical manifestation of dementia neuropathology some evidence suggests that , risk for both m'ci and dementia is proportionate to cumulative depression burden in terms of symptom severity , lifetime duration of depression , or number of major depressive episodes ( m'drs ) . in the cardiovascular health study , severity of depressive symptoms independently predicted diagnosis of mci 6 years later . a large danish case - registry study found the number of prior mdes predicted dementia diagnosis , with a hazard ratio increasing by 13% per mde . finally , several studies suggest the longer the interval between onset of first depressive episode and time of assessment for dementia , the greater the risk of dementia ( also see meta - analyses in refs 10,11 ) . overall , the weight , of available evidence suggests that , depression , including related pathophysiologic processes , may act as a true risk factor for mci and dementia . however , there is no consensus as yet on this point . other studies have found that the shorter the interval between depression onset , and assessment for dementia , the greater the risk , while others found no such relationship between depression and cognitive impairment or subsequent , dementia . such findings suggest that when depressive and cognitive symptoms appear close in time they likely arise from common neuropathologic processes . this is an important , competing hypothesis to the concept of depression as a risk factor for dementia . overall , these findings emphasize the heterogeneity of late - life depression , its cognitive manifestations , and possible cognitive sequelae . many authors emphasize the importance of determining whether depression is a true risk factor versus an early symptom occurring in the prodromal phase of dementia , particularly ad . this report , does not resolve this issue ; rather , we review evidence for several specific pathways by which depression may be linked to subsequent , cognitive decline and dementia and present , two related models that accommodate and reconcile many of the seemingly disparate research findings . one model is shown in figure 1 and presents three interacting links which affect brain and cognitive reserve thereby moderating the relationship between underlying ad neuropatholgy and its expression as clinical dementia . in the sections that follow depression is associated with neuroendocrine changes similar to those observed in animal models of chronic stress , including abnormalities within the hypothalamicpituitary - adrenal ( hpa ) axis . most notably , depressed subjects have been shown to exhibit , increased hpa central drive with elevated corticotrophin - releasing hormone ( crh ) and vasopressin production by cells of the hypothalamic paraventricular nucleus ( pvn ) ; impaired negative feedback regulation due to decreased expression of corticosteroid receptors in the hypothalamus and pituitary as well as upstream cns regulatory centers ; and adrenal hypertrophy ( reviewed in ref 25 ) . the net effect of these changes in hpa function is chronic elevation of adrenal glucocorticoid production with impaired negative feedback and abnormal homeostatic regulation . such hpa dysregulation is clinically detectable ( via dexamethasone nonsuppression or elevated 24-hour urinary cortisol ) in about , half of patients with major depression . hpa dysregulation may be more common among older depressed individuals , as suggested by the finding of a significant correlation between age and post - dexamethasone cortisol levels in individuals with late - life depression . adrenal glucocorticoid / cortisol regulates hpa activity through both direct , negative feedback at the pituitary and hypothalamus and indirect , mechanisms involving higher central nervous system ( cns ) centers . the human hippocampus , for example , contains large numbers of corticosteroid receptors and plays a critical role in downregulating crh release via a multisynaptic pathway terminating in y - aminobutyric acid ( gaba)-ergic output to the paraventricular nucleus ( reviewed in ref 28 ) . at . the same time , hpa disturbances causing prolonged hypercortisolemia may promote hippocampal atrophy and functional decline , such that hpa regulation is further compromised . ' iliis interaction may underlie the observed association between hypercortisolemic disease states such as cushing 's syndrome and depression , and both hippocampal atrophy and impairment , in the verbal and spatial memory functions subserved by the hippocampus . animal studies suggest that high - stress conditions or exogenous glucocorticoids can cause hippocampal neuronal damage and memory impairment . these changes have been observed concurrent with stress or exogenous glucocorticoid administration , and appear to progress over a lifetime of stress or glucocorticoid excess ( see review in ref 33 ) . human studies in older adults likewise suggest that hippocampal size and function are diminished in the setting of elevated glucocorticoids , and in proplemiaortion to duration of prior hypercortisolemia . on the basis of these findings , many have hypothesized that glucocorticoids may promote hippocampal cell injury and death when chronically elevated , as in the setting of hypercortisolemica associated with major depression . glucocorticoid - induced cellular damage may be mediated through effects on several biochemical substrates . postulated mechanisms include decreased glucose uptake and atp generation , elevated intracellular calcium with increased free radical production and degradative enzyme activity , and impaired uptake of glutamate from hippocampal synapses resulting in excitotoxicity . in addition , hypercortisolemia has been linked to a decrease in neurogenesis in the dentate gyrus . while the combination of cell death and decreased neurogenesis may theoretically contribute to hippocampal cell loss over time , recent , evidence suggests at . most a minor role for this mechanism in hypercortisolcmic human subjects in the absence of cooccurring insults . animal and human studies support the idea that glucocorticoids contribute to hippocampal atrophy and functional deficits predominantly through more subtle alterations , including reduced synapse number , atrophy of pyramidal cell dendrites , derangement , of glial cells , and other changes . neuroimaging studies have generally shown reduced hippocampal volumes in late - life depression subjects relative to age - matched controls ( see meta - analysis by videbech and ravnkilde ) , although this finding is not universal . furthermore , many studies find a significant association between hippocampal atrophy and greater lifetime duration of depression , as assessed by number of depressive episodes , total days depressed , total days of untreated depression , duration since first depressive episode , or early - onset as opposed to late - onset depression . although these findings differ somewhat , most studies support , to some degree the notion that depression - related hypercortisolemia can compromise hippocampal structure and function , especially in the context of old age and comorbid disease . the impact of glucocorticoids on brain structures can be expected to vary not only with age and disease status , but also with individual genetic and environmentallyimparted differences influencing hippocampal volume and connectivity , hpa reactivity , and other neurobiologie factors . besides the possibility of persistent synapse or neuron loss induced directly by prolonged hypercortisolemia , glucocorticoid - related derangement of hippocampal physiology , as described above , may increase vulnerability to damage through other pathophysiologic mechanisms . this latter effect may become clinically relevant in older persons with co - occurring neuronal insults such as accumulating ad pathology or cerebrovascular disease , promoting synapse or neuron loss through a synergistic relationship with these factors . the loss of hippocampal volume and memory function observed in some elders with late -life depression suggests the possibility that depression may be a predispositional risk factor for ad in particular . indeed , lower hippocampal volumes independently predict subsequent ad in groups of mci and cognitively normal elderly subjects . likewise , deficits in verbal learning and memory , similar to those described in cuthymic patients with history of major depression , also predict ad ( eg , ref 53 ) . while a primary causal role for depression in ad pathogenesis seems unlikely , depression - associated hypercortisolemia leading to decline in hippocampal size , connectivity and cognitive function may represent one of multiple links between depression and dementia as described below ( also see figure 1 ) . the association between depression and dementia suggested by epidemiologic data may be partially explained by one or more direct , mechanistic links between late - life depression - related processes and adspecific neuropathology ( focal and diffuse cortical neuronal loss , p - amyloid plaques , and neurofibrillary tangles ) . emerging evidence from neuroimaging studies , postmortem neuropathology analyses , and animal models provides support , for such links . some structural magnetic resonance imaging ( mri ) studies find that hippocampal atrophy is more strongly associated with late - onset than early - onset depression , suggesting that early ad - related pathophysiology could generate both hippocampal atrophy and depressive symptoms in some elderly persons . in addition , one of these studies failed to find a significant correlation between hippocampal volume and cortisol level among elders with depression . furthermore , the late - life depression subjects showed persistent memory and cognitive impairment at 6-month follow - up despite effective treatment of mood symptoms and normalization of cortisol levels . all of these data are consistent with the idea that ad pathology is a major cause of hippocampal atrophy in some ( but not all ) individuals with late - life depression , and their depressive symptoms may represent prodromal ad . several recent , animal and human studies suggest , a possible direct pathophysiologic link between late - life depression and the neuropathologic hallmarks of ad . postmortem studies report greater hippocampal amyloid plaque and neurofibrillary tangle pathology in ad patients with lifetime history of depression compared with those without , such history , and more severe cortical neurofibrillary tangle pathology in the brains of ad subjects who suffered from comorbid depression . the hippocampal findings , combined with the observation of marked hippocampal neurofibrillary pathology early in the course of ad , provoke speculation that depression - associated hypercortisolemia may facilitate ad pathogenesis by rendering hippocampal neurons and glia vulnerable to toxic insults , as discussed in the previous section . neurobiologie interaction or overlap between late - life depression and ad is further suggested by the discovery of glial changes consistent with a cns inflammatory process in both older depressed individuals ( eg , ref 60 ) and those with neurodegenerative diseases such as ad . the prolonged hypercortisolemia associated with both diseases may partially account for these findings , as glucocorticoids can induce proinflammatory changes within the cns . overall , these findings provide associational evidence for a link between late - life depression and ad , yet offer little insight into whether depression history may act as a true etiologic risk factor for ad , or , conversely , whether late - life depression arises secondary to adrclatcd neuropathologic changes . however , two recent animal studies suggest the existence of a direct mechanistic link between hypercortisolemic depression and ad pathology . green and colleagues found dexamethasonc administration increased ( i - amyloid production in a transgenic mouse model of ad , and traced this effect to increased expression of amyloid precursor protein and the p - secrctase enzyme . this group also found increased tau aggregation within neuronal cell bodies and dendrites of dexamethasone - treated animals . kang and colleagues demonstrated increased hippocampal interstitial p - amyloid levels in another mouse model of ad following acute restraint or chronic isolation stress . this finding was reproduced through direct , infusion of crh into the hippocampus , and blocked by pretreatment with crh antagonists . in conclusion , diverse findings from structural mri and human or animal histopathologic studies suggest a direct , relationship between late - life depression and ad - specific pathology . reports of a cross - sectional association between later age of depression onset and hippocampal atrophy support , the notion that early ad - related pathophysiology is causing depressive symptoms in these study groups and correlate with epidemiologic reports of elevated dementia risk in subjects with depression onset . in the recent , versus distant past , ( eg , refs 19,20 ) . conversely , postmortem ad studies suggest an association between more severe plaque and tangle pathology and lifetime depression history preceding ad diagnosis , offering support for the idea that , prior depression is a true , etiologic risk factor for ad , as suggested by other epidemiologic data ( eg , ref 11 ) . furthermore , both stress and exogenous glucocorticoids increase p - amyloid production in rodent , models of ad , consistent , with a direct . given the tremendous heterogeneity of late - life depression , various dementia pathologies , and the other clinical or subclinical disease inevitably present in older individuals , depressive symptoms should be expected to bear an inconsistent relationship with cognitive decline , dementia in general , and ad specifically . such symptoms in a given elderly individual may potentially represent either prodromal ad , or an independent process interacting with ad - related pathophysiology . as discussed in this manuscript , depression may furthermore contribute to cognitive decline and ad through glucocorticoid - relatcd hippocampal toxicity and interrelationships with other types of pathology such as vascular disease . substantial data exist showing an association between latelife depression and cerebrovascular changes . in separate reports , alexopoulos and krishnan pointed to the thennascent evidence that a subgroup of individuals with latelife depression showed evidence of cerebrovascular changes . alexopoulos coined the term vascular depression , positing that , a subgroup of individuals experience disruption of prefrontal systems that mediate both mood and executive functions , by either single vascular lesions or accumulation of lesions . ' ihc concept of vascular depression has subsequently been supported and expanded by a growing literature . depression increases risk for first - ever myocardial , infarction ( mi ) and stroke , and has been shown to predict worse outcomes in a wide range of concurrent vascular disease states ( reviewed in ref 67 ) . notably , clinical diagnosis of major depression confers significant relative risk for mi , stroke , and post - mi cardiac mortality . moreover , major depression confers greater relative risk than diagnosis of dysthymia or indices of self -reported depressive symptoms , suggesting a possible dose - response relationship between severity of depressive illness and excess cardiovascular risk . diverse mechanisms have been proposed to explain the link between prior depression and subsequent vascular disease . depressed individuals exhibit poor treatment compliance and other behaviors such as smoking , substance abuse , and inactivity , which may cause or worsen comorbid disease . elevated cortisol levels independently predict several features of the metabolic syndrome including abdominal obesity , low high - density lipoprotein ( hdl ) levels , and hypertriglyceridemia . they disrupt normal endothelial function and may contribute over time to the development , of hypertension in some cases . depressed subjects with coronary artery disease ( cad ) exhibit , autonomic dysfunction with decreased heart rate variability , a condition that likely predisposes to both cardiac arrhythmias and episodic hypoperfusion . cad show greater baseline platelet activation than nondepressed control subjects , suggesting greater susceptibility to thromboembolic events . finally , cross - sectional studies have linked depression to elevations in proinflammatory cytokines . while the causal relationship between such immunologic changes and depression is unknown , a similar proinflammatory cytokine shift c - reactive - protein is directly atherogenic , and high levels of several proinflammatory cytokines have been found to predict cardiovascular events . in a reciprocal fashion , many acute and chronic vascular disease states may promote depression . mi and stroke substantially increase risk for depression during the immediate postacute period , with depressive symptoms reported in 25% to 50% of individuals one study comparing cumulative 1 -year incidence of major and minor depression immediately following stroke or mi found no difference between these groups . this finding suggests that the loss of specific neuronal populations is less important than more global postischemic vascular or inflammatory mechanisms in the pathogenesis of poststroke depression . accordingly , depression is more frequent and severe in vascular dementia than ad , despite widespread neuronal loss in both dementia syndromes . studies of individuals with chronic cardiovascular diseases show that diabetes mcllitus ( sec meta - analysis in ref 89 ) and cad , each approximately double risk for depression . not all studies of older subjects indicate a longitudinal association between vascular disease / risk and subsequent depression . several prospective studies in elderly subjects report that clusters of cardiovascular risk factors or pre - existing cad independently predict incident depression , while at least , one large prospective study found no relationship between an index of generalized atherosclerosis and incident depression . in old age , the observed association between vascular disease and depression may be attenuated by the fact that persons with severe or long - standing disease of either type incur substantial morbidity and mortality . surviving individuals with significant , vascular or depressive pathology might , actually be expected to possess protective biopsychosocial factors which interrupt the positive bidirectional relationship described above . strong supporting evidence for the notion that vascular disease contributes to late - life depression comes from structural mri studies showing a robust association between ischemic brain lesions and depression diagnosis or selfreported symptoms in older persons . large communitybased studies have demonstrated independent cross - sectional relationships between late - life depression and small basal ganglia lesions and white matter abnormalities visualized as hyperintense regions on t2-weighted m'ri ( wmhs ) in deep or subcortical areas . longitudinal studies suggest white matter changes may both predate and independently predict late - life depression . the ischemic etiology of wmhs is suggested by several lines of evidence , including post - mortem histopathologic studies in patients with late - life depression and in the general population , correlating wmhs with both evidence of cerebrovascular disease and systemic hypotensive , or hypoxemic disease . ischemic damage to frontostriatal brain regions may explain the executive dysfunction , psychomotor slowing and resistance to treatment common in late - life depression . the few studies examining wmhs and cognition in late - life depression have found associations with psychomotor slowing , memory , language , and executive functioning . the relationship between wmhs and executive function may be particularly strong in individuals with late - onset depression . taken together , these studies suggest a relationship among late - onset depression , ischemic wmhs ( especially in the frontostriatal region ) and executive dysfunction , raising the possibility that ischemic structural changes in the brain are a common etiologic factor of both the depression and the associated cognitive dysfunction . the cognitive impairment related to this ischemic damage may be severe enough to culminate in a clinical diagnosis of dementia . high prevalence in the population ( up to 44% of all dementia ) . in accordance with the bidirectional relationship described here , prior depression independently predicts subsequent vascular dementia ( or = 2.1 5 ) and individuals with late - life depression who develop clinical ad have high rates of cerebrovascular pathology upon postmortem examination . indeed , prospective community - based studies report associations between baseline systemic vascular disease / risk and both higher rates of incident ad , and more rapid cognitive decline in established ad . moreover , rapid progression of cerebrovascular disease as inferred from serial mri predicts subsequent dementia diagnosis . in sum , mounting evidence suggests factors associated with late - life depression may predispose to persistent cognitive impairment and dementia . plausible moderators of this relationship include glucocorticoid - related hippocampal damage , an interaction between depression and ad neuropathology , and increased vascular disease , but . the potential importance of other factors ( eg , neurotransmitter and immunologic abnormalities ) can not , be excluded . moreover , in reality there appear to be abundant , interactions between the three distinct links described here and depicted in figure 1 . hypertension , for instance , is associ ated with diminished regional cerebral blood flow in the hippocampus and related limbic and paralimbic structures of cognitivcly normal older adults . together these findings suggest ischemic and inflammatory insults related to cerebrovascular disease may affect the same neuronal populations endangered by hypercortisolemia and ad . it is conceivable that hippocampal insults related to vascular disease , hypercortisolemic depression or prodromal ad which are insufficient to cause significant , cellular damage or death by themselves may produce cell death through synergistic interaction with co - occurring insults . in the context of neurodegenerative disease , cerebral ischemia may contribute to cell death outside the hippocampus , as suggested by an independent association between wmh volume and cortical grey matter atrophy in ad . notably , plasma levels of p - amyloid predict the extent , of ischemic white matter damage in mci and ad , suggesting a reciprocal interaction between cerebrovascular and ad pathophysiology . together these examples suggest that , particular combinations of insults arising from different pathophysiologics may play a crucial role in promoting cognitive decline and progressive dementia subsequent to depression , an effect , related to extensive crosstalk between links and synergism of insults at . the cellular level . clearly , many factors influence the impact , a particular risk or disease factor will have on expression of dementia . in the following section , we describe how the concepts of brain and cognitive reserve can be used to explain this multifactorial process and account for the highly variable clinical course , cognitive course and neuropathology associated with late - life depression . depression is associated with neuroendocrine changes similar to those observed in animal models of chronic stress , including abnormalities within the hypothalamicpituitary - adrenal ( hpa ) axis . most notably , depressed subjects have been shown to exhibit , increased hpa central drive with elevated corticotrophin - releasing hormone ( crh ) and vasopressin production by cells of the hypothalamic paraventricular nucleus ( pvn ) ; impaired negative feedback regulation due to decreased expression of corticosteroid receptors in the hypothalamus and pituitary as well as upstream cns regulatory centers ; and adrenal hypertrophy ( reviewed in ref 25 ) . the net effect of these changes in hpa function is chronic elevation of adrenal glucocorticoid production with impaired negative feedback and abnormal homeostatic regulation . such hpa dysregulation is clinically detectable ( via dexamethasone nonsuppression or elevated 24-hour urinary cortisol ) in about , half of patients with major depression . hpa dysregulation may be more common among older depressed individuals , as suggested by the finding of a significant correlation between age and post - dexamethasone cortisol levels in individuals with late - life depression . adrenal glucocorticoid / cortisol regulates hpa activity through both direct , negative feedback at the pituitary and hypothalamus and indirect , mechanisms involving higher central nervous system ( cns ) centers . the human hippocampus , for example , contains large numbers of corticosteroid receptors and plays a critical role in downregulating crh release via a multisynaptic pathway terminating in y - aminobutyric acid ( gaba)-ergic output to the paraventricular nucleus ( reviewed in ref 28 ) . at . the same time , hpa disturbances causing prolonged hypercortisolemia may promote hippocampal atrophy and functional decline , such that hpa regulation is further compromised . ' iliis interaction may underlie the observed association between hypercortisolemic disease states such as cushing 's syndrome and depression , and both hippocampal atrophy and impairment , in the verbal and spatial memory functions subserved by the hippocampus . animal studies suggest that high - stress conditions or exogenous glucocorticoids can cause hippocampal neuronal damage and memory impairment . these changes have been observed concurrent with stress or exogenous glucocorticoid administration , and appear to progress over a lifetime of stress or glucocorticoid excess ( see review in ref 33 ) . human studies in older adults likewise suggest that hippocampal size and function are diminished in the setting of elevated glucocorticoids , and in proplemiaortion to duration of prior hypercortisolemia . on the basis of these findings , many have hypothesized that glucocorticoids may promote hippocampal cell injury and death when chronically elevated , as in the setting of hypercortisolemica associated with major depression . glucocorticoid - induced cellular damage may be mediated through effects on several biochemical substrates . postulated mechanisms include decreased glucose uptake and atp generation , elevated intracellular calcium with increased free radical production and degradative enzyme activity , and impaired uptake of glutamate from hippocampal synapses resulting in excitotoxicity . in addition , hypercortisolemia has been linked to a decrease in neurogenesis in the dentate gyrus . while the combination of cell death and decreased neurogenesis may theoretically contribute to hippocampal cell loss over time , recent , evidence suggests at . most a minor role for this mechanism in hypercortisolcmic human subjects in the absence of cooccurring insults . animal and human studies support the idea that glucocorticoids contribute to hippocampal atrophy and functional deficits predominantly through more subtle alterations , including reduced synapse number , atrophy of pyramidal cell dendrites , derangement , of glial cells , and other changes . neuroimaging studies have generally shown reduced hippocampal volumes in late - life depression subjects relative to age - matched controls ( see meta - analysis by videbech and ravnkilde ) , although this finding is not universal . furthermore , many studies find a significant association between hippocampal atrophy and greater lifetime duration of depression , as assessed by number of depressive episodes , total days depressed , total days of untreated depression , duration since first depressive episode , or early - onset as opposed to late - onset depression . although these findings differ somewhat , most studies support , to some degree the notion that depression - related hypercortisolemia can compromise hippocampal structure and function , especially in the context of old age and comorbid disease . the impact of glucocorticoids on brain structures can be expected to vary not only with age and disease status , but also with individual genetic and environmentallyimparted differences influencing hippocampal volume and connectivity , hpa reactivity , and other neurobiologie factors . besides the possibility of persistent synapse or neuron loss induced directly by prolonged hypercortisolemia , glucocorticoid - related derangement of hippocampal physiology , as described above , may increase vulnerability to damage through other pathophysiologic mechanisms . this latter effect may become clinically relevant in older persons with co - occurring neuronal insults such as accumulating ad pathology or cerebrovascular disease , promoting synapse or neuron loss through a synergistic relationship with these factors . the loss of hippocampal volume and memory function observed in some elders with late -life depression suggests the possibility that depression may be a predispositional risk factor for ad in particular . indeed , lower hippocampal volumes independently predict subsequent ad in groups of mci and cognitively normal elderly subjects . likewise , deficits in verbal learning and memory , similar to those described in cuthymic patients with history of major depression , also predict ad ( eg , ref 53 ) . while a primary causal role for depression in ad pathogenesis seems unlikely , depression - associated hypercortisolemia leading to decline in hippocampal size , connectivity and cognitive function may represent one of multiple links between depression and dementia as described below ( also see figure 1 ) . the association between depression and dementia suggested by epidemiologic data may be partially explained by one or more direct , mechanistic links between late - life depression - related processes and adspecific neuropathology ( focal and diffuse cortical neuronal loss , p - amyloid plaques , and neurofibrillary tangles ) . emerging evidence from neuroimaging studies , postmortem neuropathology analyses , and animal models provides support , for such links . some structural magnetic resonance imaging ( mri ) studies find that hippocampal atrophy is more strongly associated with late - onset than early - onset depression , suggesting that early ad - related pathophysiology could generate both hippocampal atrophy and depressive symptoms in some elderly persons . in addition , one of these studies failed to find a significant correlation between hippocampal volume and cortisol level among elders with depression . furthermore , the late - life depression subjects showed persistent memory and cognitive impairment at 6-month follow - up despite effective treatment of mood symptoms and normalization of cortisol levels . all of these data are consistent with the idea that ad pathology is a major cause of hippocampal atrophy in some ( but not all ) individuals with late - life depression , and their depressive symptoms may represent prodromal ad . several recent , animal and human studies suggest , a possible direct pathophysiologic link between late - life depression and the neuropathologic hallmarks of ad . postmortem studies report greater hippocampal amyloid plaque and neurofibrillary tangle pathology in ad patients with lifetime history of depression compared with those without , such history , and more severe cortical neurofibrillary tangle pathology in the brains of ad subjects who suffered from comorbid depression . the hippocampal findings , combined with the observation of marked hippocampal neurofibrillary pathology early in the course of ad , provoke speculation that depression - associated hypercortisolemia may facilitate ad pathogenesis by rendering hippocampal neurons and glia vulnerable to toxic insults , as discussed in the previous section . neurobiologie interaction or overlap between late - life depression and ad is further suggested by the discovery of glial changes consistent with a cns inflammatory process in both older depressed individuals ( eg , ref 60 ) and those with neurodegenerative diseases such as ad . the prolonged hypercortisolemia associated with both diseases may partially account for these findings , as glucocorticoids can induce proinflammatory changes within the cns . overall , these findings provide associational evidence for a link between late - life depression and ad , yet offer little insight into whether depression history may act as a true etiologic risk factor for ad , or , conversely , whether late - life depression arises secondary to adrclatcd neuropathologic changes . however , two recent animal studies suggest the existence of a direct mechanistic link between hypercortisolemic depression and ad pathology . green and colleagues found dexamethasonc administration increased ( i - amyloid production in a transgenic mouse model of ad , and traced this effect to increased expression of amyloid precursor protein and the p - secrctase enzyme . this group also found increased tau aggregation within neuronal cell bodies and dendrites of dexamethasone - treated animals . kang and colleagues demonstrated increased hippocampal interstitial p - amyloid levels in another mouse model of ad following acute restraint or chronic isolation stress . this finding was reproduced through direct , infusion of crh into the hippocampus , and blocked by pretreatment with crh antagonists . in conclusion , diverse findings from structural mri and human or animal histopathologic studies suggest a direct , relationship between late - life depression and ad - specific pathology . reports of a cross - sectional association between later age of depression onset and hippocampal atrophy support , the notion that early ad - related pathophysiology is causing depressive symptoms in these study groups and correlate with epidemiologic reports of elevated dementia risk in subjects with depression onset . in the recent , versus distant past , ( eg , refs 19,20 ) . conversely , postmortem ad studies suggest an association between more severe plaque and tangle pathology and lifetime depression history preceding ad diagnosis , offering support for the idea that , prior depression is a true , etiologic risk factor for ad , as suggested by other epidemiologic data ( eg , ref 11 ) . furthermore , both stress and exogenous glucocorticoids increase p - amyloid production in rodent , models of ad , consistent , with a direct . given the tremendous heterogeneity of late - life depression , various dementia pathologies , and the other clinical or subclinical disease inevitably present in older individuals , depressive symptoms should be expected to bear an inconsistent relationship with cognitive decline , dementia in general , and ad specifically . such symptoms in a given elderly individual may potentially represent either prodromal ad , or an independent process interacting with ad - related pathophysiology . as discussed in this manuscript , depression may furthermore contribute to cognitive decline and ad through glucocorticoid - relatcd hippocampal toxicity and interrelationships with other types of pathology such as vascular disease . substantial data exist showing an association between latelife depression and cerebrovascular changes . in separate reports , alexopoulos and krishnan pointed to the thennascent evidence that a subgroup of individuals with latelife depression showed evidence of cerebrovascular changes . alexopoulos coined the term vascular depression , positing that , a subgroup of individuals experience disruption of prefrontal systems that mediate both mood and executive functions , by either single vascular lesions or accumulation of lesions . ' ihc concept of vascular depression has subsequently been supported and expanded by a growing literature . depression increases risk for first - ever myocardial , infarction ( mi ) and stroke , and has been shown to predict worse outcomes in a wide range of concurrent vascular disease states ( reviewed in ref 67 ) . notably , clinical diagnosis of major depression confers significant relative risk for mi , stroke , and post - mi cardiac mortality . moreover , major depression confers greater relative risk than diagnosis of dysthymia or indices of self -reported depressive symptoms , suggesting a possible dose - response relationship between severity of depressive illness and excess cardiovascular risk . diverse mechanisms have been proposed to explain the link between prior depression and subsequent vascular disease . depressed individuals exhibit poor treatment compliance and other behaviors such as smoking , substance abuse , and inactivity , which may cause or worsen comorbid disease . depression is also associated with systemic physiologic derangements which may contribute to vascular pathology . as mentioned above , hpa axis dysregulation with hypercortisolemia is common in depressed individuals . elevated cortisol levels independently predict several features of the metabolic syndrome including abdominal obesity , low high - density lipoprotein ( hdl ) levels , and hypertriglyceridemia . they disrupt normal endothelial function and may contribute over time to the development , of hypertension in some cases . depressed subjects with coronary artery disease ( cad ) exhibit , autonomic dysfunction with decreased heart rate variability , a condition that likely predisposes to both cardiac arrhythmias and episodic hypoperfusion . cad show greater baseline platelet activation than nondepressed control subjects , suggesting greater susceptibility to thromboembolic events . finally , cross - sectional studies have linked depression to elevations in proinflammatory cytokines . while the causal relationship between such immunologic changes and depression is unknown , a similar proinflammatory cytokine shift c - reactive - protein is directly atherogenic , and high levels of several proinflammatory cytokines have been found to predict cardiovascular events . in a reciprocal fashion , many acute and chronic vascular disease states may promote depression . mi and stroke substantially increase risk for depression during the immediate postacute period , with depressive symptoms reported in 25% to 50% of individuals ( reviewed in ref 67 ) . one study comparing cumulative 1 -year incidence of major and minor depression immediately following stroke or mi found no difference between these groups . this finding suggests that the loss of specific neuronal populations is less important than more global postischemic vascular or inflammatory mechanisms in the pathogenesis of poststroke depression . accordingly , depression is more frequent and severe in vascular dementia than ad , despite widespread neuronal loss in both dementia syndromes . studies of individuals with chronic cardiovascular diseases show that diabetes mcllitus ( sec meta - analysis in ref 89 ) and cad , each approximately double risk for depression . not all studies of older subjects indicate a longitudinal association between vascular disease / risk and subsequent depression . several prospective studies in elderly subjects report that clusters of cardiovascular risk factors or pre - existing cad independently predict incident depression , while at least , one large prospective study found no relationship between an index of generalized atherosclerosis and incident depression . in old age , the observed association between vascular disease and depression may be attenuated by the fact that persons with severe or long - standing disease of either type incur substantial morbidity and mortality . surviving individuals with significant , vascular or depressive pathology might , actually be expected to possess protective biopsychosocial factors which interrupt the positive bidirectional relationship described above . strong supporting evidence for the notion that vascular disease contributes to late - life depression comes from structural mri studies showing a robust association between ischemic brain lesions and depression diagnosis or selfreported symptoms in older persons . large communitybased studies have demonstrated independent cross - sectional relationships between late - life depression and small basal ganglia lesions and white matter abnormalities visualized as hyperintense regions on t2-weighted m'ri ( wmhs ) in deep or subcortical areas . longitudinal studies suggest white matter changes may both predate and independently predict late - life depression . the ischemic etiology of wmhs is suggested by several lines of evidence , including post - mortem histopathologic studies in patients with late - life depression and in the general population , correlating wmhs with both evidence of cerebrovascular disease and systemic hypotensive , or hypoxemic disease . ischemic damage to frontostriatal brain regions may explain the executive dysfunction , psychomotor slowing and resistance to treatment common in late - life depression . the few studies examining wmhs and cognition in late - life depression have found associations with psychomotor slowing , memory , language , and executive functioning . the relationship between wmhs and executive function may be particularly strong in individuals with late - onset depression . taken together , these studies suggest a relationship among late - onset depression , ischemic wmhs ( especially in the frontostriatal region ) and executive dysfunction , raising the possibility that ischemic structural changes in the brain are a common etiologic factor of both the depression and the associated cognitive dysfunction . the cognitive impairment related to this ischemic damage may be severe enough to culminate in a clinical diagnosis of dementia . high prevalence in the population ( up to 44% of all dementia ) . in accordance with the bidirectional relationship described here , prior depression independently predicts subsequent vascular dementia ( or = 2.1 5 ) and individuals with late - life depression who develop clinical ad indeed , prospective community - based studies report associations between baseline systemic vascular disease / risk and both higher rates of incident ad , and more rapid cognitive decline in established ad . moreover , rapid progression of cerebrovascular disease as inferred from serial mri predicts subsequent dementia diagnosis . in sum , mounting evidence suggests factors associated with late - life depression may predispose to persistent cognitive impairment and dementia . plausible moderators of this relationship include glucocorticoid - related hippocampal damage , an interaction between depression and ad neuropathology , and increased vascular disease , but . the potential importance of other factors ( eg , neurotransmitter and immunologic abnormalities ) can not , be excluded . moreover , in reality there appear to be abundant , interactions between the three distinct links described here and depicted in figure 1 . hypertension , for instance , is associ ated with diminished regional cerebral blood flow in the hippocampus and related limbic and paralimbic structures of cognitivcly normal older adults . these findings suggest ischemic and inflammatory insults related to cerebrovascular disease may affect the same neuronal populations endangered by hypercortisolemia and ad . it is conceivable that hippocampal insults related to vascular disease , hypercortisolemic depression or prodromal ad which are insufficient to cause significant , cellular damage or death by themselves may produce cell death through synergistic interaction with co - occurring insults . in the context of neurodegenerative disease , cerebral ischemia may contribute to cell death outside the hippocampus , as suggested by an independent association between wmh volume and cortical grey matter atrophy in ad . notably , plasma levels of p - amyloid predict the extent , of ischemic white matter damage in mci and ad , suggesting a reciprocal interaction between cerebrovascular and ad pathophysiology . together these examples suggest that , particular combinations of insults arising from different pathophysiologics may play a crucial role in promoting cognitive decline and progressive dementia subsequent to depression , an effect , related to extensive crosstalk between links and synergism of insults at . the cellular level . clearly , many factors influence the impact , a particular risk or disease factor will have on expression of dementia . in the following section , we describe how the concepts of brain and cognitive reserve can be used to explain this multifactorial process and account for the highly variable clinical course , cognitive course and neuropathology associated with late - life depression . brain and cognitive reserve are often used interchangeably , but in fact , have subtle but . distinct differences in meaning . nevertheless , either may account , equally well for the relationship between depression and dementia . tltic concept of brain reserve capacity , first proposed by satz varies across individuals such that those with greater neuronal redundancy are able to tolerate more cell loss than those with less redundancy , before manifesting clinical symptoms . the concept of redundancy refers to the notion that , circuits contain more than the minimum number of neurons needed to perform an operation . redundancy is evident when individuals incur substantial neuronal loss before the appearance of clinical symptoms . thus , brain reserve capacity posits that individual differences in neural redundancy translate into differences in thresholds for vulnerability to or protection from clinical symptoms after brain damage . the concept of cognitive reserve developed by stern ( eg , refs 121 , 122 ) is similar but rather than being based on differences in brain size or neuronal count , emphasizes differences in the efficiency or manner in which tasks are performed or information is processed . both brain reserve and cognitive reserve explain the role of risk and protective factors for cognitive impairment ( including progressive decline into dementia ) , associated with brain damage . for example , higher educational attainment , larger head size , larger brain volume , social engagement , physical activity , and leisure cognitive activity may result in greater redundancy and/or efficiency and therefore reserve , thereby offering protection against exhibiting clinical symptoms of dementia . similarly , lower levels of these protective factors may reduce neuronal or functional redundancy leading to earlier dementia symptom onset for a given level of cns damage . while certain mechanisms may alter an individual 's risk to develop ( or change the rate of development of ) adrelated pathology ( eg , p - amyloid deposition ) , other mechanisms alter the strength of association between these biological changes and the time to develop clinical disease . we propose that depression alters an individual 's risk of cognitive dysfunction , shortening the latent period between the development , of ad neuropathology and the onset , of clinical dementia , thus increasing the incidence and prevalence of ad among older adults with depression . we propose that the reserve threshold theory is the key explanatory mechanism behind the late - life depression / dementia association . that is , through a number of processes ( several described here ) , depression injures neurons , thus lowering reserve such that cognitive impairment is expressed earlier and/or more frequently than it would otherwise . as depicted in , depression is linked to vascular disease , especially in the frontostriatal area . depression also is linked to elevated glucocorticoid production , as well as amyloid deposition and neurofibrillary formation , each of which may lead to hippocampal injury . bach of these processes adds to the total brain injury burden , lowering reserve and vulnerability to express cognitive impairment . these links and processes are not mutually exclusive ; many are likely synergistic , so that , they act to varying degrees across groups of individuals . this accounts for the substantial heterogeneity of the mood disorder and the presence ( or absence ) of a cognitive disorder and its clinical course . for example , it is possible that the diminished hippocampal volume identified in group studies could be the result , of more than one underlying process . individuals with early - onset , recurrent , depression may have hippocampal volume loss due to the repeated stress associated with multiple depressive episodes . many individuals with later - onset depression may be in the prodromal stage of ad , their hippocampi having already sustained substantial neuronal injury due to cumulative ad neuropathology . there may be additional pathologic processes , independent of depression , which can affect cognition . for example , amyloid plaques and neurofibrillary tangles commonly accumulate in aging brains , and it is likely that in some cases ad pathology represents an independent , co - occurring process ( ie , depression is the first manifest symptom of ad ) . vascular disease accompanying ad pathology in the absence of depression , promotes cognitive decline and an earlier expression of dementia ( eg , refs 111 - 115,131 ) . in fact , the growing evidence that ad and cerebrovascular pathology co - occur with high frequency has led some to conclude that the strict distinction between ad and vascular dementia is artificial . social isolation , physical inactivity , and lack of leisure cognitive activity may result , in lowered reserve and therefore confer additional risk for exhibiting clinical symptoms of dementia . moreover , late -life depression frequently occurs in the context of chronic medical illness , and major organ system dysfunction is frequently associated with cognitive impairment , acting to further lower reserve . thus , each of the processes mentioned above and depicted in figure 1 , independently adds to the total brain injury burden , lowers reserve , and strengthens the association between the neurodegenerative process and the clinical change in cognitive functions . we believe that this explanation underlies the relationship between latelife depression and dementia in general , and ad in particular ( see figure 1 ) . this conceptualization de - emphasizes the importance of the distinctions between early and late - onset depression and the relative risk for ad vs vascular dementia in the context of late - life depression . the cognitive outcome of any given individual who has late - life depression depends largely on the predominance or particular mix of pathophysiology in that individual . the additive or synergistic effects of vascular disease , glucocorticoid - related brain injury , and intrinsic ad pathophysiology are refl.ect.ed in the empirical findings of heterogeneous neuropathology in late - life depression and dementia . this framework , by focusing on the key concept , of reserve threshold , delineates testable ( and falsifiable ) links between depression and subsequent dementia . figure 2 depicts various pathways through which the key processes outlined in figure 1 may lead to the heterogeneous cognitive and disease outcomes reported in the literature . ill e pathways include ( in order of figure presentation ) : ( i ) individuals who develop depression at any point in their lives , sustain minimal or no depression - related neuropathology ( eg , glucocorticoid neurotoxicity ) , and who have stable , normal cognitive functioning ; ( ii ) individuals who develop depression at any point and who experience depression - related neuropathology that results in mci that is stable ( unless they experience additional depressive episodes ) ; ( iii ) individuals who accumulate ad neuropathology over many years and who develop late - life depression ( related or unrelated to ad pathology ) , that lowers brain reserve capacity , and results in expression of mci earlier than otherwise would be the case , and given the underlying neuropathology , progress to ad ; ( iv ) individuals who accumulate ad neuropathology over many years along with co - occurring cerebrovascular disease , which damages the frontostriatal circuitry , leading to late - life depression . the total neuropathologic burden , combined with depressed mood , lowers brain reserve capacity , leads to expression of mci ( eg , memory and executive dysfunction ) earlier than otherwise would be the case , and , given the underlying neuropathology , progresses to ad along with co - occurring cerebrovascular disease ; and ( v ) individuals who develop cerebrovascular disease ( with variable neuropathologic burden ) , that damages the frontostriatal circuitry , leading to late - life depression and mci ( eg , executive dysfunction ) , that , will follow the course of the underlying cerebrovascular disease . based on the weight of the findings in the published literature and consistent with our model depicted in figure 1 , we suggest that pathway # 4 ( figure 2 ) leading to ad with co - occurring cerebrovascular disease is the most frequently occurring pathway among individuals with late - onset depression . understanding the pathways through which individuals with late - life depression develop progressive dementia in general , and ad in particular , is critical as novel treatment may prevent , forestall , or slow cognitive and/or disease progression . we propose that the reserve threshold theory is the key explanatory mechanism behind the late - life depression / dementia association . that is , through a number of processes ( several described here ) , depression injures neurons , thus lowering reserve such that cognitive impairment is expressed earlier and/or more frequently than it would otherwise . as depicted in , depression is linked to vascular disease , especially in the frontostriatal area . depression also is linked to elevated glucocorticoid production , as well as amyloid deposition and neurofibrillary formation , each of which may lead to hippocampal injury . bach of these processes adds to the total brain injury burden , lowering reserve and vulnerability to express cognitive impairment . these links and processes are not mutually exclusive ; many are likely synergistic , so that , they act to varying degrees across groups of individuals . this accounts for the substantial heterogeneity of the mood disorder and the presence ( or absence ) of a cognitive disorder and its clinical course . for example , it is possible that the diminished hippocampal volume identified in group studies could be the result , of more than one underlying process . individuals with early - onset , recurrent , depression may have hippocampal volume loss due to the repeated stress associated with multiple depressive episodes . many individuals with later - onset depression may be in the prodromal stage of ad , their hippocampi having already sustained substantial neuronal injury due to cumulative ad neuropathology . there may be additional pathologic processes , independent of depression , which can affect cognition . for example , amyloid plaques and neurofibrillary tangles commonly accumulate in aging brains , and it is likely that in some cases ad pathology represents an independent , co - occurring process ( ie , depression is the first manifest symptom of ad ) . vascular disease accompanying ad pathology in the absence of depression , promotes cognitive decline and an earlier expression of dementia ( eg , refs 111 - 115,131 ) . in fact , the growing evidence that ad and cerebrovascular pathology co - occur with high frequency has led some to conclude that the strict distinction between ad and vascular dementia is artificial . social isolation , physical inactivity , and lack of leisure cognitive activity may result , in lowered reserve and therefore confer additional risk for exhibiting clinical symptoms of dementia . moreover , late -life depression frequently occurs in the context of chronic medical illness , and major organ system dysfunction is frequently associated with cognitive impairment , acting to further lower reserve . thus , each of the processes mentioned above and depicted in figure 1 , independently adds to the total brain injury burden , lowers reserve , and strengthens the association between the neurodegenerative process and the clinical change in cognitive functions . we believe that this explanation underlies the relationship between latelife depression and dementia in general , and ad in particular ( see figure 1 ) . this conceptualization de - emphasizes the importance of the distinctions between early and late - onset depression and the relative risk for ad vs vascular dementia in the context of late - life depression . the cognitive outcome of any given individual who has late - life depression depends largely on the predominance or particular mix of pathophysiology in that individual . the additive or synergistic effects of vascular disease , glucocorticoid - related brain injury , and intrinsic ad pathophysiology are refl.ect.ed in the empirical findings of heterogeneous neuropathology in late - life depression and dementia . this framework , by focusing on the key concept , of reserve threshold , delineates testable ( and falsifiable ) links between depression and subsequent dementia . figure 2 depicts various pathways through which the key processes outlined in figure 1 may lead to the heterogeneous cognitive and disease outcomes reported in the literature . ill e pathways include ( in order of figure presentation ) : ( i ) individuals who develop depression at any point in their lives , sustain minimal or no depression - related neuropathology ( eg , glucocorticoid neurotoxicity ) , and who have stable , normal cognitive functioning ; ( ii ) individuals who develop depression at any point and who experience depression - related neuropathology that results in mci that is stable ( unless they experience additional depressive episodes ) ; ( iii ) individuals who accumulate ad neuropathology over many years and who develop late - life depression ( related or unrelated to ad pathology ) , that lowers brain reserve capacity , and results in expression of mci earlier than otherwise would be the case , and given the underlying neuropathology , progress to ad ; ( iv ) individuals who accumulate ad neuropathology over many years along with co - occurring cerebrovascular disease , which damages the frontostriatal circuitry , leading to late - life depression . the total neuropathologic burden , combined with depressed mood , lowers brain reserve capacity , leads to expression of mci ( eg , memory and executive dysfunction ) earlier than otherwise would be the case , and , given the underlying neuropathology , progresses to ad along with co - occurring cerebrovascular disease ; and ( v ) individuals who develop cerebrovascular disease ( with variable neuropathologic burden ) , that damages the frontostriatal circuitry , leading to late - life depression and mci ( eg , executive dysfunction ) , that , will follow the course of the underlying cerebrovascular disease . based on the weight of the findings in the published literature and consistent with our model depicted in figure 1 , we suggest that pathway # 4 ( figure 2 ) leading to ad with co - occurring cerebrovascular disease is the most frequently occurring pathway among individuals with late - onset depression . understanding the pathways through which individuals with late - life depression develop progressive dementia in general , and ad in particular , is critical as novel treatment may prevent , forestall , or slow cognitive and/or disease progression .

there is a strong association between late - life depression , cognitive impairment , cerebrovascular disease , and poor cognitive outcomes , including progressive dementia , especially alzheimer 's disease . while neuroimaging evidence suggests that cerebrovascular disease plays a prominent role , it seems that depression alone may also confer substantial risk for developing alzheimer 's disease . the relationships between the prominent cerebrovascular changes , other structural abnormalities , specific forms of cognitive dysfunction , and increased risk for developing alzheimer 's disease among those with late - life depression have been difficult to reconcile . the varied findings suggest that there are likely multiple pathways to poor cognitive outcomes . we present a framework outlining multiple , non - mutually exclusive etiologic links between depression , cognitive impairment , and progressive decline , including dementia . importantly , the model is both testable and falsifiable . going forward , using models such as this to inform research should accelerate knowledge acquisition on the depressionldemeniia relationship thai may be useful for dementia prevention , monitoring the impact of depression treatment on clinical status and course of illness .

Does depression increase risk of subsequent cognitive decline and dementia? Neurobiologie substrates mediating the depression-cognitive decline-dementia links Glucocorticoids contribute to hippocainpal atrophy and learning/episodic memory impairment Biologic relationships between depression and Alzheimer's disease Role of vascular disease in late-life depression, cognitive decline, and dementia Brain and cognitive reserve; the final common pathway linking depression to dementia Proposed multiple pathways model

myocardial infarction ( mi ) is one of the leading causes of death in developed countries and it is characterized by several associated symptomatologies and a poor quality of life . features of the mi include an increase of cardiac sympathetic nerve activity to elicit inotropic positive response for the maintenance of cardiac output ; this compensatory mechanism plays a major role in the progression of heart failure . recent data showed the role of the hypothalamic paraventricular nucleus ( pvn ) , a key neurohumoral integrative nucleus in the brain , responsible for the connections to sympathetic preganglionic motor neurons [ 3 , 4 ] . moreover , high percentage of mi patients undergo late depressive state . brain inflammation , driven by nonneuronal cells such as astrocytes and microglia , has been demonstrated to be associated with several neurological diseases including depression and anxiety , with neuropsychiatric pathologies such as mood disorders and psychosis , and with neurodegenerative diseases such as parkinson , alzheimer , and huntington diseases [ 79 ] . indeed , all these pathologies are also associated with plastic changes which involve the activation of glia and microglia in the early sensitization and the subsequent neuronal suffering . moreover , glial and microglial cells have been shown to be associated also with the chronicity of pain that could be consequence of heart failures such as myocardial infarction [ 1012 ] . recent reports have analyzed the possible changes in microglial phenotypes after myocardial infarction at different time stages , demonstrating a microglial cytoskeletal rearrangement in a late phase after mi induction in the pvn whereas no significant differences were observed in the cortex [ 13 , 14 ] . this microglia activation seems to be mediated by the p2x7 receptor and also an attenuation of microglial and neuronal activation in the brain by icv minocycline following myocardial infarction has been reported . previous studies have demonstrated the beneficial effect of exercise training on deterioration in cardiac function after mi [ 17 , 18 ] . in this study we analyzed in sedentary and trained rats the microglia and astrocytes 48 hours after mi in pvn , thalamus , prefrontal cortex , and hippocampus through immunofluorescence approach . all the experimental procedures were approved by the animal ethics committee of the second university of naples . all efforts were made to minimize animal suffering and to reduce the numbers of animals used . sixty rats ( 225250 g ; n = 60 ; harlan italy ) were randomly assigned to two main groups : sedentary ( sed ; n = 30 ) and exercise trained ( tr ; n = 30 ) . briefly , trained rats were acclimated to training by walking at a speed of 10 min / day on a treadmill for 2 weeks ( panlab / harvard apparatus treadmills , holliston , ma , usa ) . from week 3 , speed and time of running were gradually increased ( 30 m / min , 45 min / day , 5 days / week , for 6 weeks ) . the sedentary rats remained in the cages for all the duration of the training protocol . twenty - four hours from the last session of exercise training 20 rats of both experimental groups were undergoing myocardial infarction by surgical occlusion of the left anterior descended ( lad ) coronary artery , according to previous described procedures . briefly , after induction with intraperitoneal injection of ketamine hydrochloride ( 100 mg / kg ) and xylazine ( 2.5 mg / kg ) supplemented as needed , the rats were intubated and ventilated with room air by a small animal ventilator ( harvard apparatus , model 623 ) . after performing the thoracotomy in the third and fourth intercostals spaces , the pericardium was incised and a 6 - 0 silk suture ( johnson & johnson ) was placed around the proximal portion of the left coronary artery . formalin - fixed , paraffin - embedded myocardial samples were cut in 5 m thick sections and stained with hematoxylin and eosin . for fluorescence imaging , tissue sections were deparaffinized and labelled with apoalert dna fragmentation assay kit ( clontech ) . subsequently , samples were incubated with an anti--sarcomeric actin ( sigma ) antibody followed by tritc - conjugated secondary antibody ( jackson immuno , west baltimore pike , west grove , pa , usa ) . samples were analyzed with a leica dm 5000b microscope and a zeiss lsm 700 confocal microscope . under pentobarbital anesthesia ( 50 mg / kg , i.p . ) , animals were transcardially perfused with saline solution followed by 4% paraformaldehyde in 0.1 m phosphate buffer . the brains were excised , postfixed for 3 h in the perfusion fixative , cryoprotected for 72 h in 30% sucrose in 0.1 m phosphate buffer , and frozen in optimal cutting temperature ( o.c.t . ) embedding compound . transverse sections ( 20 m ) were cut using a cryostat and thaw - mounted onto glass slides . slides were incubated overnight with primary antibody solutions for the microglial cell marker iba-1 ( rabbit anti - ionized calcium binding adapter molecule-1 ; 1 : 1000 ; wako chemicals , germany ) and gfap ( rabbit polyclonal antiglial fibrillary acidic protein ; 1 : 1000 ; dako cytomation , denmark ) , according to previously reported protocols [ 21 , 22 ] . possible nonspecific labeling of mouse secondary antibody was detected by using secondary antibody alone . following incubation , sections were washed and incubated for 2 h with secondary antibody solution ( donkey anti - rabbit or igg - conjugated alexa fluor 488 ; 1 : 1000 ; molecular probes , usa ) . slides were washed , coverslipped with vectashield mounting medium ( vector laboratories , usa ) , and visualized under a leica fluorescence microscope . the number of cells positive for iba-1 or gfap was determined within a box measuring 2 10 m that was placed in the lateral , central , and medial areas of cortex , hippocampus , thalamus , and hypothalamic paraventricular nucleus . eight sections were assessed from one animal and three animals were used for each group . to avoid cell overcounting , gfap - positive cells were identified as resting ( with small somata bearing long , thin , and ramified processes ) , activated microglia and astrocytes ( with hypertrophy together with retraction of processes to a length shorter than the diameter of the somata ) , or dystrophic microglia ( no dystrophic astrocytes were detected ) . dystrophic microglia was recognized by debris consisting of several cells displaying fragmented processes and an irregularly shaped cell body as previously demonstrated in humans . forty - eight hours after coronary artery ligation , the presence of myocardial infarction was confirmed by histological analysis . this was confirmed by the detection of apoptotic cardiomyocytes with a terminal deoxynucleotide transferase- ( tdt- ) mediated dutp nick - end labeling ( tunel ) assay and confocal microscopy ( figure 1 ) . mi procedure did not change the number of both total iba-1 positive cells in the cortex . however , mi animals showed a significant increase of the number of activated as well as dystrophic microglia cells in the same area . these effects were significantly prevented by applying the exercise training protocol ( figures 2(a ) and 2(b ) ) . interestingly , the exercise alone exhibited significantly effects on cells morphological changes , as compared with sedentary animals . in particular , it reduced the number of activated microglia cells and modulated the number of astrocytes , by increasing the total number of gfap - positive cells , and reduced the number of hypertrophic cells ( figures 2(c ) and 2(d ) ) . we observed a significant increase of total , activated , and dystrophic microglia cells in the hippocampus of mi rats . the exercise counteracted the microglia activation and reduced the number of dystrophic cells , without affecting the total cells number in the same area . moreover , the exercise reduced per se the number of activated microglia as compared with sedentary animals ( figures 3(a ) and 3(b ) ) . no changes in astrocytes the mi induction did not change the number of the total or activated microglia cells in the thalamus as compared with control . however , the number of dystrophic microglia dramatically increased in mi animals 2 days after surgery . exercise trained animals showed a significantly attenuated number of dystrophic microglia induced by mi by more than 50% . interestingly , the exercise erased per se the number of activated microglia in sedentary rats ( figures 4(a ) and 4(b ) ) . the total numbers of astrocytes counted in the thalamus of the different treatment groups were similar . however , mi induced an increase in the number of hypertrophic gfap - positive cells that were significantly reduced in the trained mice . moreover , the exercise training abolished the number of hypertrophic astrocytes in sedentary rats ( figures 4(c ) and 4(d ) ) . mi and sedentary rats did not differ in the number of total microglia as well as astrocytes in the pvn . furthermore , mi surgical procedure did not change the number of activated microglia cells found in the control animals ( ~10% ) . however , mi rats showed an increased number of dystrophic microglia cells as well as hypertrophic astrocytes which were not affected by exercise training ( figures 5(a)5(d ) ) . this study provides the first evidence that the possible changes in glia and microglia brain areas after myocardial infarction could be , at least in part , prevented by the prolonged moderate exercise . in this study we analyzed the morphological changes of the microglia and astrocytes in four brain areas involved in the integration of external emotional stimuli , learning and memory , sensorial perception , and metabolism , before and 48 hours after myocardial infarction and with or without 8-week training protocol . in particular , we observed that in the prefrontal cortex the 48 h mi induced an increased number of hypertrophic and dystrophic - like microglia in sedentary mice . both microglia phenotypes were significantly reduced in the prefrontal cortex in the 48 h mi trained group . no significant changes in the hypertrophic / resting astrocytes ratio were observed in the prefrontal cortex in all experimental groups except for the trained mice without mi in which we found an increase and decrease in the total and hypertrophic astrocytes , respectively . in the thalamus and hippocampus , which represent key brain areas in the sensorial and pain - associated synaptic plasticity , we observed an increased number of dystrophic microglia . furthermore , in the thalamus , microglial changes were also accompanied by an augmented number of hypertrophic astrocytes , in the mi group as compared to the sedentary animals . the prolonged exercise significantly reduced both the dystrophic microglia and reactive astrocytes induced by mi . finally , in the pvn we observed increased number of dystrophic microglia and hypertrophic astrocytes in the sedentary animals that were not changed by the exercise . the latter data are interesting also regarding the role of the pvn which represents an important neuroendocrine and preautonomic output nucleus and is considered as the important central site for integration of sympathetic nerve activity [ 3 , 4 ] . this could be explained , at least in part , assuming that the effect of exercise is mainly acting on those brain areas involved in the sensorial and rewarding processes such as thalamus and cortical areas rather than those brain structures directly involved in the interface between cns and periphery . intriguingly , previous data showed no changes in activated microglia in the pvn and cortex 24 hours after myocardial infarction induction [ 13 , 14 ] . our data demonstrated that after 48 hours postmyocardial infarction there are changes in microglia phenotypes . in particular , we observed the appearance of a dystrophic - like phenotype of microglia mainly in the thalamus and prefrontal cortex . this phenotype of microglia is predictive of a brain malaise that could , in turn , mirror in a malfunction of the neurons . indeed , dystrophic ( senescent ) rather than activated microglia seem to be associated with tau pathology and likely precede neurodegeneration in alzheimer 's disease in human . this microglia phenotype is not well characterized yet in terms of the specific marker expression . recent data in rodents highlighted for the first time dystrophic - like microglia phenotype in transgenic mouse model lacking for the d - aspartate oxidase , the enzyme responsible of the d - aspartic acid degradation , that showed high glutamate levels which , in turn , could be in part responsible of those microglia changes . moreover , a recent report showed a possible microglia change from a reactive to an age - like phenotype with the time in culture . according to the early appearance of dystrophic microglia all these changes could be due to a glutamate spillover possibly induced by an overstimulation of afferent fibers after the surgically - induced mi , rather then to the mi per se . despite this hypothesis , which needs to be verified , it is intriguing that the prolonged exercise prevents these microglia and astrocyte alterations in the prefrontal cortex , thalamus , and prefrontal cortex . the present study suggests that myocardial infarct may be correlated with a supraspinal synaptic reorganization in which microglia and astrocytes might play a role . indeed , in our experiments the scale of infarction was strongly correlated with the proportion of dystrophic or activated microglia and hypertrophic astrocytes in the brain . it is well known that , upon activation , morphological cells rearrangements are correlated with the synthesis and secretion of cytokines , by leading critical neuronal activity modifications [ 14 , 26 ] . thus , one can speculate that supraspinal nonneuronal cells may significantly contribute to the functional as well as behavioral alterations observed following myocardial infarction . however , further analysis will be necessary to more conclusively evaluate the phenotypical cells profiles induced by mi . in this context , the exercise seems to drive down the possible glial / microglial contribution to the functional alterations associated with myocardium infarct .

myocardial infarction ( mi ) is one of the leading causes of death in developed countries and it is characterized by several associated symptomatologies and poor quality of life . recent data showed a possible interaction between infarction and brain inflammation and activity . previous studies have demonstrated the beneficial effect of exercise training on deterioration in cardiac function after mi . in this study we analyzed in sedentary and trained rats the microglia and astrocytes 48 hours after mi in pvn , thalamus , prefrontal cortex , and hippocampus through immunofluorescence approach . we found significant changes in specific microglia phenotypes in the brain areas analyzed together with astrocytes activation . prolonged exercise normalized these morphological changes of microglia and astrocytes in the prefrontal cortex , hippocampus , and thalamus but not in the pvn . our data suggest that there is an early brain reaction to myocardial infarction induction , involving nonneuronal cells , that is attenuated by the prolonged exercise .

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

breast cancer is one of the most frequently diagnosed cancers and a leading cause of death among women worldwide . in 2008 , the worldwide incidence of female breast cancer was approximately 1.4 million , and the estimated number of deaths as a result of breast cancer was more than 450,000.1 breast cancer is the second most common cancer diagnosed in us women.2 approximately 75% of primary breast cancers test positive for a hormone ( estrogen or progesterone ) receptor protein.3,4 endocrine therapies such as tamoxifen , fulvestrant , and aromatase inhibitors that interfere with signaling through the estrogen receptor have revolutionized the treatment of hormone receptor - positive ( hr ) breast cancer . despite significant advances in the treatment of hr breast cancer , primary or acquired resistance to endocrine therapy is a major obstacle in this treatment and a frequent cause of disease recurrence . deregulation or aberrant signaling in the phosphatidylinositol-3-kinase ( pi3k)/protein kinase b ( akt)/mammalian target of rapamycin ( mtor ) signal transduction pathway through activation of mutations in pi3k or inactivation of mutations in protein tyrosine phosphatase is thought to contribute to the development of breast cancer.57 mtor is a serine / threonine protein kinase located immediately downstream of the pi3k / akt pathway and upstream of several key mediators of cell growth , proliferation , survival , metabolism , and angiogenesis ( figure 1).810 activation of the mtor pathway results in the phosphorylation of two downstream targets , the ribosomal p70 s6 kinase and the eukaryotic translation initiation factor 4e - binding protein , which mediate the translation of proteins involved in regulation of cell growth and proliferation.8,9 in addition , activation of mtor may result in phosphorylation of several downstream effectors and transcription factors that regulate survival , metabolism , and angiogenesis.8,9 therefore , mtor is the central nexus in a series of signaling pathways that integrate intracellular nutrient , energy , and redox needs with extracellular amino acid , nutrient , growth factor , and cytokine availability.8 hormone - independent breast cancer cell growth is associated with increased pi3k / mtor signaling and inhibition of pi3k and mtor - induced apoptosis.11 additionally , activation of the pi3k pathway after endocrine therapy was shown to be predictive of poor disease outcome.11 mtor inhibition restores sensitivity to endocrine therapy in resistant breast cancer cells expressing aberrant akt activity.12,13 altogether , these preclinical observations suggest that mtor plays a central role in endocrine resistance . as a strategy to overcome endocrine resistance , mtor inhibitors have been studied in several clinical trials in combination with endocrine therapies , and recent evidence from these trials will be discussed here . a phase ii study ( nct00062751)14 of temsirolimus in postmenopausal women with locally advanced or metastatic breast cancer showed that intermittent dosing of temsirolimus ( 30 mg daily for 5 days , every 2 weeks ) plus daily letrozole ( 2.5 mg ) improved the clinical benefit rate ( 80% versus 69% ) and median progression - free survival ( 13.2 months versus 11.6 months ) , compared with daily letrozole alone.15 based on this finding , intermittent dosing of temsirolimus was studied in combination with letrozole as first - line therapy in the phase iii horizon trial ( nct00083993)16 in postmenopausal women with aromatase inhibitor - nave , hr , locally advanced or metastatic breast cancer.17 data from an interim analysis showed that the objective response rate ( 27% each ) and median progression - free survival ( hazard ratio [ hr ] 0.90 ; 95% confidence interval [ ci ] 0.761.07 ; p=0.25 ) were similar between the temsirolimus plus letrozole arm ( n=556 ) and the letrozole alone ( n=556 ) arm . no difference in overall survival ( hr 0.89 ; 95% ci 0.651.23 ) was noted between the two arms.17 therefore , lack of efficacy led to the early termination of this trial . the most frequently reported grade 34 adverse events in the phase iii trial were consistent with those observed in the phase ii trial with temsirolimus.17 despite the lack of efficacy observed in the main trial , an exploratory subgroup analysis showed that , in patients 65 years of age , but not in those > 65 years , temsirolimus plus letrozole improved median progression - free survival from 5.6 months to 9.0 months ( hr 0.75 ; 95% ci 0.600.93 ; p=0.009),17 suggesting that temsirolimus plus letrozole might be beneficial in younger women ; however , larger trials are necessary to confirm this finding . a phase ii trial in patients with hr , human epidermal receptor 2-negative ( her2 ) metastatic breast cancer evaluated the efficacy and safety of sirolimus in combination with tamoxifen.18 in patients whose disease progressed while they were receiving prior tamoxifen or an aromatase inhibitor , treatment with sirolimus plus tamoxifen significantly improved median progression - free survival from 3.3 months to 11.7 months ( hr 0.43 ; 95% ci 0.250.92 ; p=0.0023).18 sirolimus plus tamoxifen improved median progression - free survival by 7.0 months in the second part of this phase ii trial , in which patients with hr , her2 metastatic breast cancer who could not afford aromatase inhibitor therapy were randomly assigned to receive sirolimus plus tamoxifen and compared with those randomly assigned to receive tamoxifen alone ( hr 0.48 ; 95% ci 0.250.93 ; p=0.0028).18 respectively , anemia ( 21% versus 18% ) , hyperglycemia ( 16% versus 8% ) , hypercholesterolemia ( 11% versus 1% ) , hypertriglyceridemia ( 11% versus 1% ) , stomatitis ( 8% versus 0% ) , rash ( 7% versus 1% ) , anorexia ( 7% versus 2% ) , and fatigue ( 6% versus 8% ) were the most frequently reported grade 34 adverse events with sirolimus plus tamoxifen or tamoxifen alone in this trial.18 although the results of this small phase ii trial are promising , larger trials are necessary before sirolimus can be used to treat patients with advanced breast cancer . phase i studies suggested that treatment with everolimus in combination with endocrine therapy was feasible for patients with hr breast cancer.19 tamrad ( nct01298713),20 a randomized , open - label , phase ii study , evaluated the efficacy and safety of everolimus 10 mg / day plus tamoxifen 20 mg / day ( n=54 ) versus tamoxifen 20 mg / day alone ( n=57 ) in postmenopausal women with hr , her2 , aromatase inhibitor - resistant metastatic breast cancer.21 randomization was stratified by primary ( relapsing during or within 6 months of stopping adjuvant aromatase inhibitor treatment or progressing within 6 months of starting aromatase inhibitor treatment in the metastatic setting ) and secondary resistance relapsing > 6 months after stopping adjuvant aromatase inhibitors or responding for 6 months to aromatase inhibitors in the metastatic setting.21 the primary end point of clinical benefit rate was 61% ( 95% ci 4774 ) among patients in the everolimus / tamoxifen arm , compared with 42% ( 95% ci 2956 ) for patients in the tamoxifen only arm ( exploratory p=0.045 , table 1).21 time to progression was significantly longer in the combination group ( 8.6 months versus 4.5 months ; exploratory p=0.002 ) , and a 55% reduction in risk of death was associated with combination therapy ( hr 0.45 ; 95% ci 0.240.81 ; exploratory p=0.007).21 the clinical benefit rate and time to progression were higher in patients receiving everolimus who had secondary hormone resistance ( table 1 ) , suggesting a potential compensatory adaptive response to long - term estrogen depletion and a potential clinical benefit from adding everolimus to hormone therapy in this subpopulation of patients.21 the safety profile of everolimus was consistent with those of previous reports ; the severity of most adverse events were grade 1 or 2 , and the adverse events could be managed without treatment interruption.21 adverse events more common in the combination group were stomatitis , rash , diarrhea , anorexia , and infection.21 the overall incidence of serious adverse events was similar in the two treatment groups.21 bolero-2 ( nct00863655),25 a randomized , international , double - blind , placebo - controlled phase iii study , evaluated the safety and efficacy of everolimus 10 mg / day plus exemestane 25 mg / day compared with exemestane 25 mg / day alone in postmenopausal women with hr , her2 advanced breast cancer with recurrence or progression after previous therapy with letrozole or anastrozole.22 patients were randomly assigned 2:1 to receive everolimus ( n=485 ) or placebo ( n=239 ) , in addition to open - label exemestane , in a blinded manner.22 randomization was stratified according to the presence of visceral metastasis ( yes or no ) and sensitivity to previous hormonal therapy ( yes or no).22 the primary efficacy analysis , reported at a median follow - up of 7.1 months , resulted in an estimated 57% risk reduction for progression - free survival ( hr 0.43 ; 95% ci 0.350.54 ; p<0.001 , local assessment , table 1).22 this corresponded to a clinically meaningful 4.1-month prolongation in median progression - free survival from 2.8 months to 6.9 months.22 at a median follow - up of 12.5 months , a 56% reduction in risk of progression was identified ( hr 0.44 ; 95% ci 0.360.53 ; p<0.0001 , local assessment ) , corresponding to a 4.2-month prolongation in median progression - free survival from 3.2 months to 7.4 months ( table 1).23 recently , at a median follow - up of 18 months , the median progression - free survival was 7.8 months compared with 3.2 months ( hr 0.45 ; 95% ci 0.380.54 , p<0.001 , local assessment ) , for the combination therapy versus exemestane only therapy ( p<0.001).24 central assessment of median progression - free survival at 7.1 months , 12.5 months , and 18.0 months was consistent with local assessments.2224 in addition , the objective response rate was significantly higher with combination therapy that with exemestane only therapy after 18 months of follow - up ( 12.6% versus 1.7% ; p<0.0001).26 respectively , the most common grade 3 or 4 adverse events were stomatitis ( 8% versus 1% ) , anemia ( 6% versus < 1% ) , dyspnea ( 4% versus 1% ) , hyperglycemia ( 4% versus < 1% ) , fatigue ( 4% versus 1% ) , and pneumonitis ( 3% versus 0% ) for everolimus plus exemestane compared with exemestane alone.22 there has been a series of exploratory subanalyses of different patient populations in the bolero-2 study . in patients younger than 65 years of age , everolimus plus exemestane was found to lower the risk of progression by 62% ( hr 0.38 ; 95% ci 0.300.47 ) compared with exemestane alone.27 in patients 65 years of age , the risk of progression was reduced by 41% ( hr 0.59 ; 95% ci 0.430.80).27 the improvement in median progression - free survival with everolimus plus exemestane in patients < 65 years of age and 65 years of age was 6.83 months and 8.31 months , respectively.27 also , an analysis of patients with visceral metastases showed that treatment with everolimus plus exemestane reduced the risk of progression by 53% in patients with visceral metastasis at baseline ( hr 0.47 ; 95% ci 0.370.60 ) , whereas the risk reduction was 59% in those without baseline visceral metastasis ( hr 0.41 ; 95% ci 0.310.55).28 everolimus plus exemestane also reduced the risk of progression by 61% ( hr 0.39 ; 95% ci 0.250.62 ) in patients who had recurrence after neoadjuvant or adjuvant therapy.29 additionally , using the european organization for research and treatment of cancer quality of life questionnaire - core 30 scale to evaluate patients health - related quality of life , the time to deterioration ( ie , worsening ) was longer with everolimus plus exemestane than with exemestane alone ( 8.3 months versus 5.8 months , p=0.0084).30 a recent randomized , double - blind , placebo - controlled phase ii study demonstrated that continuation with everolimus 10 mg / day was beneficial in patients with her2 breast cancer and bone metastases.31 an exploratory analysis of the bolero-2 study showed that everolimus plus exemestane significantly lowered bone turnover marker levels at 6 and 12 weeks relative to baseline , irrespective of the presence or absence of baseline bone metastasis , whereas treatment with exemestane alone increased the levels of these markers.32 even though progression of disease in bone was more severe in patients with baseline bone metastasis than in the overall population , progression in bone was significantly less severe in patients treated with everolimus plus exemestane than in those treated with exemestane alone.32 patients with bone - only metastasis had a significant improvement in median progression - free survival with everolimus plus exemestane than with exemestane alone ( 12.88 months versus 5.29 months).28 following these analyses , the long - term effects of mtor inhibition with everolimus plus exemestane on bone health in postmenopausal women with hr locally advanced or metastatic breast cancer were assessed in the phase iiib , multicenter , open - label 4ever study ( nct01626222).33 the preliminary results of 4ever supported the findings of bolero-2 , suggesting that everolimus provides a bone - protective effect that includes reduction of bone turnover and a reversal in the increased bone resorption associated with exemestane therapy.33 two additional ongoing phase ii studies investigating the efficacy and safety of everolimus combined with endocrine therapy in postmenopausal women with hr advanced or metastatic breast cancer are currently being conducted . the multicenter , open - label bolero-4 ( nct01698918)34 trial will assess the safety and efficacy of first - line therapy with everolimus 10 mg / day plus letrozole 2.5 mg / day in postmenopausal women with er , her2 metastatic breast cancer , and the beneficial effects of continuing everolimus plus endocrine therapy ( exemestane 25 mg / day ) beyond initial disease progression.35 the multicenter , open - label , randomized bolero-6 ( nct01783444)36 trial is also underway and will compare the efficacy and safety of everolimus plus exemestane and everolimus or capecitabine monotherapy in postmenopausal women with hr , her2 advanced breast cancer who progressed on prior anastrozole or letrozole therapy . 37 these studies are expected to be completed in 2015 , and their results should provide further evidence supporting the use of mtor inhibitors , such as everolimus , in combination with endocrine therapy to improve outcomes in postmenopausal women with hr , her2 breast cancer . although the clinical trial with temsirolimus did not show any benefit , potentially because of issues related to dosing or patient selection , the clinical trials with everolimus and sirolimus provide important insight into the clinical usefulness of targeting the mtor pathway to enhance the efficacy of available hormonal therapies and to overcome or minimize endocrine resistance in breast cancer . therefore , use of mtor inhibitors might delay the need for chemotherapy . despite clinical evidence demonstrating the clinical usefulness of mtor inhibitors in the treatment of patients with breast cancer , limited research has been done on identification of biomarkers that can be used to identify patients who are most likely to benefit from these agents . recent results from a translational study of the tamrad trial indicated that the benefit derived from everolimus therapy was greater in patients with low pi3k , low liver kinase b1 , or high phosphorylated 4e binding protein 1 expression.38 additionally , a retrospective exploratory biomarker analysis of the bolero-2 trial used next - generation sequencing to analyze numerous cancer - related genes and to determine if there were correlations between exon sequence or gene copy number variations and the efficacy of everolimus ( ie , improvement in progression - free survival).39 the four most frequently altered genes / pathways were not predictive of everolimus efficacy when assessed individually , but a greater benefit from everolimus was seen in patients with minimal genetic alterations in the pik3ca / phosphatase and tensin homolog / cyclin d1 or fibroblast growth factor receptor 1/2 genes combined ( 76% of the next - generation sequencing population).39 the findings of tamrad and bolero-2 provide useful information that may have important implications for the future clinical management of patients with breast cancer ( ie , patient selection and identification of combinations of novel targeted therapies ) . however , these results will need to be validated in prospective studies of independent patient cohorts . first - generation mtor inhibitors , such as everolimus , appear to exert their inhibitory effects on the mtor pathway by specifically targeting mtor complex 1 ( mtorc1 ) without binding to mtor complex 2 ( mtorc2).10 however , specific inhibition of mtorc1 by these agents may result in induction of prosurvival feedback loops , such as the pi3k - akt pathway ; this may explain why the antitumor activity of first - generation mtor inhibitors is modest when administered alone.10 this limitation of first - generation mtor inhibitors has led to development of the second generation of mtor inhibitors , which includes adenosine triphosphate - competitive inhibitors that block both mtorc1 and mtorc2 , as well as dual pi3k / mtor inhibitors . dual mtorc1/2 inhibitors currently in the early stages of clinical development include ink128 , cc-223 , osi-027 , azd8055 , azd2014 , and palomid 529 . however , caution should be taken with the use of second - generation mtor inhibitors , because global inhibition of the pi3k / akt / mtor pathway may result in greater toxicity . with the us food and drug administration approval of everolimus to treat postmenopausal women with advanced hr , her2 breast cancer in combination with exemestane after failure of treatment with letrozole or anastrozole , inhibiting mtor when treating patients with hr , her2 breast cancer is expected to improve treatment outcomes and might help to maintain quality of life .

breast cancer is a leading cause of cancer - related death worldwide . approximately 75% of breast cancer is hormone receptor - positive ( hr+ ) and is managed with endocrine therapies . however , relapse or disease progression caused by primary or acquired endocrine resistance is frequent . phosphatidylinositol-3-kinase ( pi3k)/protein kinase b ( akt)/mammalian target of rapamycin ( mtor)-mediated signaling is one of the molecular mechanisms leading to endocrine resistance . mtor inhibitors that target the pi3k / akt / mtor pathway are the first of the targeted therapies to be evaluated in clinical trials to overcome endocrine resistance . although the clinical trial with temsirolimus , an mtor inhibitor , did not show any benefit when compared with endocrine therapy alone , a phase ii clinical trial with sirolimus has been promising . recently , everolimus was approved in combination with exemestane by the us food and drug administration for treating postmenopausal women with advanced hr+ breast cancer , based on the results of a phase iii trial . therefore , everolimus represents the first and only targeted agent approved for combating endocrine resistance .

Introduction Temsirolimus Sirolimus Everolimus Conclusion and future directions

many deleterious mutations may then be harmless , because even if one gene suffers a mutation , the redundant gene copy can provide a back - up function . put differently , after gene duplication - which can arise through polyploidization ( whole - genome duplication ) , non - homologous recombination , or through the action of retrotransposons - one or both duplicates should experience relaxed selective constraints that result in elevated rates of evolution . this hypothesis originated as least as early as ohno 's seminal book , which emphasized the importance of gene duplications in organismal evolution . but for decades any test of the hypothesis had to rely on small numbers of gene duplicates ; doubts thus remained over whether conclusions derived from such case studies were representative of all genes in a genome . such sequence information can address not only this question but also many others related to the influence of selection on gene families . for instance , does one duplicate evolve faster and thus acquire new functions more rapidly than the other ? ? and how frequent is gene conversion of duplicate genes , in which recombination and dna repair between very similar genes convert the sequence of one to that of the other ? to address such questions , one can use nucleotide alignments of duplicates to calculate two key parameters of molecular evolution : the fractions per nucleotide site , first , of synonymous ( silent ) nucleotide substitutions , and ks , second , of non - synonymous nucleotide substitutions ( which change the encoded amino acid ) , ka ( see box 1 ) . the ratio ka / ks provides a measure of the selection pressure to which a gene pair is subject . if a duplicate gene pair shows a ka / ks ratio of about 1 , that is , if amino - acid replacement substitutions occur at the same rate as synonymous substitutions , then few or no amino - acid replacement substitutions have been eliminated since the gene duplication . in other words , the gene pair is said to be under ' purifying selection ' if ka / ks < 1 : some replacement substitutions have been purged by natural selection , presumably because of their deleterious ratio is , the greater the effects . the smaller the ka / ks number of eliminated substitutions and the greater the selective constraint under which the two genes have evolved . the converse case , ka / ks > 1 , indicates that replacement substitutions occur at a rate higher than expected by chance alone , so advantageous mutations have occurred in the evolution of the two duplicates . two recent studies analyzed these ratios in multiple fully sequenced and several partially sequenced genomes . even very closely related gene duplicates , no older than a few million years , experience selective constraints - the ratio ka / ks is smaller than one even in these cases . recent duplicates appear to tolerate more replacement amino - acid substitutions than older duplicates , however . for duplicates that differ at less than 5% of synonymous sites , between one in two and one in three substitutions are amino - acid replacement substitutions . for old duplicates , this number falls to between one in ten and one in twenty replacement substitutions . even a fine - grained statistical model that allows for differences in ka / ks among young and old duplicates may explain only 50% of the variance in evolutionary rates . in addition , there may be species - specific differences in ka / ks , but detection of such differences is sensitive to how information on gene duplicates is extracted from genomes and on how ka and ks are estimated . for example , one of the above studies suggests that recent mammalian duplicates ( ka / ks = 0.45 for genes with ks between 0.05 and 0.5 ) appear to be under lower selective constraints than recent duplicates of drosophila melanogaster , caenorhabditis elegans , or arabidopsis thaliana , where ka / ks < 0.3 , whereas the other study suggests no such differences . to determine whether one duplicate evolves faster than the other , one can compare the sequences of both duplicates with that of a related but distant ' outgroup ' gene and determine whether one duplicate has diverged to a greater extent than the other . for example , in bacteria and mammals fewer than 10% of duplicates seem to evolve at different rates . in contrast , a recent study focusing on ancient zebrafish duplicates - most of them developmental genes - found that about 50% of duplicates differ in their rates of evolution . despite such differences , these results show that it is not generally the case that one duplicate ' holds down the fort ' , and retains the original function while the other can evolve freely . tandemly duplicated genes are known to be subject to gene conversion events that homogenize their sequences . if rampant , gene conversion could substantially distort inferences of selection pressures after gene duplication . one group of genes with extremely slow rates of evolution , the histone h3 genes , has received recent attention in this regard . with only three amino - acid differences between animal and plant histone h3 proteins , for example , if so , one would expect that values of ks between histone gene duplicates would be small - reflecting recent gene conversion - and not dramatically greater than values of ka . but in organisms ranging from fungi to mammals , ka and ks differ by as much as a factor of 60 between non - tandemly clustered histone h3 genes , so evolution by gene conversion is unlikely to be frequent in this family . another study asked whether yeast ( saccharomyces cerevisiae ) gene duplicates show evidence of gene conversion . part of the assay in this study was based on the observation that measures of codon - usage bias are strongly correlated with the rate of synonymous divergence of yeast genes ( because mutations in a highly expressed gene to a synonymous codon for which the respective transfer rna is rare are deleterious ) . only 4 out of 160 yeast duplicates had a synonymous divergence ( ks ) less than expected on the basis of their codon - usage bias , showing that gene conversion is rare . in summary , although gene conversion is potentially rampant for some genes , it is most likely to be rare for the vast majority of genes . perhaps the most difficult questions about the influence of selection after gene duplication is how frequently beneficial mutations occur . large amounts of genome sequence information lend themselves to the establishment of databases that document the gene families that have elevated ka / ksratios . mere sequence analysis will probably have a limited impact on answering this question , however , because finding genes with ka / ks > 1 is usually not quite enough to make a case for positive selection . although a particular genome may contain many duplicates with ka / ks apparently above one , the observed difference from unity often does not withstand statistical scrutiny . it does not , because positively selected amino - acid substitutions often occur only in a small region of the coding region , too small to be detectable by an elevated ka / ks ratio . and several case studies suggest the existence of positive selection for individual gene families , including the opsin visual pigments , primate ribonuclease genes , and triosephosphate isomerases . these studies also show that a strong case for positive selection generally requires integration of information on gene divergence , phylogeny , and protein structure and function . in summary , genome - scale surveys of gene duplication have the great merit of answering questions about molecular evolution without lingering doubts of statistical bias caused by small samples . they can assess to what extent selection is relaxed after gene duplication , to what extent gene duplicates diverge at different rates , and how abundant gene conversion events are . but their biggest strength - providing summary information about thousands of gene pairs - is also their biggest weakness . some questions , such as the abundance of beneficial mutations , generally require more information than a crude view of the whole genome can provide . genome - scale surveys thus draw our attention to their own limitations , which call for an integration of a variety of approaches to understand genome evolution .

immediately after a gene duplication event , the duplicate genes have redundant functions . is natural selection therefore completely relaxed after duplication ? does one gene evolve more rapidly than the other ? several recent genome - wide studies have suggested that duplicate genes are always under purifying selection and do not always evolve at the same rate .

None Purifying or completely relaxed selection? Gene conversion Figures and Tables

proteins of this target class are classified into readers , writers , and erasers of marks on histones or other nuclear proteins and dna . the complex combinations of these posttranslational marks regulate gene expression and have been termed the histone code . bromodomains represent one of the readers of these marks , specifically recognizing acetyl - lysine ( kac ) through an architecturally conserved interaction module . sixty - one unique bromodomains have been identified from the human genome , each containing a conserved tertiary structure as described by mutjaba et al . , with the hydrophobic kac binding site at one end formed between the z short helix , the za loop , and the bc loop ( figure 1a ) . this binding site is primarily hydrophobic , with the carbonyl oxygen of the acetyl - group forming two hydrogen bonds , one to a donor from either asparagine or threonine and the other to a conserved water molecule at the base of the pocket ( figure 1b , c ) . ( a ) conserved protein fold of bromodomains comprising the four canonical helices z , a , b , and c . all illustrated by falz ( pdb 3qzs ) . through discovery of potent small molecule inhibitors ( figure 2 ) , bet family members , a definition that will be used throughout the paper : proteins able ( or predicted to be able ) to bind drug - like molecules ( not necessarily a drug ) . a short hairpin rna screen suggested that inhibition of the bet family may be a therapeutic strategy for aml . through discovery of pan - bet family inhibitor gsk1210151a from the isoxazole class , it has been suggested that inhibition of the bet family may be a therapeutic strategy for mll - fusion leukemia , and pan - bet family inhibitor gsk525762a , from the benzodiazepine class , has demonstrated anti - inflammatory potential in mouse models of inflammatory disease and sepsis . inhibitors of other bromodomains ( crebbp and pcaf ) have been found ( figure 2 ) , but none show the submicromolar inhibition reported for bet family inhibitors so far . bromodomains are currently an underexplored protein family in both basic biology and drug discovery , however , therapeutic potential is becoming increasingly recognized . with many bromodomain structures publicly available , this led us to investigate the structure - based druggability across the protein family . selected published bromodomain inhibitors . from an initial inspection of various bromodomain binding sites , we hypothesized that not all bromodomains would be as druggable as the bet family and a wide range of druggabilities would be observed . further , we wanted to identify variations in the amino acids within the binding site that correlated with predicted druggability . prediction of the druggability of a novel protein target allows realistic expectations of hit rates before any screening effort is undertaken . for a less druggable target , the acceptable potencies and associated ligand efficiencies are likely to be lower than for a more druggable one and there is an associated risk of not finding tractable hit matter . in this scenario , alternative strategies may be sought such as higher screening concentrations , the use of larger and more diverse libraries , or the choice of screening technique employed . one analysis of the druggability across a protein family was performed by campagna - slater et al . on another epigenetic target family , the histone methyltransferases . in this study , sitemap was used alongside the degree of buried surface area of the bound cofactor to assess the druggability . all of the histone methyltransferases were predicted to be druggable with dscores from sitemap ranging from 0.96 to 1.13 but with the degree of buried surface area of the cofactor showing some variability . another study has also recently been published , performed by santiago et al . primarily on methyl - lysine sitemap was also used in this work , and the authors suggest the methyl - lysine binding proteins to be less druggable than bromodomains . however , they only consider the eight members of the bet family that may not be representative of the family as a whole . many structure - based druggability prediction methods have been published in recent years , these include dlid , dogsitescorer , the ebi s drugebility , drugpred , fpocket , mappod , screen , and sitemap . cover a number of these methods and some of the challenges in computational druggability assessment . to assess the druggability of bromodomain proteins , we required a tool that is readily available but more importantly allows water molecules to be included in the analysis . this is necessary as we have identified five water molecules that appear to be conserved for most bromodomains and reduce the overall volume of the pocket . to our knowledge , the use of sitemap is consistent with the analyses of the histone methyltransferases and methyl lysine binding proteins highlighted above . a detailed validation of this method has been published , with sitemap accurately identifying 86% of the ligand binding sites from a set of 538 complexes of the pdbbind database as the top scoring site . , demonstrating comparable performance of sitemap to fpocket on their nonredundant data set ( nrdd ) . sitemap uses the same definition of druggable as we are using here and uses contributions from the volume of the pocket , the enclosure , and the degree of hydrophobicity to assess druggability . the main output from sitemap is two druggability assessment scores : sitescore ( eq 1 ) and dscore ( eq 2 ) where n is the number of site points , e is the enclosure score , and p is the hydrophilic score.12 both scores take contributions from the same properties but with different coefficients . both scores use a cap of 100 for the number of site points ( for our analysis only two structures reach this cap ) , and sitescore uses a cap of 1.0 for the hydrophilic score , whereas dscore is not capped . for our data set , the two scores have high correlation , with r equal to 0.92 . because of the high correlation with sitescore and the suggestion that it is more discriminatory of druggable and undruggable sites , dscore was selected to be used alone in our analyses . sitemap was applied to a filtered set of the published bromodomain structures extracted from the pdb , and a wide range of predicted druggabilities was observed , from difficult to druggable . from this initial druggability assessment , the dscores were compared with the clustering generated from whole sequence similarity of structure - based alignments by filippakopoulos et al . this analysis showed that whole sequence similarity alone did not sufficiently explain the trends in the druggability that were observed , therefore we inspected the binding sites and identified unique amino acid signatures that showed better correlation with observed druggabilities . we propose that this new classification is more relevant to small molecule binding than whole sequence similarity due to its focus on the binding site residues . it allows druggability prediction of bromodomains without structural characterization and will aid the selection of templates for homology models by comparison to members within the same classification . crucially , it also enables the medicinal chemist to identify family members that are likely to bind the same inhibitors as the targeted bromodomain , which can be explored either for lead hopping or selectivity screening . many bromodomain - containing proteins possess multiple bromodomains but also exist as different sequence isoforms . when referring to a single bromodomain of one isoform , we have used this format : bromodomain - containing protein name , followed by isoform if present ( a / b / c if isoforms are identical ) , followed by the bromodomain number . for example , the second bromodomain of the b isoform of brd8 would be shortened to brd8b(2 ) , and the single bromodomain of baz1a , which is identical between isoforms a and b , would be shortened to baz1a(a / b ) . when referring to different chains within a pdb file , we have used this format : pdb code followed by a letter corresponding to the number of the protein chain within the file . for example , the second chain of the protein brd1 in pdb 3rcw would be shortened to 3rcw_b . protein chains within each pdb file were separated , ligands and nonconserved water molecules removed , and protonation states assigned using protonate3d in moe . forty - six chains from 14 pdb files with unresolved binding site residues were filtered out ( supporting information , table s1 ) . bound state , resolution , presence of unresolved side chains , and presence of conserved water molecules were recorded for each chain . for taf1(a / b ) , whereby both bromodomains have been crystallized within one peptide chain , these were separated and treated individually . individual chains were then preprocessed using the protein preparation wizard in maestro with , create disulfide bonds , and convert selenomethionines to methionines options selected . the preprocessed chains were submitted to sitemap using default parameters and with identify top - ranked potential receptor binding sites to avoid any bias from using ligands / peptides to define pockets . the minimum number of site points per pocket identified needed to be reduced to 14 from 15 for pb1(a / b / c)(1 ) . kac binding sites were then selected from all identified sites and all outputted values recorded . module and the blosum62 matrix with default settings . as used in the full sequence alignment by filippakopoulos et al . , we have also used brd4(a / b)(1 ) as a reference sequence for numbering of the residues . surfaces are color - coded using the pocket coloring from moe with green indicating enclosed surface of the protein and white indicating exposed . having selected sitemap to assess druggability , the next step was to collect the available crystal structures from the pdb . this yielded 105 different pdb entries covering 33 of the 61 unique human bromodomains . these pdb entries were then separated into the separate protein chains , as each protein chain within a crystal structure can be of a different conformation , and any chains with unresolved residues in the binding site were removed . through inspection of available bromodomain structures , it was apparent that five water molecules are conserved across most bromodomain kac binding sites . no publicly available structures demonstrate the displacement of any of these by a ligand ( figures 1a , 3 , and supporting information , figure s1 ) , suggesting that the water molecules are an important feature of the binding pocket . frequently , water molecules are removed prior to druggability assessment and we decided to determine druggability in the presence and absence of these conserved water molecules to assess their effect on the dscore . all five water molecules could be identified in structures of 23 of the 28 unique bromodomains passing the requirement of a structure without unresolved binding site residues , although not all of the water molecules were always present in the same structure due to limitations of protein crystallography ( most frequently in low resolution structures ) . to maximize our coverage of the observed protein conformations while ensuring that all assessed structures contained the same number of water molecules , for structures with missing water molecules , structures of the same protein with the missing water molecules were aligned and the missing water molecules were included from the other structure(s ) . for smarca4 , a high resolution ( 1.50 ) structure and one bound with nmp were available and both of these structures demonstrate only four of the five water molecules present , so the druggability assessment has been assessed as such , raising the number of bromodomains initially considered to 24 . details of all the water molecules included can be found in supporting information , table s3 . conserved water molecules in the binding site of brd4(a / b)(1 ) ( pdb 3mxf ) . the absence of these water molecules led to a larger identified pocket and consequently a higher druggability score , with most of the bromodomains classified in the druggable range ( dscore > 0.85 ) . crucially , without the water molecules , a smaller range of scores was observed , making the assessment less discriminative between sites ( figure 4 ) . given that these water molecules enclose the pocket and have a significant effect on the druggability , all subsequent analysis was performed with the water molecules present . ( a ) plot of dscores obtained when conserved water molecules were included in analysis against the same structures with all water molecules removed . linear line of best fit added to plot . ( b ) histogram of same data showing distribution of scores with normal distribution fitted to this data . colors indicate druggability classification : red , druggable ; yellow , intermediate ; white , difficult . the 178 qualifying protein chains ( 24 of the 61 unique human bromodomains ) were prepared and submitted to sitemap druggability assessment . a wide range of druggabilities was observed for the bromodomains from difficult ( dscore < 0.75 ) ( e.g. , baz2b pdb 3q2f , dscore = 0.52 ) to druggable ( dscore > 0.85 ) ( e.g. , pcaf pdb 3gg3_b , dscore = 1.08 ) . scores in between these two have been classified as intermediate ( e.g. , crebbp pdb 3p1e_b , dscore = 0.82 ) ( figure 5 ) . details of all outputted scores from this assessment can be found in supporting information , table s3 . box - plots showing range and distribution of druggability for each bromodomain across available structures passing imposed filters ( including presence of binding site water molecules ) colors indicate druggability classification : red , druggable ; yellow , intermediate ; white , difficult . ( a ) four outliers removed from druggability assessment ( see group 4 text ) . most bromodomains contain a small and tight binding site to recognize kac of the protein substrate . this conveys a basic level of druggability as demonstrated by baz2b ( pdb 3q2f , dscore 0.52 ) and the potential to bind a small fragment with acceptable ligand efficiency ( nmp , baz2b le 0.29 ) . the differences between the sites stem from the environments around that small and tight pocket . for the bromodomain family as a whole , even the lowest scoring bromodomain ( pb1(a / b / c)(1 ) ) would be classed as more druggable than a protein protein interaction with a large ( > 1000 ) and fairly featureless interface . sitemap would fail to identify binding sites such as this ( e.g. , siah1 pdb 2a25 ) . at the other end of the druggability scale , some bromodomains have demonstrated comparable predicted druggability to what are currently considered druggable targets such as protein kinases ( e.g. , aurora a , pdb 1mq4 , dscore = 0.96 ) or hsp90 ( pdb 1am1 , dscore = 0.99 ) . when a ligand or peptide binds , the observed conformation of the protein may change to potentially induce a more druggable pocket . this has been seen for the bcl-2 family of proteins whereby small molecule inhibitors bind to pockets not observed in the apo or peptide bound structures and show improved potency . this is in line with an increase in the sitemap predicted druggabilities of the bcl - xl binding sites from apo ( pdb 1r2d_a , dscore = 0.76 ) to ligand bound ( abt-737 , pdb 2yxj , dscore = 0.95 ) . to assess whether this could be the case for bromodomains , those with both apo and ligand or peptide bound ( holo ) structures available were collated and the dscores from sitemap compared ( table 1 ) . dscores after four outliers were removed ( see group 4 text for details ) . colors : white , bromodomains for which a more druggable pocket was not observed for the holo structures ; green , bromodomains for which a more druggable pocket was observed for the holo structures but a comparably druggable pocket was observed in the ensemble of apo structures ; red , bromodomains for which a more druggable pocket was observed for the holo structures and a comparably druggable pocket was not observed in the ensemble of apo structures . from table 1 , it can be seen that only crebbp of the 12 bromodomains with apo and holo structures available show evidence of a more druggable pocket ( 0.050.1 increase in median dscore ) in the presence of a ligand or peptide that is not observed in the ensemble of apo structures . the median dscore for the entire ensemble of structures was classed as intermediate at 0.75 , but the highest scoring structure was classed as druggable at 0.89 ( pdb 3p1c_b ) when bound with kac . three bromodomains ( brd2(2 ) , baz2b , and pb1(a / b / c)(5 ) ) unexpectedly show reduced median druggability for the ligand or peptide bound structures when compared to the apo . comparably druggable conformations of the holo structures for brd2(2 ) and pb1(a / b / c)(5 ) within the range of the apo are observed in the full ensemble , but this is not the case for baz2b . these effects will be discussed later , in the context of the similarity of each bromodomain with other members of the family . other than for crebbp , in general it does not appear that ligand binding is able to induce a significantly more druggable structure than is observed in the apo ensemble for the bromodomain family . having completed the initial computational druggability assessment , the next step was to identify trends within the data set . when compared with the clustering generated from whole sequence similarity performed by filippakopoulos et al . , a lack of correlation was observed such as the first and second bromodomains of taf1 being placed in the same cluster despite dscores at either end of the scale . it is not surprising to see a lack of correlation between druggability and whole sequence similarity , as when dealing with a druggability assessment it is the nature of the binding site that affects the score , not the rest of the domain . for this reason , we decided to inspect the structures for binding site for features that vary across the bromodomain family and can be used to order the members of the family into groups . this led to the identification of eight groups characterizing 49 of the 61 unique bromodomains . each of these groups is defined by the presence of a unique signature of up to three amino acid residues that is shared by all members of that particular group and gives a characteristic shape to the kac binding site ( table 2 , figure 9 , and supporting information , figure s1 ) . taken together , the amino acid residues of all group signatures span seven residues that enclose the kac binding site . these were position 81 , which is a tryptophan in the bet family and forms what has been termed the za - channel , the two residues facing the binding pocket on the za - loop , both leucine at position 92 and 94 in the bet family , the residue at position 140 , which is most commonly asparagine and forms the key hydrogen bond donor interaction with the kac carbonyl , residues 144 and 146 on the bc - loop and c - helix , which enclose the hydrophobic shelf in the bet family , and residue 149 , which although not enclosing the pocket does influence the position of residue 81 , which can have a large effect on both the za - channel and hydrophobic shelf . residue 145 on the c - helix has also been included in the analysis , which although not part of any of the signatures has been used in further differentiating some of the bromodomains within each of the groups . thus , in total , eight residues have been used to characterize the bromodomain binding sites ( figure 6 ) . number of bromodomains with available structures passing the filters out of the number of members in the group . eight residues around the binding site used in analysis ( brd4(a / b)(1 ) used as reference , pdb 3mxf ) . ( b ) binding site residues shown with transparent surface representation . to determine which residues were present at each position , available bromodomain structures were overlaid with the reference structure , brd4(a / b)(1 ) ( pdb 3mxf ) , and the eight binding site residues were recorded that best aligned with the brd4(a / b)(1 ) residues ( supporting information , table s5 and figure s1 ) . for five of the eight identified residues ( 140 , 144 , 145 , 146 , and 149 ) , the spacial alignment corresponded with the sequence alignment , making the identification of the residues for the bromodomains without a structure straightforward . for the other three residues , due to the variation in length and position of the za - loop , spatial alignment did not always correspond with the sequence alignment . here we have used the residue that aligned best in space with the brd4(a / b)(1 ) structure , as this should be more relevant to the nature of the binding pocket . for bromodomains without a structure , the matching residue from a protein within the same grouping from the alignment of filippakopoulos et al . was used ( e.g. , between brpf1b and brpf3 ) . in a few cases , this was not always possible due to the lack of a sufficiently homologous bromodomain with a structure being available ( e.g. , mll1 or trim28 ) . these bromodomains have been excluded from groups that are characterized by the za - loop residues ( 81 , 92 , and 94 ) . using the binding site groupings obtained , a qualitative classification tree was generated ( figure 7 ) . this allowed plotting of the predicted druggabilities to visualize where the most druggable groups are as well relationships between the groups . these included the relationship between crebbp and ep300 with the bet family as they share the extended length of the za - loop , the relationship between groups 2 and 3 ( y or f at position 146 ) , and between groups 5 and 6 ( y or f at position 94 ) ( see group texts for more details ) . bromodomain classification tree generated on the basis of eight binding site amino acid signatures showing bromodomain druggability . druggabilities of ash1l , atad2b , brpf1b , pb1(a / b / c)(3 ) , pb1(a / b / c)(4 ) , and smarca2b assessed using structures failing imposed filters included ( see group text ) . furthermore , we further divided several groups into subclassifications such as the separation of the baz family within group 4 by exploring changes in whole sequence similarity that may have an effect on the overall fold of the bromodomains such as the za loop position or more subtle changes in the binding site that have smaller effects on the pockets than the signature residues . we believe that this grouping better explains the trend in druggability assessment than whole sequence similarity , but also that this grouping will predict small molecule selectivity patterns more accurately due to the focus on the binding site . this should prove useful when determining selectivity of inhibitors and the potential to identify possibilities to transfer hit matter from one bromodomain to another . another potential use of this grouping is for the building of a homology model . if the use of the model is to predict the binding mode of a small molecule inhibitor or to select compounds in a virtual screening approach , then the choice of template is very important . the bromodomains grouped together here share binding site features , and thus members of the same group should represent the best templates for building homology models for binding mode prediction . when comparing the clustering based on whole sequence similarity to the grouping performed here , differences can be observed ( figure 8) . the two classifications are not dramatically different ( 42/61 placed in the same group ) , which is not surprising , but there is sufficient difference to suggest that when dealing with small molecule inhibitors the binding site classification may be more informative . groups from left to right in same number order as table 2 with the same coloring . whole sequence classification colors generated from binding site classification group which shares highest percentage similarity . an example of the differences is baz1a(a / b ) that shares whole sequence similarity with the bet family but does not share binding site similarity with this family . it is therefore unlikely to bind similar ligands and likely possesses druggability similar to that of the group it has been placed in by binding site classification ( group 4 ) . another example is that atad2a and atad2b are placed in the same cluster as group 3 by whole sequence similarity but do not share binding site similarity with this group or any other bromodomain . each of the binding site classification groups will now be discussed individually , commenting on their druggability , but also any trends or inconsistencies within the groups . the bet family of bromodomains was classified as druggable , which correlates with the fact that a number of potent small molecule inhibitors have been found . the comparatively high druggability for this family can be explained by a more enclosed upper part of the pocket and thus additional surface area for interaction with small molecules not present in other less druggable bromodomains . this is predominantly due to the presence of a tryptophan residue at position 81 and a methionine residue at position 149 that influences the position of the tryptophan residue , forming the za channel . on the other side of the pocket , the za loop is longer than most other bromodomains , providing additional surface that can be utilized for interaction with small molecules . these features result in above - average druggability of the sites ( figure 9a ) . bromodomains aligned with brd4(a / b)(1 ) pdb 3mxf and colors generated using moe pocket coloring : green = enclosed and white = exposed . pocket colors are used to highlight binding sites and does not represent pockets identified by sitemap used for druggability assessment . images captured from the same viewpoint except image g at the same orientation as figure 6 . ( a ) ( g ) pb1(a / b / c)(1 ) pdb 3iu5 structure representative of group 8 . ( h ) crebbp pdb 3p1c_b structure . given the high similarity of the pockets , it is not surprising that nonselective bet family inhibitors have been found , although there are subtle differences between the first and second bromodomains of the bet family that could be exploited for selectivity . the entire bet family has the same za loop residues facing the pocket , but the first bromodomains possess an aspartate at position 144 whereas the second bromodomains possess a histidine . at position 145 , the entire bet family has an acidic residue but this changes between aspartate and glutamate . we have separated this group in the classification tree into the first and second bromodomains to reflect these small changes . an outlier in the druggability assessment was seen for a peptide bound structure of brd2(2 ) ( pdb 2e3k_b , dscore = 0.64 ) . the reason for this low score was the position of the tryptophan at position 81 . for the rest of the bet family ( and other brd2(2 ) structures ) , this residue is directed toward the binding site creating the za channel ( figure 10a ) . in this outlier , the tryptophan residue is directed away from the pocket , opening the pocket significantly ( figure 10b ) and inducing a pocket more like crebbp or ep300 ( leucine at position 81 ) ( figure 10c ) , greatly reducing the druggability . for the 56 bet family structures passing the initial filters , this is the only one for which the tryptophan is oriented away from the binding site , suggesting that this is an unusual conformation and does not appear relevant to small molecule inhibitor binding . ( c ) crebbp pdb 3dwy_a structure showing similarity to brd2(2 ) atypical conformation . four of these ( cecr2 , falz(a / b ) , gcn5l2 , and pcaf ) were classified at the high end of the druggability scale by sitemap and are within the same cluster by whole sequence similarity . the key features of these pockets are the aromatic residue at position 146 compared with a small hydrophobic residue in many other bromodomains and a tryptophan at position 81 . together , these signature residues provide a significant amount of hydrophobic surface on this side of the pocket . the za loop is two amino acids shorter than the bet family , but this part of the pocket is still sufficiently enclosed to provide high druggability ( figure 9b ) . these bromodomains represent a family that demonstrate high predicted druggability but to date have not been exploited with high affinity compounds . two outliers were seen for the bromodomain falz(a / b ) ( pdb 2f6n_a and 2fsa_c ) , which both scored significantly less than the other nine structures passing the imposed filters of this bromodomain . when inspecting the structures and comparing them with more druggable conformations of falz(a / b ) , no obvious changes could be seen , as was the case with brd2(2 ) . however , when examined more closely , it could be seen that both the za loop and the bc loop are moved slightly away from the pocket , inducing a more open conformation and thus reducing the druggability . the four structures that are peptide bound do not demonstrate this more open conformation and may be stabilized in the closed conformation by the presence of the peptide . interestingly , the remaining two members of group 2 ( taf1(a / b)(2 ) and taf1l(2 ) ) possess the same signature residues but are not present in the same whole sequence classification as the other members of group 2 . taf1(a / b)(2 ) also scored in the druggable range ( dscore = 0.89 ) , however , taf1l(2 ) scored in the difficult range ( dscore = 0.73 ) , possibly due to the za loop and tryptophan 81 positions opening the binding site . with only one structure available , this could be an example of a false negative , with an effect similar to the outliers of falz(a / b ) ( pdb 2f6n_a and 2fsa_c ) , and with further conformational sampling of the za loop and tryptophan 81 , a more druggable conformation could be observed . taf1(a / b)(2 ) and taf1l(2 ) differ to the other members of group 2 by the residues at position 94 , 145 , and 149 within the binding site as well as having reduced sequence similarity ; for these reasons they have been given their own subclassification in the classification tree . group 3 contains six bromodomains , which all fall into the same classification from the whole sequence similarity and are related to the group 2 by the presence of the aromatic residue at position 146 , enclosing this part of the pocket . they differ by the lack of the tryptophan at position 81 opening the za channel , so the druggability scores are somewhat lower , placing them in the intermediate category ( figure 9c ) . within the group , brd7 and brd9 have been given their own subclassification due to the changes in za loop residues and having tyrosine rather than phenylalanine at position 146 . although a crystal structure for brpf1b was not available , an nmr structure ( pdb 2d9e ) was and passed the filters other than the presence of the conserved water molecules . when sitemap druggability assessment was applied to the ensemble of structures , a median dscore of 1.04 was obtained ( supporting information , table s4 ) and is slightly higher but in a similar range to the dscore values obtained from the other members of the group without water present . using the lines of best fit from figure 4a and a subset of the values from this group , estimates of the dscore with water molecules were achieved of 0.91 and 0.97 respectively . these values are higher than other members of the group and places this bromodomain in the druggable category . the four members of the baz family cluster together by binding site similarity within group 4 , unlike the whole sequence similarity classification . the group is characterized by a shorter za loop than the bet family , with no residue overlapping with leucine 92 from brd4(a / b)(1 ) in space , making the pocket fairly open and reducing druggability . the baz family share the tryptophan at position 81 with the bet family , but this does not form the same za channel due to the change in residue at position 149 ( figure 9d ) . for the bet family , this is a methionine , which restricts the movement of the tryptophan forming the za channel , but in the baz family , this residue is small ( alanine or cysteine ) , which results in movement of the tryptophan toward residue 149 , removing the za channel and hydrophobic shelf present in the bet family and heavily reducing the druggability into the difficult category . the baz family is joined by trim24 , trim33a , and trim66 in this group , and although these do not possess a tryptophan at position 81 , they share a very similar za loop , with no residue overlapping with the leu92 from the bet family . this open part of the pocket , and the lack of a za channel , give these bromodomains similar pockets to the baz family . they have been given their own subclassification due to this change in position 81 from tryptophan to a leucine or valine . four structures of trim24 score highly in the druggability assessment and appear to be outliers ( supporting information , table s4 ) . when inspecting the sites identified by sitemap , it was apparent that the favorable score is not solely due to the kac binding site but also an extended site ranging from the kac binding site to the interface between the bromodomain and the adjacent phd . for this reason , the analysis performed here has excluded these data points . the kac binding site is better assessed by the other generated scores , placing it in the difficult range , but there may be small pockets close to the kac binding site which could be exploited by using fragments followed by a linking effort . baz2b surprisingly indicated reduced druggability of the ligand bound structure when compared to the apo ( 0.18 reduction in dscore ) . from the initial definition of druggability , it would be expected that in general , holo structures should be as druggable if not more so than their apo counterparts . when comparing the two baz2b structures ( pdb 3g0l and 3q2f ) , there are only subtle differences between the two conformations of the binding site , namely that for the ligand bound structure the pocket is slightly narrower due to movements of the za loop and bc loop , increasing the enclosure score ( 0.610.69 ) . this narrowing most likely occurs to maximize contact with the flat heteroaromatic part of the ligand , but in doing this reduces the volume of the most enclosed part of the pocket ( 105 to 92 ) . for sitemap druggability assessment ( particularly for low druggability sites ) , the reduction in volume counts more toward the dscore than the increase in enclosure , so the overall effect is to reduce the predicted druggability of the ligand bound structure relative to the apo . group 5 is characterized by the presence of an aromatic residue at position 94 , the effect of this is to provide a lid to the pocket , thus increasing the enclosure and therefore the druggability ( figure 9e ) . structures of pb1(a / b / c)(3 ) and pb1(a / b / c)(4 ) were available ( pdb 3k2j and 3tlp ) , but these structures were excluded from the initial analysis due to them missing some of the conserved water molecules . to include them in the analysis and to allow direct comparison of the dscores , water molecules from the highly similar pb1(a / b / c)(2 ) were included through alignment of the structures . this yielded median druggabilities for the two bromodomains of 0.57 and 0.70 , respectively , placing them in the difficult category ( supporting information , table s4 ) . phip(2 ) scored highest and was placed in the druggable range . the second , third , fourth , and sixth bromodomains of pb1 also fall into this grouping but none show as high a druggability as phip(2 ) and also show less whole - sequence similarity , and this has been indicated with a different subclassification with pb1a(6 ) given its own subclassification due to a four - residue shorter za loop . the phip(2 ) structure does have a ligand bound , so this reduced druggability of the pb1 members could either be due to lack of protein conformational sampling ( za loop position ) and be a false negative or could be due to more subtle effects influencing the overall conformation of the protein and therefore the druggability . the members of group 6 also share this aromatic residue at position 94 , but without any available structures it is difficult to say whether these would be druggable like phip(2 ) or more challenging like many of the pb1 bromodomains . unlike group 5 , group 6 members cluster together by whole sequence similarity , however , there are six other bromodomains that share whole sequence similarity with group 6 but do not appear to share binding site similarity . the four proteins in this group all fall into the same classification by whole sequence similarity . by whole sequence similarity , group 7 is joined by four other bromodomains ( pb1(a / b / c)(24 ) and pb1a(6 ) ) but do not share the signature residues and do not fall into this group . pb1(a / b / c)(5 ) was classified in the intermediate druggability range but smarca4 as difficult . all of these proteins possess a shorter za loop than the bet family , reducing the surface available for interaction with small molecules . the shape of the kac binding pockets are also different to those of the bet family in the available structures , with the location of the aromatic residue at position 139 moved toward the pocket and leucine at position 87 rather than the valine present in most other bromodomains . this induces a wider entrance , opening the tight binding pocket at the base of the binding site ( figures 9f and 11a ) . for this reason , it is expected that this group could bind ligands differently to the other groups , as the tightest , most conserved part of the binding site is significantly different . ( a ) pb1(a / b / c)(5 ) structure representative of group 7 . ( c ) overlaid backbones of usual conformation ( pdb 3g0j_a ) in green and unusual ( pdb 3g0j_b ) in blue . orientation and coloring consistent with figure 9 . one structure of pb1(a / b / c)(5 ) that failed the original filters on the presence of the conserved water molecules ( pdb 3g0j_b ) showed a particularly unusual conformation that is unlike any conformation of this bromodomain or any other bromodomain ( figure 11b ) . the za loop is moved toward the z helix , which is not possible in many other bromodomains due to the presence of alanine at position 81 ( figure 11c ) but may also be allowed due to the change in shape of the binding site discussed previously . the effect of this is to close this part of the pocket , which for many of the other bromodomains is the location of the za channel . this results in an increase in hydrophobicity of the remaining pocket and reduced preference for the conserved water molecules . when sitemap druggability assessment was applied to this structure ( with a single water molecule at the base of the pocket ) a dscore of 0.87 was achieved , which is significantly higher than any other conformation of this protein and places it in the druggable range . this unusual conformation of the protein may represent an opportunity for inhibiting this bromodomain selectively over any other , as this unusual conformation is not expected to be common and may be unique to pb1(a / b / c)(5 ) . this conformation may also allow for substitution of the water molecules , which appear to be highly conserved for most other bromodomains . as was the case for brpf1b , no crystal structure was available for smarca2b , but an nmr structure was available that passed the filters imposed apart from the presence of the conserved water molecules ( pdb 2dat ) . when sitemap druggability assessment was applied to the ensemble of structures , a median dscore of 0.81 was achieved ( supporting information , table s4 ) , which is higher than the dscore for smarca4 without water present but lower than the dscore for pb1(a / b / c)(5 ) . when converted , as with brpf1b , using the dscore values with and without water molecules of the other bromodomains and the other members of the group from figure 4a , estimates of the dscore with water molecules of 0.53 and 0.64 were obtained . this places smarca2b in the difficult range with comparable predicted druggability to smarca4 , which shares high whole sequence similarity . for the binding of kac to bromodomains , a key hydrogen bond is formed between the carbonyl of the acetyl group and a donor from either an asparagine or threonine residue at position 140 . for group 8 , this key residue is replaced with tyrosine ( eight bromodomains ) or aspartic acid ( mll1 : although protein construct has been engineered and may not be true for full length protein ) . this changes the nature of the pocket significantly , and it has been suggested that these domains may not bind kac , or if they do , the manner in which they do would be unlike most other bromodomains . mll1 has been given its own subclassification due to it possessing an aspartate at position 140 as have the sp family due to their high sequence similarity to each other over the other members . a structure for pb1(a / b / c)(1 ) is available which shows how tyrosine 140 reduces the size of the pocket ( figure 9 g ) , and sitemap assessed this site as the least druggable of the bromodomains with only a very small pocket being identified . with such a low assessed druggability , the only opportunity to target this site with a small molecule inhibitor would be to displace the conserved water molecules . but , even with the water molecules removed , the site only achieves a dscore in the low end of the intermediate range suggesting that pb1(1 ) would be very challenging to bind small molecules to the equivalent of the kac binding site of other bromodomains . a structure for ash1l ( pdb 3mqm ) , another member of this group , is also available , but the conserved water molecules are not present as is the case for pb1(a / b / c)(1 ) , so it was removed by the initial filter . the site scored comparably to pb1(a / b / c)(1 ) without water molecules with a median dscore of 0.74 , suggesting that this bromodomain would be similarly difficult to target with a small molecule inhibitor . the remaining bromodomains failed to be placed into any groups larger than two , with little similarity to any of the other groupings described here . of those with available structures , none showed any particular druggability as assessed by sitemap , except crebbp , which was classified in the intermediate range . crebbp is interesting as it possesses the same longer za loop as the bet family , with similar residues facing the binding site that provide similar interaction potential . however , the tryptophan at position 81 in the bet family , which forms the za channel and hydrophobic shelf , is a considerably smaller leucine , resulting in a loss of these features and a decrease in predicted druggability ( figure 9h ) . another unusual feature is the presence of an arginine at position 145 , which provides the potential to form charged interactions with this strongly basic center . flexibility of both the za loop and the unusual arginine could explain the large changes in predicted druggability of crebbp , with the highest scoring protein conformation being placed in the druggable category and the lowest in the difficult . with high sequence similarity and binding site similarity , the bromodomain of ep300 would be expected to bind similar ligands to crebbp and have similar potential for a more druggable pocket to be induced . similarly to pb1(a / b / c)(3 ) and pb1(a / b / c)(4 ) , a structure of atad2b ( pdb 3lxj_d ) was available that was filtered out due to missing two of the conserved water molecules . all five water molecules were present in a structure of the similar atad2a ( pdb 3dai ) , and through aligning the two structures , the missing water molecules of atad2b were included . sitemap druggability assessment yielded a score of 0.64 , placing this bromodomain in the difficult category . when targeting any protein with small molecule inhibitors , selectivity for the bromodomains , the highly conserved small and tight binding site ( binding acetyl part of kac ) at the base of the pocket makes prediction of selectivity for a low molecular weight ( < 200 da ) fragment challenging as it is the environment around this site which will determine the selectivity for larger molecules . from this analysis , the first proteins that should be tested for selectivity issues would be those within the same group ( figure 7 ) . there are , however , some similarities between groups that may give rise to comparable binding of small molecules . the groups that are related to each other that have been previously discussed ( groups 2 and 3 and groups 5 and 6 ) may bind similar ligands , but there are differences that may be exploited for selectivity . also as discussed , crebbp and ep300 show some similarity through the za loop to the bet family , but also phip(2 ) and wdr9(a / b)(2 ) show some similarity in the shape of the binding site with the same hydrophobic shelf and above average druggability . other than these , selectivity would be expected between groups for molecules larger than small fragments . adequately assessing the full ensemble of protein conformations is an issue that affects any prediction that uses crystal structures , as by their nature a static image is observed . to address this issue , we have used as many experimentally observed conformations of the bromodomains as possible , and scores generated by the druggability assessment do vary between conformations of the same protein , including different protein chains within the same crystal structure . for this reason , we can not rule out that some proteins that were classified as difficult or intermediate may be false negatives and may have the potential to be druggable with additional conformational sampling . however , the range of scores observed for different conformations of the same bromodomain appear to be less than those between different bromodomains due to changes in the eight selected residues identified here ( figure 5 ) . for this reason , it is possible that new bromodomain conformers may show slightly increased druggability than predicted from the currently available structures ( e.g. , intermediate instead of difficult or druggable instead of intermediate ) . however , it is unlikely that large leaps will occur ( e.g. , for a protein classified as difficult to move into the druggable category ) , and other than a few special cases discussed in the text ( brd2(2 ) , falz(a / b ) and crebbp ) , these have not so far been observed . predicted druggabilities of available bromodomain structures were assessed and a range of scores observed . the bet family members were predicted to be druggable , consistent with literature evidence . one group ( group 2 ) showed comparable or increased predicted druggability relative to the bet family and represents a currently unexplored group of proteins that may have relevance in drug discovery as their biology is revealed . many of the other bromodomains showed lower predicted druggability and some of these were classed as difficult based on their dscore . the comparatively low score suggests that these will show lower hit rates in screening efforts and that it will be more challenging to identify and optimize hit matter . however , it should be noted that even these bromodomains are far more druggable than featureless protein protein interactions . trends within the data set were then sought and rationalized by unique signatures characterizing the binding pockets , leading to a new classification of the bromodomains into groups with similar amino acids in key positions and similar predicted druggabilities . this classification showed significant differences to the whole sequence classification , suggesting that it may prove more useful to drug discovery directed toward the acetyl - lysine binding site . our proposed classification also allows medicinal chemists who work on a particular bromodomain to identify other family members that are likely to bind similar inhibitors . this information can be explored to select proteins for counterscreening or to identify bromodomain inhibitors that can be explored in a target hopping approach . furthermore , our results highlight the significance of water molecules in the computational analysis of bromodomain binding sites . a number of conserved water molecules occupy the base of the pocket and so far no example has been reported in which these have been replaced by small molecules . for this reason , all bromodomains have been treated equally with all of these water molecules kept as part of the binding site and the druggability assessment performed as such . the corresponding assessment without the water molecules present has also been performed , which places more of the bromodomains in the druggable category and , crucially , appears to increase the observed score more for the less druggable sites , making it less discriminatory between druggable and difficult sites . this work represents the first analysis of this type for the bromodomain family and will prove useful for drug discovery projects aiming to identify inhibitors of the acetyl - lysine binding site of bromodomains .

bromodomains are readers of the epigenetic code that specifically bind acetyl - lysine containing recognition sites on proteins . recently the bet family of bromodomains has been demonstrated to be druggable through the discovery of potent inhibitors , sparking an interest in protein protein interaction inhibitors that directly target gene transcription . here , we assess the druggability of diverse members of the bromodomain family using sitemap and show that there are significant differences in predicted druggability . furthermore , we trace these differences in druggability back to unique amino acid signatures in the bromodomain acetyl - lysine binding sites . these signatures were then used to generate a new classification of the bromodomain family , visualized as a classification tree . this represents the first analysis of this type for the bromodomain family and can prove useful in the discovery of inhibitors , particularly for anticipating screening hit rates , identifying inhibitors that can be explored for lead hopping approaches , and selecting proteins for selectivity screening .

Introduction Materials and Methods Results and Discussion Conclusion

the workshop was hosted by the health technology assessment unit at the university calgary , who are actively engaged with the provincial ministry of health to develop a htr model for alberta , canada . attendees came from a variety of contexts including representation from provincial , national , and international hta agencies , ministries of health , not - for - profit health agencies , academia , and industry . the workshop began with a brief presentation to establish a common understanding of htr and its development as a field . the first panel focused on methodological challenges and consisted of a health economist , an expert in implementation science and an international expert in htr . the second panel focused on current practices with representation from three jurisdictions ( alberta , australia , and united kingdom ) each at different stages with their htr programs . during the panel sessions , each panelist was given 5 minutes to provide an introduction followed by audience discussion . all data gathered , including presentations and rich discussion transcripts , were analyzed for key themes emerging in the field of htr using constant comparative analysis . table 1.definitionsdisinvestment : the process of withdrawing ( partially or completely ) health resources from those existing healthcare practices , procedures , technologies , and pharmaceuticals that are deemed to deliver no or low health gain and are thus not efficient health resource allocations ( 8).health technology : any intervention that may be used to promote health , to prevent , diagnose or treat disease or for rehabilitation or long - term care . this includes the pharmaceuticals , devices , procedures and organizational systems used in health care ( 24).health technology assessment : a multi - disciplinary field of policy analysis that examines the medical , economic , social and ethical implications of the incremental value , diffusion and use of a medical technology in health care ( 24).health technology reassessment : a structured , evidence - based assessment of the clinical , social , ethical and economic effects of a technology currently used in the healthcare system , to inform optimal use of that technology in comparison to its alternatives ( 4 ) . table 2.summary of themesdisinvestment is difficult : limited success has been achieved internationally.focus on clinical areas in addition to specific technologies : changes in utilization of one technology may impact utilization of a competing technology . efficiencies in process may be achieved by reviewing clinical areas as opposed to single technologies.clear goals of the htr agenda are required to prioritize candidate technologies : a common understanding of the goal supports transparent prioritization of htr candidates.emphasize integration over segregation : embed htr into existing processes.focus on development of htr methods and processes : methodology to measure costs , benefits , and values is necessary for non - drug technologiesprocesses are often context - specific : learnings from other jurisdictions must be tempered by local contextual and system realities.build capacity in synergistic interdisciplinary fields : innovative , interdisciplinary teams of highly skilled people from disciplines such as economics , implementation science , kt , health services research , policy analysis , change management , evaluation , and stakeholder engagement are required.meaningful stakeholder engagement : an effective strategy should grab both the hearts and the minds of stakeholders . thoughtful consideration of patient and public engagement should be undertaken.strengthen post - implementation monitoring and evaluation : post - implementation monitoring and evaluation must be undertaken with an emphasis on active monitoring for unintended consequences . the united kingdom and australia report the most experience in htr . at the national ( medicare ) level in australia , htr has been integrated under the responsibility of the department of health and ageing , including the medical services advisory committee ( msac ) , which makes recommendations to the health minister about what medical services offer sufficient safety and ( cost)effectiveness to warrant public subsidy . since 2009 , a quality framework for australia 's medicare benefits schedule was developed under the auspices of the msac ( 16 ) . as part of this initiative , the health minister commissioned a review of hta in australia , with one outcome being the development of a postmarket surveillance system that included a parallel approach to htr . this parallel approach involved a review of individual items ( 17 ) , which dovetailed with existing hta processes , as well as a whole - of - specialty review ( 17 ) . both proved to be novel , effective approaches to engaging clinicians from the beginning of the htr process . for the review of individual items , a proposed scanning process was adapted to identify appropriate candidates for htr ( 15 ) . using this process , 156 possible candidates were identified a nd provided to government for consideration , from which they identified fifteen for initial rapid review and potential full htr ( 16 ) . outcomes from review include : an amendment to the item description such that it better captures the patient group / s most likely to benefit from any procedure , for example ; an increase , decrease or maintenance of the fee ; or a complete stop to public funding of the item . one example of an individual technology that underwent a complete review was vertebroplasty for osteoporotic vertebral fractures . the recommendation from msac was that government stop public funding of this procedure because of a lack of evidence of effectiveness . some clinicians noted their dissent to msac 's decision yet concomitantly , multiple reports from around the world have been developed supporting msac 's position ( 18 ) . in ophthalmology , a whole - of - specialty review was conducted . the initial impetus for the review was a government decision to cut the fee for cataract surgery , which resulted in dissent from ophthalmologists . the government 's observation was that advances in cataract surgery technology delivered on its promise : procedures could be done far more quickly and safely then when it was first introduced ( and priced ) , so a decrease in fee seemed the appropriate market response . the ensuing whole - of - specialty ( ophthalmology ) review of the medical benefits schedule was approached collaboratively with ophthalmology stakeholders , asking the field what in the schedule they thought required some modification . of the sixty - one item descriptors on the schedule only twenty remained unchanged through the process . items were either clarified , modified ( e.g. , refinement of indications and/or eligible patient groups ) , split , merged , or entirely removed ( 17 ) . in all respects , this was considered a success with good outcomes for all including patients . the whole - of - specialty approach is now rolling out more broadly . in the united kingdom , the national health service ( nhs ) , through the national institute for health and clinical excellence ( nice ) , has launched a htr program . the recommendations are identified during the process of guidance development where nice 's independent advisory bodies often identify nhs clinical practices that they recommend should be discontinued completely or should not be used routinely . each record contains the do not do recommendation and includes additional information including the intervention , health topic , the guidance it comes from ( with a link to the relevant paragraph in the guidance ) , and the other do not do each recommendation also includes the healthcare setting that describes the main clinical environments in which the intervention or investigation may be initiated . in addition to the these are based on cochrane reviews with the goal of helping the nhs identify practices that could be reduced or stopped freeing up cash and resources that could reinvested in more effective practices . nice 's experience over the past 12 years , however , is that although reassessment and potential disinvestment has the potential to increase efficiency and quality , actual cost savings , and therefore the potential for reinvestment has been less than anticipated . in the year 20112012 , half of the efficiency savings came from the freezing of health professionals salaries , not disinvestment in technologies ( 19 ) . an analysis of the shared international experiences resulted in three common themes emerging : even in the presence of very strong evidence that a particular technology is harmful and/or ( cost)ineffective , withdrawing the technology from the healthcare system is difficult . players , both within and outside the healthcare system , must be prepared for political and professional fall - out . it is difficult to tease out the appropriate scope of use of a particular technology from the broader pathway of care . very little done in health care is done in isolation , but rather procedures and devices are used in combination with other treatments within a pathway of care . a change in scope of use of a technology is likely to have ripple effects , resulting in unintended consequences , such as an increase in use of an alternative treatment . a clear understanding of the care pathway within which the technology is embedded , along with social and ethical implications , is critical . the unintended consequences may be mitigated by focusing on clinical areas where multiple comparators can be assessed side - by - side in any care treatment algorithm . in addition , this approach provides a natural way for engaging clinicians as one could begin by approaching a particular department or specialty . if the focus is actual cost savings , then it is likely that much could be achieved by focusing on redesigning service delivery mechanisms , and optimizing the human resources within the healthcare system . however , if the goal is to optimize the use of technology , which may include cost - savings , focus must move to technologies which are misused in the system . analyzing the audience discussion in the context of current literature and expert presentations , several key themes emerged to move the field of htr forward . integrating htr with on - going work such as the development of clinical practice guidelines and care pathways , as well as quality improvement initiatives will likely result in the greatest uptake of htr . advocating for the integration of economic considerations into guideline and care pathway development will support value for money as one criterion to inform decisions about what should and should not be done . methodologies must maximize usefulness for decision makers , and incorporate outcomes of importance to both clinicians and patients . in addition , they must represent feasible tools to use within a policy / decision - making context ; they must be relevant , timely , and interpretable . although there is much to be learned from processes developed in various jurisdictions , processes ought to be context - specific . learnings from other jurisdictions must be tempered by contextual and system realities , and processes are likely to require local adaptation . this will ensure that processes developed are supported by , and meet the needs of , the system using the outcomes to inform their decisions . highly skilled people are required in a variety of disciplines including : economics , implementation science , kt , health services research , policy analysis , change management , evaluation , and stakeholder engagement . it will take innovative teams that are interdisciplinary and cross institutional in nature to take on the htr challenge . essential elements to engage stakeholders are : transparent processes for identifying and prioritizing potential htr candidates , involvement throughout the process of evidence generation and review , and authentic meaningful engagement ( 11;13;14;20 ) . stakeholders should be defined broadly to include any group impacted by the resulting decision . as clinicians are likely to be an important stakeholder group in any htr , an effective strategy should grab both the hearts and the minds of clinicians . htr must include a developed postimplementation monitoring and evaluation process with an emphasis on active monitoring for unintended consequences . that is to say , there are things that we know we do n't know . 22 ) active monitoring for these unknown unknowns is critical to ensure that health outcomes , access , and equity are not adversely impacted . for example , within a surgical program , the operation suite will never be un - used . thus , if one procedure is stopped , the operational suite time could be filled with another procedure perhaps equally as , or more , harmful and wasteful . active monitoring for these situations will ensure that the vacuum created by removing one procedure is back - filled by a more effective procedure resulting in better value for money . this point emphasizes the need for buy - in by clinicians to mitigate unintended consequences . monitoring for these consequences will require a variety of evaluation techniques drawing on both quantitative and qualitative research methodologies . if htr is to be successful it must generate useful knowledge to inform real world decisions around the optimal use of technologies . the term inform is deliberately used here , as it is recognized that research evidence is only one of many kinds of knowledge that decision makers use when making these decisions , and that these decisions are always informed by values . in addition , decision making is neither rational nor linear , and as jonathon lomas has noted is not an event ; rather it is a generally diffuse , haphazard & somewhat volatile process that is ethereal in nature ( 23 ) . as one panelist noted , evidence - based guidance can be viewed as a practical manifestation of social contracts in deliberative democracies to ensure the most efficient and ethical allocation of finite healthcare resources . to achieve this goal , social values as well as technical issues need to be considered and guidance developed from htr should reflect the social / political milieu . healthcare budgets will not continue to increase and we need to shift our thinking about how we can use these dollars differently with the goal of optimizing managing technologies throughout their lifespan is required to ensure that technologies continue to achieve optimal value for money . htr is required to support evidence - informed continued management . to move this field forward , we must continue to build on international experiences with a focus on developing novel methodological approaches to generating , incorporating , and implementing evidence into policy and practice . gail mackean , phd , research consultant , health technology assessment unit , department of community health sciences , institute of public health , faculty of medicine , university of calgary , calgary , alberta , canada tom noseworthy , md , mph , professor , health technology assessment unit , department of community health sciences , institute of public health , faculty of medicine , university of calgary , calgary , alberta , canada adam g. elshaug , phd , associate professor , menzies centre for health policy , school of public health , the university of sydney , nsw , australia laura leggett , bsc . , research assistant , health technology assessment unit , department of community health sciences , institute of public health , faculty of medicine , university of calgary , calgary , alberta , canada peter littlejohns , phd , professor , division of health and social care research , school of medicine , king 's college london , capital house , london joan berezanski , assistant deputy minister , clinical advisory and research branch , alberta health , edmonton , alberta , canada fiona clement , ( [email protected] ) , phd , assistant professor , health technology assessment unit , department of community health sciences , institute of public health , faculty of medicine , university of calgary , calgary , alberta , canada

background : health technology reassessment ( htr ) is a structured , evidence - based assessment of the clinical , social , ethical , and economic effects of a technology currently used in the healthcare system , to inform optimal use of that technology in comparison to its alternatives . the purpose of this study is to describe the key themes in the context of current htr activities and propose a way forward for this newly emerging field.methods : data were gathered from a workshop held as part of the 2012 canadian agency for drugs and technology in health ( cadth ) symposium . the workshop consisted of two panel presentations followed by discussion ; data gathered , including presentations and rich audience discussion transcripts , were analyzed for key themes emerging in the field of htr using constant comparative analysis.results : the language chosen to describe htr will set the tone for engagement . the identification of champions at multiple levels and political will are essential . key lessons from international experience are : disinvestment is difficult , focus on clinical areas not specific technologies , identify clear goals of the htr agenda . six key themes were identified to move the htr agenda forward : emphasize integration over segregation , focus on development of htr methods and processes , processes are context - specific but lessons must be shared , build capacity in synergistic interdisciplinary fields , develop meaningful stakeholder engagement , strengthen postimplementation monitoring and evaluation.conclusions : to move this field forward , we must continue to build on international experiences with a focus on developing novel methodological approaches to generating , incorporating , and implementing evidence into policy and practice .

METHODS RESULTS CONCLUSION CONTACT INFORMATION CONFLICTS OF INTEREST

the main causes are injury to the nervous system , including the central and peripheral nervous systems , or musculoskeletal abnormalities . in rare cases in particular , paralysis of the limbs can be caused by a conversion disorder after surgical procedures , including laparoscopic operations . we found a patient who became paralyzed in her lower extremity after undergoing laparoscopic appendectomy . our findings indicate that psychological problems can be related to limb paralysis without organ damage in patients who have undergone laparoscopic surgical procedures . a 13-year - old patient visited the department of general surgery complaining of sudden abdominal pain after experiencing intermittent nausea and vomiting for a few days . tenderness and rebound tenderness in the right lower portion of her abdomen was observed upon physical examination . the operation was performed with the patient in a supine position while under general anesthesia . an incision was made 1 cm below the umbilicus , and a trocar 11 cm in length was inserted after pneumoperitoneum was induced using a veress needle . next , 5 mm trocars were inserted around the suprapubic area and anti - mcburney 's point . the appendix was extracted with laparoscopic forceps and the mesoappendix was removed by laparoscopic electric cautery . the base of the appendix was ligated with two endo - loops ( covidien co. ltd . , mansfield , ma , usa ) and a 10 mm distal region of the base was also clipped with a laparoscopic clip . two days after the operation , the patient complained of hypesthesia and weakness in the right lower extremity . the patient was transferred to the department of rehabilitation medicine , and a neurological examination was performed . the ability to sense pain and temperature were normal , but the sense of light touch was abnormal between the t11 and l2 dermatome , indicative of hypesthesia . muscle strength of right hip flexor , knee extensor , and knee flexor was reduced to 2/5 , but the strength of other muscles was unchanged according to a manual muscle test . pelvic and lumbar magnetic resonance imaging ( mri ) was performed in order to evaluate the cause of lower limb paralysis . the patient complained of pain around the surgical site , and paralysis of the right lower extremity persisted for 10 days after surgery . muscle strength of the right hip flexor , knee extensor , and knee flexor was still decreased in the follow - up manual muscle test . an electrodiagnostic study ( emg ) nerve conduction and needle electromyography results appeared normal , except that the interference pattern of motor unit action potential in the muscles was decreased at maximal volition . rehabilitation training was performed by the patient for 40 minutes twice a day with a physical therapist since being transferred to the department of rehabilitation medicine . the patient indicated that she liked ' taekwondo ' and desired to become a taekwondo athlete . therefore , functional training , including balance and posture exercises related to taekwondo , was performed continuously . . the sense of balance when in specific positions also improved while functional training was performed . on postoperative day 14 , tenderness around the right lower quadrant of the abdomen was still observed and the degree of the pain was 6 on the visual analogue scale . because the pain around the operation site and right lower abdomen persisted , a general surgery consultation was performed to evaluate the presence or absence of laparoscopic complications . however , the operation wound was found to be well healed , and no abscesses or mass lesions were found by follow - up abdominal computed tomography . on postoperative day 20 , a follow - up emg was performed in which no significant interval changes were observed , and the results were the same as the previous emg . therefore , peripheral nerve lesions and myopathy were ruled out as the cause of paralysis . brain , cervical , and thoracic mri was performed in order to detect disorders of the central nervous system , but definite abnormal findings were not observed . we subsequently decided to explore the possibility of psychological problems after organ damage , including nerve or muscle injury , was ruled out as the cause of paralysis . an interview with the patient 's parents was conducted on postoperative day 21 . according to the results of the interview , the patient was having trouble adjusting to school and her parents had visited the school several times to meet with her teacher . the patient hoped to move to another school that has a taekwondo club in order to be an athlete , but the patient 's parents were against the idea . on postoperative day 22 , significant improvement of the leg strength was not observed but the patient could independently walk from the general ward to the rehabilitation clinic . although right hip flexor strength was still 2/5 the normal level , her lower extremities moved well without claudication from the stance to swing phase during gait training . on postoperative day 23 , we interviewed with the patient 's parents and explained that a conversion disorder could be a possible cause of paralysis . the parents were encouraged to resolve the conflict concerning the patient 's career path through continuous psychiatric consultation . the patient was discharged . paralysis of the patient 's lower extremity improved gradually and the patient returned to normal life . laparoscopic surgery is widely performed instead of open appendectomy due to many advantages , such as diagnostic help , shorter hospitalization periods , reduced post - operative pain , decreased need for analgesics , fewer cases of wound infection , and improved cosmetic results.1 ) however , direct organ injuries , vascular injury , wound infection , hernia formation , tumor seeding , and endometriosis resulting from laparoscopic surgery have been reported.2 ) although rare , paralysis due to nerve injury may also occur owing to positional injury , trocar injury , peritoneal adhesion , or pneumoperitoneum after laparoscopic surgery.3 ) if organ problems or physical disorders are ruled out as the cause of sensorimotor dysfunction appearing after laparoscopic surgery , a conversion disorder might be the cause of limb paralysis.4,5 ) we came across a case of paralysis of the lower extremity after laparoscopic appendectomy . although a complete workup was performed in order to uncover the cause , no organ problems could be found . therefore , we investigated the possibility that the paralysis may be due to psychological problems . we were aware that the patient was coming into conflict with her parents over the patient 's career path and was having trouble adjusting to school . according to the diagnostic criteria of conversion disorder in the dsm - iv,6 ) we diagnosed the patient as having conversion disorder due to the motor weakness of the right lower extremity and decision of her career . in addition , there were no organ injuries causing limb paralysis in spite of multilateral evaluations , absences from school due to gait disturbance , and points that were not explained by other symptoms of mental illness . conversion disorder is a type of somatoform disorder that causes dyssynergia , tremor , dystonia , or paralysis of one or more extremities . this condition is also known as chronic neurosis , psychosomatic disorder , functional disorder , or functional overlay.7 ) conversion disorder is characterized by motor or sensory symptoms accompanied by psychological factors . functional loss is not intentional or related to general medical problems.8 ) conversion disorder must be distinguished from malingering , in which symptoms occur intentionally and are related to secondary gain . one characteristic of conversion disorder is that somatic symptoms are not provoked by intent.9 ) ten percent to 23% of patients with conversion disorder have paralysis of limbs as a major symptom.10 ) patients with conversion disorder are more likely amicable , cooperative , and reliant than malingerers and accept evaluation and treatment gladly . patients with conversion disorder are more likely to report historical gaps , incorrect , and unpredictable changes , whereas malingerers are more likely to provide extremely thorough and definite explanations of their experiences precipitating their illness.11 ) the patient cooperated well with medical examination and there were vagaries about her symptoms and signs . these factors were helpful in differentiating the diagnosis from malingering . for patients with paralytic symptoms due to conversion disorder , hospitalization is required for a few day or months , and functional recovery is good.4 ) not much research has been conducted on the outcomes or long - term prognosis of conversion disorder in children and adolescents . it has been reported that many conversion disorder symptoms spontaneously improve after a few days or weeks or require minimal intervention . it was also found that the rates of clinical improvement are good , ranging from 56% to 100% . in particular , a good prognosis is associated with young age , fast diagnosis , rapid identification of stressors , and a good relationship with family members.12 ) however , it must be noted that the clinical symptoms may be perpetuated if diagnostic tests were performed aggressively in order to identify the causes , but the cause is not detected at an early stage . symptoms are also more likely to be chronic by excessive concern from family or medical staff.13 ) in the current study , we only considered the possibility of organic problems soon after the onset of symptoms and thus conducted several diagnostic tests . however , the cause was not identified with these methods and the patient 's family became increasingly concerned . fortunately , we determined that the cause was a stressor related to a conflict regarding the patient 's career path . furthermore , a close relationship was formed between the family members and the parents with respect to the patient 's desire to become a ' taekwondo ' athlete . this is thought to be an important step as the patient 's symptoms were not chronic and completely resolved within 2 months . treatments for conversion disorder include group or individual psychotherapy , medications , and rehabilitation therapies.4 ) rehabilitation therapy for patients with paralysis generally includes strengthening exercises for the paralytic muscles , stretching exercises to maintain range of motion for each joint , and functional training to improve gait or transfer . it is important to provide this treatment while not showing excessive concern or reinforcing abnormal gait patterns caused by paralysis that is associated with conversion disorder . in particular , kanarek et al.4 ) reported that it is important to plan task - oriented goals based on functional training while considering the patient 's interests in order to improve the patient 's function . rehabilitation training that included muscle - strengthening exercises and functional gait training was conducted for 40 minutes twice a day with a physical therapist . functional training , with balance and posture exercises relating to taekwondo , was also performed continuously considering the patient 's interest . we believe that a good rapport with the patient through these therapies prevented the development of sequelae associated with conversion disorder and promoted functional improvement .

limb paralysis can develop for various reasons . we found a 13-year - old patient who became paralyzed in her lower extremities after laparoscopic appendectomy . some tests , including electrodiagnostic studies and magnetic resonance imaging , were performed to evaluate the cause of lower limb paralysis . none of the tests yielded definite abnormal findings . we subsequently decided to explore the possibility of psychological problems . the patient was treated with simultaneous rehabilitation and psychological counseling . paralysis of the patient 's lower extremity improved gradually and the patient returned to normal life . our findings indicate that psychological problems can be related to limb paralysis without organ damage in patients who have undergone laparoscopic surgical procedures .

INTRODUCTION CASE REPORT DISCUSSION

bufuralol , 1-hydroxybufuralol , 7-benzyloxy-4-trifluoromethylcoumarin ( bfc ) , 7-methoxy-4-trifluoromethylcoumarin ( mfc ) , 7-hydroxy-4-trifluoromethylcoumarin , and hydroxytolbutamide were purchased from bd gentest ( cowley , uk ) . midazolam , 1-hydroxymidazolam , and 4-hydroxymidazolam were kind gifts from roche applied science , and 1-hydroxymetoprolol and o - demethylmetoprolol were generous gifts from astra hsle ( mlndal , sweden ) . a targeting vector was constructed from an 18-kb dna fragment , produced by fusing overlapping pcr fragments generated from mouse 129/ola genomic dna ( supplemental fig . a cassette , flanked by same orientation loxp sites and containing a selectable marker ( neomycin ) , driven by the herpes simplex thymidine kinase promoter , was cloned into a bcli site in intron 1 , and a third loxp site was cloned into a kpni site in intron 5 . the construct was checked by pcr and sequencing and transfected into gk129/1 embryonic stem cells by electroporation ; the embryonic stem cells were subsequently cultured in 96-well plates under g418 selection . g418-resistant clones were screened for specific homologous recombination by southern blot analysis , using bglii and an 800-bp pcr fragment generated using 5-ggcacaacaccaattatttgtc-3 and 5-gacagtccttaacacaagctc-3 as forward and reverse primers , respectively . two correctly targeted embryonic stem cell clones ( cytb 5 ) were expanded , injected into c57bl/6 blastocysts , and transferred into pseudopregnant mice . male chimeric mice were bred to c57bl/6 mice , and heterozygous offspring were screened by southern blot and multiplex pcr to confirm germ line transmission of the cytb 5 genotype ( supplemental fig . 1b ) using the following primer set : 1 ) forward primer , 5-ccaatggtctctccttggtc-3;2 ) lox / neomycin reverse primer , 5-caatagcagccagtcccttc-3 ; 3 ) wild - type reverse primer , 5-gatggagttccccgatgat-3. mouse breeding and maintenance cytb5 mice were crossed to produce homozygous cytb5 mice and maintained by random breeding on a 129p2 c57bl/6 genetic background . cytb5 mice were crossed with a transgenic mouse line expressing cre recombinase under the control of the hepatocyte - specific rat albumin promoter ( cre ) ( 22 ) on a c57bl/6 background , and cytb5::cre offspring were backcrossed with cytb5 mice to generate liver - specific microsomal cytochrome b5 conditional knock - out mice ( hbn ; cytb5::cre ) and control ( wild - type , cytb5 ) mice . the presence of the cre transgene was determined as previously described ( 23 ) . all mice were maintained under standard animal house conditions , with free access to food and water , and a 12-h light/12-h dark cycle . all animal work was carried out on male 10-week - old mice in accordance with the animal scientific procedures act ( 1986 ) and after local ethical review . in vivo drug treatments hbn and wild - type mice were administered the following drugs either concomitantly as a mixture or individually : chlorzoxazone ( 5 mg / kg ) , metoprolol ( 2 mg / kg ) , midazolam ( 5 mg / kg ) , phenacetin ( 5 mg / kg ) , and tolbutamide ( 5 mg / kg ) , dissolved in mixture buffer ( 5% ethanol , 5% dmso , 35% polyethylene glycol 200 , 40% phosphate buffered saline , and 15% water ) , by intravenous injection or orally by gavage . preparation of microsomes microsomes were prepared from wild - type and hbn mouse tissues , using 0.30.5 g of tissue , by a modified method of meehan et al . ( 24 ) , using sonication instead of mechanical homogenization ( 25 ) . por activity was estimated by nadph - dependent cytochrome c reduction ( 26 ) . western blot analysis was carried out as previously described using polyclonal antisera raised against human por ( 27 ) ; rat cytochrome b5 ; rat cyp2a1 , cyp2b1 , cyp2c6 , cyp3a1 , and cyp4a1 ( 28 ) ; or human full - length cyp2a4 , cyp2d6 , and cyp2e1 ( 25 , 29 ) . polyclonal antiserum to rat cytochrome b5 reductase and a monoclonal antibody raised against rat cyp1a1 were also used.3 the polyclonal antiserum to cytochrome b5 oxidoreductase was a kind gift from dr . immunoreactive proteins were detected using polyclonal goat anti - rabbit , anti - mouse , or anti - sheep horseradish peroxidase immunoglobulins as secondary antibodies ( dako , ely , uk ) and visualized using immobilon chemiluminescent horseradish peroxidase substrate ( millipore , watford , uk ) and a fujifilm las-3000 mini - imaging system ( fujifilm uk ltd . ) . densitometric analysis was performed using multi gauge version 2.2 software ( fujifilm uk ltd . ) . microsomal incubations were carried out in triplicate in 50 mm hepes , ph 7.4 , 30 mm mgcl2 containing mouse liver microsomes and substrate prewarmed to 37 c before initiation of the reaction by the addition of either nadph or nadh to a final concentration of 0.5 or 1 mm , respectively . assays were performed in a final volume of 150 l using white 96-well plates and the following substrate and microsome concentrations : bfc , 50 m substrate , 20 g of mouse liver microsomes ; efc , 40 m substrate , 15 g of mouse liver microsomes ; mfc , 180 m of substrate , 15 g of mouse liver microsomes ; ethoxyresorufin ( er ) and benzoxyresorufin ( br ) , 1 m substrate , 11.25 g of mouse liver microsomes . reactions were measured in real time for 3 min , using the recommended excitation and emission wavelengths for each probe using a fluroskan ascent fl plate reading fluorimeter ( labsystems , uk ) . turnover rates were calculated using authentic metabolite standards ( 7-hydroxy-4-trifluoromethylcoumarin for bfc , efc , and mfc assays and resorufin for er and br assays ) . nadh - mediated incubations for hplc or liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) analysis nadh - mediated reactions were performed in triplicate using the following conditions : bufuralol , 300 m ( final concentration ) , 20 g of mouse liver microsomes in a final volume of 150 l for 6 min ; chlorzoxazone , 1 mm ( final concentration ) and 20 g of mouse liver microsomes in a final volume of 150 l for 15 min ; midazolam , 50 m ( final concentration ) and 25 gof mouse liver microsomes in a final volume of 150 l for 9 min ; metoprolol , 800 m ( final concentration ) and 30 g of mouse liver microsomes in a final volume of 150 l for 60 min ; phenacetin , 200 m ( final concentration ) and 20 g of mouse liver microsomes in a final volume of 100 l for 9 min ; tolbutamide , 800 m ( final concentration ) and 30 g of mouse liver microsomes in a final volume of 150 l for 60 min . assays were stopped by the addition of either 0.5 assay volumes ( for bufuralol , chlorzoxazone , and tolbutamide assays ) or 1 assay volume ( for midazolam and phenacetin assays ) of ice - cold methanol and incubated on ice for 10 min . assays to determine the apparent kinetic parameters were performed in triplicate with wild - type and hbn liver microsomes under conditions of linearity for time and protein ( data not shown ) using the same buffer / nadph conditions as described above , with the following concentrations of substrates : chlorzoxazone , 101000 m ; phenacetin , 1.7150 m ; midazolam , 0.975 m ; metoprolol , 101000 m ; tolbutamide , 101000 m ( 12 concentration points / determination ) . metabolites were detected by lc - ms / ms as described in the supplemental materials . the data generated were analyzed by nonlinear regression using the michaelis - menten equation ( chlorzoxazone , phenacetin , midazolam , and metoprolol ) , whereas the hill equation ( tolbutamide ) ( equation 1 ) was used to determine the kinetic parameters vmax , s50 , and the hill coefficient ( n ) . ( eq . 1)\documentclass[10pt]{article } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{pmc } \usepackage[euler]{upgreek } \pagestyle{empty } \oddsidemargin -1.0 in \begin{document } \begin{equation*}\;\;v=\frac{v_{{\mathrm{max}}}{\times}s^{n}}{s_{50}^{n}+s^{n}}\end{equation*}\end{document } in vivo pharmacokinetics whole blood ( 10 l ) was taken from the tail vein at intervals after drug administration and transferred into a tube containing heparin ( 10 l , 15 iu / ml ) . internal standard solution ( 10 l ; 500 ng of caffeine and 500 ng of resorpine ) was added to each tube . protein precipitation was carried out by adding methanol ( 75 l ) , followed by 8% sulfosalicylic acid ( 55 l ) . samples were mixed for 1 min and centrifuged at 13,000 rpm for 5 min , and the supernatant was analyzed by hplc . the range of concentrations for the standard curves was constructed for quantifying blood levels by spiking blank blood samples with known amounts of chlorzoxazone , metoprolol , midazolam , phenacetin , and tolbutamide . extraction and protein precipitation were carried out as outlined above for the test samples . analysis of in vitro and in vivo data average rates of metabolism were calculated for each triplicate incubation of mouse liver microsomes from each genotype ( n = 6 ) , and these data were then used to calculate p values using an unpaired t test ( available on the world wide web ) . a simple noncompartmental model was used to calculate area under the curve ( auc ) , terminal half - life , maximum plasma concentration ( cmax ) , and clearance . details of assays and separation conditions for hplc and lc - ms / ms are given in the supplemental materials . hepatic microsomal cytochrome b5 null mice mice nulled for hepatic microsomal cytochrome b5 ( cytb5::cre ) were born at the expected mendelian ratio , developed normally , and displayed no overt phenotype when compared with their wild - type littermates ( cytb5 ) . livers from adult hbn mice looked similar to those of wild - type animals , with no gross changes in general morphology or pathology noted . to establish whether hepatic microsomal cytochrome b5 protein had been deleted specifically , microsomes from liver , lung , kidney , and small and large intestine were immunoblotted for cytochrome b5 expression ( fig . cytochrome b5 protein was virtually undetectable in microsomes from the livers of hbn mice ; however , occasionally , a very faint band was observed in some samples , possibly originating from liver cells other than hepatocytes . there were no discernible differences in cytochrome b5 expression between the hbn and wild - type mice in the other organs examined . no cytochrome b5 protein could be detected in microsomes from the large intestine of either genotype ( data not shown ) . in order to investigate whether concomitant changes in p450 expression had occurred , three representative liver microsomal samples of each genotype were analyzed for p450 enzyme profiles , por , and cytochrome b5 reductase levels ( fig . although the expression of some p450s was slightly elevated in the hbn mice , these changes were not statistically significant . cytochrome b5 reductase level was slightly increased when the immunoreactive bands were quantified by densitometry ( fig . a slight reduction in cyp2d expression was also observed . as can be seen from supplemental fig . 2a , there was no difference observed in the amount of spectrally active p450 in the hbn as compared with wild - type liver microsomes , with both samples containing 0.15 nmol of p450/mg of microsomal protein . por protein levels were also essentially unchanged in hbn compared with wild - type mice , and although a slight increase in por activity in hbn mice was measured using cytochrome c as substrate ( 228 65 versus 180 26 nmol cytochrome c reduced / min / mg ) , this change was not significant . a , immunoblot analysis for cytochrome b5 expression in liver , kidney , lung , and small intestine microsomes ( 5 g of protein / lane ) from cytb5::cre ( hbn ) and cytb5 ( wild type ) , as detailed under experimental procedures . b , immunoblot analysis for expression of cytochrome p450-dependent monooxygenase components in liver microsomes ( 5 g of protein / lane ) from cytb5::cre ( hbn ) and cytb5 ( wild type ) , as detailed under experimental procedures . wild - type expression was fixed at 100% , and percentage difference was calculated for cytochrome b5 liver null ( hbn ) samples . a , immunoblot analysis for cytochrome b5 expression in liver , kidney , lung , and small intestine microsomes ( 5 g of protein / lane ) from cytb5::cre ( hbn ) and cytb5 ( wild type ) , as detailed under experimental procedures . b , immunoblot analysis for expression of cytochrome p450-dependent monooxygenase components in liver microsomes ( 5 g of protein / lane ) from cytb5::cre ( hbn ) and cytb5 ( wild type ) , as detailed under experimental procedures . wild - type expression was fixed at 100% , and percentage difference was calculated for cytochrome b5 liver null ( hbn ) samples . figure 2.nadph- and nadh - dependent cytochrome p450 activities in hepatic microsomes from wild - type and microsomal cytochrome b5 hepatic null mice . black bars represent nadph - mediated , and white bars nadh - mediated activities , respectively . * , p 0.05 ; * * , p 0.005 ; * * * , p 0.001 . nadph- and nadh - dependent cytochrome p450 activities in hepatic microsomes from wild - type and microsomal cytochrome b5 hepatic null mice . black bars represent nadph - mediated , and white bars nadh - mediated activities , respectively . * , p 0.05 ; * * , p 0.005 ; * * * , p 0.001 . in vitro cytochrome p450 activities six commonly used p450 model substrates were used to establish if there were any in vitro differences in metabolism between liver microsomes from hbn and wild - type mice : bfc ( cyp3a ) , mfc ( cyp2c ) , efc ( cyp1a/2b ) , er ( cyp1a1/2 , cyp1b1 ) , br ( cyp2b/3a ) , and bufuralol ( cyp2d ) ( 3036 ) . assays were performed at substrate concentrations of 5 times km ( mouse ) , as defined by the literature , or data were generated with recombinant mouse p450s ( data not shown ) . with nadph as electron donor , significantly lower turnover rates were measured in hbn mice relative to wild - type samples for all substrates tested . the nadph - mediated rates in hbn liver microsomes , expressed relative to wild - type samples , were as follows : er ( 73% ) , bufuralol ( 44% ) , br ( 34% ) , bfc ( 19% ) , mfc ( 11% ) , and efc ( 10% ) ( fig . the addition of recombinant cytochrome b5 to hbn microsomes led to a concentration - dependent increase in bfc or chlorzoxazone metabolism , restoring activities to wild - type levels , demonstrating that the change in metabolism was due to the absence of cytochrome b5 . nadh could support monooxygenase activity for all model substrates in the wild - type samples , although the activity relative to nadph was highly substrate - dependent , ranging from 4% ( br ) to 74% ( er ) ( fig . 2 and supplemental table 3 ) . all nadh - catalyzed reaction rates with the exception of br were significantly lower in the hbn samples compared with wild type , with barely detectable activities using bfc ( 0.66% ) , efc ( 0.79% ) , and mfc ( 0.28% ) as substrates ( fig . 2 and supplemental table 3 ) . the metabolism of er and bufuralol was also reduced , to 52 and 28% of wild - type values , respectively . the nadh - mediated in vitro turnovers of the probe drugs chlorzoxazone ( cyp2e1 ) , metoprolol ( cyp2d ) , midazolam ( cyp2c , cyp3a ) , tolbutamide ( cyp2c ) , and phenacetin ( cyp1a ) ( 3741 ) were also examined with liver microsomes from both hbn and wild - type mice . nadh supported the metabolism of all substrates examined in wild - type liver microsomes , with the exception of tolbutamide . rates observed were 601 152 ( chlorzoxazone 6-hydroxylation ) , 128 60 ( metoprolol o - demethylation ) , 17.6 0.2 ( metoprolol -hydroxylation ) , 17.1 8.0 ( midazolam 1-hydroxylation ) , 9.1 3.8 ( midazolam 4-hydroxylation ) , and 244 63 pmol / min / mg ( phenacetin hydroxylation ) . these rates observed in wild - type liver microsomes were significantly lower than those observed using nadph as co - factor , ranging from 7 to 29% ( o - demethylmetoprolol and acetaminophen production , respectively ) of the nadph rate . furthermore , the nadh - dependent turnover rates observed in hbn liver microsomes ( 177 64 ( chlorzoxazone 6-hydroxylation ) , 90 21 ( metoprolol o - demethylation ) , 7.6 2.2 ( metoprolol -hydroxylation ) , 4.3 2.0 ( midazolam 1-hydroxylation ) , 2.1 2.7 ( midazolam 4-hydroxylation ) , and 67 18 pmol / min / mg ( phenacetin hydroxylation ) ) were significantly lower than the nadh - dependent wild - type rates ( chlorzoxazone ( 29% reduction ) , metoprolol ( o - demethyl 71% , -hydroxy 44% , acid 83% reduction ) , midazolam ( 1- and 4-hydroxy 25% reduction ) , and phenacetin ( 29% reduction ) ) . figure 3.pharmacokinetic disposition of an intravenously administered p450 drug mixture in wild - type and hepatic microsomal cytochrome b5 null mice . a p450 drug mixture containing chlorzoxazone ( 5 mg / kg ) , metoprolol ( 2 mg / kg ) , midazolam ( 5 mg / kg ) , phenacetin ( 5 mg / kg ) , and tolbutamide ( 5 mg / kg ) was administered intravenously at the indicated doses to wild - type ( black circles ) or hbn ( open circles ) mice , and blood samples were taken at timed intervals to determine the in vivo pharmacokinetic parameters of the parent compounds . * , p 0.05 ; * * , p 0.005 ; * * * , p 0.001 , where n = 5 . pharmacokinetic disposition of an intravenously administered p450 drug mixture in wild - type and hepatic microsomal cytochrome b5 null mice . a p450 drug mixture containing chlorzoxazone ( 5 mg / kg ) , metoprolol ( 2 mg / kg ) , midazolam ( 5 mg / kg ) , phenacetin ( 5 mg / kg ) , and tolbutamide ( 5 mg / kg ) was administered intravenously at the indicated doses to wild - type ( black circles ) or hbn ( open circles ) mice , and blood samples were taken at timed intervals to determine the in vivo pharmacokinetic parameters of the parent compounds . * , p 0.05 ; * * , p 0.005 ; * * * , p 0.001 , where n = 5 . in vitro kinetics the apparent kinetic parameters of the probe drugs using nadph as co - factor were determined in wild - type and hbn liver microsomes , and metabolism of chlorzoxazone , phenacetin , midazolam , and metoprolol followed standard michaelis - menten kinetics ( table 1 ) . the michaelis constant for chlorzoxazone 6-hydroxylation , midazolam 1-hydroxylation , phenacetin oxidation , and metoprolol -hydroxylation were unchanged by the removal of microsomal cytochrome b5 . conversely , for those reactions , the vmax was markedly reduced in the hbn samples , ranging from a 2-fold ( phenacetin oxidation ) to a 6-fold ( metoprolol -hydroxylation ) decrease . midazolam 4-hydroxylation and metoprolol o - demethylation reactions again showed decreased vmax values for the hbn samples ( 20- and 3-fold , respectively ) ; however , the km values were marginally affected , with the hbn samples exhibiting a 1.7-fold reduction in km for midazolam 4-hydroxylation and a 1.8-fold increase in km for metoprolol o - demethylation as compared with wild type . the data obtained for tolbutamide hydroxylation could not be fitted using the michaelis - menten equation but did conform to the hill equation . interestingly , the hbn sample exhibited 68-fold higher km for tolbutamide as well as a 5-fold reduction in vmax as compared with wild type . the hill coefficient for both wild type and hbn were similar ( 0.38 versus 0.29 ) and indicative of tolbutamide binding in a negatively cooperative manner . the alternative of a 2 km,2 vmax fit was considered for these data ; however , when plotted in such a manner , the km1 parameter for both wild type and hbn was subject to such a large error as to render it meaningless . table 1kinetic analyses of the metabolism of probe drugs by hepatic microsomes from hbn mice and wild - type controlsassays were performed in triplicate for each concentration of substrate . values given are from the fit of the curve as calculated using the michaelis - menten equation ( grafit version 5 ( erithacus software , horley , uk ) ) . for tolbutamide , the hill equation ( grafit version 5 ) was used to fit the data , giving hill coefficients ( n ) of 0.38 and 0.29 for wild type and hbn , respectively.substrate metabolite wild type hbn kmvmaxkmvmax mpmol / min / mg mpmol / min / mg chlorzoxazone 6-hydroxychlorzoxazone 47 9 1930 100 53 6 387 11 midazolam 1-hydroxymidazolam 2.1 0.3 389 15 2.0 0.3 116 4 midazolam 4-hydroxymidazolam 13.3 0.4 146 2 7.7 0.9 7.3 0.3 phenacetin acetaminophen 5.6 1.0 133 6 4.1 0.7 77 3 metoprolol -hydroxymetoprolol 84 7 483 14 99 9 76 3 metoprolol o - desmethylmetoprolol 86 9 1650 2 157 14 531 21 tolbutamide hydroxytolbutamide 11200 3400 211 17 165 48 44 165 kinetic analyses of the metabolism of probe drugs by hepatic microsomes from hbn mice and wild - type controls assays were performed in triplicate for each concentration of substrate . values given are from the fit of the curve as calculated using the michaelis - menten equation ( grafit version 5 ( erithacus software , horley , uk ) ) . for tolbutamide , the hill equation ( grafit version 5 ) was used to fit the data , giving hill coefficients ( n ) of 0.38 and 0.29 for wild type and hbn , respectively . to ensure that the observed changes in p450 activity were not a consequence of perturbations of the phospholipid bilayer , fatty acid profile analysis was carried out on endoplasmic reticulum membrane fractions from the livers of both wild type and hbn mice . in addition , hepatic lipid levels were unchanged , as determined by oil red o staining ( supplemental fig . 3 ) . in vivo pharmacokinetics of probe drugs in wild - type and hbn mice we next investigated the effect of the absence of hepatic microsomal cytochrome b5 on the in vivo pharmacokinetics of the probe drugs in wild - type and hbn mice . initially , mice were administered chlorzoxazone , metoprolol , midazolam , tolbutamide , and phenacetin either as an intravenous or oral mixture . previous work with these drugs in wild - type mice , individually and as a mixture , had demonstrated that simultaneous delivery did not result in altered pharmacokinetics as compared with single administration ( not shown ) . following dosing of the drug cassette to wild - type and hbn mice , quantification of individual drugs and their metabolites was carried out by lc - ms / ms . the complete set of pharmacokinetic parameters for each drug is summarized in supplemental tables 4 ( intravenous ) and 5 ( oral ) , and the elimination profiles of the drugs are shown in fig . following intravenous administration of the drug mixture , wild - type mice showed a higher concentration of parent drug in the blood at the early time points ( 060 min ) compared with the hbn mice for all drugs in the mixture . in contrast , from 2 h , the average concentration of parent drug was statistically much lower in the wild - type than in hbn mice for most of the compounds and remained so for the duration of the experiment . pharmacokinetic analysis for each drug showed that the clearance was consistently faster , and the auc was consistently lower ( except in the case of midazolam ) in wild - type compared with hbn mice . furthermore , the terminal half - life for phenacetin , tolbutamide , chlorzoxazone , and midazolam was statistically different between the two lines , being significantly longer in hbn mice , increasing from 53 to 168 min , 197 to 321 min , 92 to 257 min , and 83 to 329 min , respectively ( table 2 , fig . 3 , and supplemental table 4 ) . for metoprolol , although a 2.7-fold increase of the terminal half - life was found in hbn mice , this did not reach statistical significance ( p = 0.1 ) . table 2 in vivo pharmacokinetic data from hbn mice and wild - type controls dosed with a drug mixtureadult male hbn and wt mice were administered a drug mixture intravenously ; serial blood samples were collected over an 8-h period and analyzed for plasma drug concentrations by lc - ms / ms and key pharmacokinetic parameters calculated . absolute oral bioavailability was calculated by dividing auc ( 0 , oral ) by auc ( 0 , intravenous ) ( supplemental tables 4 and 5).drug terminal half - life ( intravenous ) bioavailability ( % ) wild typehbnwild typehbn midazolam 83 15 329 132 14 55 phenacetin 53 11 168 56 0.6 0.8 metoprolol 96 13 260 65 0.3 0.3 chlorzoxazone 92 19 257 76 3.7 1.9 tolbutamide 197 37 321 25 95 ( 154)a 47 ( 135)aacomparative data where tolbutamide was dosed as a single agent . in vivo pharmacokinetic data from hbn mice and wild - type controls dosed with a drug mixture adult male hbn and wt mice were administered a drug mixture intravenously ; serial blood samples were collected over an 8-h period and analyzed for plasma drug concentrations by lc - ms / ms and key pharmacokinetic parameters calculated . absolute oral bioavailability was calculated by dividing auc ( 0 , oral ) by auc ( 0 , intravenous ) ( supplemental tables 4 and 5 ) . comparative data where tolbutamide was dosed as a single agent . the major metabolites of metoprolol ( o - demethylmetoprolol ) , phenacetin ( acetaminophen ) , and midazolam ( 1-oh midazolam ) were also measured in the serum of hbn and wild - type mice after intravenous administration ; the results are presented in supplemental fig . interestingly , the overall profiles of metabolite production in hbn mice were shifted to the right , consistent with a delayed metabolism of the parent compound ( fig . 3 and supplemental fig . tolbutamide and chlorzoxazone metabolites were below the limit of detection at all time points . oral pharmacokinetics following oral administration of the drug mixture , differences in the elimination profiles of all of the drugs were observed between wild - type and hbn mice . in the case of midazolam , phenacetin , and metoprolol , auc and cmax were consistently higher in hbn mice , whereas the clearance was slower ( supplemental fig . were significantly increased from 34 to 124 ming / ml and from 0.4 to 1.0 g / ml , respectively , whereas clearance was decreased from 149 to 41 ml / min / kg ( supplemental table 5 ) . the phenacetin elimination profile also showed significant changes , with cmax increasing from 0.06 to 0.09 g / ml in hbn and clearance decreasing from 688 to 550 ml / min / kg . although the phenacetin auc increased from 1.5 to 1.8 ming / ml , this change was not significant . in the case of metoprolol , similar trends were observed in cmax and auc ; however , these did not reach statistical significance . in contrast to the observations above , the alterations in the pharmacokinetic parameters for tolbutamide gave significantly lower auc and cmax values and a ( nonsignificant ) higher clearance rate , relative to wild - type animals ( supplemental fig . 4 and supplemental table 5 ) . this unexpected effect appeared to be a consequence of using the drug mixture ( see below ) . interestingly , although the terminal half - life for chlorzoxazone was significantly decreased in hbn mice from 194.0 to 46 min , the decreased auc and cmax and increased clearance in hbn mice were not significantly different ( supplemental table 5 ) . additionally , there were no significant differences between hbn and wild - type mice in the plasma concentration of chlorzoxazone at any of the time points analyzed ( supplemental fig . administration of the probe drugs individually to hbn and wild - type mice yielded essentially the same elimination profiles for phenacetin , metoprolol , midazolam , and chlorzoxazone , compared with those obtained from use of the drug mixture ( data not shown ) . 4a , inset ) , the elimination profiles were found to be reversed in comparison with that obtained from the drug mixture ( i.e. significantly increased auc and cmax and significantly decreased clearance in hbn mice ) . similar to data obtained with the drug mixture administered intravenously , the pattern of metabolite production in the hbn animals showed a delay in maximal levels of circulating metabolites ( supplemental fig . hbn animals showed a 4-fold increase in midazolam bioavailability , whereas for phenacetin , metoprolol , and chlorzoxazone , bioavailability was very low in the wild - type animals ( < 4% ) and unaltered in the nulls . when dosed as part of the drug mixture , tolbutamide bioavailability in the hbn mice was decreased by 50% as compared with wild type ; however , when orally dosed as a single agent and recalculated , both wild - type and hbn animals showed > 100% bioavailability . all previously published work on the effects of cytochrome b5 on nadph - dependent p450 activity has been carried out in vitro . in order to establish the in vivo role of this hemoprotein in drug disposition , we have generated a mouse where this enzyme has been conditionally inactivated in the liver . to exclude the possibility that a global deletion of microsomal cytochrome b5 would be lethal , mice deleted for hepatic microsomal cytochrome b5 developed normally , with the cytb5::cre genotypes being found in mendelian proportions . no alterations in liver morphology , hepatic lipid content , or hepatic serum markers were observed . the hepatic deletion of microsomal cytochrome b5 was shown to be both specific and essentially complete by western blotting ( fig . 1a ) and , importantly , unlike the hepatic deletion of cytochrome p450 reductase ( 23 , 42 ) , did not result in significantly increased expression of hepatic p450 proteins ( fig . 1 , b and c ) . these data indicate that at the time points studied , a reduction in p450 activity , as a consequence of microsomal cytochrome b5 deletion , is not compensated by an increase in p450 expression . the use of a range of both model substrates and probe drugs clearly demonstrated that the absence of microsomal cytochrome b5 significantly attenuated p450-mediated metabolism in vitro ( fig . 2 and table 1 ) . the extent of suppression of enzyme activity varied markedly between substrates , as has been previously reported ( 2 , 6 ) . for all fluorocoumarin substrates employed , there was a highly significant decrease in nadph - mediated p450 metabolism in liver microsomes from the hbn mice ( as high as 90% for efc ) , whereas for the two resorufin analogues and bufuralol , the change was still significant ( at least 50% decrease for each compound ) but less marked . this variability in cytochrome b5 effects was further illustrated by the use of a series of probe drugs , where the kinetic parameters defining the in vitro metabolism were markedly changed ( table 1 ) . for all substrates , with the exception of tolbutamide , the data followed michaelis - menten kinetics , and km values were essentially unchanged in the hbn samples . this suggests that cytochrome b5 binding does not cause a conformational change in the structure of the p450s responsible for this metabolism . in contrast , in both wild type and hbn samples , the data for tolbutamide oxidation did not fit michaelis - menten kinetics , exhibiting negatively cooperative binding . furthermore , the removal of cytochrome b5 had a pronounced effect on tolbutamide binding , decreasing the km by 68-fold , indicating that the presence of cytochrome b5 does affect tolbutamide binding . in all cases , vmax values were substantially reduced by the removal of cytochrome b5 ( range 1.720-fold lower for acetaminophen and 4-hydroxymidazolam production , respectively ) , implying that the presence of cytochrome b5 is critical for optimal turnover in the p450s investigated . interestingly , the effects of cytochrome b5 were different dependent on the site of metabolism ( e.g. with midazolam , the vmax for oxidation at the 1- versus 4-hydroxy sites was 29 versus 5% of the control , respectively ) . a similar effect was also seen for metoprolol oxidation ( table 1 ) , indicating that cytochrome b5 effects show specificity for different sites of oxidation . these findings provide strong genetic evidence that the modulatory effects of cytochrome b5 are both substrate- and p450-dependent and that the activities of many p450 enzymes are affected by this protein . similar reductions in p450 activities ( 6070% ) have also been demonstrated using antibodies to cytochrome b5 , both in microsomal fractions and with purified protein ( 43 , 44 ) . furthermore , the addition of recombinant membrane - bound cytochrome b5 to hbn liver microsomes led to a concentration - dependent increase of catalytic activities toward bfc and chlorzoxazone , indicating that the loss of activity in hbn samples was indeed due to the absence of cytochrome b5 ( supplemental fig . the in vitro data generated from the probe drugs suggests that in general , the absence of cytochrome b5 results in reduced rates of p450-mediated metabolism . it is interesting to note that in some cases the fluorocoumarin substrates and tolbutamide nadh - mediated metabolism were essentially abolished in hbn mice , whereas for the other compounds , residual activity remained ( e.g. in the case of er or br ) , indicating the existence of an alternative metabolic pathway involving nadh but independent of cytochrome b5 ( fig . 2 ) . one possible explanation for the residual activity could be the presence of an alternate electron donor , such as the recently described outer mitochondrial cytochrome b5 , which is derived from a gene locus on chromosome 8 ( 45 , 46 ) and encodes for a protein of 16.3 kda , slightly larger than microsomal cytochrome b5 at 15.2 kda and 45% identical at the amino acid level . however , to date , there is no evidence for the interaction of outer mitochondrial cytochrome b5 with the microsomal p450 system . recently , a novel , soluble , nad(p)h reductase ( nad(p)h cytochrome b5 oxidoreductase ) , located in the lumen of the endoplasmic reticulum , was reported ( 47 ) , which contains both cytochrome b5 and cytochrome b5 reductase domains . in order to confirm that this enzyme was not induced in the hbn mice , and potentially compensating for the loss of microsomal cytochrome b5 , we obtained antiserum to nad(p)h cytochrome b5 oxidoreductase ; immunoblotting liver samples from wild - type and hbn mice found no difference in expression levels ( data not shown ) . li and porter ( 48 ) have also described a second microsomal reductase , which is active with squalene monooxygenase and apparently capable of supporting up to 40% of the activity of this enzyme , although the identity of this reductase and its interactions with other enzymes remain unclear . in addition , due to its role in fatty acid desaturation , it is possible that changes in the phospholipid bilayer , as observed in stearoyl - coa - desaturase null mice ( 49 ) , are responsible for the observed effects of cytochrome b5 on p450 metabolism . however , no changes in the fatty acid composition of endoplasmic reticulum membranes from wild - type and hbn mice were found ( supplemental fig . 3 ) . in order to establish the in vivo effects of the hepatic cytochrome b5 deletion , we administered a series of probe drugs by both intravenous and oral routes to control and hbn mice . these drugs were chosen because they have been shown to be metabolized in a number of species by different cytochrome p450s . the interpretation of such experiments is inevitably complex , particularly because in the mouse there is little background historical information on the key factors that define their pharmacokinetic parameters . however , consistent with the in vitro studies , in general , it was evident that the deletion of hepatic cytochrome b5 caused marked changes in drug pharmacokinetics independent of the route of administration . when administered intravenously , the terminal half - life was increased in hbn mice for all drugs used ( table 2 ) . this increase is consistent with decreased liver metabolism as a consequence of hepatic microsomal cytochrome b5 deficiency and provides the first evidence that strongly supports the conclusion that cytochrome b5 plays a role in in vivo p450-mediated drug metabolism . in addition , the profiles of metabolite production for midazolam , phenacetin , and metoprolol were different between hbn and wild - type mice ; for example , maximal metabolite levels of 1-hydroxymidazolam occurred 3 times more slowly in hbn mice than in wild type ( 120 versus 40 min ) when dosed intravenously ( supplemental fig . these differences in metabolite production are consistent with the in vitro kinetic data shown in table 1 . for example , the vmax values for 1-hydroxymidazolam were 3 times higher in wild type as compared with hbn . no major differences in the formation of the demethyl metabolite of metoprolol were observed at the early time points between hbn and wild - type mice , despite major in vitro differences ( supplemental fig . the interpretation of pharmacokinetic parameters is more complex due to factors such as the absorption across gastrointestinal tract and hepatic blood flow . this added complexity was manifest in the fact that for some drugs , such as midazolam , microsomal cytochrome b5 deficiency and the consequent reduction in hepatic metabolism can lead to increased bioavailability ( table 2 ) as well as a decreased rate of elimination . these alterations in pharmacokinetics could therefore potentially lead to an exaggerated response to the drug . however , for the other drugs , first pass liver metabolism does not appear to be important in determining bioavailability , which is presumably dependent primarily on efficiency of gastrointestinal absorption or metabolism . when dosed orally , the cmax appears to be the most reliable indicator of a change in metabolizing capacity and acts as a valuable corollary to the increased half - life observed following intravenous administration . in summary , mice with a hepatic deletion of microsomal cytochrome b5 display a significant change in the metabolism of a range of substrates by a number of cytochrome p450 enzymes in vitro and , more importantly , in vivo . the data presented in this report suggest that the level and/or activity of hepatic microsomal cytochrome b5 can play a significant role in the metabolism , disposition , and therapeutic effectiveness of clinically used drugs . the extrapolation of in vitro data to in vivo and the relative importance to drug pharmacokinetics and disposition are very complex and involve a wide range of parameters that could include the cytochrome p450/cytochrome b5 ratio , the cytochrome p450 enzyme involved , the substrate concentration , the p450 turnover number , and the overall role of cytochrome p450 metabolism as a rate - limiting factor in drug disposition .

in vitro , cytochrome b5 modulates the rate of cytochrome p450-dependent mono - oxygenation reactions . however , the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution , metabolism , excretion , and toxicity are unclear . in order to resolve this issue , we have carried out the conditional deletion of microsomal cytochrome b5 in the liver to create the hepatic microsomal cytochrome b5 null mouse . these mice develop and breed normally and have no overt phenotype . in vitro studies using a range of substrates for different p450 enzymes showed that in hepatic microsomal cytochrome b5 null nadh - mediated metabolism was essentially abolished for most substrates , and the nadph - dependent metabolism of many substrates was reduced by 5090% . this reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs , including midazolam , metoprolol , and tolbutamide . in the case of chlorzoxazone , elimination was essentially unchanged . for some drugs , the pharmacokinetics were also markedly altered ; for example , when administered orally , the maximum plasma concentration for midazolam was increased by 2.5-fold , and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b5 null mice . these data indicate that microsomal cytochrome b5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a p450- and substrate - dependent manner .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION Supplementary Material

there are few studies that have investigated the relationship between the acute inspiration of very high co2 levels and the time delay and magnitude of the resulting change in ventilation ( ) . at lower , more physiological , levels it is generally presumed that the magnitude of the response is directly related to the concentration ; however the time delay is determined by a number of factors that need not be related to the concentration , such as : ( a ) lung to peripheral and central chemoreceptor circulation time , ( b ) nerve conduction velocity from receptor to brain to effecter organs and ( c ) baseline alveolar ventilation that determines the initial alveolar pco2 ( paco2 ) and po2 ( pao2 ) . two factors that are potentially related to the co2 concentration are ( a ) the rate of rise of pco2 in the lung and receptor sites and ( b ) the associated stimulation of other receptors ( nociceptors ) in the larynx , airway , lungs or pulmonary veins by high co2 . in order to determine whether the latter may be involved , these experiments were undertaken to measure the acute responses to inspired co2 levels from 20 to 100% in anesthetized dogs . measurements were made before and after right and left vagotomy and superimposed stimulation of the right vagus nerve . all experiments were performed on two female mongrel dogs , weighing 8.4 kg each , on separate days , after which the animals were sacrificed . the trachea was cannulated and the right and left vagus nerves isolated at the neck . gas mixtures were prepared in a douglas bag and valve , attached to one arm of the y - connector placed in the tracheal cannula . following a period of ~30 s , when baseline was recorded , the valve from the bag was opened just prior to an inspiration , a time event marker indicated time zero for establishing the time of subsequent inspirations . expiration took place through the other arm of the y - connector that had a one - way valve that closed during inspiration . recordings were continued until the tidal volume ( vt ) response to the test gas appeared maximal on the tracing . breath - by - breath chest expansion was obtained by an impedance pneumograph , and ventilation frequency ( fr ) and timing were obtained from subsequent measurements from polygraph recordings ( 1.0 cm / s ) and time event markers . as the pneumograph recordings were uncalibrated , the height was expressed in units ( u ) to represent vt because the position of the impedance band may have moved and the contribution of the diaphragm to true vt may have varied between trials . breath - by - breat was calculated as u / min for each breath from the product of vt and fr calculated from the time between a given inspiration at the measured time and the previous one . was then re - plotted on a time base relative to baseline ( /b ) and averaged at specific times for the same conditions ( up to six ) at 0.5 , 1.0 and 2.0 s intervals . then average time delays from repeated trials with the various test gases were compared from the time of the first inspiration to where was doubled ( 2/b ) and quadrupled ( 4/b ) from baseline . time delays from time zero to where interpolated exceeded 3 sd of baseline were also noted as response times . all co2 mixtures were given in alternating order from low to high co2 , ( 20 , 40 , 70 and 100% ) , with balance o2 ( 80 , 60 , 30 and 0% , respectively ) . hypoxic mixtures , 10% o2 - 90% n2 and 0% o2 - 100% n2 , and air controls were interspersed randomly with the co2 trials . after these trials with both vagi intact , the 100% co2 and 0% o2 - 100% n2 trials were repeated with the right vagus and then both vagi cut . in addition the latter were repeated with the peripheral ( efferent ) end of the right vagus stimulated ( 60 hz , 10 v , 5 ms ) with a square wave generator at the same time that the gas was inspired to induce asystole to curtail the circulation . stimulation of the cut right vagus , with and without the left vagus cut , resulted in a 13 s asystole ( range : 5 to 17 s ) , with vagal escape occurring over the next 10 s and fh then stabilizing at ~44/min during stimulation . the time course of paco2 and pao2 following inspiration of the test gas was estimated breath - by - breath from a ) the mixing of measured vt and functional residual capacity ( frc ) with the bag o2 and co2 concentrations , assuming baseline paco2 = 35 mmhg and paco2 = 99 mmhg and a baseline vt of 120 ml and frc of 600 ml , as measured in dogs of similar weight by muggenburg et al . and b ) an o2 consumption of 5 ml / min / kg and baseline respiratory exchange rate of 0.80 . the change in ph value ( dph ) corresponding to changes in paco2 was calculated from the henderson - hasselbalch equation , assuming instantaneous equilibration between arterial pco2 and paco2 , a pk of 6.1 , a fixed bicarbonate ( hco3 ) concentration of 24 mmol / l and no co2 storage in lung tissue ; the latter would tend to buffer changes in paco2 . the average /b responses to the four co2 concentrations , two hypoxic mixtures and air controls are depicted in figure 1 , with values in table 1 . the response time and time delay for the four co2 levels was inversely related to concentration and the magnitude was directly related ; the inverse relationship between the highest three concentrations and time delays to 2/b and 2/b was linear . the responses to the two levels of hypoxia were similarly related to reduced o2 , but attenuated and slower than those for co2 . for 100% co2 the response time was 0.3 s , with time delays of 0.5 s and 2.0 s at 2/b and 4/b , respectively ; the rise above baseline occurred on the first inspiration , with a greater vt ( 124% ) and fr ( 8% ) than baseline . figure 2 shows the responses to the four co2 levels , along with the estimated paco2 at each inspiration . the 100% co2 trial ( fig . the values for the instantaneous change / time of paco2 and /b are shown at 2/b and 4/b , assuming no time delay between paco2 and /b . the clear pattern is that paco2/s markedly increased with inspired co2 level as the time delay decreased , whereas /b / s was not markedly affected . /b was greater at equivalent times as co2 concentration increased mainly because of the shorter time delays . the average of the individual time delays to 2/b and 4/b were significantly shorter for the 70 and 100% trials than for the 20 and 40% trials ( 1.8 vs. 23.5 and 3.4 vs. 32.8 s , respectively ) , with response times of 0.3 and 13.4 s ( p<0.01 for all ) . pao2 at 100% co2 is indicated by solid circles in panel d. instantaneous values / time for paco2 and /b are indicated at 2/b and 4/b . the percentage changes in fr and vt and estimated paco2 , pao2 and ph values at 2/b and 4/b for all trials are included in table 1 . with vagi intact most of the increase to 2/b for all co2 levels and 0% and 10% o2 were due to increased vt , with an increasing , but still negligible contribution by fr at 4/b . pao2 was ~70 mmhg when 2/b was reached for the 10% and 0% o2 trials , but the time delay at 0% o2 was 10 s less than at 10% . a comparison of baseline ventilatory components before and after vagotomy shown in table 1 shows a significant reduction in baseline fr , an increase in vt and no change in after cutting the right vagus . bilateral vagotomy further reduced fr , resulting in a significant reduction in compared to right vagotomy alone . the effects of right and bilateral vagotomy and superimposed right vagus stimulation on the response to 100% co2 and 0% o2 are summarized in figure 3 . stimulation of the cut right vagus by itself caused a small increase in /b ( figs . 3b and 3c ) , mainly resulting from a greater fr , with the left vagus intact or cut ( table 1 ) . inspiring 0% o2 after bilateral vagotomy increased the time delays to 2/b and 4/b and by some 36 s ( fig . 3a ) , with the increased /b resulting predominantly from increasing fr , whereas vt was the main contributor in the intact trials . 3b ) resulted in a vigorous response that was delayed about 4 s compared to that with the vagus intact , with vt still the main contributor . when the peripheral end of the right vagus was stimulated as co2 was inspired , the vigorous response was delayed an additional 5 s , with fr now the main contributor to the /b increase . the effect of bilateral vagotomy on the response to co2 was qualitatively similar , but magnified ( fig . 3c ) . the time delay to 2/b was extended an additional 2 s after both vagi were cut , with /b reaching a plateau at ~12 s. the relatively greater contribution of vt to the increase to 2/b remained about the same as with intact vagi , similar to hypoxia ( fig . the response was greatly attenuated and the time delay to 2/b and 4/b increased by an average of 15 s , with the fr contribution increasing compared with no stimulation . mean /b responses to 100% co2 and 0%-100% n2 before and after bilateral vagotomy ( panel a ) . panel b shows mean responses to 100% co2 before vagotomy , after rt vagotomy while electrically stimulating the right ( rt ) vagus and stimulation alone . panel c indicates mean responses to 100% co2 before vagotomy , after bilateral vagotomy while stimulating the right vagus and stimulation alone . numbers in italics indicate percentage change in fr and vt above baseline at 4/b . : time : response time for interpolated to exceed baseline mean + 3 sd ; : inspired ventilation divided by baseline ventilation ; time : time from onset of first inspiration to 2/b and 4/b from average curve ; fr and vt : percentage change in fr and vt from baseline to 2/b and 4/b ; pao2 and paco2 : estimated at 2/b and 4/b assuming baseline values of 99 and 35 mmhg , respectively ; ph : change estimated from paco2 change from baseline ( 35 mmhg ) assuming fixed hco3 ; parentheses : s.e.m . ; * : value significantly ( p<0.05 ) different from that with vagi intact ; # : value significantly different ( p<0.05 ) from value with rt vagotomy these experiments strongly suggest that ventilation increases and the time delay decreases as the inspired co2 level approaches 100% . at levels 70% the time delay is shorter than reported for aortic arch and carotid body chemoreceptor response times from previous and subsequent studies . vagotomy delayed the response to 100% co2 and restricting the circulation delayed it further . that our limited experimental set - up was reasonable is partly supported by the following : ( a ) the changes in fr and vt with vagotomy during baseline ( table 1 ) agree closely with those reported in anesthetized dogs by anrep and samaan , who concluded that the slowing of respiration was due to denervation of the lungs and not the peripheral chemoreceptors , ( b ) the /b response to hypoxia ( fig . 1 and table 1 ) was not far removed from the 10 s time delay reported in humans and dogs and occurred at estimated pao2 values close to those reported for steady state breathing , ( c ) the /b response leveled off with 100% co2 after vagotomy ( fig . 3c ) , as reported in dogs and ( d ) the response was greatest and time delay shortest with 100% co2 when pao2 fell most rapidly ( fig . 2d ) , demonstrating the well - known enhanced ventilatory sensitivity to co2 when combined with hypoxia . studies of ventilatory responses to co2 and hypoxia in humans and mammals have typically utilized inspired concentrations of < 10% co2 ( inspired pco2<71 mmhg at sea level ) and > 10% o2 . ventilatory studies using non - physiological concentrations > 20% co2 have rarely been reported ; when breathing concentrations > 35% for some minutes it is an effective anesthetic in dogs . in humans , repeated applications of 12 inspirations of a 30% co2 - 70% o2 gas mixture were utilized by meduna some 6 decades ago to treat psychoneuroses and anxiety disorders with some success . the reaction to a mixture of 35% co2 - 65% o2 has also been used as a trait marker for panic disorders . barcroft and margaria compared the ventilatory effect of co2 inhalation and exercise on themselves and stated , the breathing of 7.5% of co2 for 20 minutes produces a shock from which the system does not wholly escape for some hours or perhaps even a longer time . they also measured the change in fr with the inspiration of 64% co2 in anesthetized cats and noted that the increase was inversely related to baseline fr and that bilateral vagotomy resulted in an erratic response . their recordings suggest a time delay of 4 to 5 s between first inspiration and ventilation increase . the existence of lung air chemoreceptors acting reflexly on the ventilatory regimen is generally not admitted , because , these results have been observed only as a result of enormous and quite unphysiological shifts of pco2 , and , the hyperventilation resulting from breathing co2-rich mixtures does not occur before a lag of many seconds . on the other hand , pi - suer , in summarizing extensive chemoreceptor research prior to the early1940s , took exception to the statement by cordier and heymans that , -authors have administered by inhalation air with concentrations of co2 which pass beyond physiological limits and even beyond the pathological . pi - suer concluded from numerous experiments , in addition to the well known action on the respiratory centers , there is exerted a parallel or perhaps previous peripheral influence due to the excitation of end - organs which are sensitive to stimuli of chemical nature by the co2 contained in the inspired air . our results support the latter in the continuing controversy regarding lung chemoreceptors , the same as many early studies based on the ventilation response to higher concentrations . the aortic arch and carotid body ( peripheral ) and central medullary chemoreceptors all respond to co2 and hydrogen ion concentration ( h+ ) to increase ventilation ; the relative contributions of these responses to this rise following the stimuli of lung or blood co2/h+ have been studied extensively and remain controversial , especially with variations in baseline arterial blood po2 ( 12 , 15 , 16 ] . recent studies with isolated carotid sinus perfusion show that the central chemoreflex can respond to an increase in paco2 in unanesthetized dogs in 6 s , but take 11 s longer when separated from peripheral receptors ; this demonstrates that the gain of the central receptors is critically dependent on the peripheral ones . it is often not clear whether reported time delays pertain to central and/or peripheral receptors , but the latter should respond first to the co2 /h+ signal . time delays result primarily from the lung - to - chemosensor circulation time . our average time delay from first inspiration to 2/b was inversely related to co2 concentration , ranging from 25.1 to 0.5 s , for 20 and 100% , respectively . the lung to brain time delay from an increase in paco2 to affect ph at the medulla oblongata in unanesthetized cats has been reported to be 5 to 7 s. in humans the peripheral response to inspiring hypoxic gas has been measured at 5 s , from lung - to ear circulation time by oximetry . mcclean et al . measured a 10 s delay to peak ventilation after a single breath of 13% co2-balance air in healthy humans and suggested this as a test for peripheral chemoreceptor function in patients . the time delay from infusion of co2-equilibated blood into the aortic arch to increase ventilation was found to be 6.6 s in unanesthetized dogs by sylvester et al . , who concluded that the circulation time from aortic arch to aortic body , carotid body and the medulla to be 1 , 3 - 4 and 5 - 6 s , respectively . definitive time delay experiments in unanesthetized dogs were reported by gonzalez et al .. they measured the time from injection of cold nahco3 to the increase in ventilation to be 2.0 and 6.9 s , when injected into the aortic arch and superior vena cava , respectively . the corresponding times for arrival of the blood to these sites were 1.9 and 3.7 s. the time from the paco2 rise in the lung , induced by the nahco3 , to arrival at the carotid body was about 1 s , implying a lung stimulus to ventilation response time of ~3 s. an important consideration is that a rapid response in fr and/or vt during the first inspirate will increase the rate of rise of paco2 to raise the alveolar / arterial blood stimulus level for the downstream arterial chemoreceptors ( fig . 2 ) . carbonic anhydrase , present in the interstitial lung tissue , would be expected to rapidly convert co2 to h+ in the pulmonary capillaries and then stimulate the downstream chemoreceptors . at 100% co2 , with concurrent hypoxia assuming that effect is negligible and with instantaneous equilibration of lung - blood paco2 and ph , the vt and fr measurements , and interpolating paco2 for the times courses in fig . 2 at 2 s , the estimated lung tissue ph decreased 0.19 , 0.41 , 0.65 and 0.79 as inspired co2 fractions increased , respectively . with the right vagus cut the ph fell 0.59 with 100% co2 and to 0.34 with both cut . this is about half the increase in h+ compared to that with the vagi intact . emphasized the difficulty of separating the chemoreceptors involved in responding to inhaled co2 vs. hypercapnic blood . they noted a vagally mediated response to inspired co2 on the first breath that was absent after vagotomy , similar to our results . 2 at 20 and 40% in intact dogs suggest stimulation of peripheral and central chemoreceptors without an initial rapid response , as the time delays are within those reported . however , at 70 and 100% the response is faster than can be explained by those . our results imply that there is a third sensing site , upstream from the aortic bodies in or near the lung that is dependent on vagal afferents . laryngeal co2 receptors have been noted in anesthetized dogs and when these myelinated and unmylenated fibers in the vagus were blocked the reflex was decreased . these sensing regions are located in the trachea and larger bronchi , where they are more chemosensitive , and can stimulate ventilation . they probably add to the response of the unmylenated c - fibers in contributing to the total reflex response . there is also evidence that the j - receptors and vagal bronchopulmonary c - fiber sensory nerves are also involved in the rapid ventilatory responses to lung irritants and may contribute to dyspnea in patients with copd . furthermore , these c - fibers have been shown to respond rapidly in dose - related fashion to h+ induced by injections of lactic acid in anesthetized rats . an increase in paco2 , acting via h+ , has been shown to augment the responses of the c - fibers to chemical stimulants . the estimated ph changes shown in table 1 exceed those reported to be effective in c - fiber stimulation in anesthetized rats . the high co2 or h+ acting as a direct irritant , could explain our results with 70 and 100% co2 ( fig . both the near instantaneous c - fiber response and part of the peripheral reflex are abolished by vagotomy , accounting for the delayed response , which then results only from central chemoreceptors . the response by the latter is further reduced when the peripheral receptor potentiation is partially removed by cutting both vagi ( fig . 3c ) and further delayed by slowing the circulation by stimulating the efferent right vagus . our indirect evidence for fast - acting chemoreceptors in the broncho - tracheal region to high co2/h+ concentrations can be criticized for having too few animals and lack of ancillary respiratory measurements . certainly a shift in baseline acid - base status because of repeated trials with co2 would have an effect on the response curves . however , the time delays were carefully measured and suggest that more experiments are required to determine the contribution of airway and lung area chemoreception to the control of ventilation when alveolar pco2 is rapidly altered .

background : the ventilation ( ) response to inspired co2 has been extensively studied , but rarely with concentrations > 10%.aims : these experiments were performed to determine whether would increase correspondingly to higher concentrations and according to conventional chemoreceptor time delays.materials and methods : we exposed anesthetized dogs acutely , with and without vagotomy and electrical stimulation of the right vagus , to 20 - 100% co2-balance o2 and to 0 and 10% o2-balance n2.results:the time delays decreased and response magnitude increased with increasing concentrations ( p<0.01 ) , but at higher concentrations the time delays were shorter than expected , i.e. , 0.5 s to double at 100% co2 , with the response to 0% o2 being ~3 s slower . right vagotomy significantly reduced baseline breathing frequency ( fr ) , increased tidal volume ( vt ) and increased the time delay by ~3 s. bilateral vagotomy further reduced baseline fr and , and reduced the response to co2 and increased the time delay by ~12 s. electro - stimulation of the peripheral right vagus while inspiring co2 caused a 13 s asystole and further reduced and delayed the response , especially after bilateral vagotomy , shifting the mode from vt to fr.conclusions:results indicate that airway or lung receptors responded to the rapid increase in lung h+ and that vagal afferents and unimpaired circulation seem necessary for the initial rapid response to high co2 concentrations by receptors upstream from the aortic bodies .

Introduction Materials and Methods Results Discussion Conclusion

in vertebrates , the process of skeletal muscle development occurs in subsequent steps that involve distinct populations of myogenic progenitors , the myoblasts , which arise from the dermomyotomal domain of somitic mesoderm ( christ and ordahl , 1995 ) . the process of myogenic differentiation is initiated in mesodermal cells by a family of basic - helix - loop - helix ( bhlh ) transcription factors , named muscle regulatory factors ( mrfs ) , that are able to activate transcription of muscle - specific markers such as the myosin heavy chain ( myhc ) isoforms ( pinney et al . , 1988 , cao et al . , 2010 ) . embryonic myoblasts sustain a first wave of myogenesis between embryonic day 10 ( e10 ) and e12 in the mouse and give rise to primary myofibers that establish the primitive shape of muscle and express high levels of the slow myhc isoform ( myhc - i , encoded by the myh7 gene ) and of the embryonic myhc isoform ( myhc - emb , encoded by myh3 ) ( schiaffino et al . , 1986 , stockdale , 1992 ) . a second wave of muscle differentiation takes place between e15 and e18 , driven by fetal myoblasts that form secondary fibers , characterized by low levels of myhc - i and high levels of neonatal myhc ( myhc - neo , encoded by myh8 ) ( eusebi et al . , 1986 , lyons et al . , 1990 , daou et al . , eventually , primary fibers conserve the slow - twitch phenotype typical of embryonic muscle , while secondary fibers lose the expression of several embryonic - specific markers such as myhc - i and acquire expression of fast - twitch markers ( ferrari et al . , 1997 , embryonic and fetal myoblasts , once isolated from the embryo , are committed to a specific fiber type , suggesting the involvement of intrinsic factors rather than nerve activity in the establishment of fiber phenotype ( page et al . , 1992 ) . these observations suggest that the proper transition of skeletal muscle from the embryonic to the fetal / post - natal phenotype requires a switch in the transcriptional status of differentiating myoblasts . in our previous study , we have shown that the transcription factor nfix , a member of the nuclear factor i ( nfi ) family , has a key role in the establishment of fetal muscle phenotype and in the downregulation of slow myhc both in fetal and adult muscles ( messina et al . , 2010 , rossi et al . , 2016 ) . we have shown that nfix is strongly expressed in fetal myoblasts and indirectly represses myhc - i expression via the transcription factor nfatc4 , a positive regulator of myhc - i in skeletal muscle ( calabria et al . we have also reported that the zebrafish ( danio rerio ) nfix ortholog nfixa has an evolutionarily conserved role in the transition from slow - twitch to fast - twitch myogenesis ( pistocchi et al . , 2013 ) . in the past few years , it has been shown that sox6 , a member of the sry - related hmg box ( sox ) factor family , which is highly conserved in vertebrates , plays a critical role in fetal fiber specification through direct repression of myhc - i by binding to the 5-upstream region in two different binding sites . the first is located 200 bp from the transcription start site ( tss ) in the proximal promoter and is sufficient for sox6-dependent myhc - i repression in fetal myotubes ( hagiwara et al . , 2007 , an et al . , 2011 ) , and the second is located 2,900 bp from the tss in a distal muscle enhancer that is required for full promoter activity ( giger et al . , sox6-null mouse muscle displays increased levels of myhc - i and a general switch toward a slower phenotype ( hagiwara et al . , studies in zebrafish have shown that sox6 is restricted to fast - twitch fibers during embryonic muscle development and that ectopic sox6 expression in adaxial cells ( the slow muscle progenitors in zebrafish ) leads to the silencing of slow - twitch genes ( such as the slow myhc isoform smyhc1 and the transcription factor prox1a ) ( von hofsten et al . , 2008 , wang et al . , 2011 , , we observed that sox6 , at variance with nfix , is expressed at comparable levels in embryonic and fetal myoblasts , despite its role of inhibitor of slow twitching program . intriguingly , we demonstrated that sox6 has opposite roles in regulating myhc - i expression between embryonic and fetal myogenesis in mouse . specifically , during embryonic myogenesis , sox6 indirectly promotes myhc - i expression via transcriptional activation of mef2c . as a consequence , sox6 deficiency in embryonic muscle leads to a strong downregulation of myhc - i . on the contrary , during fetal myogenesis , sox6 cooperates with nfix to repress myhc - i in a complex in which nfix is necessary for the proper binding of sox6 to the myhc - i promoter in fetal myotubes . finally , we show that nfixa and sox6 together regulate smyhc in zebrafish embryos , revealing an evolutionarily conserved mechanism that is required for the acquisition of normal muscle phenotype . sox6 has been intensively studied as an inhibitor of slow muscle phenotype during the fetal period . however , we observed that sox6 is also expressed during embryonic myogenesis , which is mainly characterized by the expression of typical slow genes such as the slow myhc isoform myhc - i . myf5 embryos and fetuses were collected at e12.5 or e16.5 , and gfp - positive myoblasts were isolated via fluorescence - activated cell sorting ( facs ) as previously described ( messina et al . , 2010 ) . using quantitative real - time pcr and western blot , we found that sox6 levels do not significantly change , whereas nfix , as known , is drastically upregulated in fetal progenitors ( figures 1a1c ) . importantly , these data show that sox6 and nfix proteins are co - expressed only during fetal myogenesis . we also performed extensive immunofluorescence analysis on frozen mouse embryo sections from e10.5 to e18.5 in order to follow sox6 protein expression throughout development . notably , sox6 is first expressed between e11.5 and e12.5 in primary fibers that express high levels of myhc - i ( figures s1a s1h ) , whereas at e17.5 , the localization of sox6 is almost completely associated with secondary fibers that are negative for myhc - i ( figures s1i s1l ) , as previously described ( an et al . , 2011 , we therefore decided to investigate the possible function of sox6 during embryonic myogenesis , a function not apparently linked to repression of myhc - i . to this aim , we performed immunofluorescence analysis on e12.5 muscle sections from homozygous mice carrying the sox6 allele ( hence referred to as sox6-null mice ) ( smits et al . , 2001 ) . surprisingly , the staining for myhc - i is strongly decreased in sox6 null in comparison to wild - type ( wt ) muscle ( figures 1d and 1e ) . importantly , no differences in total myhc content were assessed in embryonic muscle groups of sox6-null embryos ( figures 1 g and 1h ) , implying that the decrease in myhc - i expression is not due to delayed or aberrant muscle differentiation . we also performed western blot and quantitative real - time pcr on embryonic muscle lysates to confirm the immunofluorescence data , and our results showed a decrease of myhc - i protein in sox6-null samples , without major changes in total myhc content ( figures 1j and 1k ) . interestingly , the phenotype of sox6-null embryonic muscle is reminiscent of the tg : mlc1f - nfix2 gain - of - function embryo ( figures 1f and 1i ) , in which the nfix2 splice variant is ectopically expressed in muscle cells from e11.5 , leading to a fetal - like muscle phenotype ( kelly et al . , 1997 , messina et al . , 2010 ) importantly , nfix expression in embryonic skeletal muscle is not altered in the absence of sox6 at both the protein and mrna levels ( figures s2a s2d ) . taken together , these results show that sox6 is expressed at equal levels in skeletal muscle during embryonic and fetal myogenesis and that deletion of sox6 during the embryonic period unexpectedly leads to downregulation of myhc - i . in order to define a possible mechanism by which sox6 regulates the transcription of myhc - i during embryonic myogenesis , we performed chromatin immunoprecipitation ( chip ) for sox6 in differentiated embryonic myoblasts . we found that sox6 does not significantly bind either to the proximal or to the distal regulative regions upstream of myhc - i ( figure 2a ) , thus suggesting that sox6 is not able to directly regulate myhc - i transcription in embryonic muscle . to confirm this hypothesis , we performed luciferase assays on wt and sox6-null embryonic differentiated myoblasts with vectors containing the 3,500-bp myhc - i full 5-upstream region ( myhc - i 3500 ) , the 408-bp myhc - i proximal promoter sequence ( myhc - i 408 ) , or the mutated forms of the distal and proximal canonical sox6 binding sites , myhc - i 3500 m and myhc - i 408 m ( figure s3a ) ( hagiwara et al . , 2007 , an et al . , 2011 ) . as expected , in the absence of sox6 , we found a significant reduction of firefly luciferase activity in all the conditions with the only notable exception of the 408 wt construct ( figure 2b ) , suggesting that sox6 is promoting myhc - i expression in embryonic myocytes without direct binding to its canonical binding sites . in order to identify a possible indirect mechanism by which sox6 enhances myhc - i expression in embryonic muscle , we focused on the transcription factor mef2c , a known positive regulator of the slow phenotype ( wu et al . , 2000 , potthoff et al . , 2007 , anderson et al . , 2015 ) . to verify the interaction between mef2c and the myhc - i promoter in embryonic myoblasts , we performed a chip assay , which showed direct binding of mef2c on the proximal myhc - i promoter ( figure s3b ) . interestingly , mef2c mrna is downregulated in e12.5 sox6-null muscle , at variance with the closely related mef2a ( figure 2c ) . by chip on embryonic differentiated myoblasts , we found that sox6 directly binds to a region located in the mef2c promoter ( figure 2d ) , indicating that sox6 is a direct activator of mef2c during embryonic myogenesis . in order to validate a possible mef2c - mediated mechanism of myhc - i regulation , we transfected wt and sox6-null unpurified embryonic myoblasts with a vector overexpressing mef2c . mef2c overexpression in sox6-deficient cells ( figure s3c ) leads to an increase of myhc - i expression when normalized on the levels of myhc - emb to account for the total number of myogenic cells ( figure 2e ) . to validate these data , we also transduced purified embryonic myoblasts with a lentiviral vector expressing small hairpin rna ( shrna ) against sox6 to achieve in vitro sox6 knockdown ( figure s3d ) . we then transfected cells with mef2c - overexpression vector ( figure s3d ) and assessed the levels of myhc - i mrna by quantitative real - time pcr ( figure 2f ) . strikingly , overexpression of mef2c was able to partially rescue myhc - i expression in shsox6 cells ( up to 30% of the levels of scramble - transduced cells ) . these results show that during the embryonic period , sox6 acts indirectly as a positive regulator of myhc - i via a mef2c - dependent mechanism , in sharp contrast with its well - known and characterized function during fetal myogenesis ( an et al . , 2011 , quiat et al . , 2011 ; see below ) . since sox6 has opposite roles in myhc - i regulation in embryonic and fetal muscle , and since nfix is only expressed during fetal myogenesis ( messina et al . , 2010 ) , we decided to investigate the possible cooperation between nfix and sox6 during fetal myogenesis . 2011 ) , fetal fiber specification is completely disrupted in the absence of sox6 . indeed , in contrast to wt , sox6-null fetal muscle displays very high levels of myhc - i by immunofluorescence , quantitative real - time pcr , and western blot ( figures s4a s4d ) . we also looked at nfix on sections from the sox6-null mice and found that despite the dramatic increase in myhc - i expression , nfix is correctly expressed in the nuclei of muscle fibers in the absence of sox6 ( figures 3a3d ) , and western blot analysis did not reveal differences in nfix protein content ( figure s4c ) . these data suggest that nfix is normally expressed in fetal muscles lacking sox6 but unable to properly repress myhc - i . to verify whether sox6 is required for nfix function , we performed chip for nfix in wt fetal myotubes , which revealed binding of nfix to myhc - i promoter in the same region of the proximal sox6 binding site ( 200 bp ) ( figure 3e ) . interestingly , we observed that in sox6-null fetal myotubes , the binding of nfix to the myhc - i promoter is significantly reduced . importantly , nfix binding to nfatc4 promoter is not impaired in the absence of sox6 ( figure 3e ) , suggesting that nfix is still able to bind to other transcriptional targets in the absence of sox6 . we further validated the repressive role of nfix on the myhc - i proximal promoter by performing a luciferase assay with myhc - i 408 and myhc - i 408 m vectors in wt and nfix - null fetal myotubes ( figure 3f ) . our results showed increased luciferase expression in the absence of nfix with the vector carrying the wt sequence , suggesting that indeed nfix represses myhc - i by acting on the proximal promoter regulative region , in spite of the absence of any nfix consensus sequences ( data not shown ) . importantly , nfix - dependent negative regulation of myhc - i is lost when the proximal sox6 binding sequence is mutated , demonstrating that sox6 is required for nfix binding to the proximal myhc - i promoter ( figure 3f ) . overall , these results suggest a possible crosstalk between nfix and sox6 in regulating myhc - i expression at the proximal promoter region . to better investigate a possible reciprocal interplay between nfix and sox6 , we performed immunofluorescence for sox6 and total myhc on frozen muscle sections from e16.5 wt and nfix - null mice ( campbell et al . , 2008 ) . we found that sox6 is expressed in both wt and nfix - null muscles ( figures s4e and s4f ) , implying that nfix is dispensable for normal sox6 expression in fetal muscle . interestingly , immunofluorescence for sox6 and myhc - i on sections from the same mice revealed that in the absence of nfix , there is a marked increase in the number of sox6-positive fibers that co - express myhc - i , in contrast to wt muscles ( figures 4a4d ) . we further validated these data by western blot and quantitative real - time pcr on differentiated fetal myotubes and observed that nfix - null myotubes express higher levels of myhc - i than wt cells ( figures 4e and 4f ) . these data show that in the absence of nfix , the repressive activity of sox6 on myhc - i is partially impaired , even if the normal expression pattern of sox6 is maintained . the results obtained in both sox6 and nfix - null fetuses led us to hypothesize a functional cooperation between nfix and sox6 in myhc - i regulation . therefore , we tested the ability of nfix and sox6 to bind to each other in a multi - protein complex . to this aim , we used fetal myotubes transiently transfected with an nfix2 hemagglutinin ( ha ) vector ( see experimental procedures ) and performed a co - immunoprecipitation ( coip ) assay from nuclear extracts for ha and sox6 . coip revealed the presence of a complex containing both sox6 and nfix2-ha , as shown in figure 5a . therefore , we wondered whether , as observed for nfix fetuses in absence of sox6 , nfix might be required for the binding of sox6 to the myhc - i promoter as well . hence , we performed chip for sox6 in wt and nfix - null fetal myotubes . our results showed a decrease in sox6 binding to the two different sites in the myhc - i 5-upstream region ( figure 5b ) . notably , the binding with the proximal promoter was completely lost in the absence of nfix , whereas the binding with the distal enhancer was reduced by 50% . interestingly , chip for sox6 on the mef2c promoter reveals that in wt fetal myotubes , sox6 is not able to bind to the mef2c promoter , at variance with what happens during the embryonic stage . on the contrary , sox6 binding on the mef2c promoter still occurs in nfix - null fetal myotubes . these data indicate that nfix is required for the proper binding of sox6 to the myhc - i promoter during fetal myogenesis . finally , we performed chip for sox6 on wt and tg : mlc1f - nfix2 embryonic myoblasts ( figure 5c ) . strikingly , nfix overexpression leads to a switch in the binding properties of sox6 ; indeed , we found that sox6 is bound to the myhc - i promoter , but not to the mef2c promoter , in tg : mlc1f - nfix2 embryonic myoblasts . these data demonstrate that nfix is necessary and sufficient for the binding of sox6 to the myhc - i proximal promoter and therefore for sox6 repressive activity on myhc - i . it was previously demonstrated that both sox6 and nfixa have an evolutionarily conserved role in the repression of slow - twitch genes in zebrafish ( von hofsten et al . , 2008 , we thus wondered whether a functional interplay between nfixa and sox6 is conserved in zebrafish myogenesis . as a preliminary analysis , we performed quantitative real - time pcr on trunk and tail regions isolated from zebrafish embryos at 1 , 2 , and 3 days post - fertilization ( dpf ) and found that the sox6 transcript is expressed at high levels at 1 dpf and is steadily downregulated up to 3 dpf ( figure s5a ) , whereas the nfixa transcript peaks at 2 dpf ( figure s5b ) , as shown previously ( pistocchi et al . , 2013 ) . additionally , we performed immunofluorescence for sox6 and total myhc ( mf20 antibody ) or the slow myhc isoform smyhc ( f59 antibody ) on 2 dpf embryos and found that sox6 protein is specifically expressed in the nuclei of fast muscle fibers that are negative for smyhc ( figures 6a and 6b ) , whereas the outer superficial fibers positive for smyhc are mostly negative for sox6 ( data not shown ) . interestingly , we observed that a minority of slow superficial fibers show cytoplasmic staining for sox6 ( figures 6c6j ) , suggesting that sox6 subcellular localization might be spatially regulated in the different muscle domains . in order to elucidate the role of sox6 in slow - twitch genes regulation , we performed morpholino ( mo)-mediated knockdown of sox6 ( von hofsten et al . , 2008 ) . by quantitative real - time pcr analysis ( figures s5c and s5d ) , we found that the expression of smyhc1 is markedly increased in sox6 morphants at 2 dpf , whereas the fast - twitch gene mylpfa ( fast myosin light chain isoform ) is expressed at equal levels . moreover , the nfixa transcript is drastically upregulated in sox6 morphants , suggesting that sox6 might negatively regulate nfixa ( figure s5e ) . we conclude that sox6 is a critical repressor of the slow - twitch phenotype in zebrafish and that nfixa is not able to compensate for sox6 knockdown . to verify a possible cooperation between nfixa and sox6 , we performed co - injections of morpholinos against sox6 and nfixa at lower doses with respect to those previously described ( von hofsten et al . , 2008 , pistocchi et al . , 2013 ) in order to minimize their effect when injected separately ( see experimental procedures ) . co - injection of these doses of mos resulted in synergistic defects in motility in touch - response assays ( figure s6 ) . control embryos and the vast majority of sox6 or nfixa morphants readily swam away when touch - stimulated . on the contrary , 66% of double partial morphants were either shivering or bending their tails before eventually moving away but within a shorter distance ( figures s6a and s6c ; movie s1 ) . the synergistic effect is more evident when lowering the doses ( see figures s6b and s6d ) . strikingly , quantitative real - time pcr results show that smyhc1 is significantly upregulated only in the double morphants at 48 hpf , whereas the level of myl1 , an early marker for differentiating fast muscle cells ( burguire et al . , 2011 ) , does not change ( figure 6k ) . moreover , we validated the increased expression of smyhc ( f59 antibody ) in sox6/nfixa double morphants by western blot ( figure 6l ) . we thus conclude that functional cooperation of sox6 and nfix is required for proper skeletal muscle development and that this cooperation is evolutionarily conserved in mouse and zebrafish . adult skeletal muscle is composed of two major fiber types presenting a wide range of physiological and biochemical differences . slow - twitch type i fibers use oxidative metabolism and express the slow myhc isoform myhc - i ; in contrast , fast - twitch type ii fibers present glycolytic or mixed metabolism and express three fast myhc isoforms ( myhc - iia , iix / d , and iib ) ( peter et al . , 1972 , schiaffino et al . , 1988 , chakkalakal et al . , 2012 ) . the phenotype of post - natal muscle fibers is strictly regulated by extrinsic signals such as muscle activity and hormones ( butler - browne et al . , 1982 , gambke et al . , 1983 , additionally , different factors controlling adult muscle plasticity have been identified , including the nfatc ( calabria et al . , 2009 ) and mef2 ( wu et al . , 2000 , potthoff et al . transcription factor families and pgc-1 ( li et al . , 2002 ) . on the contrary , the molecular mechanisms by which muscle fiber diversity is achieved during pre - natal development are still poorly understood . it was shown that the intrinsic transcriptional properties of embryonic and fetal myogenic progenitors are important to set the fiber type in the absence of nerve activity ( cho et al . , 1993 , cusella - de angelis et al . , 1994 ) . moreover , in recent years , several transcription factors contributing to developmental muscle fiber specification have been identified , including sox6 ( hagiwara et al . , 2007 , an et al . , 2011 ) , nfix ( messina et al . , 2010 ) , six1/six4 ( richard et al . , 2011 ) , and nfatc2 ( daou et al . , 2013 ) . however , the network of transcription factors controlling fiber specification during embryogenesis is still far from being fully characterized , and until now , functional interactions among the different regulators were completely unknown . in this work , we provided evidence for functional interplay between nfix and sox6 in controlling expression of the slow myhc isoform during mouse pre - natal muscle development . in contrast to nfix , which is a specific marker of fetal myogenesis ( messina et al . , 2010 , mourikis et al . , 2012 ) , sox6 is expressed in both embryonic and fetal purified myoblasts at the mrna and protein levels . consistently , we found that sox6 protein is expressed in skeletal muscle in vivo starting between e11.5 and e12.5 . this was unexpected , since sox6 is known to be a repressor of myhc - i , which along with myhc - emb is expressed in all embryonic fibers ( hagiwara et al . it is known that sox6 transcript is absent in mouse primary myotome between e9.5 and e10.75 ( vincent et al . , 2012 ) , suggesting that sox6 is quickly activated in embryonic myoblasts at the beginning of primary myogenesis . unexpectedly , sox6 deficiency during primary myogenesis leads to a transient faster muscle phenotype with low levels of myhc - i . this is followed in sox6-null fetuses by dramatic upregulation of myhc - i at the transcription level , consistently with previous characterizations of the sox6-null phenotype ( hagiwara et al . our results demonstrate that sox6 plays opposite roles in myhc - i expression during development . importantly , in embryonic myogenic cells , sox6 does not bind to the two canonical myhc - i binding sites , which are both dispensable for sox6-dependent embryonic regulation of myhc - i , at least in our culturing conditions . we found that sox6 directly binds to the promoter of mef2c and activates its expression in primary myofibers . mef2c is part of a transcription factor family that is constitutively expressed in muscle cells since early embryogenesis ( edmondson et al . , 1994 ) and plays an important role in the specification and maintenance of type i fibers ( chin et al . , 1998 , wu et al . , 2000 , anderson et al importantly , conditional deletion of mef2c in skeletal muscle leads to a drastic reduction in slow - twitch fibers ( potthoff et al . , 2007 ) . our rescue experiment , although partial , clearly shows that one of the sox6-dependent effects on myhc - i transcription in embryonic myofibers is mediated by mef2c , which is directly targeted by sox6 in embryonic , but not fetal , muscle . this function led us to hypothesize that the binding ability of sox6 is differentially regulated in discrete myogenic progenitor populations or at different times during development according to different co - factors , which contribute to the high versatility of sox6 functions . it is known that soxd factors , lacking trans - acting functional domains , have a critical requirement for co - factors in order to regulate transcription of target genes ( reviewed in hagiwara , 2011 ) . therefore , it is likely that the reversal in sox6 function is due to different factors that are progressively recruited and activated during muscle development . indeed , we found that during fetal myogenesis , nfix acts as a fundamental co - factor of sox6 and is able to form a complex with sox6 , which is no longer able to bind to the mef2c promoter but can bind to myhc - i regulative regions , in particular the proximal promoter that was shown to be critical for sox6-dependent fetal repression of myhc - i ( hagiwara et al . , 2007 ) . in our previous study , we showed that nfix negatively regulates myhc - i by repressing nfatc4 , a positive regulator of myhc - i interestingly , we have now found that during fetal myogenesis , nfix is also present at the myhc - i proximal promoter along with sox6 , suggesting that a physical association between these two proteins may be required for proper myhc - i downregulation . since nfix presents both a trans - repression domain and a trans - activation domain , we hypothesized that formation of a complex with sox6 might provide the basis for the transcriptional repression at the myhc - i locus ( figure 7 ) . indeed , nfix and sox6 were found to be part of the same complex in fetal myotubes and are both present at the myhc - i proximal promoter . moreover , our study on nfix - null and sox6-null fetuses clearly shows that sox6 and nfix are independently expressed during secondary myogenesis and that neither sox6 nor nfix is able to correctly downregulate myhc - i expression when the other one is not present . a scheme of the scenario in which sox6 and nfix behave and cooperate during pre - natal muscle development is shown in figure 7 . in this work , we have also provided evidence for a conserved transcriptional cooperation of sox6 and nfixa in zebrafish . we have shown that sox6 is crucial for the regulation of smyhc1 , in line with the findings of jackson et al . it was shown that sox6 expression is silenced in zebrafish slow fibers by the combined effects of prdm1a and mir-499 ( von hofsten et al . , 2008 , we found that a minority of smyhc - positive cells display cytoplasmic staining for sox6 , suggesting that sox6 might also be regulated through subcellular localization in different fiber types . notably , our double partial knockdown performed with suboptimal doses of sox6-mo ( von hofsten et al . , 2008 ) and nfixa - mo ( pistocchi et al . , 2013 ) caused a severe impairment in the touch - evoked escape response , suggesting that sox6 and nfixa cooperate in proper muscle function . molecularly , our data suggest that the two transcription factors can act together to repress smyhc1 expression in zebrafish embryos , whereas the expression of a typical fast - twitch gene , such as myl1 ( burguire et al . , 2011 ) , , we have presented a complex model of regulation of embryonic to fetal myhc - i regulation that involves a functional interplay between nfix and sox6 that is conserved in mammals and teleosts . the following murine lines were used : myf5 ( kassar - duchossoy et al . , 2004 ) , nfix null ( campbell et al . , 2008 ) , sox6 ( smits et al . , 2001 ) , and tg : mlc1f - nfix2 ( messina et al . , 2010 ) . for each of these lines , the genotyping strategy has been described in the references . mice were kept in pathogen - free and controlled conditions , and all procedures conformed to italian law ( d. lgs n 2014/26 , implementation of the 2010/63/ue ) and were approved by the animal welfare body of the university of milan and the italian minister of health ( 1212/2015pr ) . the following line was used : ab ( from carole wilson , ucl , london , uk ) . mouse or zebrafish embryos were fixed in 4% paraformaldehyde , extensively washed in pbs , and incubated overnight in pbs containing 15% sucrose . samples were then frozen in nitrogen - chilled isopentane and kept at 80c until use . 0.2% triton x-100 in pbs for 30 min at room temperature and then incubated for 1 hr in blocking solution ( 10% goat serum in pbs ) and overnight with the primary antibody or with mock pbs . after incubation for 45 min with the fluorescent - conjugated secondary antibody ( jackson immunoresearch laboratories ) , sections were washed in pbs 0.2% triton x-100 and mounted , and fluorescent immunolabeling was recorded with a dm6000 leica microscope . the following primary antibodies were used : rabbit anti - sox6 ( abcam , 1:300 ) , rabbit anti - nfix ( novus biological , 1:200 ) , mouse anti - myhc - i ( sigma , 1:200 ) , bad5 ( monoclonal , 1:2 ) , mf20 ( monoclonal , 1:2 ) , and f59 ( monoclonal , 1:10 ) . nuclei were stained with hoechst ( 1:1,000 ) . dissected myf5 embryonic or fetal muscles were digested by 0.15 mg / ml collagenase ( sigma ) , 1.5 mg / ml dispase ( gibco ) , and 0.1 mg / ml dnase i ( sigma ) for 30 min at 37c in agitation as described in biressi et al . after centrifugation and filtration , cells were collected in dmem , 20% fetal bovine serum ( fbs ) , 2 mm edta , and 20 mm hepes . for cell gfp - positive cells were collected for mrna and protein extraction . for the preparation of unpurified fetal myoblasts after digestion of tissue , cells were pre - plated for 30 min on a plastic dish to lose fibroblasts , which normally adhere to plastic . unpurified cells were kept in incubation at 37c in 20% hs ( 20% horse serum in dmem ) and 24 hr later allowed to differentiate in dm ( 2% horse serum in dmem ) for 48 hr . preparation of viral particles were performed by co - transfecting plko.1-shsox6 vectors ( thermo fisher scientific ) or non - targeting shrna vectors ( 30 g ) , together with the packaging plasmids pmdlg / p ( 16.25 g ) , pcmv - vsvg ( 9 g ) , and prsv - rev ( 6.25 g ) , in hek293 t cells . transfection was performed using the calcium phosphate transfection method . viral particles were collected 40 hr after transfection and subjected to ultracentrifugation at 20,000 rpm for 2 hr at 20c ( beckman coulter , optima l-100 xp ) . the concentrated viral particles were re - suspended in pbs and stored in aliquots at 80c until further use . these preparations were used to transduce embryonic myoblasts at an moi of 10 . the day after transduction , embryonic myoblasts were transfected with mef2c ( pcdnai / a - mef2c ) or the empty vector as a negative control using lipofectamine ltx ( invitrogen ) . cultured cells were washed twice in ice - cold pbs and then lysed ( 30 min in ice ) with ripa buffer ( 10 mm tris - hcl [ ph 8.0 ] , 1 mm edta , 1% triton - x , 0.1% sodium deoxycholate , 0.1% sds , and 150 mm nacl in deionized water ) plus protease inhibitors ( 1 mm pmsf ) . for zebrafish , only trunk and tail regions were used , cutting out the head , and protein was extracted in laemmli buffer ( 3 l per embryo ) . protein was harvested after centrifugation , quantified by absorbance reading at 750 nm , and stored at 80c . 30 g protein was resolved on 8%12% polyacrylamide gels or on miniprotean tdx gels ( bio - rad ) after denaturation at 95c for 5 min with sds - page loading sample buffer 2x ( 100 mm tris [ ph 6.8 ] , 4% sds , 0.2% bromophenol blue , 20% glycerol , and 10 mm dithiothreitol ) . for western blot analysis , proteins were transferred onto nitrocellulose with the iblot dry blotting system ( invitrogen ) . following transfer , membranes were blocked in 5% milk in tris - buffered saline ( tbs)-t ( tbs plus 0.02% tween20 ) for 1 hr at room temperature . the primary antibodies used were rabbit anti - sox6 ( abcam ; 1:1,000 ) , mouse anti - myhc - i ( sigma ; 1:5,000 ) , mouse anti--tubulin ( santa cruz biotechnology ; 1:5,000 ) , rabbit anti - nfix ( novus biologicals ; 1:500 ) , mouse anti - ha ( covance ; 1:500 ) , mouse anti - vinculin ( sigma ; 1:1,000 ) , and mouse anti - smyhc ( f59 , hybridoma bank ) anti - mouse sarcomeric myhc ( mf20 , hybridoma bank ) . horseradish peroxidase ( hrp)-conjugated antibodies ( bio - rad ) were used as secondary antibodies , and the signal was revealed with the chemidoc mp system ( bio - rad ) . for transfection of fetal myoblasts , wt or sox6-null cells were plated on 90-mm dishes and allowed to reach 80% confluence in proliferating conditions . cells were transfected with pch - nfix2-ha or pch - ha plasmids with lipofectamine ltx ( invitrogen ) overnight at 37c . cells were kept in dm for 36 hr after transfection , and then nuclear extracts were prepared by collecting 5 10 cells in 400 l ice - cold hypotonic buffer a ( 10 mm hepes [ ph 7.9 ] , 10 mm kcl , 0.1 mm egta , 1 mm dtt , protease inhibitors , 1 mm pmsf , and phosphatase inhibitors ) for 15 min in ice . then , np40 was added to a final concentration of 0.625% , and cells were centrifuged at 5,000 rpm for 5 min at 4c . the supernatant was discarded as cytoplasmatic extract , while the pelleted nuclei were resuspended in 100 l immunoprecipitation lysis buffer containing 50 mm tris - hcl ( ph 7.5 ) , 1% np40 , 1 mm edta , 100 mm nacl , and protease and phosphatase inhibitors . hr with gentle rotation at room temperature with 5 g rabbit anti - sox6 antibody ( abcam ) , rabbit anti - ha antibody ( santa cruz ) , or non - related rabbit immunoglobulin g ( igg ) ( santa cruz ) . then , the bead - antibody complex was incubated with gentle rotation for 2 hr at 4c with 1.5 mg total protein lysates per condition . the eluted proteins were denatured with non - reducing sds - page loading sample buffer ( 100 mm tris [ ph 6.8 ] , 4% sds , 0.2% bromophenol blue , and 20% glycerol ) and loaded onto a gel . chip for sox6 and luciferase assays were performed as previously published ( an et al . , 2011 ) . rna from homogenized mouse or zebrafish embryos and isolated cells was extracted with nucleospin rna kits ( macherey - nagel ) following the manufacturer s instructions . eluted rna was checked on agarose gels , quantified with a nanodrop spectrophotomer , and stored at 80c . approximately 0.5 g rna was used with the improm - ii reverse transcriptase kit ( promega ) . real - time pcr was performed on cdna using sybr green supermix ( bio - rad ) and the cfx connect real time system ( bio - rad ) . after amplification , relative mrna expression levels were calculated using standard curves from cdna dilutions and normalized on the gapdh expression levels . for quantitative real - time pcr in zebrafish the antisense mos ( gene tools ) used in this study , sox6-mo1 ( von hofsten et al . , 2008 ) and nfixa - mo ( pistocchi et al . , 2013 ) , were previously described . mos , diluted in danieau buffer ( nasevicius and ekker , 2000 ) , were injected at the one- to two - cell stage . escalating doses of each mo were tested for phenotypic effects ; as control for unspecific effects , each experiment was performed in parallel with an std - mo ( standard control oligo ) with no target in zebrafish embryos . we usually injected 0.25 pmol sox6-mo per embryo . for combined knockdown experiments , we injected sox6-mo and nfixa - mo at 0.1 and 0.25 or 0.08 and 0.125 pmol per embryo , respectively . statistical significance was assessed by unpaired student s t test with prism 5 software . statistical significance with a probability of less than 5% , 1% , or 0.1% is indicated in each graph with a single , double , or triple asterisk , respectively , followed by the number of independent experiments ( n ) . conceptualization , g. maroli , v.t . , and g. messina ; methodology , s.c . ; writing original draft , g. maroli and v.t . ; writing review & editing , v.t . , m.b . , and g. messina ; supervision , m.b . and g. messina ; funding acquisition , g. messina .

summarysox6 belongs to the sox gene family and plays a pivotal role in fiber type differentiation , suppressing transcription of slow - fiber - specific genes during fetal development . here , we show that sox6 plays opposite roles in myhc - i regulation , acting as a positive and negative regulator of myhc - i expression during embryonic and fetal myogenesis , respectively . during embryonic myogenesis , sox6 positively regulates myhc - i via transcriptional activation of mef2c , whereas during fetal myogenesis , sox6 requires and cooperates with the transcription factor nfix in repressing myhc - i expression . mechanistically , nfix is necessary for sox6 binding to the myhc - i promoter and thus for sox6 repressive function , revealing a key role for nfix in driving sox6 activity . this feature is evolutionarily conserved , since the orthologs nfixa and sox6 contribute to repression of the slow - twitch phenotype in zebrafish embryos . these data demonstrate functional cooperation between sox6 and nfix in regulating myhc - i expression during prenatal muscle development .

Introduction Results Discussion Experimental Procedures Author Contributions

delirium is a neuropsychiatric disorder secondary to a general medical condition and represents a serious complication of the underlying disease or its treatment . three subtypes of delirium can be distinguished : ( 1 ) hypoactive delirium , which is characterized by apathy , decreased responsiveness , and withdrawal ; ( 2 ) hyperactive delirium , which is characterized by restlessness , emotional lability , and agitation ; ( 3 ) mixed delirium , which is a combination of the two other subtypes . the condition is frequently seen in critically ill adult and geriatric patients and is associated with poor prognosis , reflected by worse functional and cognitive outcome , prolonged hospital stay , and a higher mortality rate after discharge from hospital [ 1 , 35 ] . although the clinical picture of delirium in adults is well known , comparatively scarce attention has been paid to its counterpart in children pediatric delirium ( pd)even though data suggest that pd is a similarly serious condition [ 2 , 68 ] . it is important to correctly diagnose delirium in children , in order to make early intervention possible . adult delirium is often missed by clinicians , because it commonly manifests as the hypoactive subtype [ 911 ] ; anecdotal evidence suggests that hypoactive forms , and misclassification thereof , may be even more prevalent in the pediatric population . as a consequence , there is an urgent need for a valid instrument which can be used by staff at pediatric intensive care units ( picu ) [ 2 , 12 ] to assess pd . the pediatric confusion assessment method for the intensive care unit ( pcam - icu ) , the pediatric anesthesia emergence delirium ( paed ) scale , the delirium rating scale ( drs-88 ) , and the delirium rating scale - revised ( drs - r-98 ) are instruments that have been developed to diagnose delirium in clinical practice . the pcam - icu has recently been validated as a highly reliable instrument in pediatric critically ill children ( 815 years ) . the paed , which only includes items covering behavioral symptoms , has been validated in children in the age range of 18 months6 years in a post - anesthesia setting . however , in that study , the diagnosis of delirium was only made if the anesthetist decided that treatment of the condition was necessary . the drs-88 and the drs - r-98 focus more on cognitive functioning and have only been validated in adults , and not in icu settings . although turkel and colleagues reported that the drs-88 could be applied to diagnose delirium in children ( 6 months19 years ) , their study was carried out in retrospect and no control group was available . thus , to our knowledge , only the pcam - icu has been tested in picu settings in children , whereas the drs / paed instruments have not been tested in a picu setting . therefore , the main objective of the present prospective panel study was to investigate , under circumstances of routine clinical care , the use of the drs / paed instruments as diagnostic tools for pd in a picu setting , taking into account both item and sum score levels of the instruments . it was hypothesized that ( 1 ) the paed would be more suitable than the drs in this setting , as it contains only behavioral items , which are easier to assess in young critically ill children and , therefore , ( 2 ) a significant negative impact of younger age was expected with regard to the proportion of rateable items for both versions of the drs ( as it relies on cognitive items that can not be assessed in the youngest or most ill children ) , but not for the paed . data pertained to a cohort of critically ill ( defined as a threatening failure of the brain , heart , or lungs ) pediatric patients , admitted from november 2006 until february 2010 . the setting was a tertiary eight - bed picu at maastricht university medical centre ( mumc+ ) in the netherlands . all non - electively admitted patients to the picu between the ages of 1 and 17 years , ventilated and non - ventilated , were included . the sampling frame also included patients who had been admitted to the picu after an elective surgical procedure and who were still at the picu after 48 h. patients were excluded if they were ( 1 ) non - dutch speakers and ( 2 ) not in need of intensive care . as the information necessary to rate the drs / paed instruments is collected as part of routine clinical care at the picu , under dutch law the study did not fall under the remit of the medical ethics committee , provided data anonymity is preserved at all times . the paed was developed by sikich and lerman in order to trace emergence delirium ( ed ) . ed is defined as a disorder occurring during awakening from anesthesia in the immediate postoperative period . it is characterized by confusion , hallucinations , delusions and can be accompanied by moaning , restlessness , and agitation . the scale consists of five items which are rated on a 5-point scale ranging from extremely to not at all. a score of 10 or higher indicates diagnosis of delirium . for the purpose of the present analyses , a maximum of two missing items ( 2/5 = 40% ) was allowed a priori ; a weighted sum score was computed in the case of one or two missing items . it consists of ten items encompassing a range of areas which can be affected by delirium such as hallucinations , psychomotor behavior , cognitive status , sleep - wake cycle disturbance , and lability of mood . a score of 10 or higher indicates a diagnosis of delirium [ 18 , 19 ] . for the purpose of the present analyses , a maximum of four missing items ( 4/10 = 40% ) was allowed a priori ; a weighted sum score was computed in the case of four or fewer missing items . the drs - r-98 is a revised form of the original drs and consists of 16 items , of which the last three are optional and scored only when there is suspicion of delirium ( score of 15.25 or higher on the first 13 items ) ; the three optional items serve to differentiate between delirium and other disorders . for the purpose of the present analyses , a maximum of five missing items was allowed a priori if only the first 13 items were scored ( 5/13 = 39% ) and a weighted sum score was computed in the case of five or fewer missing items . if all 16 items were scored , a maximum of 6 missing items was allowed ( 6/16 = 38% ) and a weighted sum score was computed in cases of 6 or fewer missing items . the picu pediatric neuropsychiatrist ( js ) routinely examined all children using the criteria of the revised fourth edition of the dsm - iv . he reviewed and discussed all available information and opinions ( from parents , nurses , and the medical charts ) with the other members of the picu team before making a final diagnosis [ 7 , 12 ] . he also made further categorizations into the hyperactive , hypoactive , and mixed states of pd . patients at our picu are routinely assessed for the presence of delirium under circumstances of quality improvement in clinical care since 2005 , using a schedule of twice - daily examinations . the paed , drs-88 , and drs - r-98 are completed during the first assessment , which is carried out by the intern over the course of their attachment to the unit , having received on - site clinical training by the pediatric neuropsychiatrist of the unit . the second assessment was carried out within 3 h by the pediatric neuropsychiatrist , who was unaware of the screening results . once pd was diagnosed , appropriate management was initiated in collaboration with the picu team . for each patient , a single assessment day during hospitalization was selected . for patients diagnosed with pd , paed , drs-88 , and drs - r-98 assessments on the first day of the pd gold standard diagnosis were selected ; for patients without pd , paed , drs-88 , and drs - r-98 assessments were randomly selected in such a way that groups were frequency - matched on number of admission days until assessment . exclusion criteria for analysis were unconsciousness or deep sedation according to the clinical judgment of the responsible intensivist treating the patient . a patient was regarded non - rateable on a scale if more than the maximum allowed number of items was missing , precluding diagnosis . non - rateability of items was examined as a function of age using one - way analysis of variance ( anova , one - tailed ) . three psychometric properties of the paed , drs-88 , and drs - r-98 ( with exclusion of the three diagnostic items of the last scale , because scoring of these items is optional ) were evaluated : ( 1 ) internal consistency , expressed as cronbach s alpha ; ( 2 ) number and proportion of items not rateable ; ( 3 ) discriminative ability , expressed as the area under the receiver operating characteristic ( roc ) curve , was calculated per instrument item and for instrument sum scores , with 95% confidence intervals ( 95% ci ) . roc curves allow for exploration of the relationship between the sensitivity and specificity of a continuous predictor ( in this case the scores for each item on a scale ) and a dichotomous outcome ( in this case pd ) . instruments with the largest area under the curve ( auc ) are most accurate in distinguishing between patients with and without delirium . given the fact that pd is a serious condition requiring immediate intervention , sensitivity was given more weight than specificity in determining the optimal cutoff scores for pd . sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) for the diagnosis of delirium were calculated , as well as the likelihood ratio for a positive test result ( the ratio between true and false positive rate ) . data pertained to a cohort of critically ill ( defined as a threatening failure of the brain , heart , or lungs ) pediatric patients , admitted from november 2006 until february 2010 . the setting was a tertiary eight - bed picu at maastricht university medical centre ( mumc+ ) in the netherlands . all non - electively admitted patients to the picu between the ages of 1 and 17 years , ventilated and non - ventilated , were included . the sampling frame also included patients who had been admitted to the picu after an elective surgical procedure and who were still at the picu after 48 h. patients were excluded if they were ( 1 ) non - dutch speakers and ( 2 ) not in need of intensive care . as the information necessary to rate the drs / paed instruments is collected as part of routine clinical care at the picu , under dutch law the study did not fall under the remit of the medical ethics committee , provided data anonymity is preserved at all times . the paed was developed by sikich and lerman in order to trace emergence delirium ( ed ) . ed is defined as a disorder occurring during awakening from anesthesia in the immediate postoperative period . it is characterized by confusion , hallucinations , delusions and can be accompanied by moaning , restlessness , and agitation . the scale consists of five items which are rated on a 5-point scale ranging from extremely to not at all. a score of 10 or higher indicates diagnosis of delirium . for the purpose of the present analyses , a maximum of two missing items ( 2/5 = 40% ) was allowed a priori ; a weighted sum score was computed in the case of one or two missing items . it consists of ten items encompassing a range of areas which can be affected by delirium such as hallucinations , psychomotor behavior , cognitive status , sleep - wake cycle disturbance , and lability of mood . a score of 10 or higher indicates a diagnosis of delirium [ 18 , 19 ] . for the purpose of the present analyses , a maximum of four missing items ( 4/10 = 40% ) was allowed a priori ; a weighted sum score was computed in the case of four or fewer missing items . the drs - r-98 is a revised form of the original drs and consists of 16 items , of which the last three are optional and scored only when there is suspicion of delirium ( score of 15.25 or higher on the first 13 items ) ; the three optional items serve to differentiate between delirium and other disorders . for the purpose of the present analyses , a maximum of five missing items was allowed a priori if only the first 13 items were scored ( 5/13 = 39% ) and a weighted sum score was computed in the case of five or fewer missing items . if all 16 items were scored , a maximum of 6 missing items was allowed ( 6/16 = 38% ) and a weighted sum score was computed in cases of 6 or fewer missing items . the picu pediatric neuropsychiatrist ( js ) routinely examined all children using the criteria of the revised fourth edition of the dsm - iv . he reviewed and discussed all available information and opinions ( from parents , nurses , and the medical charts ) with the other members of the picu team before making a final diagnosis [ 7 , 12 ] . he also made further categorizations into the hyperactive , hypoactive , and mixed states of pd . patients at our picu are routinely assessed for the presence of delirium under circumstances of quality improvement in clinical care since 2005 , using a schedule of twice - daily examinations . the paed , drs-88 , and drs - r-98 are completed during the first assessment , which is carried out by the intern over the course of their attachment to the unit , having received on - site clinical training by the pediatric neuropsychiatrist of the unit . the second assessment was carried out within 3 h by the pediatric neuropsychiatrist , who was unaware of the screening results . once pd was diagnosed , appropriate management was initiated in collaboration with the picu team . the paed was developed by sikich and lerman in order to trace emergence delirium ( ed ) . ed is defined as a disorder occurring during awakening from anesthesia in the immediate postoperative period . it is characterized by confusion , hallucinations , delusions and can be accompanied by moaning , restlessness , and agitation . the scale consists of five items which are rated on a 5-point scale ranging from extremely to not at all. a score of 10 or higher indicates diagnosis of delirium . for the purpose of the present analyses , a maximum of two missing items ( 2/5 = 40% ) was allowed a priori ; a weighted sum score was computed in the case of one or two missing items . it consists of ten items encompassing a range of areas which can be affected by delirium such as hallucinations , psychomotor behavior , cognitive status , sleep - wake cycle disturbance , and lability of mood . a score of 10 or higher indicates a diagnosis of delirium [ 18 , 19 ] . for the purpose of the present analyses , a maximum of four missing items ( 4/10 = 40% ) was allowed a priori ; a weighted sum score was computed in the case of four or fewer missing items . the drs - r-98 is a revised form of the original drs and consists of 16 items , of which the last three are optional and scored only when there is suspicion of delirium ( score of 15.25 or higher on the first 13 items ) ; the three optional items serve to differentiate between delirium and other disorders . for the purpose of the present analyses , a maximum of five missing items was allowed a priori if only the first 13 items were scored ( 5/13 = 39% ) and a weighted sum score was computed in the case of five or fewer missing items . if all 16 items were scored , a maximum of 6 missing items was allowed ( 6/16 = 38% ) and a weighted sum score was computed in cases of 6 or fewer missing items . the picu pediatric neuropsychiatrist ( js ) routinely examined all children using the criteria of the revised fourth edition of the dsm - iv . he reviewed and discussed all available information and opinions ( from parents , nurses , and the medical charts ) with the other members of the picu team before making a final diagnosis [ 7 , 12 ] . he also made further categorizations into the hyperactive , hypoactive , and mixed states of pd . patients at our picu are routinely assessed for the presence of delirium under circumstances of quality improvement in clinical care since 2005 , using a schedule of twice - daily examinations . the paed , drs-88 , and drs - r-98 are completed during the first assessment , which is carried out by the intern over the course of their attachment to the unit , having received on - site clinical training by the pediatric neuropsychiatrist of the unit . the second assessment was carried out within 3 h by the pediatric neuropsychiatrist , who was unaware of the screening results . once pd was diagnosed , appropriate management was initiated in collaboration with the picu team . data were analyzed using stata , version 11.0 . for each patient , a single assessment day during hospitalization was selected . for patients diagnosed with pd , paed , drs-88 , and drs - r-98 assessments on the first day of the pd gold standard diagnosis were selected ; for patients without pd , paed , drs-88 , and drs - r-98 assessments were randomly selected in such a way that groups were frequency - matched on number of admission days until assessment . exclusion criteria for analysis were unconsciousness or deep sedation according to the clinical judgment of the responsible intensivist treating the patient . a patient was regarded non - rateable on a scale if more than the maximum allowed number of items was missing , precluding diagnosis . non - rateability of items was examined as a function of age using one - way analysis of variance ( anova , one - tailed ) . three psychometric properties of the paed , drs-88 , and drs - r-98 ( with exclusion of the three diagnostic items of the last scale , because scoring of these items is optional ) were evaluated : ( 1 ) internal consistency , expressed as cronbach s alpha ; ( 2 ) number and proportion of items not rateable ; ( 3 ) discriminative ability , expressed as the area under the receiver operating characteristic ( roc ) curve , was calculated per instrument item and for instrument sum scores , with 95% confidence intervals ( 95% ci ) . roc curves allow for exploration of the relationship between the sensitivity and specificity of a continuous predictor ( in this case the scores for each item on a scale ) and a dichotomous outcome ( in this case pd ) . instruments with the largest area under the curve ( auc ) are most accurate in distinguishing between patients with and without delirium . given the fact that pd is a serious condition requiring immediate intervention , sensitivity was given more weight than specificity in determining the optimal cutoff scores for pd . sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) for the diagnosis of delirium were calculated , as well as the likelihood ratio for a positive test result ( the ratio between true and false positive rate ) . from november 2006 to february 2010 , 182 acute , non - electively admitted patients were included . twenty - eight patients without a diagnosis of delirium were excluded because of deep sedation or coma on the assessment day selected for the analysis . of the remaining 154 patients , 26 ( 16.9% ) mean age in pd patients was 7.2 years [ sd ( standard deviation ) = 5.4 ] compared to 6.7 years ( sd = 5.0 ) in non - pd patients . two routinely used illness severity indicators are the pediatric index of mortality ( pim ) and the pediatric risk of mortality ( prism ii ) . mean pim score in the current sample was 8.8 ( sd = 16.7 ) in patients with and 3.6 ( sd = 5.6 ) in patients without delirium . mean prism ii score was 8.8 ( sd = 19.2 ) in patients with and 3.6 ( sd = 5.8 ) in patients without delirium . mean length of stay in the pd group was 7.4 days ( sd = 4.9 ) compared to 3.9 days ( sd = 3.3 ) in the non - pd group . in both groups , the most common primary picu indication was a respiratory disorder , followed by neurological disorders ( table 1).table 1population characteristics ( n = 154)characteristicspatients with delirium ( n = 26)patients without delirium ( n = 128)n%n%male1453.88163.3female1246.24736.7mechanical ventilation1453.81511.7sedation1557.71310.2primary picu indication respiratory disorder830.84232.8 neurological disorder726.92922.7 circulatory disorder623.12015.6 metabolic disorder0075.5 surgical311.5107.8 multiple13.8118.6 others13.897.0retrospective data311.53124.2picu pediatric intensive care unitpost - surgical interventionsmultiple medical indications for picu admission population characteristics ( n = 154 ) picu pediatric intensive care unit post - surgical interventions multiple medical indications for picu admission of 154 patients , the paed could be rated , applying the a priori criteria for rateability specified above , in 144 ( 93.5% ) , the drs-88 in 103 ( 66.9% ) , and the drs - r-98 in 73 ( 46.8% ) . for the drs-88 , the mean age of rateable ( 8.0 years ) and non - rateable patients ( 4.3 years ) differed significantly from each other ( f ( 1,152 ) = 20.39 ; sd = 5.05 ; p < 0.001 ) . this also held for the drs - r-98 ( rateable patients 9.6 years , non - rateable patients 4.3 years , f ( 1,152 ) = 57.68 ; sd = 5.05 ; p < 0.001 ) . for the paed , the difference between rateable and non - rateable groups was in the same direction albeit smaller and not significant ( 6.9 and 4.9 years respectively ; f ( 1,152 ) = 1.45 ; sd = 5.05 ; p > 0.05 ) . owing to non - rateability , the paed missed 3 , the drs-88 12 , and the drs - r-98 22 of the 26 diagnoses of delirium made by the gold standard . of the 144 cases in which the paed could be assessed , 23 were diagnosed with delirium by the gold standard , and 21 of these were correctly identified by the paed ( table 2 ) . the lr for a positive diagnosis was 55.2 and the auc was 0.99 ( fig . 1 ) . the optimum cutoff score was 8 ( sensitivity = 100% ; specificity = 92.6% ) . secondly , out of 103 cases in which the drs-88 could be assessed , 12 were diagnosed with delirium by the gold standard . the ppv and npv were 98.9 and 100% , respectively ( table 2 ) . finally , out of 73 cases in which the drs - r-98 could be assessed , 4 were diagnosed with delirium by the gold standard . the ppv and npv were 100 and 98.6% , respectively ( table 2).table 2patients diagnosed with delirium patients ( n)fn ( n)sensitivity ( % ) fp ( n)specificity ( % ) pediatric neuropsychiatrist diagnosis26100100 hyperactive delirium18 hypoactive delirium4 mixed delirium4paed outcome , delirium21291.3298.3 hyperactive delirium1511 hypoactive delirium31 mixed delirium31drs-88 outcome , delirium11191.70100 hyperactive delirium51 hypoactive delirium4 mixed delirium2drs - r-98 outcome , delirium3175.00100 hyperactive delirium31 hypoactive delirium0 mixed delirium0fn false negatives , fp false positivesfig . the 15th dot from the right is at the paed value of 8 and higher patients diagnosed with delirium fn false negatives , fp false positives roc curve for paed . the 15th dot from the right is at the paed value of 8 and higher cronbach s alpha was 0.89 for the paed , 0.69 for the drs-88 , and 0.57 for the drs - r-98 . all items of the paed had a low proportion of missing values and high discriminative ability , as evidenced by a high auc ( table 3 ) . the cognitive items of both the drs-88 and drs - r-98 had the highest proportion of missing values . additional data regarding the item characteristics of drs-88 and drs - r-98 are shown in tables 4 and 5 . however , in cases in which these items could be rated , they demonstrated high discriminative ability , with the exception of two items : delusions ( both scales ) and long - term memory ( drs - r-98).table 3item characteristics for paed ( n = 154)paedmissing valuesaucn%95% cieye contact159.70.930.890.98goal directedness of movements2516.20.930.861.00awareness of surroundings1912.30.900.830.98restlessness10.60.970.950.99inconsolability74.50.900.840.96table 4item characteristics for drs-88 ( n = 154)drs-88missingaucn%95% cicognition perceptual disturbances7246.80.850.671.00 hallucination type7045.50.860.681.00 delusions8051.90.630.380.87 cognitive status during formal testing7246.80.930.801.00affect lability of mood5435.10.760.580.94dsm - iv criteria temporal onset of symptoms128.40.930.870.99 variability of symptoms1811.70.930.861.00 physical disorder00.0sleep - wake cycle sleep - wake cycle disturbance2918.80.830.710.96behavior psychomotor behavior95.80.940.900.97physical disorder was always scored as therefore , the auc and ci of this item could not be estimatedtable 5item characteristics for drs - r-98 ( n = 154)drs - r-98missingaucn%95% cicognition perceptual disturbances and hallucinations7045.50.820.621.00 delusions8051.90.630.380.87 orientation6844.20.980.971.00 attention5636.40.930.811.00 visuospatial ability11272.7 language7649.40.810.601.00 thought process abnormalities8353.91.01.001.00 short - term memory8857.11.0 long - term memory10064.90.49affect lability of affect5435.10.760.580.95sleep - wake cycle sleep - wake cycle disturbance2918.80.830.710.96behavior motor agitation53.20.930.880.98 motor retardation1912.30.490.420.57visuospatial ability was either scored as being normal or unrateable ( due to sedation , severity of illness , and young age ) . therefore , the auc and ci of this item could not be estimatedshort - term and long - term memory were scored as being normal or unrateable ( due to sedation , severity of illness , and young age ) and rarely as affected . therefore , their standard error ( se ) and thus their ci could not be estimated item characteristics for paed ( n = 154 ) item characteristics for drs-88 ( n = 154 ) physical disorder was always scored as severe because all patients at the picu are critically ill . therefore , the auc and ci of this item could not be estimated item characteristics for drs - r-98 ( n = 154 ) visuospatial ability was either scored as being normal or unrateable ( due to sedation , severity of illness , and young age ) . therefore , the auc and ci of this item could not be estimated short - term and long - term memory were scored as being normal or unrateable ( due to sedation , severity of illness , and young age ) and rarely as affected . therefore , their standard error ( se ) and thus their ci could not be estimated from november 2006 to february 2010 , 182 acute , non - electively admitted patients were included . twenty - eight patients without a diagnosis of delirium were excluded because of deep sedation or coma on the assessment day selected for the analysis . of the remaining 154 patients , 26 ( 16.9% ) mean age in pd patients was 7.2 years [ sd ( standard deviation ) = 5.4 ] compared to 6.7 years ( sd = 5.0 ) in non - pd patients . two routinely used illness severity indicators are the pediatric index of mortality ( pim ) and the pediatric risk of mortality ( prism ii ) . mean pim score in the current sample was 8.8 ( sd = 16.7 ) in patients with and 3.6 ( sd = 5.6 ) in patients without delirium . mean prism ii score was 8.8 ( sd = 19.2 ) in patients with and 3.6 ( sd = 5.8 ) in patients without delirium . mean length of stay in the pd group was 7.4 days ( sd = 4.9 ) compared to 3.9 days ( sd = 3.3 ) in the non - pd group . in both groups , the most common primary picu indication was a respiratory disorder , followed by neurological disorders ( table 1).table 1population characteristics ( n = 154)characteristicspatients with delirium ( n = 26)patients without delirium ( n = 128)n%n%male1453.88163.3female1246.24736.7mechanical ventilation1453.81511.7sedation1557.71310.2primary picu indication respiratory disorder830.84232.8 neurological disorder726.92922.7 circulatory disorder623.12015.6 metabolic disorder0075.5 surgical311.5107.8 multiple13.8118.6 others13.897.0retrospective data311.53124.2picu pediatric intensive care unitpost - surgical interventionsmultiple medical indications for picu admission population characteristics ( n = 154 ) picu pediatric intensive care unit post - surgical interventions multiple medical indications for picu admission of 154 patients , the paed could be rated , applying the a priori criteria for rateability specified above , in 144 ( 93.5% ) , the drs-88 in 103 ( 66.9% ) , and the drs - r-98 in 73 ( 46.8% ) . for the drs-88 , the mean age of rateable ( 8.0 years ) and non - rateable patients ( 4.3 years ) differed significantly from each other ( f ( 1,152 ) = 20.39 ; sd = 5.05 ; p < 0.001 ) . this also held for the drs - r-98 ( rateable patients 9.6 years , non - rateable patients 4.3 years , f ( 1,152 ) = 57.68 ; sd = 5.05 ; p < 0.001 ) . for the paed , the difference between rateable and non - rateable groups was in the same direction albeit smaller and not significant ( 6.9 and 4.9 years respectively ; f ( 1,152 ) = 1.45 ; sd = 5.05 ; p > 0.05 ) . owing to non - rateability , the paed missed 3 , the drs-88 12 , and the drs - r-98 22 of the 26 diagnoses of delirium made by the gold standard . of the 144 cases in which the paed could be assessed , 23 were diagnosed with delirium by the gold standard , and 21 of these were correctly identified by the paed ( table 2 ) . the lr for a positive diagnosis was 55.2 and the auc was 0.99 ( fig . 1 ) . the optimum cutoff score was 8 ( sensitivity = 100% ; specificity = 92.6% ) . secondly , out of 103 cases in which the drs-88 could be assessed , 12 were diagnosed with delirium by the gold standard . the ppv and npv were 98.9 and 100% , respectively ( table 2 ) . finally , out of 73 cases in which the drs - r-98 could be assessed , 4 were diagnosed with delirium by the gold standard . the ppv and npv were 100 and 98.6% , respectively ( table 2).table 2patients diagnosed with delirium patients ( n)fn ( n)sensitivity ( % ) fp ( n)specificity ( % ) pediatric neuropsychiatrist diagnosis26100100 hyperactive delirium18 hypoactive delirium4 mixed delirium4paed outcome , delirium21291.3298.3 hyperactive delirium1511 hypoactive delirium31 mixed delirium31drs-88 outcome , delirium11191.70100 hyperactive delirium51 hypoactive delirium4 mixed delirium2drs - r-98 outcome , delirium3175.00100 hyperactive delirium31 hypoactive delirium0 mixed delirium0fn false negatives , fp false positivesfig . the 15th dot from the right is at the paed value of 8 and higher patients diagnosed with delirium fn false negatives , fp false positives roc curve for paed . cronbach s alpha was 0.89 for the paed , 0.69 for the drs-88 , and 0.57 for the drs - r-98 . all items of the paed had a low proportion of missing values and high discriminative ability , as evidenced by a high auc ( table 3 ) . the cognitive items of both the drs-88 and drs - r-98 had the highest proportion of missing values . additional data regarding the item characteristics of drs-88 and drs - r-98 are shown in tables 4 and 5 . however , in cases in which these items could be rated , they demonstrated high discriminative ability , with the exception of two items : delusions ( both scales ) and long - term memory ( drs - r-98).table 3item characteristics for paed ( n = 154)paedmissing valuesaucn%95% cieye contact159.70.930.890.98goal directedness of movements2516.20.930.861.00awareness of surroundings1912.30.900.830.98restlessness10.60.970.950.99inconsolability74.50.900.840.96table 4item characteristics for drs-88 ( n = 154)drs-88missingaucn%95% cicognition perceptual disturbances7246.80.850.671.00 hallucination type7045.50.860.681.00 delusions8051.90.630.380.87 cognitive status during formal testing7246.80.930.801.00affect lability of mood5435.10.760.580.94dsm - iv criteria temporal onset of symptoms128.40.930.870.99 variability of symptoms1811.70.930.861.00 physical disorder00.0sleep - wake cycle sleep - wake cycle disturbance2918.80.830.710.96behavior psychomotor behavior95.80.940.900.97physical disorder was always scored as severe because all patients at the picu are critically ill . therefore , the auc and ci of this item could not be estimatedtable 5item characteristics for drs - r-98 ( n = 154)drs - r-98missingaucn%95% cicognition perceptual disturbances and hallucinations7045.50.820.621.00 delusions8051.90.630.380.87 orientation6844.20.980.971.00 attention5636.40.930.811.00 visuospatial ability11272.7 language7649.40.810.601.00 thought process abnormalities8353.91.01.001.00 short - term memory8857.11.0 long - term memory10064.90.49affect lability of affect5435.10.760.580.95sleep - wake cycle sleep - wake cycle disturbance2918.80.830.710.96behavior motor agitation53.20.930.880.98 motor retardation1912.30.490.420.57visuospatial ability was either scored as being normal or unrateable ( due to sedation , severity of illness , and young age ) . therefore , the auc and ci of this item could not be estimatedshort - term and long - term memory were scored as being normal or unrateable ( due to sedation , severity of illness , and young age ) and rarely as affected . therefore , their standard error ( se ) and thus their ci could not be estimated item characteristics for paed ( n = 154 ) item characteristics for drs-88 ( n = 154 ) physical disorder was always scored as severe because all patients at the picu are critically ill . therefore , the auc and ci of this item could not be estimated item characteristics for drs - r-98 ( n = 154 ) visuospatial ability was either scored as being normal or unrateable ( due to sedation , severity of illness , and young age ) . therefore , the auc and ci of this item could not be estimated short - term and long - term memory were scored as being normal or unrateable ( due to sedation , severity of illness , and young age ) and rarely as affected . therefore , their standard error ( se ) and thus their ci could not be estimated to our knowledge , this is the first prospective study evaluating the utility of the drs and the paed as assessment tools for pd in a picu setting . the paed was rateable most often ( 93.5% ) and proved to be excellent in discriminating between patients with and without pd ( auc = 0.99 ) . this is in line with the findings of two recent studies which both found an auc of 0.98 for the paed in detecting ed in a post - anesthesia setting [ 22 , 23 ] . it seems , therefore , that the paed can be used in both settings . both versions of the drs could not be rated as frequently as the paed , as these scales contain numerous items that are difficult to assess in young children , especially regarding cognition . as the largest part of the picu population consists of children under the age of 3 , this is a severe drawback . in addition , these tools require substantially more time to score . taken together , in this study , the drs scales were found to be far less suitable for routine pd assessment at a picu . however , the paed is not flawless : there were two false negatives ( 1 hypoactive and 1 hyperactive ) that were probably due to the fluctuating nature and/or the subtle presentation of delirium and two false positives ( 1 hyperactive and 1 mixed ) . in these cases , it subsequently appeared that the presentations ( emotional and/or behavioral disturbances ) were better explained by other somatic complications . taken together , the strengths of this study are ( 1 ) it was executed during daily regular clinical care , and ( 2 ) it also included the ( near ) majority of the picu children , namely those in the age range greater than 1 and less than 5 years . this broad inclusion could explain the incidence of pd ( 16.9% ) in our study compared to the 13% found by smith and colleagues who only included children above the age of 5 years . however , because the pcam - icu is relatively new we did not have sufficient data points to evaluate its use . firstly , data for 40 of 182 patients ( 22% ) were assessed in retrospect , because patients were not observed during weekends or holidays . in these cases , we had to rely on collateral information from parents , nurses , intensivists , and child neurologists to reconstruct ratings . however , there were no significant differences between the results with or without retrospective data ( data not shown ) . second , given the fluctuating nature of delirium , it could be that some cases were missed . thirdly , only one observer rated the paed each day . because the paed contains an element of subjectivity , depending on what a given observer defines as a little bit or extreme in rating a given symptom , inflated random error may have been the result . unfortunately , reliability analysis was not possible given that the picu staff over the study period was not yet participating as rater of the different instruments this is the topic of a current study . fourth , this is one of the first psychometric studies conducted in a picu setting where a higher proportion of missing data can be expected because assessments are complicated by factors such as severity of critical illness , young age , mechanical ventilation , sedation , and limited amount of time available for assessments . because listwise deletion would lead to too much data loss we used a conservative strategy and deleted subjects for which less than 60% of the items were scored for a specific scale . fifth , the findings suggest that the incidence of delirium is much lower than in adults , especially with respect to the hypoactive subtype . the difficulties with the assessment of delirium in young and/or non - verbal patients could be an explanation for the relatively low rate of pd ; in addition , the exclusive reliance of the paed on subtle behavioral alterations may explain the relatively low rate of hypoactive delirium . finally , replications of the current findings are necessary as it remains to be seen whether results can be generalized to other picu settings . a sufficient level of involvement of child psychiatrists and their trainees is therefore recommended . given ( 1 ) the potentially dangerous nature of delirium , ( 2 ) the high workload at the picu , ( 3 ) a shortage of pediatric neuropsychiatrists , and ( 4 ) the fact that 75% of the children at the picu are below the age of 3 years , an instrument is needed that is easy to use at the bedside , not time - consuming , and suitable for young children as well . it is therefore concluded that the paed should be used for pd screening at the picu , preferably multiple times per day . whenever a score of 8 or higher ( or other scores , depending on the patient mix of the picu in question ) is obtained for a patient , a pediatric neuropsychiatrist should be consulted to assess whether the child is delirious or not . in this way , the likelihood that pd will be missed may be reduced . future studies could be directed towards the development of an improved screening instrument for pd , based on the paed .

purposedelirium is a poor - prognosis neuropsychiatric disorder . pediatric delirium ( pd ) remains understudied , particularly at pediatric intensive care units ( picu ) . although the pediatric anesthesia emergence delirium ( paed ) scale , the delirium rating scale ( drs-88 ) , and the delirium rating scale - revised ( drs - r-98 ) are available , none have been validated for use in picu settings . the aim of the present study was to investigate the use of the drs / paed instruments as diagnostic tools for pd in the picu.methodsa prospective panel study was conducted , under circumstances of routine clinical care , investigating the diagnostic properties of the paed , drs-88 , and drs - r-98 in picu patients at a tertiary university medical center . a total of 182 non - electively admitted , critically ill pediatric patients , aged 117 years , were included between november 2006 and february 2010 . sensitivity , specificity , and receiver operating characteristic ( roc ) curves were calculated . three psychometric properties were analyzed : ( 1 ) internal consistency ( 2 ) proportion of items not rateable , and ( 3 ) discriminative ability.resultsthe paed could be completed in 144 ( 93.5% ) patients , much more frequently than either the drs-88 ( 66.9% ) or the drs - r-98 ( 46.8% ) . compared with the clinical gold standard diagnosis of delirium , the paed had a sensitivity of 91% and a specificity of 98% ( auc 0.99 ) . the optimal paed cutoff score as a screening instrument in this picu setting was 8 . cronbach s alpha was 0.89 ; discriminative ability was high.conclusionsthe paed is a valid instrument for pd in critically ill children , given its reliance on routinely rateable observational signs and symptoms .

Introduction Method Setting and patients Measurement procedures The Pediatric Anesthesia Emergence Delirium (PAED) scale The Delirium Rating Scale The Delirium Rating Scale-Revised (DRS-R-98) Clinical/expert judgment of the gold standard Procedure Statistical analysis Results Population characteristics Sum score analysis Per-item analysis Discussion Conclusion

rickets is still a common health problem and is considered to be a challenge even in developed countries . epidemiological data are not available in many countries , etiology is sometimes difficult to establish and vitamin d supplementation and rickets treatment have been debated for a long time . a child aged 10 months old was admitted in pediatric emergency department with focal seizures with secondary generalization . the child has , delay in motor developmental mile stones and hypotonia and poor gain in height . biochemical and radiologic signs were suggestive for rickets . biochemical profile showing low serum calcium-6.1 mg / dl ( 9.2 - 11 mg / dl ) , low phosphorus-3.9 mg / dl ( 3.4 - 6.2 mg / dl ) , elevated alkaline phosphatase-4116 u / l ( < 269 u / l ) parathyroid hormone was 233.6 pg / ml ( 15 - 65 pg / ml ) 25-oh vitamin d was 40 ng / dl . radiographic images of the wrist showed fraying and cupping of the distal radius and ulna , as well as bone demineralization ; the metaphyses are widened , irregular and cupped . physical examination , biochemical data ( hypocalcemia , mild hypo - phosphatemia , high serum concentration of alkaline phosphatase ) and rachitic changes on x - ray , were compatible with the diagnosis of rickets . the child was started on sodium valproate 20 mg / kg / day , calcium 500 mg / day and vitamin d 6 lakh iu following which there was no improvement in the serum calcium level and there was recurrence of seizures . then endocrinologist opinion sought following which the child was initiated on 0.5 g / day of active vitamin d ( cacidiol ) along with 1000 mg of calcium . there was no seizure recurrence in the last 15 months and serum calcium normalized . in view of response to 1 calciferol 1,25 oh vitamin d and enzyme studies were not done in view of financial constraints and lack of availability . a total of 10 different allelic variants of vddr have been identified , exhibiting little or no clinical differences . prominent clinical manifestations of vddr - i include growth failure , short stature , skeletal abnormalities , genu valgum , rachitic rosary , open fontanels , pathologic fractures , muscle weakness and convulsions . radiologic findings usually include fractures , generalized osteopenia , growth failure and arched or curved legs . the most common laboratory findings include hypocalcemia , hypophosphatemia , elevated parathormone levels and high alkaline phosphatase . moreover , the serum levels of 25(oh ) vitamin d are normal or raised while the levels of 1,25(oh ) vitamin d are low or undetectable . vitamin d is intimately involved in a wide variety of biologic processes , including calcium homeostasis , bone formation and cellular differentiation . in order to exert its effects , vitamin d must be activated by a metabolic pathway that requires the enzymatic activity of 25-hydroxylase and 1--hydroxylase . the gene for vddri has been recently mapped to the long arm of chromosome 12 , region one bands 2 - 4 ( 12q12-q14 ) , representing the location of the 1--hydroxylase gene . hence , it has been suggested that the disease should be referred to as 1--hydroxylase deficiency . stong clinical suspicion is required for the clinical diagnosis of vddr and therapeutic challenge with 1 calciferol will prove the diagnosis .

there are two types of vitamin d dependent rickets ( vddr ) that cause rickets in children . vddr type 1 ( vddr - i ) is caused by an inborn error of vitamin d metabolism , which interferes with renal conversion of calcidiol ( 25ohd ) to calcitriol ( 1,25(oh)2d ) by the enzyme 1--hydroxylase . patients with vddr - i have mutations of chromosome 12 that affect the gene for the enzyme 1--hydroxylase , resulting in decreased levels of 1,25(oh ) vitamin d. clinical features include growth failure , hypotonia , weakness , rachitic rosary , convulsions , tetany , open fontanels and pathologic fractures . we report a case of vddr - i in 14-month - old male child . establishing an early diagnosis of these genetic forms of rickets is challenging , especially in developing countries where nutritional rickets is the most common variety of the disease where genetic diagnosis is not always possible because of financial constraints . a prompt diagnosis is necessary to initiate adequate treatment , resolve biochemical features and prevent complications , such as severe deformities that may require surgical intervention .

I C D C

behet 's disease ( bd ) is a chronic , multisystemic , relapsing inflammatory disorder , characterized by recurrent oral ulcers , genital ulcers , uveitis , and skin lesions . it also attacks other systems involving the musculoskeletal system , blood vessels , nervous system , and gastrointestinal tract . bd is predominantly found in eastern mediterranean countries and the eastern rim of asia , including china . when bd patients have predominantly gastrointestinal symptoms and show gastrointestinal lesions with certain objective measures , the disease is defined as intestinal bd . the prevalence of intestinal bd varies from 0% to 60% of all bd patients with geographic and ethnic differences . the ileocecal region is the most commonly involved region , and abdominal pain , diarrhea , and gastrointestinal bleeding are common symptoms . crohn 's disease ( cd ) is a relapsing and transmural inflammatory disease which can affect the entire gastrointestinal tract . typical presentations include the presence of longitudinal ulcers with a cobblestone appearance , skip lesions , and the development of complications such as strictures and fistulas . cd also has extraintestinal manifestations , such as oral ulcers , arthralgia , erythema nodosum , and autoimmune hepatic diseases . both diseases commonly have a young age of onset , nonspecific gastrointestinal symptoms , similar extraintestinal manifestations , and chronic , waxing , and waning course . there are only a few comparative studies regarding the differential diagnosis , which were conducted in korean patients , and no comparative study has been done in chinese patients . we carried out this retrospective study to elucidate the clinical and colonoscopic features in chinese patients with intestinal bd or cd , and to identify valuable strategies for the differential diagnosis . from january 1983 to january 2010 , 50 intestinal bd patients and 219 cd patients were hospitalized in peking union medical college hospital , a tertiary academic hospital , in beijing , china . among them , patients ( 35 intestinal bd patients and 106 cd patients ) who had ulcerative lesions in the terminal ileum or colon under colonoscopy and had no history of gastrointestinal operation except appendectomy were enrolled in this study . patients diagnosed with ulcerative colitis , celiac disease , intestinal tuberculosis , malignancy , or other recognized causes of intestinal inflammation were excluded . behet 's disease was diagnosed based on the criteria from the bd research committee of japan in 1987 . briefly , the criteria include four major features ( recurrent oral aphthous ulcers , eye lesions , genital lesions , and skin lesions including erythema nodosum , subcutaneous thrombophlebitis , acne - like lesions , and cutaneous hypersensitivity ) and five minor features ( arthritis , gastrointestinal lesions characterized by ileocecal ulcers , epididymitis , vascular lesions , and lesions of the central nervous system ) . diagnosis of complete type bd requires all four major features . the incomplete type needs three major features , or two major plus two minor features , or typical ocular symptoms plus one major or two minor features . the suspected type needs two major features , or one major plus two minor features . when patients with bd had predominantly gastrointestinal symptoms with endoscopic , histologic , and/or radiographic evidence of gastrointestinal lesions , they were diagnosed as intestinal bd . in this study , 3 ( 8.6% ) , 26 ( 74.3% ) , and 6 ( 17.1% ) patients were diagnosed as complete , incomplete , and suspected bd , respectively . morphological features include : ( 1 ) discontinuous and asymmetrical mucosal involvement ; ( 2 ) deep mucosal longitudinal ulcers ; ( 3 ) transmural inflammation ; ( 4 ) a rigid intestinal wall ; and ( 5 ) presence of an enterocutaneous or entero - enteric fistula and/or chronic perianal disease . pathological features include : ( 1 ) normal mucus content in the goblet cells of the inflamed region ; ( 2 ) lymphocyte aggregation in the mucosa and submucosa ; ( 3 ) noncaseating granulomas ; ( 4 ) longitudinal ulcers ; and ( 5 ) transmural inflammation . for definite diagnosis of cd , the following criteria were used : the presence of at least three different histologic features ; or the presence of noncaseating granulomas on histology with at least one other feature ; or the resolution of symptoms and morphologic features after 312 months treatment . data were collected including demographics ( gender , age and disease duration ) , clinical manifestations ( disease distribution , gastrointestinal symptoms , extraintestinal manifestations , and gastrointestinal complications ) , and colonoscopic findings . the disease distribution was determined by endoscopic and radiologic examinations . gastrointestinal complications included perforation , fistulae , intestinal obstruction and massive gastrointestinal hemorrhage which were associated with hemodynamic instability , acute anemia , and/or the need for blood transfusion . colonoscopic features included the ulcer distribution ( focal involvement , segmental involvement ) , the number of ulcers in a local segment ( 1 , or 2 ) , ulcer type ( longitudinal ulcer , circumferential ulcer , aphthous ulcer or irregular ulcer ) , cobblestone appearance , pseudo - polyps , and stricture . focal involvement was defined as one local area with ulcerative lesions or two adjacent areas involved by continuous ulcerative lesions . a circumferential ulcer was defined as a round or transverse , deep and well - demarcated ulcer . an aphthous ulcer was defined as a small ( < 1 cm ) , punched out , raised or a flat lesion with a white center [ figure 1a c ] . the cobblestone appearance was defined as a mucosal pattern with raised nodules , resembling the paving of the roman road . pseudo - polyps are defined as polypoid lesions which are usually small and isolated or multiple and scattered , though they can sometimes be giant in size . all images of colonoscopic examinations were reviewed by an expert endoscopist who was blinded from other information . histologic features of biopsy specimens , which were paraffin - embedded and stained with hematoxylin and eosin , were evaluated for the presence of epithelioid granulomas and the appearance of vasculitis . ( a ) a longitudinal ulcer in a patient with crohn 's disease ( cd ) . ( b ) a circumferential ulcer in a patient with intestinal behet 's disease . ( c ) an aphthous ulcer in a patient with cd . differences in quantitative data between the two groups were examined statistically through univariate analysis using an independent t - test and chi - squared test for continuous and categorical variables , respectively . p values were two - tailed , and the significance level was set at p < 0.05 . for multivariate logistic regression analysis , predictors with a p < 0.3 were entered to select independent discriminating predictors . receiver operating characteristic ( roc ) curves were constructed to evaluate the discriminating ability of predictors . statistical analyses were performed using statistical package for the social sciences ( spss ) 19.0 statistical software ( spss inc . from january 1983 to january 2010 , 50 intestinal bd patients and 219 cd patients were hospitalized in peking union medical college hospital , a tertiary academic hospital , in beijing , china . among them , patients ( 35 intestinal bd patients and 106 cd patients ) who had ulcerative lesions in the terminal ileum or colon under colonoscopy and had no history of gastrointestinal operation except appendectomy were enrolled in this study . patients diagnosed with ulcerative colitis , celiac disease , intestinal tuberculosis , malignancy , or other recognized causes of intestinal inflammation were excluded . behet 's disease was diagnosed based on the criteria from the bd research committee of japan in 1987 . briefly , the criteria include four major features ( recurrent oral aphthous ulcers , eye lesions , genital lesions , and skin lesions including erythema nodosum , subcutaneous thrombophlebitis , acne - like lesions , and cutaneous hypersensitivity ) and five minor features ( arthritis , gastrointestinal lesions characterized by ileocecal ulcers , epididymitis , vascular lesions , and lesions of the central nervous system ) . the incomplete type needs three major features , or two major plus two minor features , or typical ocular symptoms plus one major or two minor features . the suspected type needs two major features , or one major plus two minor features . when patients with bd had predominantly gastrointestinal symptoms with endoscopic , histologic , and/or radiographic evidence of gastrointestinal lesions , they were diagnosed as intestinal bd . in this study , 3 ( 8.6% ) , 26 ( 74.3% ) , and 6 ( 17.1% ) patients were diagnosed as complete , incomplete , and suspected bd , respectively . morphological features include : ( 1 ) discontinuous and asymmetrical mucosal involvement ; ( 2 ) deep mucosal longitudinal ulcers ; ( 3 ) transmural inflammation ; ( 4 ) a rigid intestinal wall ; and ( 5 ) presence of an enterocutaneous or entero - enteric fistula and/or chronic perianal disease . pathological features include : ( 1 ) normal mucus content in the goblet cells of the inflamed region ; ( 2 ) lymphocyte aggregation in the mucosa and submucosa ; ( 3 ) noncaseating granulomas ; ( 4 ) longitudinal ulcers ; and ( 5 ) transmural inflammation . for definite diagnosis of cd , the following criteria were used : the presence of at least three different histologic features ; or the presence of noncaseating granulomas on histology with at least one other feature ; or the resolution of symptoms and morphologic features after 312 months treatment . data were collected including demographics ( gender , age and disease duration ) , clinical manifestations ( disease distribution , gastrointestinal symptoms , extraintestinal manifestations , and gastrointestinal complications ) , and colonoscopic findings . the disease distribution was determined by endoscopic and radiologic examinations . gastrointestinal complications included perforation , fistulae , intestinal obstruction and massive gastrointestinal hemorrhage which were associated with hemodynamic instability , acute anemia , and/or the need for blood transfusion . colonoscopic features included the ulcer distribution ( focal involvement , segmental involvement ) , the number of ulcers in a local segment ( 1 , or 2 ) , ulcer type ( longitudinal ulcer , circumferential ulcer , aphthous ulcer or irregular ulcer ) , cobblestone appearance , pseudo - polyps , and stricture . focal involvement was defined as one local area with ulcerative lesions or two adjacent areas involved by continuous ulcerative lesions . a circumferential ulcer was defined as a round or transverse , deep and well - demarcated ulcer . an aphthous ulcer was defined as a small ( < 1 cm ) , punched out , raised or a flat lesion with a white center [ figure 1a c ] . the cobblestone appearance was defined as a mucosal pattern with raised nodules , resembling the paving of the roman road . pseudo - polyps are defined as polypoid lesions which are usually small and isolated or multiple and scattered , though they can sometimes be giant in size . all images of colonoscopic examinations were reviewed by an expert endoscopist who was blinded from other information . histologic features of biopsy specimens , which were paraffin - embedded and stained with hematoxylin and eosin , were evaluated for the presence of epithelioid granulomas and the appearance of vasculitis . ( a ) a longitudinal ulcer in a patient with crohn 's disease ( cd ) . ( b ) a circumferential ulcer in a patient with intestinal behet 's disease . ( c ) differences in quantitative data between the two groups were examined statistically through univariate analysis using an independent t - test and chi - squared test for continuous and categorical variables , respectively . p values were two - tailed , and the significance level was set at p < 0.05 . for multivariate logistic regression analysis , predictors with a p < 0.3 were entered to select independent discriminating predictors . receiver operating characteristic ( roc ) curves were constructed to evaluate the discriminating ability of predictors . statistical analyses were performed using statistical package for the social sciences ( spss ) 19.0 statistical software ( spss inc . , chicago , il , usa ) . table 1 shows the differences in demographics and clinical features between intestinal bd patients and cd patients . there were a higher prevalence of females and a slightly earlier onset in intestinal bd patients compared to cd patients ( p = 0.010 and 0.136 , respectively ) . there was no significant difference in the duration from the onset of gastrointestinal symptoms to diagnosis between two groups ( p = 0.738 ) . comparison of the demographic and clinical manifestations between intestinal bd and cd bd : behet s disease ; cd : crohn s disease ; sd : standard deviation . although the involvement of both the ileum and colon was most common in both intestinal bd patients ( 91.4% ) and cd patients ( 82.1% ) , there was still a significant difference in the disease distribution between two groups ( p = 0.005 ) . there was an increased tendency for intestinal bd patients to have involvement of the upper gastrointestinal tract ( 17.1% ) compared to cd patients ( 5.7% ) ( p = 0.073 ) . the most frequent gastrointestinal symptom of both diseases was abdominal pain ( p = 0.517 ) . massive gastrointestinal hemorrhage and fever were more frequent in intestinal bd patients ( p = 0.022 and 0.048 , respectively ) , while diarrhea , intestinal obstruction , and perianal lesions including perianal abscesses , fistula , and/or anal fissure were more common in cd patients ( p = 0.002 , 0.010 , and 0.027 , respectively ) . intestinal bd , as a multisystemic disease , involved more extraintestinal symptoms compared to cd ( p = 0.001 ) . for example , genital ulcers and skin lesions were very common in intestinal bd patients , while they were rare in cd patients ( p < 0.001 ) . furthermore based on multivariate logistic regression analysis of demographics and clinical features , diarrhea and extraintestinal manifestations were found to be two independent discriminating predictors ( p = 0.048 and 0.008 , respectively ) . based on colonoscopy , there were significant differences in the ulcer distribution ( p < 0.001 ) , ulcer size ( p = 0.017 ) , ulcer type ( p < 0.001 ) , and ulcer number ( p = 0.014 ) in a local area between the two groups [ table 2 ] . focal involvement , ileocecal valve deformity , solitary ulcer , large ulcers ( ulcer size > 2 cm ) , and circumferential ulcers were more common in intestinal bd patients ( p = 0.003 , 0.003 , 0.014 , 0.013 , and 0.003 , respectively ) , while segmental involvement , longitudinal ulcers , a cobblestone or nodular appearance , and pseudo - polyps were more common in cd patients ( p = 0.003 , 0.008 , 0.023 , and 0.002 , respectively ) . there was no significant difference in the frequency of aphthous ulcers between the two groups ( p = 0.679 ) . comparison of the colonoscopic features between intestinal intestinal bd and cd ( n ( % ) ) * ileocecal region represents terminal ileum and/or cecum . bd : behet s disease ; cd : crohn s disease . according to multivariate logistic regression analysis of colonoscopic features , the ulcer distribution , ulcer size , ulcer type and pseudo - polyps were independent discriminating predictors ( p = 0.006 , 0.021 , 0.002 , and 0.041 , respectively ) . the histology of mucosa biopsies in 11.4% cd patients revealed noncaseating epithelioid granulomas . neither the appearance of vasculitis nor granulomas was found in the mucosa biopsies of intestinal bd patients . no dysplasia or cancer was found in the mucosa biopsies of any patient . among the independent discriminating predictors , seven predictors were selected for evaluation of their discriminating ability by using the roc analysis [ table 3 ] . for the diagnosis of intestinal bd , genital ulcers and skin lesions had a higher area under the roc curve ( auc ) ( 0.900 and 0.853 , respectively ) with high specificity ( 94.3% and 93.4% , respectively ) . circumferential ulcers had a high specificity ( 92.5% ) , but very low sensitivity ( 28.9% ) . the classification algorithm composed of seven predictors had the highest auc of 0.987 , and high sensitivity ( 94.3% ) and specificity ( 97.2% ) . discriminating potential of independent factors for the diagnosis of intestinal bd * the sensitivity and specificity were obtained with the conventional cut - off value of 0.5 . auc : area under the receiver operator characteristic curve ; bd : behet s disease . table 1 shows the differences in demographics and clinical features between intestinal bd patients and cd patients . there were a higher prevalence of females and a slightly earlier onset in intestinal bd patients compared to cd patients ( p = 0.010 and 0.136 , respectively ) . there was no significant difference in the duration from the onset of gastrointestinal symptoms to diagnosis between two groups ( p = 0.738 ) . comparison of the demographic and clinical manifestations between intestinal bd and cd bd : behet s disease ; cd : crohn s disease ; sd : standard deviation . although the involvement of both the ileum and colon was most common in both intestinal bd patients ( 91.4% ) and cd patients ( 82.1% ) , there was still a significant difference in the disease distribution between two groups ( p = 0.005 ) . there was an increased tendency for intestinal bd patients to have involvement of the upper gastrointestinal tract ( 17.1% ) compared to cd patients ( 5.7% ) ( p = 0.073 ) . the most frequent gastrointestinal symptom of both diseases was abdominal pain ( p = 0.517 ) . massive gastrointestinal hemorrhage and fever were more frequent in intestinal bd patients ( p = 0.022 and 0.048 , respectively ) , while diarrhea , intestinal obstruction , and perianal lesions including perianal abscesses , fistula , and/or anal fissure were more common in cd patients ( p = 0.002 , 0.010 , and 0.027 , respectively ) . intestinal bd , as a multisystemic disease , involved more extraintestinal symptoms compared to cd ( p = 0.001 ) . for example , genital ulcers and skin lesions were very common in intestinal bd patients , while they were rare in cd patients ( p < 0.001 ) . furthermore based on multivariate logistic regression analysis of demographics and clinical features , diarrhea and extraintestinal manifestations were found to be two independent discriminating predictors ( p = 0.048 and 0.008 , respectively ) . based on colonoscopy , there were significant differences in the ulcer distribution ( p < 0.001 ) , ulcer size ( p = 0.017 ) , ulcer type ( p < 0.001 ) , and ulcer number ( p = 0.014 ) in a local area between the two groups [ table 2 ] . focal involvement , ileocecal valve deformity , solitary ulcer , large ulcers ( ulcer size > 2 cm ) , and circumferential ulcers were more common in intestinal bd patients ( p = 0.003 , 0.003 , 0.014 , 0.013 , and 0.003 , respectively ) , while segmental involvement , longitudinal ulcers , a cobblestone or nodular appearance , and pseudo - polyps were more common in cd patients ( p = 0.003 , 0.008 , 0.023 , and 0.002 , respectively ) . there was no significant difference in the frequency of aphthous ulcers between the two groups ( p = 0.679 ) . comparison of the colonoscopic features between intestinal intestinal bd and cd ( n ( % ) ) * ileocecal region represents terminal ileum and/or cecum . according to multivariate logistic regression analysis of colonoscopic features , the ulcer distribution , ulcer size , ulcer type and pseudo - polyps were independent discriminating predictors ( p = 0.006 , 0.021 , 0.002 , and 0.041 , respectively ) . the histology of mucosa biopsies in 11.4% cd patients revealed noncaseating epithelioid granulomas . neither the appearance of vasculitis nor granulomas was found in the mucosa biopsies of intestinal bd patients . among the independent discriminating predictors , seven predictors were selected for evaluation of their discriminating ability by using the roc analysis [ table 3 ] . for the diagnosis of intestinal bd , genital ulcers and skin lesions had a higher area under the roc curve ( auc ) ( 0.900 and 0.853 , respectively ) with high specificity ( 94.3% and 93.4% , respectively ) . circumferential ulcers had a high specificity ( 92.5% ) , but very low sensitivity ( 28.9% ) . the classification algorithm composed of seven predictors had the highest auc of 0.987 , and high sensitivity ( 94.3% ) and specificity ( 97.2% ) . discriminating potential of independent factors for the diagnosis of intestinal bd * the sensitivity and specificity were obtained with the conventional cut - off value of 0.5 . auc : area under the receiver operator characteristic curve ; bd : behet s disease . the differential diagnosis of intestinal bd and cd is challenging due to their similarities in mucocutaneous and gastrointestinal manifestations . both diseases have variable epidemiological and clinical manifestations among different ethnicities . as the first comparative study in chinese intestinal bd and cd patients , this study revealed that diarrhea , extraintestinal systemic manifestations , and certain colonoscopic features including ulcer distribution , ulcer type , ulcer size , and pseudo - polyps were independent discriminating predictors . consistent with other studies , this study showed the following similarities between two diseases : ( 1 ) both diseases had a young age of onset ( 2040 years ) and long disease duration from symptom onset to diagnosis . ( 2 ) the ileocecal region was the most commonly involved site , and multiple gastrointestinal tract sites could be involved . ( 3 ) abdominal pain was the most common gastrointestinal symptom , and severe complications including perforation , fistulae , and massive gastrointestinal bleeding were not rare in either disease . ( 4 ) both diseases could have extraintestinal systemic manifestations such as recurrent oral ulcers , musculoskeletal lesions , and skin lesions . beyond the similarities , intestinal bd patients were more likely to be female while cd patients were more likely to be male . more upper gastrointestinal tract involvement occurred in intestinal bd patients while more perianal lesions occurred in cd patients . gastrointestinal bleeding , especially massive hemorrhage , and fever were more common in intestinal bd , while diarrhea and intestinal obstruction were more common in cd . extraintestinal systemic manifestations were the most powerful discriminating factors between two diseases , especially genital ulcers and skin lesions . the diagnostic value of endoscopic features for both diseases has always been promising . in 2009 , kim et al . compared the colonoscopic findings between 115 intestinal bd patients and 135 cd patients , and found that ulcer shape , ulcer distribution , ulcer number , absence of aphthous lesions , and a cobblestone appearance were independent discriminating predictors . our study confirmed that there were significant differences with respect to these five predictors except the presence of aphthous lesions . we also found that ulcer size and pseudo - polyps were two additional independent discriminating predictors . in the korean study , aphthous lesions occurred in 74.7% of cd patients , and in 11.3% of intestinal bd patients , while aphthous ulcers were rare in the intestinal bd and cd patients from our study . aphthous ulcers may be the earliest lesions which then become larger and deeper following the disease progression . all patients in our cohort were hospitalized due to advanced and complicated illness , so these early stage aphthous ulcers might have been missed . korman et al . found that the prevalence of aphthous ulcers in bd patients was 83.3% based on enteroclysis , which is contrary to the results of the former study and our findings based on colonoscopy . kim et al . also developed a simple decision tree by classification and regression tree analysis which was based on the ulcer shape and distribution . this was validated in the korean cohort and proved to have high sensitivity ( 94.3% ) and specificity ( 90.0% ) . however , when this same decision tree was applied to our cohort , the sensitivity for the diagnosis of intestinal bd was 85.7% , while the specificity was only 60.3% . the difference in the diagnostic accuracy of the decision tree may be due to these factors : ( 1 ) different definitions of ulcer type . in our study , we defined a circumferential ulcer as a deep round ulcer with a discrete margin , but in the korean study round ulcers were only defined by shape . the prevalence of focal involvement in cd patients was only 23.1% in the korean study , while it was 47.2% in our study . as mentioned above , the patients in our cohort had advanced disease , which could be indicated by the high incidence of operation ( 58% ) over the course of their follow - ups . ( 3 ) in our study , circumferential ulcers and longitudinal ulcers were both present in 5 patients , which made it difficult to categorize them according to the decision tree . until now , no worldwide consensus has been established for the diagnosis of intestinal bd . in 2009 , kim et al . developed a novel set of diagnostic criteria for intestinal bd and validated it in korean patients with ileocolonic ulcers . the criteria have two aspects : extraintestinal systemic manifestations and typical ulcers upon colonoscopy , defined as < 5 deep ulcers with an oval shape , discrete borders and locations in the ileocecal area . in their cohort , 86.2% intestinal bd patients had typical ulcers , compared to only 1.5% of nonintestinal bd patients . using the criteria , the overall sensitivity was 98.6% , the specificity was 83% , and the accuracy was 91.1% . according to the criteria , 35 intestinal bd patients in our study would be classified as definite intestinal bd ( 30 ) or suspected intestinal bd ( 5 ) , while 106 cd patients would be classified as probable intestinal bd ( 2 ) , suspected intestinal bd ( 33 ) , or nonintestinal bd ( 71 ) . thus , when we apply these criteria for the differential diagnosis of patients with ileocecal ulcers , the suspected intestinal bd patients should be carefully examined by regular check - ups . first , since this study was a retrospective cohort study in a single center , and all patients enrolled were hospitalized , there was definite selection bias . intestinal bd is still rare , and only patients with ulcerative lesions based on colonoscopy and without any history of operation on the gastrointestinal tract except appendectomy were enrolled in the study , which also contributed to the selection biases . third , intestinal bd and cd patients were most often diagnosed and treated by rheumatologists and gastroenterologists , respectively . due to their different perspectives , some valuable information might have been missed . for example , a gastroenterologist might have paid more attention to the gastrointestinal manifestations and ignored extraintestinal manifestations . further large prospective multi - center or population - based studies are needed to explore the differential markers between these two diseases and develop a worldwide consensus on the diagnosis of intestinal bd . extraintestinal systemic manifestations and some colonoscopic features including focal involvement , large ulcers , and circumferential ulcers favored the diagnosis of intestinal bd . on the contrary , diarrhea and the endoscopic features , longitudinal ulcers , and pseudo - polyps favored the diagnosis of cd .

background : the distinction between intestinal behet 's disease ( bd ) and crohn 's disease ( cd ) is always challenging due to many overlapping clinical features . we conducted a retrospective study to reveal valuable strategies for the differential diagnosis between intestinal bd and cd in chinese patients based on their clinical and colonoscopic features.methods:thirty-five intestinal bd patients and 106 cd patients hospitalized from january 1983 to january 2010 , who had ulcerative lesions in the terminal ileum or colon under colonoscopy and no history of gastrointestinal operation except appendectomy before admission , were enrolled . univariate and multivariate logistic regression analyses were conducted to find discriminating predictors among demographic data , clinical manifestations , and colonoscopic findings.results:based on univariate analysis , massive gastrointestinal hemorrhage , fever , and extraintestinal systemic manifestations were more common in intestinal bd patients ( p = 0.022 , 0.048 and 0.001 , respectively ) , while diarrhea , intestinal obstruction , and perianal lesions were more common in cd patients ( p = 0.002 , 0.010 , and 0.027 respectively ) . based on colonoscopy , focal involvement , ileocecal valve deformity , solitary ulcers , large ulcers ( ulcer size > 2 cm ) , and circumferential ulcers were more common in intestinal bd patients ( p = 0.003 , 0.003 , 0.014 , 0,013 , and 0.003 , respectively ) , while segmental involvement , longitudinal ulcers , a cobblestone or nodular appearance , and pseudo - polyps were more common in cd patients ( p = 0.003 , 0.008 , 0.023 , and 0.002 , respectively ) . based on multivariate logistic regression analysis , diarrhea , extraintestinal manifestations , ulcer distribution , size , and type , and pseudo - polyps were independent discriminating predictors between the two groups ( p = 0.048 , 0.008 , 0.006 , 0.021 , 0.002 , and 0.041 , respectively ) . the discriminating algorithm composed of the above independent predictors had the highest area under the curve of 0.987 for distinguishing between the two diseases.conclusions:extraintestinal systemic manifestations and the characteristic colonoscopic features , such as ulcer distribution , size and type , helped to distinguish intestinal bd from cd .

I M Patients Diagnostic criteria Clinical and colonoscopic features Statistical analysis R Demographics and clinical features Colonoscopic features The discriminating potential of independent predictors D C

in most emergency departments , nursing personnel or medical technicians attempt initial intravenous catheter placement . if the initial attempts are unsuccessful , physicians are then asked to either assist or attempt a line placement . in cases where initial attempts have been unsuccessful , ultrasound can be used as an adjunctive technique . ultrasound - assisted vascular access has been shown to provide a greater margin of safety in central venous cannulation and is an efficient means to obtain both peripheral and central venous access [ 13 ] . ultrasound - guided vascular access is an acquired skill , with a steep learning curve . there are few studies looking at the best approach to ultrasound - guided vascular access . traditionally this procedure is taught and performed in the short axis with a standard needle . manufacturing technology has the ability to produce intravenous catheter needles with an enhanced ultrasonographic image . an echo - enhanced needle has a tip that produces a high - intensity echo so that it creates a very bright echogenic focus at the needle tip on the ultrasound screen . commercially available echo - enhanced needle tips are used in radiology for ultrasound - guided procedures to assist localizing the needle on the ultrasound screen by enhancing the reflectivity of the needle itself . the theoretical advantage of the echo tip is that enhanced visualization , compared to standard needles , should therefore improve procedural ease and success . while ultrasound - guided vascular access has been encouraged in emergency departments , there have been few studies evaluating echo - enhanced needles and their usefulness in performing vascular access . our purpose was to perform a prospective , randomized , observational study to evaluate the effect of echo - enhanced needles on venous cannulation in a vascular access phantom . we presumed that participants using an echo - enhanced needle would have a significant reduction in time to cannulate because the needle should have been easier to visualize . to determine if these needles provided any benefit we utilized a standard vascular access model , standard short - axis vessel visualization , and measured the number of needle sticks , number of redirects , and time to dye flash . this was a prospective , randomized , observational study evaluating ultrasound - guided vascular access in the short axis of the vessel using an echo - enhanced needle ( echotip needle , cook , bloomington , in , usa ) compared to a standard needle using a phantom vascular access model ( blue phantom , advanced medical technologies , llc , kirkland , wa , usa ) . this study was reviewed and approved by the institutional review board for verbal consent for all participants . this study was conducted in the emergency departments of two midwestern urban tertiary care centers with a shared emergency medicine residency program . as part of their training , emergency medicine residents are instructed in ultrasound techniques through a combination of didactic lectures , lab time , and a 1-month dedicated rotation in emergency medicine ultrasound . following this baseline training , skills in ultrasound - guided vascular access are taught through lectures and direct supervision by attending physicians certified in the technique . a random number generator with a ten - subject block design was used to randomly assign which needle each participant would use first . each subject was then shown a 10-min video describing ultrasound - guided vascular access ( produced by blue phantom , advanced medical technologies , llc , kirkland , wa , usa ) . they then attempted to obtain vascular access with both standard and echo - enhanced needles . ultrasound imaging was performed with the sonosite titan ultrasound machine ( sonosite , bothell , wa , usa ) , with a 38-mm broadband ( 10 - 5 mhz ) linear array transducer ( titan l38 , sonosite , bothell , wa , usa ) . the blue phantom vascular access model is a durable anthropomorphic phantom that images similar to real tissue with ultrasound and contains simulated blood vessels . for this study , the phantom was modified to further obscure the embedded vessel such that there were no visual or palpable anatomic clues for vascular access . this was done by placing a separate rectangular flap made of the same material as the phantom over the phantom obscuring any visualized vessels . this added approximately 1.0 cm extra depth , an insignificant amount , but provided excellent camouflage of the vessel . we did our best to reposition the flap after multiple uses to hide obvious needle sticks . the vessel was 8 mm in diameter and was filled with a blood simulation material ( water colored with red food dye ) . participants attempted to obtain vascular access with the 18-gauge standard needle and echo - enhanced needle in the short axis view . residents and staff members were not allowed to observe others attempting to gain vascular access . physicians were directly observed by a trained research assistant during all of their attempts to gain vascular access . using a stopwatch , time from ultrasound probe placement on the phantom until dye flash within the cannula was measured . the research assistant also recorded the number of needle sticks required and the number of needle redirections . median differences between echo - enhanced and standard needles for the variables time to dye flash , number of sticks , and number of redirects were calculated by subtracting the standard needle tip value from the echotip value . a positive value corresponded to higher echo - enhanced needle values and negative values indicated a higher standard needle value . wilcoxon rank sum testing was used for analysis with a p < 0.05 taken to indicate statistical significance . all analyses were done using the sas software , version 9.1 ( cary , nc , usa ) . this was a prospective , randomized , observational study evaluating ultrasound - guided vascular access in the short axis of the vessel using an echo - enhanced needle ( echotip needle , cook , bloomington , in , usa ) compared to a standard needle using a phantom vascular access model ( blue phantom , advanced medical technologies , llc , kirkland , wa , usa ) . this study was reviewed and approved by the institutional review board for verbal consent for all participants . this study was conducted in the emergency departments of two midwestern urban tertiary care centers with a shared emergency medicine residency program . as part of their training , emergency medicine residents are instructed in ultrasound techniques through a combination of didactic lectures , lab time , and a 1-month dedicated rotation in emergency medicine ultrasound . following this baseline training , skills in ultrasound - guided vascular access are taught through lectures and direct supervision by attending physicians certified in the technique . a random number generator with a ten - subject block design was used to randomly assign which needle each participant would use first . each subject was then shown a 10-min video describing ultrasound - guided vascular access ( produced by blue phantom , advanced medical technologies , llc , kirkland , wa , usa ) . they then attempted to obtain vascular access with both standard and echo - enhanced needles . ultrasound imaging was performed with the sonosite titan ultrasound machine ( sonosite , bothell , wa , usa ) , with a 38-mm broadband ( 10 - 5 mhz ) linear array transducer ( titan l38 , sonosite , bothell , wa , usa ) . the blue phantom vascular access model is a durable anthropomorphic phantom that images similar to real tissue with ultrasound and contains simulated blood vessels . for this study , the phantom was modified to further obscure the embedded vessel such that there were no visual or palpable anatomic clues for vascular access . this was done by placing a separate rectangular flap made of the same material as the phantom over the phantom obscuring any visualized vessels . this added approximately 1.0 cm extra depth , an insignificant amount , but provided excellent camouflage of the vessel . we did our best to reposition the flap after multiple uses to hide obvious needle sticks . the vessel was 8 mm in diameter and was filled with a blood simulation material ( water colored with red food dye ) . participants attempted to obtain vascular access with the 18-gauge standard needle and echo - enhanced needle in the short axis view . residents and staff members were not allowed to observe others attempting to gain vascular access . physicians were directly observed by a trained research assistant during all of their attempts to gain vascular access . using a stopwatch , time from ultrasound probe placement on the phantom until dye flash within the cannula was measured . the research assistant also recorded the number of needle sticks required and the number of needle redirections . median differences between echo - enhanced and standard needles for the variables time to dye flash , number of sticks , and number of redirects were calculated by subtracting the standard needle tip value from the echotip value . a positive value corresponded to higher echo - enhanced needle values and negative values indicated a higher standard needle value . wilcoxon rank sum testing was used for analysis with a p < 0.05 taken to indicate statistical significance . all analyses were done using the sas software , version 9.1 ( cary , nc , usa ) . overall there were 69 participants with a median of 3 years [ interquartile range ( iqr ) : 27 ] of clinical experience ( including residency ) . of the participants , 46.4% ( 32/69 ) were staff emergency medicine physicians and 53.6% ( 37/69 ) were residents . most [ 82.6% ( 57/69 ) ] reported that they had little or no experience ( zero to nine actual attempts ) with ultrasound - guided vascular access before participating in this study ; 100% of placement attempts were successful , and no placement required more than three attempts . the overall median time from needle stick to flash was 17.56 s ( iqr : 12.3733.15 ) for the standard needle and 19.22 s ( iqr : 10.1931.10 ) for the echo - enhanced needle . there was no difference for number of needle sticks , redirects , or time to dye flash ( tables 1 and 2 ) . table 1median number of sticks , redirects , and time to dye flash nmedian25th , 75th percentileecho sticks691.001.00 , 1.00standard sticks691.001.00 , 1.00echo redirects691.000.00 , 2.00standard redirects691.000.00 , 2.00echo time to dye flash6919.2212.37 , 33.15standard time to dye flash6917.5610.19 , 31.10table 2median differences between needle sticks , redirects , and time to dye flash nmedian25th , 75th percentilep valuestick diff.690.000.00 , 0.000.99redir . diff.690.00 - 1.00 , 0.000.35time diff.692.06 - 9.22 , 14.500.35median of the difference between echo and standard ( calculated by echostandard ) . thus , positive numbers correspond to higher echo values and negative values indicate higher standard valuesp value from wilcoxon rank sum test , testing the median difference ( echostandard ) = 0 median number of sticks , redirects , and time to dye flash median differences between needle sticks , redirects , and time to dye flash median of the difference between echo and standard ( calculated by echostandard ) . thus , positive numbers correspond to higher echo values and negative values indicate higher standard values p value from wilcoxon rank sum test , testing the median difference ( echostandard ) = 0 our study showed that there was no significant difference in vascular access metrics for ultrasound - guided intravenous catheter placement between echo - enhanced or standard needles using a vascular access surrogate with the vessel in the short axis . as ultrasound becomes more prevalent in emergency departments , physicians will increasingly be using this device for both peripheral and central vascular access . some of these procedures may benefit from having a needle tip that is more easily localized . there are several commercially available needles that have been altered to improve needle visibility [ 7 , 8 ] . the potential to facilitate placement of a needle into a vessel with a needle that shows up brighter on the ultrasound screen seems obvious . when performing ultrasound - guided procedures using a non - echo - enhanced needle tip , it is often challenging to visualize the needle tip especially in the short axis . visualization of the needle is one of the most common difficulties encountered during ultrasound - guided vascular access . the steeper the angle of the needle to the probe the more difficult it is to visualize . studies have shown that at a decreasing angle of insonation , it is more difficult to visualize the needle ; some echogenic needles are advantageous and show significantly improved visibility . optimization of conditions , like angle of insertion , are sometimes limited because of anatomy and when unfavorable conditions exist , utilizing a needle that has been echo - enhanced can make it more visible . often the physician placing the needle estimates its location by watching for changes in the soft tissue or vessel itself during insertion , never fully seeing the needle tip as a reflection on the screen . the advantages of an echo - enhanced needle tip for ultrasound localization , especially for teaching purposes , seem obvious . many practitioners of ultrasound , like interventional radiologists and gynecologists , have been using echo - enhanced products when performing their ultrasound - assisted procedures . echo enhancement can come in a number of different categories including a chemical coating or even etches in the wall of the needle [ 11 , 12 ] . most of the participants had little or no experience with ultrasound . despite this fact and with minimal training all participants successfully cannulated the vascular access phantom . moreover , despite a core curriculum that includes ultrasound lectures , we did not control or document who actually completed the lectures . most of the residents ( especially upper level ) had some previous ultrasound education / lectures while the emergency medicine staff did not , but all participants watched the same brief video . a group of experienced ultrasound - guided vascular access participants may have performed better with the echo - enhanced needle knowing how to better localize the tip . the ultrasound phantom , although a good approximation of human tissue , does not in fact mimic actual human tissue , and this needs to be taken into consideration when extrapolating the data . the visibility of the needle is much more obvious in the phantom than during ultrasound - guided vascular access in human tissue . we used a vascular access phantom for ease of study and to avoid subjecting patients to training procedures . the results found here may have been different had we studied real patients or cadavers . while this phantom represented our best available vascular access model , the differences with human tissue and the location of deeper vessels make extrapolation of this study difficult in a live human setting . however , it still appears that inexperienced ultrasound users may not benefit from the advantages of an echo - enhanced needle until further along in their training and understanding of ultrasound - guided vascular access . in a group of mostly ultrasound inexperienced participants attempting ultrasound - guided vascular access visualizing the vessel in the short axis , the echo - enhanced needle did not improve measures of successful vascular access .

backgroundvascular access is a critical skill for emergency physicians . however , it can be unpredictably challenging in some patients . while ultrasound - guided vascular access has been encouraged in emergency departments , there have been few studies evaluating echo - enhanced needles and their usefulness in performing vascular access.aimsour purpose was to determine if the use of an echo - enhanced needle tip results in faster vascular access times , with fewer needle sticks , fewer redirections , and improved needle visualization in ultrasound - guided vascular access with the vessel in the short axis.methodsthis is a prospective , randomized , observational study of ultrasound - guided vascular access on a vascular phantom comparing an echo - enhanced needle with a standard needle . each participant viewed a teaching video demonstrating typical ultrasound - guided vascular access and then attempted ultrasound - guided vascular access using both a standard and an echo - enhanced needle with the vessel in the short axis . the numbers of needle sticks , redirections , and time to dye flash were measured.resultsthe 69 participants attempted 69 short - axis ultrasound - guided vascular cannulations with no difference in time to dye flash between needle types : the median time from needle stick to flash was 17.56 s [ interquartile range ( iqr ) : 12.3733.15 ] for the standard needle and 19.22 s ( iqr : 10.1931.10 ) for the echo - enhanced needle . there was no difference between needle types for number of needle sticks or redirects.conclusionecho-enhanced needles did not provide objective performance improvement compared to standard needles during ultrasound - guided vascular access with a vascular access model in the short axis .

Introduction Methods Study design Study setting and population Study protocol Measurements/outcome measures Data analysis Results Discussion Conclusion