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numerous studies show that boys play more computer games, compared to girls. a study conducted in usa (2005) on the level of use of computer games by the americans showed that in the houses of 75% of the subjects, the device to play computer games was found. the prevalence of adolescents playing computer games was found to be 66.4% in one of the towns in isfahan province. such games are vastly played, as sharifi reported that adolescents averagely played such games 2 - 4 h per day. in iran, some studies reported such games played for 1 - 5 h per week or somewhat more than 6 h in a week. with regard to the impressive effects of computer games on lifestyle, these games have been named as a revolution and not just as the games to pass leisure time. this idea has attracted the researchers to this field and has resulted in two different opinions about such games. some believe age or time of communication and consider the positive and negative functions of them similar to other games, while some other researchers explore the negative functions of computer games. their positive effects are innovation and imagination skills, eye - hand coordination, and a change in abstract thinking, while their negative effects include sore eyes, headache, propensity to violence, a personality change, anti - social behavior, and addiction. showed that normal adolescents had higher social skills, compared to those addicted to computer games. meanwhile, among the negative effects of playing computer games at night, the probability of depression has been reported. adolescents and teenagers, who are the valuable wealth of the society and the human resources to make future, undergo numerous physical and mental changes, due to which constant evaluation of physical, psychological, mental, emotional, and social health is of great importance in health provision service. in addition, paying close attention to the health of this specific age group, especially during school years, from aspects including the critical role of their health in their academic performance, promotion of the personal and social abilities, and ultimately, having a healthy soul and body in their future life is inevitable. adolescent period is the most critical period in the formation of personality and adaptation. according to the latest census conducted in 2011,. therefore, evaluation of the effects of their usual and current behavior on their health is essential. although research in this field is not older than 20 years, prevalence and popularity of computer games is notable among iranian adolescents in recent years. this issue has been concurrently accompanied by their contact with other factors that are effective on their health, such as internet and application of other mass media. therefore, in the current socio - cultural background, playing computer games may be associated with socio - mental health and needs research. the present study aimed to find the association between playing computer games and the mental and social health among male adolescents in iran in 2014. the study population comprised all 1314-year - old adolescents studying in governmental high schools of isfahan. firstly, the list of all boys high schools in six educational districts was prepared. then, through random cluster sampling, six boys high schools were selected (one from each district). next, through distributional sampling (with regard to the number of adolescents in each school), 210 students were selected by test power of 80% (z = 0.84) and keeping possible dropouts into consideration. the parents were called to give their consent for their children 's attendance in the study. to collect the data, goldberg general health questionnaire (ghq) and kiez social health questionnaire were adopted. demographic characteristics and computer games associated factors such as the length of games, type of games, type of device, and location and mode of playing games the ghq, designed by goldberg and hillier (1979), was adopted to investigate subjects general health status with the goal of distinguishing healthy individuals from the diseased ones. these dimensions include physical signs, anxiety and insomnia, social dysfunction, and depression. the questionnaire is valid (0.93) and its reliability is moderate (0.87). up to now, over 70 studies have been conducted on its validity all over the world. for instance, danesh reported its validity as 0.91 through test re - test method. adolescents social health was evaluated through kiez social health questionnaire that contains 20 items on five components of social prosperity, social unity, social cohesion, social acceptance, and social participation. this questionnaire was scored based on a five - point likert 's scale, with total score of 100. scores 20 - 46 show poor social health, 47 - 74 show moderate social health, and 75 - 100 show appropriate social health. the questionnaire is valid (0.83) and the validity of its subscales of cohesion, acceptance, participation, social prosperity, and unity was calculated by chronbach alpha values of 0.75, 0.85, 0.83, 0.75, and 0.79, respectively. conditions of playing games included length of games (once or twice a week, and 1 h, 2 h, 3 h, 4 h, or more in a day) ; type of games which was categorized by mehrabifar. in seven categories of events, combat, educational, sports, simulations, internet, and mystery ; type of device used for playing games (computer, mobile, tablet, and play station) ; location of playing games (home, a coffee net, or a game net) ; and mode of playing games (single, two partners, and group). questionnaires were filled by the students at school during their recess time with coordination of the school principal. data were analyzed by pearson and spearman correlation coefficients, one - way analysis of variance (anova), and independent t - test in spss20. results showed that most of the subjects fathers and mothers were self - employed and homemakers, respectively. mean (sd) of subjects previous year average was 18.40 (1.47), and mean (sd) of their family members number was 4.22 (0.9). most of the subjects played computer games (96.7%) mostly at home and 1 h in a day. most of the subjects played games alone on a computer (58.9%). among the types of games (events, combat, educational, sports, simulation, internet, and mystery), the subjects preferences were sports (48.1%), combat (29.9%), events (6.1%), internet (5.1%), simulations (4.2%), educational (4.1%), and mystery (0.5%), in this order. with regard to mean scores of mental health domains, the highest mean (sd) was for social dysfunction [9.97 (3.6) ] and the lowest was for depression [3.32 (3.3) ]. mean (sd) of overall score was 22.15 (9.66). among the domains of social health, social unity had the lowest mean (sd) [8.28 (2.67) ] and social participation had the highest mean (sd) [17.52 (4.3) ]. subjects social health status was graded as poor (4.7%), moderate (83.2%), and appropriate (12.1%), which shows that most of the subjects had moderate social health. pearson test showed no significant association between the length of games and subjects mental health. meanwhile, it showed a significant inverse association with the score of social health [table 1 ], which was more in the domain of social unity. spearman test showed a poor significant inverse association between events games and mental health scores (r = 0.16, p = 0.01) in the domains of physical condition, anxiety, and depression [table 2 ]. one - way anova showed no significant association between the device used for playing games and the mental health score, although a significant inverse association was observed between the type of device (tablet) and mental health [table 3 ]. spearman test showed no significant association between the mode of game and mental health (p = 0.51) and social health (p = 0.33). independent t - test showed no significant association between the location of playing games and mental health score (p = 0.49) and social health score (p = 0.27). association between the length of playing games and subjects mental and social health association between events games and mental health compass mean score of mental and social health according to the type of device used and anova this study aimed to investigate the association of conditions of playing computer games and the mental and social health with a limited sample size. the findings showed that most of the subjects played games alone on a computer at home for 1 h a day, and sports, combat, and events games were among their priorities, respectively. with regard to mental health and its domains, the dimensions of social dysfunction and depression had the highest and the lowest means, respectively. with regard to the obtained results, as the cutoff point score in ghq (2008), it can be concluded that the subjects had relatively appropriate mental health. amini reported the subjects mean score of mental health (using similar ghq) as 48.29. the difference in subjects mental health in these two studies could have resulted from the difference in subjects age and geographical locations. with regard to social health and its domains, the dimensions of social participation and unity had the highest and the lowest scores, respectively. subjects overall social health was observed as moderate, which needs improvement. in the study of ganji. comparison of social health components in their study and in the present study showed that subjects mental health had a better condition in their study, possibly due to the age difference (with regard to the critical age of adolescence period that overlaps transition from childhood to adulthood) and the gender of the subjects (as girls generally have a better mental health, compared to boys). with regard to the association between subjects mental and social health and the length of playing computer games, there was an inverse significant association between the length of playing games and social health. in fact, the more is the time spent on such games, the less social communications the adolescents make with their family members and relatives, and consequently, more are the negative effects on their social health. previous research in this field showed the effect of length of games on their users, including the study of saffarian., conducted in golestan province, iran, on the association between use of computer games and adolescents mental health and educational function and reported a significant association between the length of games and subjects mental health and educational function.. also showed an association between the length of games and negative outcomes in 13 - 18-year - old subjects in sweden. in can be generally concluded that the length of modern communicational technologies (internet, computer games, and satellite programs) is associated with adolescents social training including adaptation. in fact, the more is the time the users of new communicational technologies spend on these issues including computer games, the lesser time they dedicate for making communication with others and they somehow get socially isolated. previous studies investigated and reported the effect of length of games on mental health, but no study was conducted on social health and computer games. the present study showed that the length of games affected social health, although its sample size was limited. further studies are suggested to be conducted with a larger sample size. with regard to the type of computer games and subjects mental and social health, there was an inverse association between events games and mental health, which was significant for the domains of mental health (physical condition, anxiety, and depression). as events games was among the first three priorities of the subjects and its association with mental health was revealed, importance of parents supervision on adolescents selection of games is felt.. showed a significant association between computer games and adolescents function and academic performance, which is in line with the finding of the present study. various studies showed that, with respect to psychological health, computer games result in aggression, computer addiction, depression, isolation, and anxiety, which is consistent with the finding of the present study. with regard to the association between the selected device to play games and the mental and social health, a significant association was observed between the device and social health (between using a tablet and social health). it can be argued that the more portable the device is, the more conveniently the user can carry that and the more time is consumed on it leading to less communication with others, negatively affecting the users social health. reported that there was a significant association between absence or presence of computer games and subjects studying. it can be concluded that the more the users play computer games, the less time they spend on making communications with others and doing their own duties. with regard to the mode of playing games (single, paired, group) and adolescents mental and social health, no significant association was observed. investigated the association between playing computer games and aggression among guidance school students in rasht, iran. their results showed no significant difference in aggression between two groups (with and without parents partnership), while with regard to physical aggression, there was a significant difference between two groups(with and without peers partnership) as the aggression was more in the group with peers partnership. the existing difference between the results of their study and those of present study can be due to the number and gender of the subjects, and the fact that although aggression is an effective element in mental health, in the present study, overall mental health was investigated. there was no significant association between the location of playing games and subjects mental and social health. doran. showed an inverse association between location and attendance of others in the location of playing games, and social skills. in fact, selection of home to play games and more history of playing games lessened the social skills. the difference between the findings of their study and those of the present study may be attributed to progression of existing devices and technologies, and the fact that nowadays, there is no limitation in playing such games in specific locations. the present study revealed a significant association between adolescents mental and social health (p = 0.001). in fact, mental health and social health have interactional effects on each other, and consequently, empowerment of one has a positive effect on the other leading to adolescents overall health. the limitation of the present study was that it just showed the correlation, while a disorder in health may lead to increased use of such games. therefore, our obtained results do not show a one - way association between computer games and social and mental health. generally, it can be noted that the type, nature, consumed time on the computer games, and to some extent, the type of device concerning its portability and convenience of use most of the times may be associated with their users mental and social health and the relevant technologies. with regard to the obtained results, it can be concluded that playing computer games can be an effective factor on users. further research with a higher sample size is needed to investigate other aspects of this issue. generally, it should be noted that there is a poor correlation between playing computer games and adolescents mental and social health, which reveals the need for more precise supervision and education of correct way of playing such games by the parents and the nurses working in schools, with regard to the type and length and the device used in playing such games. further studies with a larger sample size are needed to investigate other aspects of playing such games. faculty of nursing and midwifery, isfahan university of medical sciences, isfahan, iran. faculty of nursing and midwifery, isfahan university of medical sciences, isfahan, iran.	background : as adolescents spend much time on playing computer games, their mental and social effects should be considered. the present study aimed to investigate the association between playing computer games and the mental and social health among male adolescents in iran in 2014.materials and methods : this is a cross - sectional study conducted on 210 adolescents selected by multi - stage random sampling. data were collected by goldberg and hillier general health (28 items) and kiez social health questionnaires. the association was tested by pearson and spearman correlation coefficients, one - way analysis of variance (anova), and independent t - test. computer games related factors such as the location, type, length, the adopted device, and mode of playing games were investigated.results:results showed that 58.9% of the subjects played games on a computer alone for 1 h at home. results also revealed that the subjects had appropriate mental health and 83.2% had moderate social health. results showed a poor significant association between the length of games and social health (r = 0.15, p = 0.03), the type of games and mental health (r = 0.16, p = 0.01), and the device used in playing games and social health (f = 0.95, p = 0.03).conclusions : the findings showed that adolescents mental and social health is negatively associated with their playing computer games. therefore, to promote their health, educating them about the correct way of playing computer games is essential and their parents and school authorities, including nurses working at schools, should determine its relevant factors such as the type, length, and device used in playing such games.
despite high vaccination coverage, pertussis continues to be a major cause of morbidity and mortality in europe. the clinical spectrum is diverse and is affected by patient age, previous exposure to the organism and immunization history [2, 3, 11 ]. pertussis appears epidemically every 2 to 5 years. however, epidemiological data are incomplete, and different reporting procedures suggest that only the tip of the iceberg is evident [2, 3, 11, 16 ]. improved disease surveillance and diagnoses are essential to determine the true burden of the disease. the situation in denmark is unique in that all children are vaccinated with a domestically produced dtap (di - te - kik) vaccine in which toxoided pertussis toxin (pt) is the only pertussis component. the pt toxoid vaccine is reported to provide about 78% protection against notified pertussis after three vaccinations given at 3, 5 and 12 months of age as part of the national child immunization program. vaccination effectiveness for two doses of pertussis toxoid vaccine in the first year of life is reported to be 59%. in september 2003, a pre - school pertussis booster vaccination at 5 years of age was implemented to reduce transmission to susceptible young children. infection results in iga and igg antibody responses to specific bordetella pertussis antigens, whereas vaccination results in only an igg response to these antigens [3, 11 ]. diagnosis of pertussis based on clinical symptoms is complicated by a number of factors : the wide spectrum of symptoms and misdiagnosis owing to similarity between symptoms of other infections [2, 3, 11, 16 ]. a who committee in 1991 developed a primary case definition to be used in vaccine efficacy trials : an illness with paroxysmal cough of 21 days and either culture - confirmed infection with b. pertussis or serological evidence of infection with b. pertussis or household contact with a case of culture - confirmed pertussis with onset of cough within 28 days before or after onset of cough. in the present study, we investigated whether there was evidence of circulation of b. pertussis in the community. this would provide baseline data on which to assess the epidemiology of b. pertussis and thus direct future use of pertussis vaccines in denmark. study subjects the study population consisted of healthy children enrolled from the postnatal ward at hvidovre hospital, denmark during a 12-month period from may 2004 to may 2005. children were originally enrolled for the study of human metapneumovirus and respiratory syncytial virus infections in infancy. approximately 20 children were enrolled each month to ensure that the children were sampled equally throughout the year. a total of 242 healthy newborns were enrolled, of whom 217 were followed throughout 1 year. study procedures children were monitored with detailed health diaries and through monthly home visits until 1 year of age. the children were scheduled to receive routine immunizations with d - t - ap - ipv - hib at 3, 5 and 12 months of age. blood samples were obtained by venous puncture from 203 of the children at age 12 months and the sera kept at 80c until further analysis. laboratory assays twofold dilutions of 50 l of serum were used for the pertussis elisa. iga and igg antibodies to pt and filamentous hemagglutinin (fha) were measured by a standardized elisa procedure and expressed in elisa units (eu / ml) with respect to reference serum 3 of the center for biologics evaluation and research / us food and drug administration (cber / fda). the lower levels of detection for the assays were 1.7 eu / ml for igg - anti - pt, 2.4 eu / ml for iga - anti pt, 1.8 eu / ml for igg - anti - fha and 1.7 eu / ml for iga - anti - fha. statistical analysis antibody concentrations and prevalence were tabulated. for differences in duration in days of coughing episodes and in infants with iga positive and iga negative samples, at the time of blood sampling, 100% of children had received one dose, 98% had received two doses and 23% had received three doses of the pt toxoid vaccine. because iga antibodies are a result of infection and not vaccination, we used their presence as an indicator of infection in the study children. ten children (4.9%) had iga antibody to pt, 18 (8.9%) had igg antibody to fha, 193 (95.1%) had igg antibody to pt and 13 (6.4%) had iga antibody to fha. seven of the ten pt iga responders had received three doses of vaccine and the remaining three pt iga responders had received two doses at 3 and 5 months of age. the median pt igg titer for fully vaccinated children was 50 eu / ml vs. 8 eu / ml for children who had only received one or two doses of the pt toxoid vaccine (p 0.05). mean age at longest coughing episode was 7.5 months. in one pt iga positive and 20 pt iga negative children, the longest coughing episode was associated with wheezing, suggesting a viral infection and not b. pertussis infection as the cause of the illnesses. excluding these 21 children from analysis did not alter the main results (median duration of cough 11 vs. 9 days, p = 0.209). no differences in other symptoms such as fever, rash, diarrhea and vomiting were observed between the groups (data not shown). table 1duration of longest coughing episode during the first year of life in 203 danish children related to pt iga antibody statusduration of coughpt iga pos (n = 10) (%) pt iga neg (n = 193) (%) 49 days03 (1.6) duration of longest coughing episode during the first year of life in 203 danish children related to pt iga antibody status infection with b. pertussis stimulates both iga and igg antibody responses to b. pertussis antigens. in an efficacy trial in germany, only 120 (52%) of 232 children with pertussis had an iga response to pt (heininger and cherry unpublished data 1998). we found serological evidence of b. pertussis infection (iga pt antibody) in 5% of our cohort of vaccinated healthy children during their first year of life. since only 50% of infections result in a pt iga antibody response, the actual infection rate in our cohort may have been 10%. it is of interest that seven of 47 (15%) with three doses of vaccine had a pt iga antibody response, whereas only three of the 156 (2%) who had less than three doses of vaccine had a pt iga antibody response. the explanation for this observation is that once a natural infection has occurred, immunization will enhance the pt iga response. this was observed following the fourth dose in the german efficacy trial mentioned above as well as in a more recent adult vaccine efficacy trial. the presence of igg and iga antibodies to fha in 8.9% and 6.4%, respectively, in the serum of study subjects is also an indicator of b. pertussis infection since the vaccine did not contain this antigen. however, antibody to fha in contrast to antibody to pt is less specific. infection with other bordetella sp. as well as other gram negative bacteria may stimulate an antibody response to fha. the duration of the most significant cough illness in our study subjects with evidence of b. pertussis infection was not significantly different from the duration in subjects without evidence of b. pertussis infection. this observation suggests that the infections that occurred in the previous vaccinees were clinically mild. with the exception of the dtap vaccine (di - te - kik) in use in denmark, which contains only toxoided pt as the pertussis component, the dtap vaccines presently in use throughout the world contain toxoided pt as well as 14 other b. pertussis antigens (fha, pertactin [prn ] and fimbriae 2/3 [fim 2/3 ]) [3, 11 ]. the reported pertussis vaccine efficacy in the 1980s and 1990s trials varied considerably based on the case definition used [37, 1115, 17 ]. the main objection to the who primary case definition of pertussis is that it eliminated many laboratory - confirmed clinically mild cases from efficacy calculations in vaccine trials. because mild cases are more common in vaccinees than in controls, efficacy is inflated. therefore, vaccine efficacy appears better than it truly is, and less effective vaccines would also seem comparable with their more effective counterparts. results from a randomized trial of acelluar pertussis vaccines in stockholm in the 1980s showed that significantly fewer recipients of a two - component vaccine (jnih-6) were diagnosed as primary or co - primary cases in households than those who received a monocomponent vaccine (jnih-7). regarding the danish pt toxoid vaccine (di - te - kik), it was used exclusively in the gothenburg region of sweden from 1996 until the spring of 1999. the rate of pertussis (in children born between january 1996 and june 1999) between october 1997 and september 2004 was determined for children in the gothenburg region and the rest of sweden [1, 8 ]. the rate of pertussis (mostly vaccine failures) in the gothenburg region was significantly higher in all children less than 8 years than the rate in the rest of sweden, where a three component dtap vaccine was predominantly used. in conclusion, our data indicate that 510% of our vaccinated cohort of healthy children had a b. pertussis infection during the first year of life, demonstrating that b. pertussis is circulating in the community even though few clinical cases were recognized. the high b. pertussis infection rate in danish infants as well as the surveillance data from sweden suggest that the pt toxoid vaccine used in denmark has less than optimal efficacy. a prospective immunization study comparing a multi - component vaccine with the present monocomponent pt toxoid vaccine should be undertaken.	we measured iga and igg antibodies to pertussis toxin (pt) and filamentous hemagglutinin (fha) in sera from 203 1-year - old children who had received one to three doses of a monocomponent pt toxoid vaccine. ten children (5%) had iga antibody to pt indicating recent infection ; seven of these children had received three doses of vaccine. pt iga responders did not have significantly longer coughing episodes than pt iga non - responders. since an iga antibody response occurs in only 50% of infected children, the actual infection rate in our cohort is estimated to 10%. the apparent high bordetella pertussis infection rate in danish infants suggests that the monocomponent pt toxoid vaccine used in denmark has limited efficacy against b. pertussis infection. a prospective immunization study comparing a multi - component vaccine with the present monocomponent pt toxoid vaccine should be undertaken.
diabetic retinopathy (dr) is one of the most common complications of diabetes and is a leading cause of blindness in people of the working age in industrialized countries. approximately 25% of type 1 diabetic patients may have signs of retinopathy after 5 years of diabetes, increasing to 60% after 10 years. eventually, after 25 years, almost all (97%) type 1 diabetic patients will develop retinopathy. type 2 diabetic patients may already have background retinopathy at the time of diagnosis, and over 60% will develop some form of retinopathy after 20 years. according to the international diabetes federation (idf - atlas, 4th ed., 2009), diabetes currently affects nearly 285 million people worldwide, and this number is expected to reach 438 million by 2030. these studies have led to the recognition of hyperglycemia, hypertension and dyslipidemia as major risk factors for dr. consequently, tight glycemic control, blood pressure control and lipid - lowering therapy have shown proven benefits in reducing the incidence and progression of dr. in clinic, laser photocoagulation and vitrectomy remain the two conventional approaches to treat sight - threatening conditions such as macular edema and proliferative dr. vascular endothelial growth factor (vegf) blockers (pegaptanib, ranibizumab and bevacizumab) in combination with laser photocoagulation represents an emerging novel therapy to reduce macular edema and induce neovascular regression.[57 ] in spite of this progress, dr remains a major clinical challenge, and the number of patients keeps growing due to difficulty in achieving tight glycemic control, metabolic memory, unresponsive to the current therapeutic approaches and significant side - effects from therapies.[56810 ] there is a great need to develop new therapeutic approaches for this devastating disease. vascular alterations in the early stage of the disease include alterations in blood flow, death of retinal pericytes (perivascular contractile cells), basement membrane thickening and subtle increases in vascular permeability. with the progression of disease, these include non - perfused vessels, microaneurysms, dot / blot hemorrhages, cotton - wool spots, venous beading, vascular loops and significant vascular leakage. the above alternations happen in the non - proliferative stage of dr, in which vision loss is mainly caused by macular edema as the consequence of increased vascular permeability. in the proliferative stage of dr, neovascularization happens on the retinal surface after vascular function is impaired by capillary occlusion, non - perfusion and degeneration. in this stage, severe vision loss or even blindness may be caused by bleeding, hemorrhage and subsequent retinal detachment because of the newly formed fragile vessels. the mechanisms by which diabetes causes microvascular complications and disease progression in the retina are not fully understood. however, studies in patient samples and animal models have shown that dr has features of chronic, subclinical inflammation. inflammation is the body 's defense against pathogens and is also a critical step in wound healing. this process involves multiple mediators such as pro - inflammatory cytokines, chemokines and adhesion molecules that initiate the interaction between leukocytes and the endothelium and guide directional leukocyte migration toward infected or injured tissue. pro - inflammatory cytokines (such as tumor necrosis factor [tnf] and interleukins) and chemokines (such as ccl2 and ccl5) released from infected / injured tissue activate the endothelium to increase expression of adhesion molecules (such as e - selectin, intercellular adhesion molecule [icam]-1, vascular cell adhesion molecule [vcam]-1) and chemokines. mediated by adhesion molecules and chemokines, leukocytes attach to the vessel wall, transmigrate through the endothelium and move to the infected or injured tissue. while normal inflammation is beneficial, excessive or uncontrolled inflammation can cause tissue injury and result in diseases. although there are no pathogens in dr, analysis of inflammatory molecules in vitreous, serum and retina form diabetic patients or experimental animals indicate that dr is associated with significant increases in pro - inflammatory cytokines, chemokines and adhesion molecules. high tnf- levels have been observed in vitreous, serum and ocular fibrovascular membranes from patients with dr and in retinas from rodent model of diabetes mellitus.[1416 ] interleukin-1 (il-1) and its downstream signaling molecule caspase 1 are significantly increased in vitreous, retinas and serum from diabetic patients and rats. chemokines such as ccl2, ccl5, cxcl8, cxcl10 and cxcl12 are also upregulated in vitreous samples from dr patients.[1921 ] increases in il-6, icam-1 and vcam-1 have been shown to be related to the progression of dr.[2124 ] correlated with increases in inflammatory molecules, dr is associated with recruitment of leukocytes. the numbers of neutrophils are significantly elevated in both retinal and choroidal vessels from diabetic patients and monkeys. the accumulation of neutrophils is correlated with upregulation of icam-1 immunoreactivity in the vessels and is associated with capillary closure. in diabetic rodent models, there is a cumulative and sustained increase in leukocyte adherence to the retinal vasculature (leukostasis) along with the progression of dr. the increase in leukostasis may be attributable to diabetes - induced increases in icam-1 and integrins in endothelial cells and leukocytes, respectively, in that blockade of icam-1 or deletion of cd18 (icam-1 receptor subunit on leukocytes) prevents diabetes - induced leukostasis.[2729 ] in addition to leukocytes in the circulation, microglia that are scattered throughout the retina are likely to be involved in dr. in dr, they are rapidly activated to release inflammatory cytokines such as tnf-. a2a adenosine receptor agonist effectively blocks tnf- production in microglia and markedly decreases hyperglycemia - induced retinal cell death (ibrahim., 2011). in summary, this evidence demonstrates that dr has all the characters of inflammatory disease, and suggests involvement of low - grade chronic inflammation in this disease. tremendous efforts have been made to understand how diabetes causes inflammatory reactions in dr in the absence of pathogens. these factors have been shown to induce inflammation by a variety of mechanisms, including oxidative stress, nf - kb activation, dysregulation of nitric oxide synthase (nos) and formation of advanced glycation endproducts (ages). oxidative stress happens when ros are overproduced or when endogenous anti - oxidant systems are impaired. mitochondria can generate ros by leakage of electrons to molecular oxygen at electron transport chain complexes i, ii and iii. in diabetes, the metabolism of glucose - derived pyruvate through the etc complexes is increased because of high glucose concentration within cells, resulting in superoxide overproduction by mitochondria. this pathway not only produces more ros by itself but also initiates other pathways, leading to a breakdown in the balance between pro - oxidant and endogenous anti - oxidant systems, such as increases in glucose flux through the aldose reductase pathway, formation of ages and activation of protein kinase c (pkc). all these changes can lead to oxidative stress by decreasing the activities of anti - oxidant enzymes or further activating the ros - generating machinery inside the cells.[3741 ] in addition to mitochondria, nicotinamide adenine dinucleotide phosphate (nadph) oxidase is another important source of ros in diabetes, and studies in other models have suggested a positive reciprocal regulation between mitochondria and nadph oxidase - derived ros. it consists of a nox catalytic subunit, 4 phox subunits (p22phox, p40phox, p47phox and p67phox) and the low - molecular weight g protein rac. assembly of the complex is initiated upon phosphorylation of p47phox, which is regulated by many intracellular signaling kinases such as pkc, mitogen - activated protein kinases and akt.[4549 ] the p67phox subunit mediates direct binding of the complex with activated rac, which initiates the electron transfer reaction that produces superoxide. these regulatory mechanisms contribute to the unique feature of nadph oxidase - derived ros as signaling mediator in many cellular responses. however, when these mechanisms are activated because of diabetes, they can cause nadph oxidase to overproduce ros. these mechanisms in fact allow nadph oxidase to function as a key link between hyperglycemia and hypertension. increased napdh oxidase activity has been found in diabetic patients and animals and high glucose - treated endothelial cells.[5154 ] nadph oxidase is also potently activated by angiotensin ii, a product of the renin overall, these changes lead to a net increase in ros, and diabetes - induced sustained oxidative stress is a major cause of retinal inflammation. ros play a key role in inflammatory gene expression by activation of redox - sensitive transcription factors such as nf - kb, signal transducers and activators of transcription proteins and activator protein 1. the activity of nf - kb is increased in high glucose - treated retinal endothelial cells, pericytes or glial cells and in diabetic retinas from patients or animal models.[5964 ] this activation is significantly blocked by inhibiting redox systems by blockade of nadph oxidase or antioxidant treatment, indicating a cause - and - effect relationship. activation of nf - kb has an essential role in diabetes - induced retinal inflammation, in that inhibition of nf - kb blocks both high glucose and diabetes - induced production of inflammatory molecules in retinal cells and retinal tissue, and attenuates leukostasis.[5961 ] similarly, studies in animal and tissue culture models have demonstrated the involvement of nadph oxidase in diabetes - induced inflammation and breakdown of the blood the results showed that diabetes - induced increases in retinal ros, vegf expression and vascular permeability are accompanied by increases in the nadph oxidase catalytic subunit nox2.[6567 ] the experiments further demonstrated that deleting the nox2 gene or inhibiting nadph oxidase prevented diabetes - induced increases in retinal permeability and icam-1 expression and leukostasis, indicating that the nox2 nadph oxidase is critically involved in the pathology of dr. correlated with the involvement of oxidative stress in diabetes - induced retinal inflammation, studies have shown that supplement of antioxidants such as vitamins c and e attenuates the development of acellular capillaries and decreases the number of pericyte ghosts in diabetic rats. such effects are further enhanced when a more comprehensive mixture of anti - oxidant diet is applied. clinical studies also show that high doses of vitamin e reverse some of the changes in the retinal vessels of diabetic patients. in addition to oxidative stress, diabetes - induced vascular inflammation is very closely related to nitric oxide (no), which is an important second messenger that regulates many physiological and pathological events, including vascular dilation and vascular inflammation. studies indicate that no has a biphasic role in vascular and inflammatory diseases, depending on the specific source and the amount produced. the constitutively expressed noss, endothelial nos (enos) and neuronal nos (nnos), are ca2-dependent and regulated to produce low levels of no. no from nnos is involved in neural signaling and is also expressed in smooth muscle cells, where it has a key role in regulating vascular responses to tissue hypoxia. in fact, deleting enos results in elevated expression of inducible nos (inos) (an nf - kb - mediated inflammatory molecule) in retina that is associated with accelerated development and increased severity of dr. in contrast with enos and nnos, inos is ca2-independent, constitutively active and produces large amounts of no. it is not expressed in normal retinas but is induced in retinal glial and microglial cells during inflammatory conditions, including dr. inhibition of inos by inhibitor or gene deletion prevents icam-1 expression, leukostasis and vascular permeability in the diabetic retina. the nos pathway is dysregulated by diabetes and contributes to the pathogenesis of dr. on the one hand, ros generated from nadph oxidase or mitochondrial oxidase can rapidly react with no to form rnos and reduce bioavailable no, which reduces vessel dilation and increases leukocyte adhesion and platelet aggregation, leading to inflammation. on the other hand, nos can be an important source of ros due to nos uncoupling, in which the enzyme generates superoxide rather than no when its substrate l - arginine is limited by increased arginase activity or when the co - factor tetrahydrabiopterin is oxidized. consistently, inhibition of arginase is shown to increase bioavailable no, reduce superoxide formation and block inflammatory reactions during retinal inflammation. although hyperglycemia has a direct impact to the pathology of dr, the byproducts of hyperglycemia, particularly ages, also have a significant role in retinal inflammation in dr. ages are formed by non - enzymatic glycation and oxidation of amino groups of proteins, lipids and dna. this process is accelerated in diabetes in the presence of high glucose and oxidative stress.[7678 ] in the diabetic retina, the level of ages is prominently increased and age immunoreactivity is localized in vitreous, internal limiting membrane and retinal vasculature. the receptor for ages (rage) is expressed in numerous cells in the retina, including muller cells, endothelial cells and neurons. the age - rage pathway can activate many downstream signaling molecules, including nf - kb and ros, to induce inflammatory reactions such as icam-1 expression and leukostasis in the retina and retinal cells. blocking age with soluble rage or inhibiting age formation with lr-90 significantly reduces diabetes - induced icam-1 expression, leukostasis and vegf expression. in summary, however, it should be noted that inflammation is not an endpoint of the above inflammatory mechanisms. many inflammatory cytokines induce oxidative stress and use the nadph oxidase pathway to induce expression of other inflammatory molecules such as ccl2. angiotensin ii - induced leukostasis in the retina but also abolishes nadph oxidase - mediated ros formation. thus, inflammation serves as a key mediator in the positive feedback loop of inflammatory cascades in dr. hyperglycemia induces formation of advanced glycation end products, generation of reactive oxygen species from multiple sources, mainly nicotinamide adenine dinucleotide phosphate oxidase and mitochondrial electron transport chain, and dysregulation of nitric oxide synthase (nos) pathway. these changes activate nf - kb and, in turn, upregulation of cytokines, chemokines and inos. because diabetes is found to cause retinal inflammation by divergent mechanisms, significant efforts have been made to understand how inflammation is involved in the microvascular complications in the disease. these studies have demonstrated important roles of inflammatory cytokines and leukocytes in vascular leakage, vessel occlusion and degeneration and pathological neovascularization [figure 1 ]. the microvascular endothelium forms an effective barrier to control the movement of blood fluid and proteins across the vessel wall. this barrier is formed by tight junctions and adhesion junctions that join endothelial cells to each other. signaling molecules, including pro - inflammatory cytokines and chemokines, which induce disorganization / redistribution of junction proteins in the retinal endothelium may lead to breakdown of the brb, resulting in an abnormal extravasation of blood components and retinal edema. tnf- is known to cause significant retinal endothelial permeability within a few hours by pkc-mediated downregulation of tight junction proteins. intravitreal injection of tnf- leads to increased retinal vascular permeability, which is prevented by pkc inhibitor. moreover, blocking tnf- with etanercept, a soluble tnf- receptor, prevents brb breakdown in the diabetic rat model. similar increases in endothelial permeability are also observed when endothelial cells are treated with other cytokines such as ccl2 or cxcl8. brb breakdown can also be caused by cytokine - induced vascular cell death and by leukocyte - mediated vascular alternations. leukocytes recruited in inflammation have a key role in causing vascular leakage by mechanisms including inducing junction protein alteration, releasing cytokines or increasing ros as well as inducing vessel occlusion and injury. mechanisms leading to capillary degeneration may involve inflammatory cytokine - induced endothelial cell death because inflammatory cytokines such as tnf- and il-1 are also known to increase caspase 3 activity and potently induce endothelial cell apoptosis. alternatively, leukostasis in dr may cause capillary occlusion when leukocytes block blood flow because of their large cell volume and high rigidity. this notion is supported by studies showing that transient leukocyte attachment to the vessel wall correlates with capillary non - perfusion, and capillary reperfusion can occur when leukocytes detach and move on. in addition to blocking blow flow, leukocytes induce endothelial death during leukostasis via fas ligand (fasl) and fas - mediated apoptosis. the level of fas is increased in retinal vascular endothelial cells during diabetes, while fasl is increased on neutrophils. blocking fas or inhibiting icam-1/cd18-mediated chronic inflammation is known as a key player in angiogenesis in many diseases such as rheumatoid arthritis and cancer. leukocytes, recruited by inflammatory cytokines produced in the disease tissue, enhance the formation of new vasculature by releasing angiogenic factors and increasing the activity of matrix metallopeptidase. direct investigation of inflammation in proliferative dr is not feasible because there are no small animal models that can develop reproducible proliferative dr, as occurs in humans. therefore, studies of retinal neovascularization are carried out in rodent models of oxygen - induced retinopathy, in which retinal vessel obliteration is induced with hyperoxia. these studies have shown that inflammatory genes are upregulated at the onset of the hypoxia as well as during the period of neovascularization. in addition to inflammatory cytokines, monocytes / macrophages are found to be present in the neovascular tufts, and depleting the monocyte lineage with clodronate - liposomes leads to the suppression of pathological but not physiological retinal angiogenesis. although there is no pathogen in the diabetic retina, dr has all the common features of inflammation, such as increase in inflammatory molecules in retina, vitreous and plasma, leukocyte recruitment and tissue edema. these changes are caused by diabetes - induced disorders such as oxidative stress, dysregulation of the nos pathway, ages formation and nf - kb activation [figure 1 ]. inflammation is now recognized as an important player in the pathogenesis of dr, and a number of studies have been designed to address whether blockade of specific inflammatory molecules can be beneficial for dr. subcutaneous administration of tnf- trap (a soluble tnf- receptor / fc construct, etanercept) significantly blocks retinal inflammation, retina cell injury and vessel leakage in diabetic rats. in one clinical study with four patients, infliximab, a tnf--neutralizing antibody, was shown to improve visual acuity and reduce macular thickness in patients who failed to improve in response to laser photocoagulation treatment. leukocyte function - associated antigen-1 (lfa-1, an integrin) expressed in leukocytes is important for leukocyte - endothelial cell interaction by binding to icam. topical delivery of sar 1118, a small antagonist of lfa-1, dose - dependently reduced leukostasis and retinal vascular leakage in a diabetic rat model. in addition to icam-1, blockade of vcam-1-mediated leukocyte attachment with anti - cd49a neutralizing antibody significantly attenuated the diabetes - induced leukostasis and vascular leakage. in spite of the rapid progress in this field, there are many issues that remain to be addressed before effective anti - inflammatory therapy can be designed to treat dr. for example, inflammation is involved in almost all diseases and, therefore, it is unclear why inflammatory reactions in most diseases do not affect retinal vasculature. is there crosstalk between diabetes - induced inflammation and other chronic disease - induced inflammation in relation to the pathogenesis of dr ? although inflammation is involved in the pathogenesis of dr, there is a higher incidence of dr in type 1 diabetic patients than in type 2 diabetic patients. is it because of the fact that type 1 and type 2 diabetes induce different inflammatory reactions ? retinal inflammation might be more severe in type 1 diabetes as this disease is associated with aberrant autoimmunity. however, there is no study to address such difference at this moment. given that diabetes induces the production of inflammatory molecules in both retinal local cells and blood leukocytes, what is the individual role of retinal local cells and leukocytes in inflammatory reactions and vascular injury in dr ? many inflammatory molecules have redundant functions and cause activation of similar downstream targets such as nf - kb. these mysteries have led to challenges when considering any of the inflammatory molecules as a drug target as blockade of any one of them may be effective if the molecule is indispensible. however, the blockade can also be ineffective, or even worse, if compensation happens or goes in an unwanted direction. further studies may resolve these issues and bring novel therapies for dr by controlling inflammatory reactions precisely and at the right time.	diabetic retinopathy (dr) is one of the most common complications of diabetes and is a leading cause of blindness in people of the working age in western countries. a major pathology of dr is microvascular complications such as non - perfused vessels, microaneurysms, dot / blot hemorrhages, cotton - wool spots, venous beading, vascular loops, vascular leakage and neovascularization. multiple mechanisms are involved in these alternations. this review will focus on the role of inflammation in diabetic retinal microvascular complications and discuss the potential therapies by targeting inflammation.
age - related macular degeneration (amd) is a macular neurodegenerative disease that nowadays constitutes one of the main socioeconomical health issues worldwide, afflicting the elderly population. the exponentially increasing prevalence of amd is linked to progressive aging of the population, and it is the leading cause of legal blindness in the developed world [16 ]. it is traditionally divided into three categories : early amd, characterized by the presence of pigmentary changes of the retinal pigment epithelium (rpe) and/or hard small drusen ; intermediate amd, characterized by the presence of soft large drusen and/or geographic atrophy (ga) of the rpe with foveal sparing ; and late amd, characterized by ga with foveal involvement and/or choroidal neovascularization (cnv) [7, 8 ]. on the other hand, the presence or absence of cnv makes the distinction between neovascular amd (presence of macular fluid and/or hemorrhage secondary to cnv) and atrophic or nonneovascular amd (presence of any other amd sign except for cnv). neovascular amd accounts for the most cases of severe vision loss, although the atrophic form is the most frequent presentation of the disease [710 ]. a variety of risk factors for amd have been described. however, the evidence and strength of such associations are widely variable, probably due to the difficulty of measuring some of these factors in clinical practice [11, 12 ]. advanced age, caucasian race, certain genetic polymorphisms, higher body mass index, excessive alcohol consumption, and a history of smoking are proven risk factors in the development of amd and progression to late amd [1319 ]. they can also be divided depending on the grade of evidence showed in the literature. age showed the highest evidence, as the odds ratio (or) increases from 1 at 5569 years to 4.428.70 at 7079 years and 18.832.3 in ages between 80 and 86 years [2022 ]. smoking (or range : 2.394.22) is the second most consistent risk factor related with amd [11, 23 ]. race and ethnicity may also play an important role, as the whites are the racial group with a higher risk of amd compared with the blacks or the hispanic whites [2022 ]. other significant risk factors are family history of amd (or range : 3.956.98), previous cataract surgery (or : 1.59), high body mass index (or range : 1.061.35), and hypertension (or range : 1.021.48) [11, 23 ]. the purpose of this review is to analyze the current scientific evidence of smoking as an independent risk factor in amd and the relevance of advising patients to quit smoking for their visual health. a systematic review of all of the peer - reviewed articles indexed in pubmed about smoking and age - related macular degeneration was performed. the analyzed data were summarized classifying them into four main headings : reported epidemiological association between smoking and amd ; studied mechanisms for toxic damage to the retina and choroid induced by smoking ; smoking and biomarkers in amd ; and treatment considerations for amd and smoking. the association between smoking and amd has been consistently demonstrated in many epidemiological studies carried out within different populations in the last decades confirming previous clinical impressions. cross - sectional studies and prospective cohort studies have described the natural history of the disease and its associations with risk factors, where smoking has been the most consistent factor associated with geographic atrophy and neovascular amd. cross - sectional studies examining the association between smoking and amd include two american, three european, and two large australian populations. further studies also provided additional information about smoking as a risk factor for amd in different ethnic groups and geographic areas (table 2). population - based epidemiologic studies have provided estimates of prevalence and incidence of advanced amd among various racial / ethnic groups : geographic atrophy and neovascular amd are rare before 55 years of age, becoming more prevalent in patients aging over 75 ; overall, the prevalence is higher in caucasian and lower in african - american patients. the beaver dam eye study recruited 4771 patients from beaver dam (wi, usa) from 1988. after controlling subjects for age and passive smoking, higher rates of neovascular amd in current - smokers compared to those who had never smoked independently of gender were evidenced. more recently, the study on the large beaver dam offspring study (boss) cohort found a prevalence of amd of 3.4%. after controlling subjects for age and gender, a history of current - smoking and greater numbers of pack - years smoked were associated with early amd. the rotterdam study is a single - center prospective study of the population aging over 55 years in rotterdam (the netherlands). a total number of 6251 participants were included from 1990 until 1993. the blue mountains eye study was conducted in sydney (australia) between 1992 and 1994. a total number of 3654 patients were included. a highly significant association between current - smokers and geographic atrophy and neovascular amd was evidenced. estimated that 20% of all cases of blindness in australia may be attributable to smoking, and they advocated a new warning for cigarette packs about the risk of blindness associated with smoking. later, the visual impairment project (vip) included 4744 australian patients and found that total duration of smoking (regardless of the amount) was significantly associated with the prevalence of amd, suggesting that 14% of all amd cases might be due to cigarette smoking for longer than 40 years. pooled data from the aforementioned trials from three different continents provided strong and consistent evidence that tobacco smoking is the main known preventable exposure associated with any form of amd. depending on the type of amd, current - smokers have 2- to 4-fold increase in risk for amd when compared to patients that never smoked. the age - related eye disease study (areds) is a longitudinal multicenter study of the natural history of amd and cataract in 4757 participants recruited between 1992 and 1998. one of the main findings of the areds was that smoking was associated with all of the three more severe amd groups. the eureye study is a cross - sectional study that included 4,750 participants in 2003 from seven centers across europe. current - smokers had increased odds of any type of late amd, and bilateral amd was also more likely to have a history of heavy smoking. the attributable fraction for amd due to smoking was 27%. the eye disease case - control study of risk factors for neovascular amd studied 421 cases and 615 controls between 1986 and 1990, finding a strong association between current cigarette smoking and incidence of amd. the pola (pathologies oculaires lies l'age) study included 2196 participants in france. current- and former - smokers were found to have an increased prevalence of late amd. this risk was higher in those patients who smoked 20 pack - years and more. the odds ratio (or) for current - smokers and late amd observed in this study (3.6) was not significantly different from the or of 4.46 in the blue mountains eye study, the or of 2.2 for neovascular amd in the eye disease case - control study, the or of 2.38 for late amd in the vip study, the or of 3.6 for neovascular amd in the rotterdam study, and the or of 2.5 for women and 3.29 in men for neovascular amd in the beaver dam eye study. other recent case - control studies show a significant association between both current- and former - smokers and early amd and polypoidal choroidal vasculopathy in asian patients [34, 35 ]. smokers had a twice - fold increased risk of amd implicating that factors other than dna sequence are involved in the etiology of amd. prospective cohort study design is most suitable in order to demonstrate that smoking precedes amd development. there are three key cross - sectional studies that were subsequently extended into longitudinal studies. the length of the follow - up period is the strength of two of them. at 5-year followup, the beaver dam eye study reported that men who smoked greater amounts of cigarettes were more likely to develop early amd. these results were confirmed over a 10-year followup and also evidenced that they were more likely to have progression of the disease [36, 37 ]. the 5-year incidence of any late amd lesions found in current, past, or never - smokers in the blue mountains eye study was 3.1%, 1.2%, and 1.4%, respectively. after adjusting for age, current - smokers had an increased risk of incident geographic atrophy (rr = 3.6 ; 95% ci : 1.111.3) and any late lesions (rr = 2.5 ; 95% ci : 1.06.2). the long - term incidence over 10 years showed that current - smokers had a 4-fold increase in the risk of late amd compared with never - smokers (rr = 3.9 ; 95% ci : 1.78.8). former - smokers had a 3-fold higher risk of geographic atrophy (rr = 3.4 ; 95% ci : 1.29.7). a further pooled analysis of the 5-year results from these three studies found a continued 3-fold association of current - smoking with the development of amd. the physicians ' health study and the nurses ' health study also evidenced that current - smokers of 20 or more cigarettes per day had a 2-fold increased risk compared with never - smokers in two different cohorts of incident cases of amd followed for at least 7 years. the areds cohort with a median followup of 6.3 years estimated that a larger amount smoked was statistically significantly associated with the incidence of geographic atrophy due to amd. more recently, coleman. found an increased risk of early amd among subjects aging 80 years or older who were smokers compared to those younger than 80 years who were not smokers. the risk of smoking has also been estimated in a meta - analysis from six prospective cohort studies, five case - control studies, and five cross - sectional studies. the odds ratio for case - control studies was 1.78 (95% ci : 1.522.09), and it was 3.58 (95% ci : 2.684.79) for cross - sectional studies. the relative risk (rr) obtained through analysis of prospective cohort studies was 1.86 (95% ci : 1.272.73). several studies have investigated the dose - response effect by comparing different levels of smoking classified as pack - years, and most of them confirmed a dose - response effect. the rotterdam study and the pola study found an increased risk of neovascular amd in persons who had smoked 10 or more pack - years (or : 7.1, 95% ci : 2.1019.0) and 20 or more pack - years (or : 4.8, 95% ci : 1.8012.9) [26, 33 ]. the physicians ' health study and the nurses ' health study find significant 2-fold higher risk of amd in persons who had smoked more than 25 cigarettes per day [16, 40 ]. the vip study results reveal that only the duration, not the amount of smoking, is associated with amd. in the 5-year follow - up results from the beaver dam eye study the relative risk of early amd and progression of early amd increased as the amount smoked increased ; the 10-year follow - up results were inconsistent with earlier findings from this and other studies, as no association of the amount smoked or the incidence of late amd was evidenced. however, the beaver dam offspring study confirmed that smoking 11 or more pack - years was associated with early amd. other studies in korea and japan also evidenced that the level of smoking was associated with early and late amd [34, 44 ]. the rotterdam study was the first to reveal that the increased risk of neovascular amd remained present up to 20 years after cessation of smoking, which was also confirmed in the pola study. the nurses ' health study reported that past - smoker women who previously smoked 25 or more cigarettes per day had a 2-fold increased risk of amd even 15 years after cessation of smoking. other prospective studies found that former - smokers compared with those who never smoked had a modest increased risk of amd. its pathological effects through different biochemical pathways and an ocular exposure to cigarette smoke may cause oxidative damage, vascular changes, and inflammation within the pathogenic cascade of amd. smoke is responsible for cellular changes at the level of the rpe in amd patients. cigarette smoke promotes pathophysiological processes that contribute to atherosclerosis, including thrombosis, vascular inflammation, and endothelial dysregulation. nicotine itself promotes angiogenesis in experimental models due to its vasculogenic properties [4850 ] and may also induce catecholamine release increasing platelet aggregability. platelets contribute to the growth of plaque through the accretion of thrombus, as well as through the release of growth factors (such as platelet - derived growth factor (pdgf)) that induce vascular smooth muscle cell proliferation. the effect of nicotine also enhances physiological angiogenesis, as observed in wound healing [49, 51 ] where smoking is known to be a risk factor to delay the wound healing process. in a murine model of cnv, areas that underwent laser - induced rupture of bruch 's membrane are larger in mice after nicotine exposure. human choroidal and retinal arterial endothelial cells express nicotinic acetylcholine receptors (nachr), and nicotine enhances their proliferation, migration, and tube - forming ability. nicotine also exerts a vasoconstrictive action via -adrenergic stimulation which may impair blood flow through the choroid. nornicotine, a metabolite of nicotine catalyses, can lead to the accumulation of lipofuscin and, therefore, also contribute to the formation of drusen in rpe. nornicotine can catalyze the alkene isomerization of key retinal intermediates through iminium - ion formation and disrupt proper retinoid homeostasis, revealing an underlying molecular mechanism for tobacco - dependent pathologies, particularly amd. most of the toxic effects of dioxins are mediated by the cytosolic dioxin receptor known as aryl hydrocarbon receptor (ahr) [54, 55 ]. dioxin acts on ocular tissues through the ahr pathway, promotes vascular endothelial growth factor (vegf) production in mouse retinal tissues and human rpe cells, and exacerbates the development of laser - induced cnv in mice. the oxidative damage to the rpe contributes to the development and progression of amd, and the alterations in the metabolic support of the rpe cause apoptosis of the photoreceptors [57, 58 ]. nicotine promotes nitric oxide (no) production, and the effect of other proangiogenic growth factors. cadmium accumulates preferentially in the rpe and choroid and may contribute to the development of amd through an increase in reactive oxygen species (ros). however, hydroquinone (hq) is the most abundant oxidant and is not only in cigarette smoke but also in processed foods, plastic containers, and atmospheric pollutants as well as its widespread occurrence in nature [61, 62 ]. the rpe cells provide support for the structure and function of the outer retina by secreting several cytokines including monocyte chemoattractant protein-1 (mcp-1) [63, 64 ]. rpe cells after exposure to hq can secrete mcp-1 during inflammatory responses promoting macrophage dysfunction. mcp-1 expression is markedly decreased in rpe cells in smoker amd patients and might play a key role in the pathogenesis of amd [65, 66 ]. both in vitro and in vivo findings suggest that hq - induced oxidative damage is unequivocally associated with an imbalance between vegf and pedf leading to pathological angiogenesis for the development of cnv. rpe cells from smoker amd patients exhibit vegf expression increase and pedf expression decrease [6567 ]. the exposure to cigarette smoke and hq results in rpe membrane blebbing and sub - rpe deposits in mice. in cultured rpe cells, hq - induced oxidative injury results in reorganization of actin cytoskeleton and blebs formation important for accumulation of deposits [6567 ]. after exposure to hq, phosphorylate heat shock protein 27 (hsp27) expression increases, and there is an f - actin reorganization required for rpe - derived bleb formation. therefore, phosphorylated hsp27 might be a key mediator in amd. cigarette smoke extract (cse) is widely used for in vitro models [6870 ]. cse causes oxidative damage to human rpe cells in vitro, cell death, significantly reduces viability in both arpe-19 cells and primary rpe cells, via alterations in mitochondrial integrity, and increases lipid peroxidation. lower concentrations of cse may induce ros release and thus cause oxidative stress in primary human rpe cells. treatment of primary human rpe cultures with cse could significantly increase the proportion of -galactosidase (sa--gal) cells. sublethal concentrations of hydrogen peroxide have been shown to induce senescence associated to sa--gal activity in primary cultured rpe cells and in vivo in rpe cells of old primate eyes. exposure of primary human rpe cells to cse may also lead to significant elevations of apo j, ctgf, and fibronectin expression, which are senescence - associated biomarkers. all three biomarkers are inducible by oxidative stress [75, 76 ] and have been previously detected in the rpe of amd donor eyes, although its role and function in the rpe remain unclear. oxidative stress is thought to be essential in lipofuscin and drusen formation [77, 78 ]. acrolein, an unsaturated aldehyde found in the gas phase of cigarette smoke, exerts an oxidant - mediated damage by inducing protein modifications. rpe cells exposed to acrolein show a decrease in viability and mitochondrial membrane potential due to oxidative stress. in the acrolein rpe model, there is a significant decrease in mitochondrial membrane potential, oxygen consumption, and activity of mitochondrial complexes, and it increased significantly the calcium - ion level. polycyclic aromatic hydrocarbons (pahs) are one of the most toxic compounds in cigarette smoke. benzo(a)pyrene (b(a)p) is a pah with toxic effects on cultured rpe and rpe / choroid from bovine exposed to chronic cigarette smoke. it causes extensive mitochondrial dna damage and increases lysosomal activity, formation of a reactive epoxide, and caspase - mediated cell apoptosis of human rpe cells perhaps through the generation of epoxides. these altered cell biological processes in the rpe may contribute to the formation of drusen in individuals who are cigarette smokers and underlie susceptibility to genetic mutations associated with amd. arpe-19 cells, a spontaneously arising human rpe cell line, are widely used for in vitro studies of cigarette smoke effect in rpe cells. these cells are treated with different toxic substances derived from cigarette smoke such as hq, acrolein [79, 85 ], cse [71, 72 ], b(a)p, cadmium, and 2,3,7,8-tetrachlorodibenzo - p - dioxin (tcdd). recently, the effect of the hq has been studied in a combination of three cellular lines : arpe-19 cells, rat retinal neurosensory cells (r-28), and human microvascular cells (hmvec), to demonstrate that nonapoptotic cell death can occur in many forms after the damage. human donor eyes obtained from the eye bank are processed to obtain human rpe cells. these cellular lines are exposed to hq and cse to demonstrate a decrease in the viability after damage. on the other hand, bovine rpe cells also are used in in vitro experiments to study the effect of exogenous b(a)p. other types of experiments include rpe / choroids from mice to analyze the treatment of hq [56, 65 ]. as in in vitro models, the same toxic compounds are used in animals to investigate the effect of cigarette smoke in the retina. hence, these mice receive hq orally in drinking water for a period of time and are injected intraperitoneally with tcdd. on the other hand, mice are placed into a smoking chamber for a period of time to analyze the effect of the cse. control mice are kept in a filtered - air environment [66, 86, 87 ]. cigarette smoke has been also studied in rpe sheets from rats exposed to nicotine in drinking water. the rpe constitutes a cell monolayer that is crucial to maintain a normal photoreceptor function. rpe cell apoptosis and basal deposits, or accumulations of heterogeneous debris in bruch 's membrane, are two critical histopathologic changes that are well recognized to occur during the development of early amd. fujihara. observed these changes in mice after chronic exposure to cigarette smoke, and espinosa - heidmann showed that shorter duration and higher concentration of cigarette smoke in old mice induce ultrastructural changes to bruch 's membrane and the choriocapillaris endothelium that are compatible with early amd. in summary, the most important alterations observed are bruch 's membrane thickening, mild basal deposits and enlargement, and loss of basolateral infoldings, which are an established marker of epithelial cell injury. in order to look for the most appropriate therapies and to individualize lifestyle recommendations, it is ideally necessary to integrate the different clinical features, the habits, and, if available, the biomarkers of a certain disease. a biomarker is a characteristic objectively measured and evaluated as indicator of physiologic / pathologic processes or pharmacologic responses., very limited information exists about biomarkers in smokers and type, stage, and progression of amd or clinical response to treatment. evaluated the association of serum c - reactive protein (crp) levels and the risk of amd, showing that in the smoking population this risk was increased more than 1.7-fold in the lower pcr levels (crp there was no association between smoking and amd in the highest level of crp (crp > 4.5 mg / l). however, the crp levels were significantly higher among participants with advanced amd (case patients) than among those with no amd (controls ; median values : 3.4 versus 2.7 mg / l ; p =.02), so the highest levels of crp seem to increase the risk of amd independently of smoking. in a subsequent study, seddon. found that smoking had a positive association with some proinflammatory cardiovascular disease biomarkers such as crp, interleukin 6, soluble tumor necrosis factor alpha receptor 2, soluble intercellular adhesion molecule-1 (icam-1), and apolipoprotein b (apob) but not with vascular cell adhesion molecule-1 (vcam-1) or lipoprotein(a) in nonexudative amd. gibson. assessed the levels of plasmatic complement component c1 inhibitor (c1inh), and they found that c1inh levels were higher in smokers compared to nonsmokers. other biomarkers have been studied separately in smokers versus nonsmokers and amd patients versus normal patients and a parallel increase (e.g., increased levels of lipid peroxidation products) and decrease (e.g., decreased levels of antioxidants) of considered markers have been found in both kinds of studies [89, 9498 ]. there is limited information about the specific treatment of dry and wet amd in smokers. the use of antioxidant supplementation consisting of vitamin c (500 mg), vitamin e (400 international units), beta - carotene (15 mg), zinc (80 mg), and copper (2 mg) demonstrated reduction of the risk of progression to advanced dry amd in the areds study with an average followup of 6.3 years. some evidence suggested that smokers taking beta - carotene supplementation had an increased risk of lung cancer [99, 100 ]. however, at the end of the study, the influence of treatment on mortality stratified by smoking status found no effect for current - smokers who took antioxidants. otherwise, the small proportion of deaths from lung cancer (0.8%) in the areds study showed no difference between treatments. more recently, the areds2 study showed that the addition of omega-3 fatty acids and/or lutein+zeaxanthin to the the original areds formulation only reduces by 10% the risk of progression to advanced dry amd or neovascular amd. moreover, there was no effect of beta - carotene elimination or lower zinc dose to the original areds formulation on progression to advanced amd. given the hypothetic risk of lung cancer due to beta - carotene supplementation, current- or former - smokers within the past year were allowed to participate in the study only in the groups not receiving beta - carotene. the incidence of lung cancer was higher in the beta - carotene (2%) group than in the non - beta - carotene group (0.9%), mainly in former - smokers (91% of participants who developed lung cancer were former - smokers). the simultaneous administration of high doses of beta - carotene and lutein+zeaxanthin may suppress serum and tissue levels of lutein+zeaxanthin due to the competitive absorption of carotenoids. the areds2 study concluded that lutein+zeaxanthin could be a safe carotenoid substitute in the areds formulation. in addition, the influence of smoking on the visual outcomes in cases of neovascular amd treated with intravitreal vascular endothelial growth factor inhibitors has also been analyzed. evidenced that smokers had a statistically significant higher risk (7-fold increase) for a poor response to intravitreal therapies, whereas other authors found better functional outcomes in nonsmokers compared with smokers but without statistical significance. the mechanism for the negative influence of cigarette smoking in the response to these therapies is poorly understood. age - related macular degeneration (amd) is the commonest cause of irreversible visual loss in the western world [16 ]. amd is a complex multifactorial disease with an uncertain etiology associated with genetic and environmental risk factors. the hypothesis that interventions trying to minimize the role of such environmental factors may reduce the incidence of amd and/or the progression to late stages has led to several studies evaluating their relevance in the clinical practice. among them, cigarette smoking is a proven risk factor for both development and progression of amd [2443 ], as well as for the clinical response in both atrophic and neovascular forms of amd [99104 ]. as has been previously described, smoking by itself promotes molecular and pathological changes that may establish an ideal macular microenvironment for the development of amd : vascular inflammation and endothelial dysregulation [4756 ], oxidative damage [5780 ], toxic damage, and histopathological changes [8187 ]. however, patients are not frequently aware of the significant role played by cigarette smoking in blindness associated with amd. quitting smoking reduces the risk of amd, and after 20 years of cessation the risk of developing amd is the same as for nonsmokers [105, 106 ]. recently, genetic testing has arisen as an option to provide patients with a certain risk profile based on their own genetic phenotypes in the high - risk genes for amd [106108 ]. this is even more relevant in smoking subjects, as a genetic high - risk profile might influence their motivation to quit smoking. in the situation described above, we believe that institutional support to disseminate the relevance of cigarette smoking in terms of visual health is warranted. very few countries show health warnings on cigarette packets related to this issue (smoking causes blindness), whereas several other health issues warnings are usually included.	age - related macular degeneration (amd) is one of the main socioeconomical health issues worldwide. amd has a multifactorial etiology with a variety of risk factors. smoking is the most important modifiable risk factor for amd development and progression. the present review summarizes the epidemiological studies evaluating the association between smoking and amd, the mechanisms through which smoking induces damage to the chorioretinal tissues, and the relevance of advising patients to quit smoking for their visual health.
drug rash with eosinophilia and systemic symptoms (dress) syndrome is an extremely serious adverse effect caused by medications, characterized by skin rash, fever, and lymphadenopathy. it generally occurs 2 to 4 weeks after treatment with the reactive medication.1 it occurs most often with the use of anticonvulsants such as phenytoin and phenobarbital, though it can also be caused by sulphasalazine, nevirapine, penicillamine, and allopurinol.1,2 allopurinol is generally considered a safe treatment medication commonly used to protect renal function by dropping proteinuria and blood pressure when administered in low doses to patients with mild degrees of chronic renal disease.3 we report a case of a patient who had been taking low - dose allopurinol for several months due to chronic renal disease, was admitted to the emergency room with a complaint of jaundice, and was diagnosed with dress syndrome after confirming there was no bile duct obstruction according to endoscopic ultrasound (eus) and magnetic resonance cholangiography (mrc). an 84 year old patient was admitted to the emergency room in our hospital with complaints of fever and jaundice. two months ago, this patient was diagnosed with stage 3 chronic renal disease and prescribed allopurinol 100 mg / day and torasemide 2.5 mg / day. his vital signs upon admission to the emergency room were 170/70 mmhg for blood pressure, 80/min for pulse rate, 20/min for respiratory rate, and 38.8 for body temperature. consciousness was clear, and severe scleral jaundice and systemic diffuse types of papule were observed (fig. 1). according to peripheral blood tests, leukocytes were 6,020/l (atypical lymphocyte 4%), eosinophil count was 840/l (13.6%), hemoglobin was 13.3 g / dl, and platelet count was 64,000/l. blood chemical tests showed a blood urea nitrogen level of 48.4 mg / dl, serum creatinine of 2.2 mg / dl, ast / alt of 140/343 u / l, alkaline phosphatase of 210 u / l, total bilirubin of 6.6 mg / dl, direct bilirubin of 4.7 mg / dl, and a prothrombin time of 55% (inr 1.52). according to serum tests, all hbsag, including anti - hbs and anti - hcv, were negative. the patient 's previously normal liver function and creatinine levels were slightly elevated (1.3 - 1.5 mg / dl). obstructive jaundice was clinically suspected ; therefore, non - enhanced abdominal computed tomography (ct) was performed to rule this out. the ct scan showed multiple calcifications along the periportal areas of both hepatic lobes and the hepatic hilum (fig. no bile duct obstructions or calcifications were detected, but multiple cysts were seen in the liver along the biliary tree (fig. an eus was then performed to rule out obstructive jaundice, but this did not reveal any evidence of biliary obstruction. the numbers of atypical lymphocytes increased to 6% 3 days after admission, and both liver function and renal function worsened, showing serum creatinine of 5.5 mg / dl, total bilirubin of 31.3 mg / dl, direct bilirubin of 21.5 mg / dl, and ast / alt of 155/303 u / l. a diagnosis of dress syndrome was made, and methylprednisolone 40 mg / day was administered. although the patient was slightly improved 8 days later, with serum creatinine of 3.0 mg / dl, ast / alt of 53/30 u / l, total bilirubin of 27.1 mg / dl, direct bilirubin of 19.4 mg / dl, and prothrombin time of 98% (inr 1.01), liver function and renal function deteriorated again 15 days later (13 days after methylprednisolone administration), and the patient complained of chest pain. dress syndrome is a relatively rare disease caused by an adverse reaction to medications, referred to sometimes as drug hypersensitivity syndrome (dhs). it is characterized by a skin rash, lymphadenopathy, fever, and single- or multiple - organ involvement. in terms of hematology, eosinophilia and atypical lymphocytes are observed, and patchy papules appear over the skin of the entire body.4 it typically manifests 4 to 6 weeks after administering the associated medication. bocquet suggested diagnosis criteria of dress syndrome (table 1) ; when all three items are satisfied, dress is diagnosed. it is reasonable to conclude the diagnosis criteria are applicable to this case the death rate is approximately 10%, and the most common cause of death is hepatic failure. the most common medications that cause dress syndrome are phenytoin, phenobarbital, carbamazepine, sulphasalazine, and allopurinal (as in this case). there is some confusion regarding the diagnosis of dress syndrome vs. stevens - johnson syndrome (sjs)/toxic epidermal necrolysis (ten),6 although the skin lesions in sjs / ten are different from those in dress. dress typically begins with a maculopapular rash, which progresses to an erythroderma or exfoliative dermatitis, often with edema on the face and limbs.7 a bullous appearance, which is caused by subepidermal edema and is not due to extensive necrosis of keratinocytes, may be observed in some cases. cell - mediated immunity (type iv reaction) directed toward allopurinol, and more importantly, to its oxypurinol metabolite, is the mechanism involved in the pathogenesis of allopurinol - induced dress syndrome.8,9 this is supported by the fact that most patients with hypersensitivity to this drug developed the illness 4 - 6 weeks after the initiation of drug therapy. the pathophysiology of allopurinol - associated dress syndrome seems to be related to the accumulation of oxypurinol in patients with renal insufficiency. hande and co - workers10 showed that the renal clearance of oxypurinol is directly proportional to the renal clearance of creatinine. patients with chronic renal failure and simultaneous use of diuretics, mainly thiazides, are at greatest risk for developing allopurinol - induced toxicity.11 by impairing allopurinol clearance, they increase allopurinol levels and may impair, as previously suggested, oxypurinol clearance.12 hyperuricemia is known as one of the aggravating factors of chronic renal disease. allopurinol administration in low doses is generally safe for patients with mild - to - moderate chronic renal disease and protects renal function by dropping proteinuria and blood pressure.13 for this reason, many chronic renal disease patients take allopurinol. however, there have been some reports14 that dress syndrome occurs in about 0.4% patients taking allopurinol, and the death rate in this case is 25%, which is significantly higher than that of dress syndrome induced by other medications.1 many physicians are unaware of serious adverse events due to allopurinol - induced dress syndrome and thus forget adequate explanations, which can lead to lots of problems. arellano reported that out of 101 cases of dress syndrome, fever occurred in 95.1% of patients, skin rash in 93.1%, and eosinophilia in 93.1%. however, because the patient in this case complained of jaundice and blood tests revealed a direct bilirubin increase, it was necessary to exclude other diseases that could cause obstructive jaundice ; consequently, eus and mrc were performed to determine whether there was a bile duct obstruction, and dress syndrome was subsequently able to be diagnosed. an report a case of dress syndrome due to antibiotics and/or antiviral treatment following hepatitis a. by comparison, in this case, jaundice - presenting dress syndrome was mistaken for a bile duct obstruction. the most important treatment for dress syndrome caused by medications is to discontinue medications as soon as possible. chopra and vittorio report that the potentially controversial use of steroids sometimes results in dramatic improvements in clinical symptoms and hematological examination findings after their use, while markel report that caution should be given regarding potential recurrence after reducing steroid dosages. although complete cure was not achieved, steroid treatment resulted in some improvements in laboratory and clinical situations during early periods of dress. even though it has been reported that most dress syndrome deaths are due to liver damage,1 there have been reported cases of death caused by cardiac attack occurring due to fulminant myocarditis.18 the patient in this case indicated chest pain on the day of his death, there was a finding of increased troponin i in blood tests right before death occurred, and the patient died from a sudden cardiac attack ; consequently, it is assumed that his death was caused by cardiac damage that occurred due to dress syndrome. in hypersensitivity myocarditis, electrocardiography evaluation often shows nonspecific st segments, t - wave abnormalities, conduction delays, or sinus tachycardia. elevation of cardiac enzymes also occurs, and echocardiography shows systolic dysfunction and occasional pericardial perfusions. sudden cardiac death has also been reported.19,20 in hypersensitivity myocarditis, an eosinophilic and a mixed lymphohistiocytic infiltrate without necrosis or fibrosis is seen.20 but, the diagnosis of myocarditis is still largely based on clinical criteria using ecg, echocardiography, and cardiac enzymes rather than biopsy. in conclusion, allopurinol is a relatively safe medication ; however, when dress syndrome occurs, it can be fatal. therefore, close observation is required, along with adequate education, after its administration. in particular, if jaundice and fever are indicated as main symptoms, as seen in this case, there may be a potential delay in the diagnosis of dress ; thus special cautions should be taken regarding the medication histories and symptoms of patients.	an 84-year - old man was admitted to our hospital with fever, jaundice, and itching. he had been diagnosed previously with chronic renal failure and diabetes, and had been taking allopurinol medication for 2 months. a physical examination revealed that he had a fever (38.8), jaundice, and a generalized maculopapular rash. azotemia, eosinophilia, atypical lymphocytosis, elevation of liver enzymes, and hyperbilirubinemia were detected by blood analysis. magnetic resonance cholangiography revealed multiple cysts similar to choledochal cysts in the liver along the biliary tree. obstructive jaundice was suspected clinically, and so an endoscopic ultrasound examination was performed, which ruled out a diagnosis of obstructive jaundice. the patient was diagnosed with dress (drug rash with eosinophilia and systemic symptoms) syndrome due to allopurinol. allopurinol treatment was stopped and steroid treatment was started. the patient died from cardiac arrest on day 15 following admission.
many stroke survivors exhibit difficulties with object manipulation when using the paretic hand for grip initiation and termination. grip termination in the paretic hand takes approximately three - fold longer than in the non - paretic hand, which results in serious difficulties in releasing a grasped object in a timely fashion. delays in initiating and terminating, i.e., relaxing, have also been observed for the paretic wrist and lower limb in stroke survivors. this delay in grip termination / release is attributed to sustained finger flexor muscle activity after grip. sustained finger flexor activity after a voluntary contraction, e.g., grip, has been consistently observed in stroke- survivors. it has been ascribed to spastic hypertonia of the finger flexor muscles and inappropriate co - activation between finger flexors and extensors during finger extension, e.g., an attempt to extend fingers paradoxically results in flexion. botulinum toxin (bt) injection is commonly used to manage focal spasticity and is recommended as the first - line treatment for post - stroke spasticity, such as finger flexor hypertonia. the toxin blocks acetylcholine release pre - synaptically and inhibits muscle contraction temporarily, consequently decreasing spasticity for several months. a recent study reported that bt type a injection is useful for improving basic upper limb tasks, such as hand hygiene and facilitation of dressing. however, the study reveals that injection is unlikely to be useful for improving active upper limb functions, such as reaching and grasping. the present study demonstrated improved voluntary motor control of the hand after bt injection to the spastic finger flexors in a chronic stroke patient, who exhibited severe finger flexor spasticity, but residual weak voluntary finger extension. a 53-year - old, right - handed female (height 1.54 m ; weight 48.2 kg) sustained a hemorrhagic right middle cerebral artery stroke 3 years earlier. the patient presented with left spastic hemiparesis involving, among others, the finger flexors. in addition, she exhibited weak voluntary finger and wrist extension. the patient underwent extensive physical and occupational therapy after the stroke and up to the time of presentation. a total of 50 units onabotulinum toxin a (botox, allergan, irvine, ca, usa) was administered to the left flexor digitorum superficialis (fds) and flexor digitorum profundus, respectively. the injection was guided by percutaneous electrical stimulation of the motor points of these muscles. after providing written consent, the subject attended three laboratory sessions before (pre = day 0), after (post 1 = 10 days), and at the follow - up (post 2 = 2.5 months) injection. delays in grip initiation and release were assessed using electromyographic (emg) signals in paretic and non - paretic sides, respectively. bilateral grip strength was measured using a hand - held dynamometer (lafayette instruments, lafayette, in, usa), and the functional independence measure (fim) was used to evaluate clinical improvement. during grip initiation and release tasks, the subject sat comfortably in a chair with a backrest to provide proper trunk support. the limb was supported and secured firmly on a customized apparatus on a desk and was placed with 45 shoulder abduction and flexion and a 90 elbow flexion. the wrist was placed and secured in a neutral position (with thumb pointing upward), and the hand was placed around a customized hand - held apparatus. the apparatus was fixed to the table and was adjusted to accommodate hand size, adjusting for comfort and a firm grip. first, the non - paretic hand was tested, followed by the impaired hand. in each 20-second trial, the subject was asked to relax the fingers for the first 10 seconds, maximally grip the apparatus and hold for 5 seconds, and then relax the hand for 10 seconds. computer - generated audible tones cued the subject when to start and stop grip efforts. the subject was instructed to grip the apparatus as quickly as possible in response to the first tone (start), and then relax the hand as quickly as possible when the second tone (stop) was played. the patient practiced five trials prior to the grip initiation and release task, which allowed for familiarizing with the setting and task. bipolar active surface electrodes (delsys, boston, ma, usa) were placed over the muscle bellies of fds and extensor digitorum communis (edc). the fds muscle is the primary mover in power grip, and edc muscle activity was recorded to monitor antagonist muscle activity. for consistency, electrode locations were recorded, and electrodes were placed at identical locations for all sessions. emgs were recorded at 1 000 hz using a delsys amplifier and data acquisition system (national instruments, austin, tx, usa) controlled by custom labview program (national instruments). emg signals were processed and analyzed offline using a custom matlab program (the mathworks, natick, ma, usa). emg signals were initially band - pass filtered (10 - 400 hz), rectified, and then root - mean - square smoothed with a 40-ms time constant to create envelopes that clearly displayed aggregated muscle activity. the teager - kaiser energy operator was used to analyze surface emg to improve the signal - to - noise ratio, as well as to optimize emg onset / offset detection. the baseline fds emg was calculated as values for the emg averaged across a 0.5-second window during the initial resting period of each trial. the fds emg onset time was identified when the emg value increased more than the mean value plus 3 standard deviations of the baseline values. grip initiation delay was calculated as the time difference between emg onset and the start audible tone (figure 1). a representative electromyography of flexor digitorum superficialis (fds) and extensor digitorum communis (edc) before and 10 days after botulinum toxin injection. the subject was asked to grip as soon and as hard as possible after the grip signal and relax after the release signal (dash lines). the release delay time decreases after injection, along with shortened edc activities. the fds emg offset time was identified as the time when emg values fell below the mean plus 3 standard deviations of the baseline values. the release delay was then calculated as the time difference between emg offset and the stop after bt injection to the finger flexor muscles (fds and flexor digitorum profundus) of the paretic left hand, grip strength decreased at 10 days post - injection and returned to pre - injection levels at 2.5 months post - injection (pre vs. post 1 vs. post 2 : 2.3 vs. 1.0 vs. 2.2 kg), while grip strength of the non - paretic hand remained stable (pre vs. post 1 vs. post 2 : 22.7 vs. 20.4 vs. 22.2 kg). in parallel, the mas scores of the affected finger flexors were as follows : pre vs. post 1 vs. post 2 : 2 vs. 0 vs. 1 for fds ; 2 vs. 0 vs. 1 for flexor digitorum profundus. fim scores remained unchanged (fim = 6 for pre, post 1, and post 2), but the subject reported an improved overall ability for handling objects, as well as bimanual activities at home while performing activities of daily living in post 1 and post 2 measures. release delay time in the paretic hand resulted in dynamic changes (pre : 2 223 ms ; post 1:1 024 ms ; post 2 : 1 333 ms), without dramatic changes in grip initiation delay times in the paretic hand (pre : 417 ms ; post 1 : 439 ms ; post 2 : 378 ms). it is noteworthy to mention that a decreased fds release delay time was paralleled by shortened edc and edc emgs (figure 1). in contrast, both grip initiation and release delay times in the non - impaired hand remained relatively stable in all measurements (pre vs. post 1 vs. post 2 : 468 ms vs. 404 ms vs. 371 ms for grip initiation delay ; 725 ms vs. 740 ms vs. 691 ms for release delay). results from the present case report demonstrated that bt injection to spastic finger flexors in a chronic stroke patient improved grip initiation and termination. the magnitude of grip initiation and release delay time in the paretic hand was within previously published ranges. the pattern of changes in initiation and release delay time after the bt injection, however, could shed light on the mechanisms underlying grip release delay in chronic stroke survivors. given the unchanged grip initiation delay time in the paretic hand after bt injection, as well as stable grip initiation and release delay time in the non - paretic hand in the follow - up measurements, improvement in grip release time was not likely related to practice / learning effect. previous results have shown that bt injections can paralyze afferent fibers, in addition to blocking acetylcholine release pre - synaptically at neuromuscular junctions. this would release the presynaptic and/or reciprocal inhibitory effect exerted by the finger flexor afferents on the motoneurons of finger extensors during attempted voluntary finger extension, i.e., reduced inhibition from paralyzed finger flexors after bt injection. this interpretation is further supported by two findings in this case study : (1) duration of extensor activity was dramatically shortened. this apparently resulted from a reduced need of the finger extensors to counteract reciprocal inhibition from the finger flexors ; (2) grip release delay time increased in conjunction with the finger flexor mas score at 2.5 months post - injection. this parallel change suggested that increased reciprocal inhibition from hypertonic finger flexors resulted in prolonged grip release delay. functional improvement of the hand after bt injection is likely to be case - specific. selected patients with spastic finger flexors, who exhibit residual voluntary finger extension, are likely to have this potential as a result of reduced reciprocal inhibition from paralyzed finger flexors after the injection. following the bt injection to spastic finger flexors in a chronic stroke patient, grip strength of the paretic hand decreased at 10 days post - injection and returned to the pre - injection level at 2.5 months post - injection. dynamic changes in paretic hand - grip strength correlated with dynamic changes in finger flexor hypertonia. the unchanging fim scores after bt injection could be explained by the ceiling effects of this measurement. however, patient subjective improvement in overall motor control correlated with laboratory measurements of improved release delay time in the paretic hand. given that the patient suffered from residual weak voluntary finger extension, it is possible that reduced reciprocal inhibition from the finger flexors after bt injection could result in increased ability to handle objects and bimanual activities. the present case report demonstrated that bt injection improved voluntary motor control of the hand in a chronic stroke patient with residual finger extension.	the effect of botulinum toxin type a injection on voluntary grip control was examined in a 53-year - old female, who sustained a hemorrhagic right middle cerebral artery stroke 3 years previously, which resulted in finger flexor spasticity and residual weak finger / wrist extension. the patient received 50 units of botulinum toxin type a injection each to the motor points (2 sites / muscle) of the left flexor digitorum superficialis and flexor digitorum profundus, respectively. botulinum toxin injection led to weakness and tone reduction in the spastic finger flexors, but improved grip release time in grip initiation / release reaction time tasks. improved release time was accompanied by shortened extensor electromyography activity, and improved release time likely correlated with blocked co - contraction of finger flexors during voluntary finger extension. this case report demonstrated that botulinum toxin injection improved voluntary motor control of the hand in a chronic stroke patient with residual finger extension.
debonding orthodontic fixed appliances is a critical stage during and at the end of comprehensive fixed orthodontic treatment. others used instruments that are not manufactured specifically for debonding such as weingart pliers, howe pliers, and straight wire cutters. multiple in vitro and some in vivo studies have assessed and compared the effect of such instruments on the debonding strength, enamel surfaces, adhesive remnants, as well as on bracket breakage, with varied results. the most common type of forces applied at debonding are shear, tension, compression, and torsion (torque). the force application type, magnitude, location, direction, and angulation are some of the major factors affecting the final bond strength value. the in vivo versus in vitro environments have also shown to give different results. the reported bond strength measured in vivo multiple finite element analysis highlighted the factor of the nonhomogenous distribution of stresses on the enamel, bracket, adhesive, alveolar bone, as well as on the periodontium with the different debonding forces applied at debonding. this could be another added attribute to the great variability of results on debonding strengths reported in the literature. the debonding pliers, which are specifically designed for debonding orthodontic brackets, are designed to produce shear / peel, compressive, or tensile forces depending on the type of pliers used, direction (occluso - gingival or mesiodistal), and location of force application (at bracket wings or bracket base). su., compared in vitro four different methods of debonding using clinically simulated models. they found that applying a debonding force on the brackets in an occluso - gingival direction at the bracket base produced significantly lower bond strength and less adhesive remnants than applying the same force in the same direction but at the bracket wings. thus, they concluded that force at bracket base in an occluso - gingival direction is the least damaging to enamel when using compressive force. however, they used different pliers for the two latter methods, which makes it difficult to conclude with regards to the best plier or debonding method to be used. furthermore, brosh. reported the opposite results in debonding strength when assessed the two compressive methods in vivo, with no significant difference in the amount of adhesive remnants prietsch., compared three debonding methods ; tensile force applied by the instron machine, shear force applied at bracket base by the instron machine, and compression force applied in an occluso - gingival direction at the bracket base by a specially designed device. they found that the mean bond strength for shear (7.71 mpa) was statistically higher than for compression (2.98 mpa) and tension (2.69 mpa) with no significant difference between the latter two methods. in their in vivo study, used a lift - off debonding instrument, straight cutter plier, how plier, and the conventional bracket removal plier. the latter instrument was used to apply a squeezing force at the bracket base in a mesiodistal direction. they found that the conventional bracket removal plier as used in their study seemed to result in a lot of adhesive remnants after debonding. the objective of the current study was to assess and compare in vitro ; the quantitative and qualitative differences in debonding behavior between compressive strengths produced by an aez debonding plier at bracket base in an occluso - gingival direction and shear bond strengths (sbss) at bracket base produced by the instron machine in debonding orthodontic metal brackets. in this in vitro study, two different debonding methods were assessed and compared quantitatively and qualitatively. the first method was the regular laboratory method that produced sbss by a load applied at bracket base by the blades of an instron universal testing machine (electroplus e1000, instron, canton, ma, usa) and directed from gingival to occlusal [figure 1 ]. the second method simulated the clinical situation where a compressive load at bracket base in an occluso - gingival direction was applied by an aez narrow blade debonding plier mounted on an instron machine, as proposed by bishara. and diagram showing the shear debonding force applied by the instron blades from gingival to occlusal direction at debonding diagram showing the compressive debonding force applied by the aez narrow blade debonding plier in an occluso - gingival direction at debonding based on 80% power of the test, 32 sound extracted premolars, with no obvious enamel cracks, were collected and stored in distilled water. teeth were then cleaned, polished with nonfluoridated pumice and rubber prophylactic cups for 15 s, rinsed with water spray for 10 s, and dried with oil - free compressed air for 10 s. the buccal surfaces were etched with 37% phosphoric acid solution for 30 s according to the manufacturer 's instructions, rinsed with water spray for 20 s, and then dried with oil - free compressed air for 20 s. all teeth were bonded with metal brackets (gemini series, 3 m unitek, usa) using transbond xt light - cured composite resin (3 m, st. excess composite was removed from around the bracket margins with the tip of a probe and then photo - polymerized from five directions : above the bracket, cervical, occlusal, mesial, and distal for 20 s each (ortholux, 3 m unitek, monrovia, ca, usa) (light output, 430480 nm). teeth were then stored in distilled water at 37c in an incubator for 1 week to ensure complete polymerization of the adhesive resin. each tooth was then embedded in an acrylic block so that only the buccal surfaces are exposed for testing. the sides of the blocks were trimmed straight to act as a flat surface at which the blocks were screwed on the attachments of the instron testing machine. this method was designed to help properly align the blades of the testing device parallel to the enamel surface and secure it in place for standardization. all teeth were then randomly divided into two groups (n = 16 each) ; compressive bond strength (cbs) and sbs groups. all teeth were debonded using an instron universal testing machine with a crosshead speed of 0.5 mm / min and a 50-kg load cell. the sbs group was subjected to a shear force produces by the blades of the instron machine. the force was applied by the blades on the bracket base and directed from gingival to occlusal until bond failure occurred. the cbs group was debonded with a custom - made attachment that was composed of a regular narrow blade orthodontic debonding plier (aez debonding orthodontic plier, ormco corporation, usa) mounted on the instron machine. forces exerted by the stainless steel blades of the pliers were applied at the bracket base with a squeezing action in an occluso - gingival direction until bond failure occurred. the force at failure, in newtons (n), was recorded in both groups. the debonding strength, in megapascal (mpa), was then calculated as follows : for the sbs group, the sbs was calculated using the formula f / a, where a is the surface area of the bracket basefor the cbs group, the debonding force by the instron was applied at a predetermined constant distance on the arms of the plier. thus, the actual compressive debonding strength was calculated using the formula ; for the sbs group, the sbs was calculated using the formula f / a, where a is the surface area of the bracket base for the cbs group, the debonding force by the instron was applied at a predetermined constant distance on the arms of the plier. thus, the actual compressive debonding strength was calculated using the formula ; (the actual debonding strength = recorded force [by the instron machine ] [b / a ]). where a is the distance between the blades of the plier at the bracket base and the fulcrum of the plier, while b is the distance from the fulcrum of the plier to where the force is applied on the plier 's arms by the instron machine [figure 3 ]. the conversion factor (diagram showing (a) the design of the aez debonding plier mounted on the instron machine, and (b) how the distances (a) and (b) used to calculate the actual debonding force are measured all teeth were examined under a stereomicroscope at 10, and 20 magnifications for the residual adhesives remaining on the buccal enamel surfaces. the amount of adhesive remnant was then scored using the modified adhesive remnant index (ari) as follows : score 0 : no retained resin (0%)score 1 : 50% retained resin on the enamel surface (> 050%)score 2 : > 50% retained resin on the enamel surface (> 50 050%) score 2 : > 50% retained resin on the enamel surface (> 50 050%)score 2 : > 50% retained resin on the enamel surface (> 50 050%) score 2 : > 50% retained resin on the enamel surface (> 50 0.05). the mean debonding strength for the sbs group was 6.17 0.77 mpa, and for the cbs was 6.68 1.67 mpa [figure 4 ]. mean and standard deviation of the debonding strength values (mpa) for the two groups assessed. using independent sample t - test ; no significant difference was found at p 0.05). no significant difference in the mean debonding strength was reported between the sbs and cbs groups (p > 0.05). the mean debonding strength for the sbs group was 6.17 0.77 mpa, and for the cbs was 6.68 1.67 mpa [figure 4 ]. mean and standard deviation of the debonding strength values (mpa) for the two groups assessed. using independent sample t - test ; no significant difference was found at p 0.05). the force direction and location applied in the current study using an aez narrow blade debonding plier was similar to the method developed by bishara and fehr, in which force was applied at the bracket base on both sides of the bracket simultaneously (bilateral load) in an occluso - gingival direction. bishara and fehr described such type of force as being similar to the force applied during a diametral compression test for tension. they also proposed that such method is an indirect way of measuring the tensile strength of materials that exhibited very limited plastic deformation such as ceramics, composites, and enamel. such method was further assessed by bishara. in 1994 for debonding ceramic brackets. they found that debonding with pliers required the application of 30% less force to the enamel surface than debonding with shear forces as tested in the laboratory, with no significant differences in the ari. thus, they hypothesized that applying the load to the two sides of the bracket increased the chances of starting a crack and propagating it in the brittle adhesive causing debonding to occur at a lower debonding force. however, they used it to assess the bond strength between different types of adhesives but did not use it for assessing debonding strengths. contrary to the findings of prietsch. and bishara., the current study reported no significant differences in the debonding strength between shear and compressive forces when applied at the bracket base. however, a significant reduction in the amount of adhesive remnants was found in the current study with the compressive group. the latter indicates that the site of bond failure was more toward the enamel - adhesive interface than the bracket - adhesive interface. it also shows that the debonding behavior of metal brackets using the current settings differs than that of ceramic brackets as reported by bishara., no significant correlation was found between the debonding strength and the ari scores in both groups. thus, the debonding force can not be used as a predictor to the bond failure site or enamel fracture. holberg. in his finite element analysis studies assessed four directions and locations of forces applied by pliers. he found that the least amount of stress on enamel occurred when applying lateral rotation force, which is a compressive force in an occluso - gingival direction but at bracket wings. however, he also found this method resulted in a significantly high stress on the periodontium. in contrast, he found that applying compressive loads in a mesiodistal direction at bracket wings produced a moderate maximum stress values on the enamel and alveolar bone, but with a negligible effect on the periodontium. the latter method of force application was also found to result in a bond failure that is at or just close to the enamel - adhesive interface in vitro which could explain the results of the finite element analysis. thus, holberg. recommended that lateral rotation force is the best method to be used for debonding orthodontic brackets in a healthy periodontium, while compressive force is the best to be used in a compromised periodontium. however, holberg did not assess the compressive force in an occluso - gingival direction at bracket base, which makes it difficult to compare with the findings of the current study. findings of this study highlight two issues ; the first is that any findings in vitro on sbs using the instron blades in an occlusal to the gingival direction at bracket base with similar laboratory conditions as our study can be generalized to represent the clinical situation of using aez debonding plier in an occluso - gingival direction at bracket base. the second issue is that using the aez debonding plier resulted in minimal adhesive remnant and thus less chair - time for cleanup after debonding, thus giving it an extra advantage over using sbs. further studies are needed to assess the debonding behavior of the aez debonding pliers using different forces, methods of applications, as well as comparing in vivo, in vitro, and finite element analysis. furthermore, with the current advances in technology and science, a standardized protocol needs to be developed for assessing the debonding problems in orthodontics to solve the big variability in debonding results in the literature, hence, aid in reaching comparable findings for conclusive meanings. as observed, but not investigated in this study, the aez pliers have thick beaks that might not always guaranteed that the pliers are applied at the enamel - adhesive interface. within the limitations of this study, in vitro testing of sbs by the instron blades in an occlusal to gingival direction was found to produce similar debonding strength as applying the aez debonding plier in a similar direction at bracket base. however, the aez debonding plier resulted in less adhesive remnant which is of great advantage for reducing chair - time during cleanup after debonding brackets.	objectives : to assess in vitro the quantitative and qualitative debonding behavior of the aez debonding plier, compared to shear debonding force, in debonding orthodontic metal brackets.materials and methods : thirty - two extracted premolars bonded with metal brackets were randomly divided into two equal groups according to the type of simulated debonding method ; compressive bond strength (cbs) group using aez debonding plier (ormco corporation, usa) attached to the instron machine, and shear bond strength (sbs) group using regular instron attachments. all teeth were subjected to debonding forces, and debonding strength was assessed. the buccal surfaces were then examined, under a stereomicroscope, and adhesive remnants were scored using adhesive remnant index (ari). debonding strengths comparison was performed using the independent sample t - test. ari score comparison was performed using the mann whitney u - test. correlation between debonding strength and ari scores was performed using the spearman correlation.results:there was no significant difference in mean debonding strength between the sbs (m = 6.17 0.77 mpa) and cbs (m = 6.68 1.67 mpa) groups (p > 0.05). the cbs group showed significantly less adhesive remnants than the sbs group (p < 0.05) ; 62.5% of cbs group had ari score 1, whereas 68.8% of sbs group had ari score 3. no significant correlation between ari and debonding strength was found (p < 0.05).conclusion : sbs was found to produce similar debonding strength to the aez debonding plier in vitro. however, the aez debonding plier resulted in less adhesive remnant which is of great advantage for reducing chair - time during cleanup after debonding brackets.
elizabethkingia meningosepticum (formerly known as chryseobacterium meningosepticum / flavobacterium meningosepticum) is a nonfermentative gram negative bacillus, ubiquitous in nature [1, 2 ]. e. meningosepticum causes meningitis, pneumonia, bacteremia, and sepsis in infants and pneumonia, endocarditis, postoperative bacteremia, and meningitis in adults [13 ]. among the different infections, a high mortality and severe postinfection sequelae including hydrocephalus, deafness, and developmental delay infections caused by e. meningosepticum are difficult to treat because of its resistance to extended spectrum -lactam agents and aminoglycosides [13 ]. in this study we report the clinical profile, antibiotic susceptibility, and treatment outcome of meningitis caused by multidrug resistant elizabethkingia meningosepticum in neonates. nine neonates with e. meningosepticum meningitis who presented to the neonatal intensive care unit of niloufer hospital for women and children, hyderabad, india, between january 2009 and december 2010, were included in the study. cerebrospinal fluid collected from the neonates was subjected to direct microscopic examination, inoculated on to blood agar, chocolate agar, and macconkey 's agar, and incubated overnight at 37c. blood cultures were done by automated blood culture systems (bact / alert, biomerieux, france). the isolates were identified by conventional biochemical reactions and vitek 2 (biomerieux, france). sequencing was performed (forward primer, 5-ttggagagtttgatcctggctc-3 ; reverse primer, 5-ggactaccagggtatctaa-3) with fluorescence - labeled dideoxynucleotide terminators using an abi 3130 xl automated sequencer, following the manufacturer 's instructions (pe applied biosystems). the sequences were analysed and identified using the megablast search program of the genbank database. the sequence of the isolate perfectly (100%) matched the sequences of e. meningosepticum deposited in genbank. antibiotic susceptibility of the isolates was done by kirby bauer 's disk diffusion method and also by vitek 2. gram 's stain of csf showed polymorphonuclear leukocytes and gram negative bacilli in all the nine patients. confluent growth of moist raised colonies was seen on blood and chocolate agar in all patients. blood culture was positive and showed the growth of moist raised colonies on subculture on blood and chocolate agar. all the isolates were non motile, and catalase and oxidase positive, indole positive, citrate and urease negative, ortho - nitrophenyl - beta - galactoside positive, gelatin was liquefied after 48 hours ; oxidative fermentative test was positive after 72 hours. antibiotic susceptibility and genbank accession numbers of the isolates were shown in table 1. clinical features and treatment outcome of the nine patients with e. meningosepticum meningitis the data on antibiotic susceptibility of e. meningosepticum is limited because it is rarely isolated from clinical specimen and there are no standard guidelines on antibiotic susceptibility testing and reporting and interpretation of the susceptibility data. according to the published literature e. meningosepticum is known to be resistant to -lactams, extended spectrum cephalosporins, carbapenems, and gentamicin, susceptible to vancomycin, trimethoprim - sulfamethoxazole, rifampicin, and ciprofloxacin, and moderately susceptible to piperacillin. in the present study we have noted that all the isolates are resistant to -lactams, extended spectrum cephalosporins, -lactam combinations, carbapenems, aztreonam, aminoglycosides, tetracyclines, and trimethoprim - sulfamethoxazole and only one isolate has been susceptible to ciprofloxacin. determination of antibiotic susceptibility by disk diffusion is not a recommended method, but there is no option in vitek to check the susceptibility of nonfermenting gram negative bacilli to vancomycin. there is no discrepancy between the susceptibility results of the isolates by vitek and disk diffusion to the remaining antibiotics. antibiotic susceptibility data of our study highlights that the majority of isolates (8/9) are multidrug resistant (resistant to all drugs except vancomycin). the underlying host factors associated with e. meningosepticum meningitis in neonates are prematurity and low birth weight and we also observed the same risk factors [4, 5 ]. based on the susceptibility of the isolates only to vancomycin, we have used vancomycin in 7/9 neonates and one neonate received combination of vancomycin and ciprofloxacin antibiotics. only two neonates recovered from infection after using vancomycin and the remaining 7 expired. according to the previous reports vancomycin is not an effective antimicrobial agent to treat the e. meningosepticum meningitis [2, 6, 7 ]. based on our study findings and other reports the efficacy of vancomycin in treating e. meningosepticum meningitis in neonates is questionable. previous authors treated neonates with e. meningosepticum meningitis successfully with piperacillin in combination with rifampicin. the mortality rate (6/9) in our study is high compared to other studies. several outbreaks of e. meningosepticum meningitis in neonates have been reported [8, 9 ]. the outbreaks reported in the literature occurred within few weeks to months except in one study, where the authors reported an outbreak with one strain of e. meningosepticum for 2 years. all the nine cases reported in the present study occurred over a period of one year. we have tried to identify the source of infection in the present study by collecting environmental specimens like water from incubators, tap water, suction fluids, the disinfectants and healthy babies were also screened for asymptomatic carriage by collecting rectal and umbilical swabs. we also carried out the gene sequencing of the isolates from seven patients and phylogenetic tree was constructed to see the genetic relatedness of the isolates (gene sequences of the isolates deposited in the genbank) and found that none of the isolates are genetically related to each other. in the present study we could not identify the source of infection. though the organism shows susceptibility in vitro to vancomycin, its efficacy in vivo is questionable and it is difficult to determine the most appropriate antibiotic for treating e. meningosepticum meningitis in neonates.	clinical and microbiological profile of 9 neonates with meningitis by elizabethkingia meningosepticum identified by 16s ribosomal gene sequencing was studied. all the clinical isolates were resistant to cephalosporins, aminoglycosides, trimethoprim - sulfamethoxazole, -lactam combinations, carbapenems and only one isolate was susceptible to ciprofloxacin. all the isolates were susceptible to vancomycin. six of nine neonates died even after using vancomycin, based on susceptibility results. e. meningosepticum meningitis in neonates results in high mortality rate. though the organism is susceptible to vancomycin in vitro, its efficacy in vivo is questionable and it is difficult to determine the most appropriate antibiotic for treating e. meningosepticum meningitis in neonates.
as a metabolic disorder, osteoporosis is characterized by low bone mass and bone microarchitectural deterioration. arteriosclerosis and atherosclerosis as a specific type of arteriosclerosis are phenomena that results with an increase of vascular stiffness and blood flow restriction. both osteoporosis and atherosclerosis share common risk factors including hypertension, smoking, diabetes, and sedentary lifestyle. osteoporosis and osteopenia have been found to be associated with higher cardiovascular events rates, arterial wall calcification, and more extensive atherosclerosis. arteriosclerosis refers to reduced arterial compliance due to increased fibrosis, loss of elasticity, and vessel wall calcification affecting the media of large and middle - sized arteries, with consecutive increase of pulse wave velocity (pwv) as measure of arterial stiffness [4, 5 ]. parameters that describe vessel stiffness include compliance and distensibility. the consequence of reduced compliance / distensibility is an increased propagation velocity of the pressure pulse along the arterial tree, called pwv. pwv reflects the elasticity of the segmental artery, stiffer segment of artery results in an increased speed of pulse wave in the artery, which means higher pwv. these facts accompany the aging process and share common increase of arterial pwv as a measure of vascular stiffness. several studies have examined associations between atherosclerosis at different sites and osteoporosis or low bone mineral density (bmd) in women, and the findings suggest that the development of osteoporosis is a risk for advanced atherosclerosis after menopause (as discussed by hak. association between bmd and vascular stiffness has been the subject of research in several studies (as discussed by mikumo. these latest data support the correlation of atherosclerosis and osteoporosis, indicating the parallel progression of two tissue (bone and vascular) destruction process with increased fatal and nonfatal cardiovascular events, as well as higher fracture risk [911 ]. the progression of these two parallel processes of destruction (bmd and arterial stiffness) was the target of research in our previous studies [1214 ]. arterial stiffening is a marker for increased cardiovascular risk, including myocardial infarction, heart failure, and total mortality, as well as stroke, dementia, and renal disease. low bmd and bone loss appear to be risk factors for cardiovascular mortality in both women and men. for years, osteoporosis and cardiovascular disease (cvd) were thought to be independent chronic diseases that increased markedly with advancing age. a variety of factors that influence bone metabolism are involved in the development of atherosclerosis and vascular calcification. interestingly, several bone - related proteins are implicated in the calcification process resulting in mineral deposition. there are limited studies on the association of bmd and arterial stiffness with cvd events rates. evidence for an association of high pwv, as a measure of arterial stiffness, with low bmd might have implications for screening decision in patients with low bone mass and vice versa. the commonly used imaging modalities to assess bone mass and vascular stiffness use the following imaging technologies of x - radiography and ultrasonography : dual - energy x - ray absorptiometry (dxa) and pulsed - doppler ultrasound. data acquired from both the femur and anterior - posterior (ap) spine dxa scans are considered gold standards for diagnosing osteoporosis. although bmd is considered a reasonable surrogate measure of bone strength, it does not capture aspects of bone geometry that may contribute to fracture risk, such as bone size, shape, and trabecular and cortical properties of bone. carotid - femoral pwv is considered to be the gold standard for assessing central arterial stiffness [10, 19, 20 ]. the aim of our study is to assess the correlation between bone strength and arterial stiffness and their impact on cardiovascular mortality in general population. this study was designed as a longitudinal prospective analysis in 558 patients from the general population (226 men and 332 women) who underwent noninvasive dxa and pwv measurements at the baseline, accompanied by 36-month (35.28 4.13) follow - up period. they had a mean age of 56.24 11.62 years, and their mean body mass index (bmi) was 27.85 4.37 one hundred and sixty - four patients were smokers, 61 were insulin - independent diabetes, and 190 were hypertensive. exclusion criteria were chronic renal disease, insulin - dependent diabetes, degenerative disease (osteoarthritis, chronic obstructive pulmonary disease, friedreich 's ataxia, marfan 's syndrome, etc.), rheumatoid arthritis, liver disease, malignancy, or any other chronic disease that might affect the skeleton or cardiovascular system (stroke, myocardial infarction, and peripheral vascular occlusion). demographic and disease history data were collected from the patient 's chart and include age, height, weight, history of hypertension, diabetes mellitus, smoking habit, and the diseases mentioned above, which might affect the bone mass and cardiovascular system. carotid - femoral pwv was assessed by pulsed - doppler ultrasound synchronized with electrocardiography (ecg). each patient was observed from a baseline during 3 year, for lethal event caused by cardiovascular disease. cardiovascular mortality was defined as death whose cause was one of the following cardiovascular events or sudden death : cerebrovascular disease (stroke), arrhythmia, peripheral vascular disease, congestive heart failure, or myocardial infarction. all participants signed an informed consent and the ethics committee of our institution approved the study. we conducted bmd testing using dxa and arterial stiffness estimation by doppler sonography in the same day at the beginning of the study (baseline). dxa is enhanced form of x - ray absorptiometry that is used to measure bmd. a dxa scanner is an electronic machine that produces two different x - ray beams, each with different energy levels : low and high. during dxa scan, the soft tissue and the bone will absorb some radiation, and some will travel through the body. measurement of bmd is based on the difference in absorption of the high and low level x - ray beams across soft and mineral density tissue. measurement of the bmd is based on the calculated difference of the bone density measurement and of the soft tissue measurement. doppler ultrasound scanner is a device, which can be used for measurement of blood flow velocities by high frequency probe. we used it to detect the flow signal in carotid and femoral artery to find time delay of blood circulation between these two sites. we conducted bmd measuring using dxa scanner qdr4500sl by hologic (hologic inc., bedford, ma, usa) in two locations : femoral neck and lumbar spine. for assessment of the femoral neck, the patient 's foot was placed in a brace that rotates the hip inward. for assessment of the spine, the patient 's legs were supported on a padded box to flatten the pelvis and lower the (lumbar) spine. in both cases, the detector was slowly passed over the area generating images on a computer monitor [10, 20 ]. patient laid on his back on a flat and open x - ray table. during the scan, which is carried out by radiographer, a large scanning arm with x - ray tube was passed over patient 's body to measure bone density in the lumbar spine and hip area. before we started scanning, we prescanned desired anatomic area to find the best femur neck or lumbar spine position in region of interest (roi). small deviations in bone position relative to the scanner were corrected without need to move the patient or mobile console on the bed. from previously done prescan we selected the right area for additional scanning with tool global roi toolbox. scan started in the moment when in scan parameters appeared in the window, with all parameters of scanned area : scan length and scan width, line spacing, scanned resolution, and energetic beam expressed in kvp (kilovolt peak) and mas (miliampers seconds). start scan in the same window. after the scan was finished, we selected the icon analyze scan to get specific data acquired by the previously made scan : area (cm), bmc (bone mineral content), bmd (bone mineral density), t - score, and z - score. absolute bmd values, z - scores (number of standard deviations [sds ] below an average person of the same age), and t - scores (number of sds below the bmd of a younger reference group) of the lumbar spine and right femoral neck were recorded as bmd (g / cm), t - score, and z - score (for femoral neck and lumbar spine from l1 to l4 region). the who (world health organization) defined the following categories based on bone density in caucasian females : normal bone, t - score greater than 1 ; osteopenia, t - score between 1 and 2.5 ; osteoporosis, t - score less than 2.5. we used linear 7.5 mhz probe (toshiba ssa-340a, toshiba medical system corporation, tokyo, japan) at sequential recording of carotid artery and femoral artery. first we recorded the flow wave form of the proximal (carotid artery) and then of the distal site (femoral artery). at both sites, the proposed method includes the following steps : first step is to acquire flow waveforms by doppler ultrasound at two locations within left common carotid artery (lcca) and left femoral artery (lfa) and next step is to detect the delay or the difference in arrival time of the flow wave at the two arterial locations. distances between the sampling sites d (m) are measured as straight lines between the points on the body surface. pwv measurements were taken in supine position after resting for at least 10 minutes, including a constant room temperature of 19c to 21c. two doppler waves were recorded transcutaneously at the base of the neck for the lcca and over the lfa in the groin. the examination began with the patient in a supine position after locating the lcca with b - mode at the supraclavicular level (1 to 2 cm of the bifurcation). although it is not possible to analyze the carotid and femoral waves simultaneously, they can be normalized separately with the ecg getting. the distance traveled by the pulse wave was measured over the body surface as the distance between the two recording sites. the distance was assessed using a tape measure located at the same place as the ultrasound probe, with two - dimensional guidance to localize the exact position of the analyzed arterial site. we calculated pwv as the ratio of distance (d) to transit time (tt). the tt was determined when we measured the time from r wave of three graphical deflections seen on typical ecg : q wave, r wave, and s wave, which occur, in rapid succession (qrs) complex to the foot of the waveform using digital measuring calipers. four to six (dependent of heart frequency) heart measurements were taken and the average was calculated. we acquired the distance (d) measured from the sternal notch to the femoral artery at the groin. dividing the distance tt we got carotid - femoral pwv [pwv = d (meters)/tt (seconds) ]. the tt is the time of travel for the onset of the wave over a known distance and it was estimated by foot to foot the foot of the wave is defined at the end of diastole, where the rise of the waveform begins. student 's t - test for paired data was used to compare the results from biomarkers. all tests were two - sided. a value of p 55 years) population (0.105 0.1373 this distinction certainly stems from the growing amount of calcification in the aorta that leads to a corresponding increase of aortic stiffness and pwv, consequently for each of the age groups (< 55, 7.960 1.1631 we found significant (p < 0.0001) inverse correlation of pwv and bmd fn (r = 0.327) and pwv and bmd spine (r = 0.150) by bivariate pearson 's correlation. that means that there is a significant correlation between the two processes, decreasing hip and vertebral bone density measured by dxa and increasing vascular stiffness as measured by carotid - femoral pwv. the vascular calcification that occurs in elderly and osteoporotic patients is likely responsible for a substantial increase in vascular stiffness as measured by pwv. in the adynamic state in osteoporosis, excessive bone resorption causes rapid accrual and removal of calcium and phosphorus from the bone. poor bone mineralization and excessive calcium and phosphorus in the circulation likely favor deposition of hydroxyapatite crystals in soft tissues. the fact that the study included relatively small number of diabetic 's participants with type 2 diabetes (10.93%) was the reason that strong and statistically significant associative connection between pwv and diabetes could not be shown. insulin - like growth factors and other cytokines may influence diabetic bone metabolism. in this study, there is an inverse correlation between bone strength (both for bmd fn and bmd spine) and hypertension also and between bone strength and pwv. hypertension is presumed to be relate to alteration in calcium metabolism leading to increased calcium losses, secondary activation of the parathyroid gland, and increased movement of calcium from bone. increased angiotensin ii levels in hypertensive patients have a harmful effect by increasing bone resorption and inhibiting mineralization. the presence of hypertension is an independent predictor of low bone density, as discussed by yazici., by linear regression we found inverse close correlation between pwv as a scalar - dependent variable and bmd fn as an explanatory variable. because of the negative value of b1 coefficient (4.729) in linear regression equation, we discuss that, with each increase of one unit (g / cm) in bmd fn, the pwv value decreases 4.729 times. 2014 discussed that the correlation between bmd and vascular stiffness has not been clearly established in epidemiology and clinical studies. age, smoking, hypertension, diabetes, hyperlipidemia, renal failure, physical activity, and menopause are the risk factors, and factors like inflammatory factor, oxidative ldl, osteopontin, vitamin d, and estrogen are predicted to have an effect on the correlation between bmd and vascular stiffness and osteoprotegerin has been highlighted as an important factor. in multiple regression analysis, we found an independent predictor with inverse correlation (bmd fn, p < the identification of a high - risk subset of population by dxa will be an important element of effective preventive strategies for bone resorption and atherosclerosis. detection of low bmd by dxa will set indication for diagnostic pwv estimation of arterial stiffness. by multiple regression analysis, we found diabetes and hypertension which increased pwv, as determinants with inverse correlation with pwv. bmd fn plays superior role as independent predictor over other variables (spine bmd, age, bmi, hypertension, diabetes, and smoking) because of his relative high coefficient of determination r (0.2899). that means one - third of pwv changes is dependent on bmd fn as the predictor only, versus two - thirds which belong to other determinants, above - mentioned variables. by cox - regression analysis we detected the predictor of cardiovascular mortality. pwv increases the hr risk for 1.2977 times (29.77%) with each unit increase in pwv. arterial stiffness is a cause of premature return of reflected waves in late systole, increasing central pulse pressure and the load on the ventricle, reducing ejection fraction, and increasing myocardial oxygen demand. arterial stiffness is correlated with atherosclerosis probably through the effects of cyclic stress on arterial wall thickening. aortic pwv may represent a surrogate end point, which may in fact indicate which patients with osteoporosis and the traditional cardiovascular risk factors translate into real risk. our finding (hr = 1.297) is close to summary comparative results from meta - analysis of the predictive value of pwv for cardiovascular events presented by vlachopoulos. importantly, the predictive value of increased arterial stiffness is larger of the patients with higher risk disease states, such as osteoporosis or renal disease. the principal finding of our study was that joint effects of pwv and bmd fn were strongly and independently predictive of outcome in the general population patients. the femoral neck bmd showed superior predictable value for cardiovascular events than pwv by cox - regression analysis (p = 0.015 versus p = 0.0318) in our study. the bmd fn shown in roc curves analysis superior sensitivity (88.2% versus 80.9%) and greater surface of the curve above the diagonal (0.777 versus 0.773 and 0.662, figure 3) emphasizes stronger diagnostic value of bmd fn in predicting cardiovascular event than of pwv and bmd spine. taking into consideration the above - mentioned arguments, it remains to be demonstrated whether bmd fn, which is a major determinant of bone strength, have any independent prognostic relevance for cardiovascular events. due to the small statistical difference of bmd and pwv as independent predictors (p = 0.9669), their role in cardiovascular risk predicting became equal. the appropriate cut - off values for pwv and bmd fn represent their highest sensitivity and specificity pair in detection of cardiovascular mortality. cardiovascular disease and osteoporosis as common age - related condition traditionally were considered unrelated and their coexistence was attributed to independent age - related process. however, an increasing body of biological and epidemiological evidence has provided support for a link between the two conditions that can not be explained by age alone. one of the main reasons for the increase in vascular stiffness is calcification of arteries. calcification of the arterial tissue is not merely a passive process of calcium phosphate precipitation or adsorption in end - stage atherosclerosis, but instead it is a highly organized process that is regulated by mechanisms similar to those involved in bone mineralization. osteoporosis and cardiovascular disease shared similar etiological factors (such as smoking, physical activity, alcohol intake, menopause, hypertension, etc.), which may simultaneously promote or inhibit atherosclerosis and bone demineralization and could partly explain the association between the two diseases. low bmd and bone loss appear to be risk factors for cardiovascular mortality at both male and female. the study of osteoporotic fractures (sof) showed that an increase in bmd loss at the hip in the order of one standard deviation (sd) was associated with a 1.3-fold increase in cardiovascular mortality among white female 65 years of age and older. the results of our study are very close to the results of above - mentioned studies : femoral neck bmd indicate hr increase by 1.116 with each unit decrease of density raised over his cut - off value (0.783 g / cm). a plot of kaplan - meier shows the survival functions approaching the true survival function in general patients at mean of covariates. evidently 70.58% (12/17) of the patients who died have bmd femoral neck below cut - off point (0.783 g / cm) and 64.70% (11/17) of the patients who died have pwv greater than cut - off point (9.4 the tendency to hold this values of bone density and pwv below the critical values (0.783 g / cm and 9.4 m / s) leads to a significant reduction of cardiovascular risk. the salient finding of our study was that bmd fn and pwv were strong - independent predictors of cardiovascular mortality with high - level performance values, assessed by simple, indirect, reproducible, and noninvasive evaluation of regional arterial stiffness and bone mineral density. in conclusion, the current study 's findings suggest that bmd fn as measure of bone strength and pwv as a measure of arterial stiffness are strong independent predictors of cardiovascular mortality in general population patients. in our study, high degree of correlation between bmd and pwv suggests that the process of atherosclerosis has many common pathophysiological and epidemiological factors with the process of osteoporosis. a standard pwv cut - off point in the general population should be further investigated and estimated by large clinical studies. by incorporating dxa bmd and carotid - femoral doppler pwv measurements into standard regular diagnostic assessments for cardiovascular estimation, the patients which are at increased cardiovascular risk can be pinpointed earlier, with a recommendation for preventive appropriate osteopenia treatment and stiffness reduction by therapy starting.	osteoporosis and increased arterial stiffness independently have been found to be associated with higher cardiovascular events rates in the general population (gp). we examined 558 patients from gp by dual - energy x - ray absorptiometry (dxa) and pulse wave velocity (pwv) measurements at baseline, with 36-month follow - up period. dxa assessed bone mineral density of femoral neck (bmd fn) and lumbar spine (bmd ls). carotid - femoral pwv was assessed by pulsed - doppler. the aim of our study is to find correlation between bone strength and arterial stiffness and their impact on cardiovascular mortality in gp. the mean sd of bmd fn, bmd ls, and pwv was 0.852 0.1432 g / cm2, 0.934 0.1546 g / cm2, and 9.209 1.9815 m / s. in multiple regression analysis we found bmd fn (st = 6.0094, p < 0.0001), hypertension (st = 1.7340, p < 0.0091), and diabetes (st = 0.4595, p < 0.0046). with cox - regression analysis, after 17 cardiovascular events, the significant covariates retained by the backward model were bmd fn (b = 2.4129, p = 0.015) and pwv (b = 0.2606, p = 0.0318). the cut - off values were pwv = 9.4 m / s, bmd fn = 0.783 g / cm2, and bmd ls = 0.992 g / cm2. the results for bmd fn and pwv hazard ratio risk were 1.116 and 1.297, respectively. bmd fn as a measure of bone strength and pwv as a measure of arterial stiffness are strong independent predictors of cardiovascular mortality in gp.
intravesical bcg is a well- recognised treatment for high - risk superficial bladder cancer [1, 2 ] and is generally well tolerated. however various local and systemic adverse effects are reported including cystitis, isolated renal tuberculosis and multi - organ failure. diagnosis of peritoneal tuberculosis poses a significant challenge to clinicians especially in patients receiving peritoneal dialysis (pd) and a high degree of clinical suspicion is essential to make the diagnosis. diagnosis is challenging due to its indolent nature, variability in presentation and inconsistent pattern of sensitivity of various diagnostic tests especially in context of renal failure. we present a case of an elderly man receiving pd for end stage renal failure and a past history of bladder cancer treated by intravesical bcg instillations. he developed recurrent pd peritonitis and was treated with standard intraperitoneal antibiotics twice before a diagnosis of peritoneal tuberculosis was suspected. this was confirmed by peritoneal fluid analysis and the isolate was identified as bcg strain. an 86-year - old male patient with a past history of muscle - invasive bladder cancer diagnosed in 2000 was treated with external beam radiotherapy and transurethral resection of the bladder tumour. a recurrence of his bladder tumour in november 2007 staged as carcinoma in situ and was treated with standard six instillations of intravesical bcg. his renal function deteriorated during the course of urology follow - up to a creatinine of 310 mol / l and an estimated glomerular filtration rate of 17 ml / min/1.73 m and a renal ultrasound showed moderate left hydronephrosis and severe chronic hydronephrosis of atrophic right. left hydronephrosis was related to recurrence of bladder cancer and was not evident on previous imaging. his renal function continued to decline and he was started on continuous ambulatory pd (capd) in july 2009. in december 2010, he presented acutely with abdominal pain, fever and cloudy dialysis bags. microscopy of his capd fluid showed 400 10/l of polymorphs with no lymphocytes and no growth on culture. clinically, he made a recovery and was discharged home only to present again 1 month later with similar symptoms. on this occasion his capd fluid showed 130 10/l polymorphs, 28 10/l lymphocytes and again no growth on standard culture. due to the recurrent nature of his peritonitis, his capd fluid was sent for acid- and alkali - fast bacilli, which was positive. his tenckhoff pd catheter was therefore removed and peritoneal tuberculosis was confirmed by extended culture of capd fluid. the isolate was later identified as bcg strain by genetic analysis (using hain genoquick technology) at the regional public health laboratory (birmingham, uk). computed tomography scan of the chest, abdomen and pelvis did not reveal any evidence of disseminated tuberculosis or lymphadenopathy. his renal replacement therapy was changed to in - centre haemodialysis and he was started on anti - tuberculosis medications. he was commenced on rifater (rifampicin 120 mg, isoniazid 50 mg and pyrazinamide 300 mg) five tablets and ethambutol 300 mg daily for 6 weeks before switching to rifinah (rifampicin 300 mg and isoniazid 150 mg) two tablets daily prescribed for a further 11 months. he had no further episodes of peritonitis during his treatment but died from withdrawal of haemodialysis 4.5 months after presentation with tuberculous (tb) peritonitis. intravesical use of bcg to treat high - risk superficial bladder cancer was introduced by morales. in 1976 and has widely been used since. bcg is thought to produce an anti - tumour effect via modulation of t - cell immunity and at the same time exerts a local inflammatory response. local side effects are common and are usually a result of bcg - induced local inflammation or contamination of urinary tract and include visible haematuria, cystitis, granulomatous prostatitis and isolated renal abscess. systemic side effects vary from malaise and flu - like symptoms to life - threatening sepsis and include fever > 38c, pneumonitis, granulomatous hepatitis, mycotic aneurysm and peritonitis consistent with development of pulmonary or extra - pulmonary tuberculosis. an hiv test was not performed in our patient, as his risk was deemed low. in our patient, the mechanism of infection is unclear ; however, it has previously been theorized that pd may increase the risk of peritonitis due to direct inoculation or bacterial translocation. the majority of patients will have lymphocytic ascites ; however, in patients on capd there tends to be a neutrophilic response as seen in our patient. the protein content of the fluid is usually > 30 g / l and the serum ascites albumin gradient is < 11 g / l. although normochromic normocytic anaemia and raised c - reactive protein are common, routine laboratory and radiologic investigations are non - specific and do not help in confirmation of diagnosis [4, 5 ]. the gold standard for diagnosis is growth of mycobacterium on ascitic fluid or peritoneal biopsy but peritoneal fluid culture may take several weeks. microbiological analysis and ziehl neelsen staining of the ascitic fluid for acid fast bacilli is positive in only 06% of cases with proven peritoneal tuberculosis. mycobacterium tuberculosis dna detection in peritoneal fluid by nucleic acid amplification using real - time polymerase chain reaction testing can provide rapid results with sensitivity reaching up to 95% in smear - positive patients ; however, sensitivity falls to 48% in smear - negative patients. various studies recommend laparoscopy as a diagnostic tool of choice for visual and histological diagnosis and confirmation of abdominal and peritoneal tuberculosis. the best treatment option for peritoneal tuberculosis following bcg therapy therefore, the treatment regimens are based upon the same principles as pulmonary tuberculosis. the prognosis is variable and mortality ranges from 8 to 50%. tb peritonitis should be considered as a differential diagnosis in any patient presenting with several weeks of abdominal pain, ascites, fever and weight loss. renal physicians and urological surgeons providing care for patients receiving capd and concurrent bladder cancer should discuss potential risks of tb peritonitis associated with intravesical bcg treatment. this clinical report has not been presented or published previously in either an abstract format or in whole or part.	intravesical bacillus calmette - gurin (bcg) is an established treatment for high - risk superficial bladder cancer [morales a, eidinger d, bruce aw. intracavitary bacillus calmette - gurin in the treatment of superficial bladder tumors.1976. j urol 2002 ; 167 : 891893, lamm dl, van der meijden apm, morales a. incidence and treatment of complications of bacillus calmette - gurin intravesical therapy in superficial bladder cancer. j urol 1992 ; 147 : 596600 ]. we describe a patient receiving peritoneal dialysis (pd), who developed peritoneal tuberculosis following treatment of bladder cancer with intravesical bcg instillations. to the best of our knowledge, this is the first case of peritoneal tuberculosis following intravesical bcg treatment in which the mycobacterium has been typed and confirmed as a bcg strain with genetic analysis.
the nhanes iii used a stratified multistage probability design to obtain a nationally representative sample of the civilian, noninstitutionalized u.s. population. in the nhanes iii, each survey participant had a household interview and a physical examination ; a subset of individuals had fasting blood sampling. blood specimens for dna collection were obtained from the nhanes iii phase 2 (19911994) participants aged 12 years. dna lysates were created from cell lines generated using epstein - barr transformed lymphocytes from these blood specimens. we limited our analyses to nondiabetic participants who were aged 20 years and had fasted between 8 and 23 h before blood collection. of 3,517 eligible participants, we excluded 162 participants who self - identified their race / ethnicity as other and also excluded 331 participants who either self - reported diabetes or had a fg level 7.0 mmol / l or who had an oral glucose tolerance test (ogtt) 11.1mmol / l, which left 3,024 participants in the study. plasma glucose levels were measured by hexokinase (cobas mira ; roche diagnostics, montclair, nj). we used homa as a surrogate measure of -cell function (homa - b ; calculated as [20 fasting insulin { iu / ml}/fg { mmol / l } 3.5 ]) (14). we defined ifg as having a fasting plasma glucose 5.6 mmol / l, but < 7.0 mmol / l and diabetes as use of antidiabetes medications (including insulin) or a fasting plasma glucose 7.0 mmol / l (4). we genotyped 16 snps that were confirmed as associated with fg levels among nondiabetic people of european ancestry in the magic gwas (10). we used snp rs573225 as a proxy (r = 1.0 in ceu) for snp rs560887 in g6pc2 and snp rs11558471 as a proxy (r = 1.0 in ceu) for snp rs13266634 in slc30a8. the minimum call rate was 98.1%, and all snps were in hardy - weinberg equilibrium (hwe) according to the national center for health statistics criterion (if p < 0.01 in two or more race / ethnicity groups, hwe is rejected). we constructed a weighted genetic risk score (grs) to examine the combined effect of 16 snps on fg levels, homa - b, and risk for ifg. we used the -coefficients from the published european ancestry gwass of these fg - associated snps (10). we multiplied these -coefficients by 0, 1, or 2, according to the number of risk alleles carried by each individual, and then summed them. to facilitate the comparison with the simple grs (summing the number of risk alleles), we divided the score by 0.948 (twice the sum of the -coefficients) and multiplied by 32 (total number of alleles) (16). although a number of snps did not show significant association with fg or homa - b in the nhanes iii, we assumed, on the basis of gwas results, that each snp is independently associated with fg for computation of a weighted grs. this assumption might not be appropriate if an index snp is correlated with the causal snp in the discovery population but not so in other racial / ethnic groups due to differences in linkage disequilibrium patterns (17). we used an additive genetic model for each snp and applied a linear weighting of 0, 1, or 2 to genotypes containing 0, 1, or 2 risk alleles (16). we fit the weighted grs as a continuous variable and categorized it into quintiles in multivariate analyses. in presenting the results we calculated weighted allele frequencies and their 95% cis, stratified by race / ethnicity (non - hispanic white, non - hispanic black, and mexican american) using the nhanes sample weights. we carried out hwe tests for each genetic variant for each racial / ethnic group. to test for differences in allele frequencies, we pooled all racial / ethnic groups and used to test the differences across the racial / ethnic groups. we used linear regression modeling to evaluate the relationship between 16 snps and fg levels and homa - b, adjusted for age and sex. we examined the adjusted marginal effect on fg by including one snp at a time in the model for each racial / ethnic group. snps were coded as 0, 1, or 2 for those who were noncarriers, heterozygous, or homozygous for the risk (fg raising) alleles, respectively. we modeled the grs in a similar fashion by testing associations of a per risk allele increase with fg. for the grs models, we calculated adjusted r, with and without the grs to determine the explained variance, by adding the grs in the models for each racial / ethnic group. we tested for homogeneity of effects of these snps in marginal and grs models across race / ethnicity by including the interaction terms of snp or grs with race / ethnicity in the regression models using the pooled dataset and reported the associated p value. we used age- and sex - adjusted logistic regression to evaluate the association of the grs with prevalence of ifg for each racial / ethnic group. we modeled the grs to test the association of a per risk allele increase with odds of ifg. we also categorized the grs into quintiles to examine the trend of effect on risk of ifg. we tested for homogeneity of the grs effect on ifg across race / ethnicity by including the interaction term using the pooled dataset. statistical tests, using linear or logistic regression modeling, were based on satterthwaite adjusted - f statistics. all tests were two sided, and a p value of < 0.05 at each locus or the grs was considered significant. to avoid nonresponse bias among nhanes iii participants for whom dna were available, the sample weights were recalculated for the nhanes iii dna dataset by using previously described methods (18). we used sudaan (version 10.0) and sas (version 9.2 ; sas institute, cary, nc) statistical software for our analyses to account for the complex sampling design. plasma glucose levels were measured by hexokinase (cobas mira ; roche diagnostics, montclair, nj). we used homa as a surrogate measure of -cell function (homa - b ; calculated as [20 fasting insulin { iu / ml}/fg { mmol / l } 3.5 ]) (14). we defined ifg as having a fasting plasma glucose 5.6 mmol / l, but < 7.0 mmol / l and diabetes as use of antidiabetes medications (including insulin) or a fasting plasma glucose 7.0 mmol / l (4). we genotyped 16 snps that were confirmed as associated with fg levels among nondiabetic people of european ancestry in the magic gwas (10). we used snp rs573225 as a proxy (r = 1.0 in ceu) for snp rs560887 in g6pc2 and snp rs11558471 as a proxy (r = 1.0 in ceu) for snp rs13266634 in slc30a8. the minimum call rate was 98.1%, and all snps were in hardy - weinberg equilibrium (hwe) according to the national center for health statistics criterion (if p < 0.01 in two or more race / ethnicity groups, hwe is rejected). we constructed a weighted genetic risk score (grs) to examine the combined effect of 16 snps on fg levels, homa - b, and risk for ifg. we used the -coefficients from the published european ancestry gwass of these fg - associated snps (10). we multiplied these -coefficients by 0, 1, or 2, according to the number of risk alleles carried by each individual, and then summed them. to facilitate the comparison with the simple grs (summing the number of risk alleles), we divided the score by 0.948 (twice the sum of the -coefficients) and multiplied by 32 (total number of alleles) (16). although a number of snps did not show significant association with fg or homa - b in the nhanes iii, we assumed, on the basis of gwas results, that each snp is independently associated with fg for computation of a weighted grs. this assumption might not be appropriate if an index snp is correlated with the causal snp in the discovery population but not so in other racial / ethnic groups due to differences in linkage disequilibrium patterns (17). we used an additive genetic model for each snp and applied a linear weighting of 0, 1, or 2 to genotypes containing 0, 1, or 2 risk alleles (16). we fit the weighted grs as a continuous variable and categorized it into quintiles in multivariate analyses. in presenting the results we calculated weighted allele frequencies and their 95% cis, stratified by race / ethnicity (non - hispanic white, non - hispanic black, and mexican american) using the nhanes sample weights. we carried out hwe tests for each genetic variant for each racial / ethnic group. to test for differences in allele frequencies, we pooled all racial / ethnic groups and used to test the differences across the racial / ethnic groups. we used linear regression modeling to evaluate the relationship between 16 snps and fg levels and homa - b, adjusted for age and sex. we examined the adjusted marginal effect on fg by including one snp at a time in the model for each racial / ethnic group. snps were coded as 0, 1, or 2 for those who were noncarriers, heterozygous, or homozygous for the risk (fg raising) alleles, respectively. we modeled the grs in a similar fashion by testing associations of a per risk allele increase with fg. for the grs models, we calculated adjusted r, with and without the grs to determine the explained variance, by adding the grs in the models for each racial / ethnic group. we tested for homogeneity of effects of these snps in marginal and grs models across race / ethnicity by including the interaction terms of snp or grs with race / ethnicity in the regression models using the pooled dataset and reported the associated p value. we used age- and sex - adjusted logistic regression to evaluate the association of the grs with prevalence of ifg for each racial / ethnic group. we modeled the grs to test the association of a per risk allele increase with odds of ifg. we also categorized the grs into quintiles to examine the trend of effect on risk of ifg. we tested for homogeneity of the grs effect on ifg across race / ethnicity by including the interaction term using the pooled dataset. statistical tests, using linear or logistic regression modeling, were based on satterthwaite adjusted - f statistics. all tests were two sided, and a p value of < 0.05 at each locus or the grs was considered significant. to avoid nonresponse bias among nhanes iii participants for whom dna were available, the sample weights were recalculated for the nhanes iii dna dataset by using previously described methods (18). we used sudaan (version 10.0) and sas (version 9.2 ; sas institute, cary, nc) statistical software for our analyses to account for the complex sampling design. there were 3,024 individuals available for this study, including 1,225 non - hispanic white, 897 non - hispanic black, and 902 mexican american participants. compared with other groups, fg was higher among mexican americans, and homa - b was lower among non - hispanic whites (table 1). characteristics of participants by race / ethnicity, nhanes iii dna bank (19911994) for continuous variables, the means were adjusted for age and sex. p values were tests for the differences across race / ethnicity groups and are based on satterthwaite adjusted f statistics for the continuous variables and for the categorical variables. all allele frequencies (16 snps) were significantly different across racial / ethnic groups (p < 0.05). risk allele frequencies varied from 17.1% (rs4607517, gck) to 86.1% (rs10885122, adra2) among non - hispanic whites, from 7.9% (rs10830963, mtnr1b) to 93.7% (rs7944584, madd) among non - hispanic blacks, and from 18.0% (rs7903146 (tcf7l2) to 86.5% (rs11920090, slc2a2) among mexican americans (table 2) (online appendix table 1, available at http://care.diabetesjournals.org/cgi/content/full/dc10-0898/dc1). weighted - risk (fg raising) allele frequencies of 16 fg - associated snps by race / ethnicity, nhanes iii dna bank (19911994) all snps were in hwe. we observed three snps (rs573225, rs780094, and rs11558471) that were significantly associated with fg levels among non - hispanic whites, one snp (rs10830963) among non - hispanic blacks, and three variants (rs573225, rs4607517, and rs10830963) among mexican americans (online appendix tables 2 and 3). however, the majority of -coefficients (37 of 48) showed positive associations in the expected direction with fg levels among all three racial / ethnic groups. we found no evidence of heterogeneity effects of these snps on fg levels across racial / ethnic groups, except for rs11885122 for which non - hispanic whites had lower fg and non - hispanic blacks and mexican americans had higher fg levels (p = 0.009). generally, similar patterns of associations of these 16 snps were seen for homa - b (online appendix table 2). the number of risk alleles at 16 snps ranged from 10 to 27 (median 19), 12 to 26 (median 20), and 10 to 26 (median 18) among non - hispanic whites, non - hispanic blacks, and mexican americans, respectively. for the weighted grs, the corresponding numbers were 827 (median 17), 1024 (median 18), and 926 (median 17), respectively. the grs was normally distributed in all three racial / ethnic groups (kolmogorov - smirnov p < 0.001) (online appendix fig. each additional risk allele in the score was associated with a 0.022, 0.036, and 0.033 mmol / l increase in fg among non - hispanic whites, non - hispanic blacks, and mexican americans, respectively (table 3). there was no evidence of a different effect of the grs on fg levels or homa - b across race / ethnicity (p = 0.291 and 0.637 for testing heterogeneity of the grs on fg levels and homa - b across race / ethnicity, respectively). in a model including age and sex, the grs explained more of the total variation in fg in mexican americans and in non - hispanic blacks than in non - hispanic whites (table 3). generally, similar patterns of associations of these 16 snps were seen for homa - b (table 3). adjusted mean fg levels and homa - b by quintiles of weighted genetic risk score and race / ethnicity, nhanes iii dna bank (19911994) data and 95% cis in parentheses. -coefficients (95% cis) of linear regression models adjusted for age and sex. p values for testing difference in fg levels or homa - b across quintiles of weighted genetic risk score are based on satterthwaite adjusted f statistics. adjusted r for regression models with and without (age and sex only) weighted genetic risk score. p = 0.291 and p = 0.637 for testing heterogeneity of weighted genetic risk score on fg levels and homa - b, respectively, across racial / ethnic groups are based on satterthwaite adjusted f statistics. the prevalence of ifg, adjusted for age and sex, was lowest among non - hispanic blacks and highest among mexican americans. the adjusted odds ratios for ifg were highest among mexican americans, comparing people in the highest with those in the lowest quintiles of the grs for all racial / ethnic groups (table 4) (online appendix fig. 2). there was no evidence of a different effect of the grs on risk for ifg across race / ethnicity (p = 0.365 for testing heterogeneity across race / ethnicity). each additional allele was associated with a significant increased risk of ifg for all racial / ethnic groups. adjusted prevalence and odds ratio for ifg by quintiles of weighted genetic risk score and race / ethnicity, third national health and nutrition examination survey dna bank (nhanes iii 19911994) data and 95% ci are in parentheses. for prevalence of ifg, p values for trend test across quintiles of weighted genetic risk score ; for adjusted odds ratio, p values for using weighted genetic risk score as a continuous variable in logistic regression models are based on satterthwaite adjusted f test. p = 0.365 for testing heterogeneity of weighted genetic risk score as a continuous variable on risk for ifg across racial / ethnic groups is based on satterthwaite adjusted f statistic. all allele frequencies (16 snps) were significantly different across racial / ethnic groups (p < 0.05). risk allele frequencies varied from 17.1% (rs4607517, gck) to 86.1% (rs10885122, adra2) among non - hispanic whites, from 7.9% (rs10830963, mtnr1b) to 93.7% (rs7944584, madd) among non - hispanic blacks, and from 18.0% (rs7903146 (tcf7l2) to 86.5% (rs11920090, slc2a2) among mexican americans (table 2) (online appendix table 1, available at http://care.diabetesjournals.org/cgi/content/full/dc10-0898/dc1). weighted - risk (fg raising) allele frequencies of 16 fg - associated snps by race / ethnicity, nhanes iii dna bank (19911994) all snps were in hwe. we observed three snps (rs573225, rs780094, and rs11558471) that were significantly associated with fg levels among non - hispanic whites, one snp (rs10830963) among non - hispanic blacks, and three variants (rs573225, rs4607517, and rs10830963) among mexican americans (online appendix tables 2 and 3). however, the majority of -coefficients (37 of 48) showed positive associations in the expected direction with fg levels among all three racial / ethnic groups. we found no evidence of heterogeneity effects of these snps on fg levels across racial / ethnic groups, except for rs11885122 for which non - hispanic whites had lower fg and non - hispanic blacks and mexican americans had higher fg levels (p = 0.009). generally, similar patterns of associations of these 16 snps were seen for homa - b (online appendix table 2). the number of risk alleles at 16 snps ranged from 10 to 27 (median 19), 12 to 26 (median 20), and 10 to 26 (median 18) among non - hispanic whites, non - hispanic blacks, and mexican americans, respectively. for the weighted grs, the corresponding numbers were 827 (median 17), 1024 (median 18), and 926 (median 17), respectively. the grs was normally distributed in all three racial / ethnic groups (kolmogorov - smirnov p < 0.001) (online appendix fig. each additional risk allele in the score was associated with a 0.022, 0.036, and 0.033 mmol / l increase in fg among non - hispanic whites, non - hispanic blacks, and mexican americans, respectively (table 3). there was no evidence of a different effect of the grs on fg levels or homa - b across race / ethnicity (p = 0.291 and 0.637 for testing heterogeneity of the grs on fg levels and homa - b across race / ethnicity, respectively). in a model including age and sex, the grs explained more of the total variation in fg in mexican americans and in non - hispanic blacks than in non - hispanic whites (table 3). generally, similar patterns of associations of these 16 snps were seen for homa - b (table 3). adjusted mean fg levels and homa - b by quintiles of weighted genetic risk score and race / ethnicity, nhanes iii dna bank (19911994) data and 95% cis in parentheses. -coefficients (95% cis) of linear regression models adjusted for age and sex. p values for testing difference in fg levels or homa - b across quintiles of weighted genetic risk score are based on satterthwaite adjusted f statistics. adjusted r for regression models with and without (age and sex only) weighted genetic risk score. p = 0.291 and p = 0.637 for testing heterogeneity of weighted genetic risk score on fg levels and homa - b, respectively, across racial / ethnic groups are based on satterthwaite adjusted f statistics. the prevalence of ifg, adjusted for age and sex, was lowest among non - hispanic blacks and highest among mexican americans. the adjusted odds ratios for ifg were highest among mexican americans, comparing people in the highest with those in the lowest quintiles of the grs for all racial / ethnic groups (table 4) (online appendix fig. 2). there was no evidence of a different effect of the grs on risk for ifg across race / ethnicity (p = 0.365 for testing heterogeneity across race / ethnicity). each additional allele was associated with a significant increased risk of ifg for all racial / ethnic groups. adjusted prevalence and odds ratio for ifg by quintiles of weighted genetic risk score and race / ethnicity, third national health and nutrition examination survey dna bank (nhanes iii 19911994) data and 95% ci are in parentheses. for prevalence of ifg, p values for trend test across quintiles of weighted genetic risk score ; for adjusted odds ratio, p values for using weighted genetic risk score as a continuous variable in logistic regression models are based on satterthwaite adjusted f test. p = 0.365 for testing heterogeneity of weighted genetic risk score as a continuous variable on risk for ifg across racial / ethnic groups is based on satterthwaite adjusted f statistic. our findings show that risk allele frequencies of the included snps discovered in populations of european ancestry differed significantly by racial / ethnic group, consistent with the findings of other studies and hapmap estimates (11,12). however, despite significant variations in allele frequencies, the patterns of influence of these snps on fg levels, homa - b, and ifg were generally consistent across racial / ethnic groups. a grs derived by the combination of these 16 snps was weakly, yet significantly, associated with an increase in fg levels, a decrease in homa - b, and an increase in risk for ifg in all racial / ethnic groups. our findings suggest that the genetic variants at these glycemic loci, discovered in the white population of european ancestry, also contribute to the elevated fg and reduced homa - b among non - hispanic blacks and mexican americans. the 16 fg - associated snps are located in or near genes involved in multiple biological pathways (10). some of these snps were also associated with type 2 diabetes (1921), and a few have been replicated in non - european populations (22,23). our study includes the most updated snps associated with fg and estimates their frequencies and effects in a nationally representative sample of the u.s. population. our results suggest that with an adequate sample size (not necessarily as large as for a gwas), these fg - associated snps would likely be replicated among non - hispanic blacks and mexican americans. although fg levels seem tightly regulated within a narrow range by a feedback mechanism that targets a particular fg set point for each person (24), fg levels vary substantially among nondiabetic populations, and an estimated 2540% of the variation may be explained by genetic factors (7). many studies have suggested that elevated fg levels are associated with multiple health conditions, including risk for type 2 diabetes and cardiovascular diseases (1,2,4,5). identification of populations at high risk of developing type 2 diabetes has great public health importance. our findings suggest that a grs, on the basis of these fg - associated snps, is significantly associated with ifg, but it is unclear if these snps can improve predictions for risks of type 2 diabetes or cardiovascular disease in the general population. further studies are also needed to examine the possible associations of these snps with type 2 diabetes and cardiovascular diseases in different races and ethnicities. however, the combined effects of these snps explain only a small percentage of the total variations in fg levels and homa - b (< 3%), suggesting that additional loci exist. these loci may be common genetic variants with small effects, rare variants with larger effects, or variants that strongly interact with each other or with environmental factors (in which the number and effect size remain unknown). these interactions are undoubtedly important but complicated to model ; their exploration is beyond the scope of our study. the major strengths of our study include the availability of fg and homa - b measurements from a nationally representative sample of the u.s. population with multiple racial / ethnic groups and the genotyping of the 16 most updated fg - associated snps. first, these fg - associated snps were discovered among populations of european ancestry and may be proxies for the causal variants. it is well known that linkage disequilibrium patterns vary significantly by race / ethnicity (17), and it is unclear if linkage disequilibrium might break down for some of these snps in other racial / ethnic groups (online appendix fig. second, we replicated a limited number of snps even among non - hispanic whites that were significantly associated with fg levels or homa - b in the nhanes iii. as indicated in the power calculation (online appendix), we had limited sample size to detect an effect size of < 0.07 mmol / l in fg per allele for each individual snp. the most likely explanation for the lack of significant association was the limited sample size of fasting individuals from the nhanes iii. since these were gwas - confirmed, fg - associated snps (at least among non - hispanic whites), we included all snps in our analysis. however, using the grs as a continuous variable, we have adequate power to detect an effect size as low as 0.016 mmol / l per risk allele. when including only the 11 snps that showed the expected direction of effects for fg and homa - b, the association of the grs appeared to be stronger, though the patterns remained unchanged (results not shown). at least for non - hispanic whites, a larger sample size would not likely change the conclusions since the majority of the snps ' effects are in the expected direction, consistent with studies in european populations (10). third, we calculated the weighted grs for the three racial / ethnic groups on the basis of the published -coefficients from populations of european ancestry because of a lack of estimates for other racial / ethnic groups (10). these -coefficients might not be appropriate for other populations. however, the consistent effects of the grs on fg, homa - b, and ifg across racial / ethnic groups suggest that the weighting was not totally inappropriate in these populations. in addition, the pattern of association between weighted and unweighted grs on fg, homa - b, and ifg were consistent (online appendix tables 4 and 5). in summary, our results suggest that the allele frequencies of fg - associated snps varied significantly by race / ethnicity. however, the patterns of combined effects of these snps on fg levels and homa - b were consistent across the different racial / ethnic groups. a grs that was based on 16 snps was significantly associated with risk of ifg among all racial / ethnic groups, which may make it useful for the identification of people who are at high risk for developing diabetes.	objectiveto estimate allele frequencies and the marginal and combined effects of novel fasting glucose (fg)-associated single nucleotide polymorphisms (snps) on fg levels and on risk of impaired fg (ifg) among non - hispanic white, non - hispanic black, and mexican americans.research design and methodsdna samples from 3,024 adult fasting participants in the national health and nutrition examination survey (nhanes) iii (19911994) were genotyped for 16 novel fg - associated snps in multiple genes. we determined the allele frequencies and influence of these snps alone and in a weighted genetic risk score on fg, homeostasis model assessment of -cell function (homa - b), and ifg by race / ethnicity, while adjusting for age and sex.resultsall allele frequencies varied significantly by race / ethnicity. a weighted genetic risk score, based on 16 snps, was associated with a 0.022 mmol / l (95% ci 0.0090.035), 0.036 mmol / l (0.0190.052), and 0.033 mmol / l (0.0200.046) increase in fg levels per risk allele among non - hispanic whites, non - hispanic blacks, and mexican americans, respectively. adjusted odds ratios for ifg were 1.78 for non - hispanic whites (95% ci 1.003.17), 2.40 for non - hispanic blacks (1.075.37), and 2.39 for mexican americans (1.374.14) when we compared the highest with the lowest quintiles of genetic risk score (p = 0.365 for testing heterogeneity of effect across race / ethnicity).conclusionswe conclude that allele frequencies of 16 novel fg - associated snps vary significantly by race / ethnicity, but the influence of these snps on fg levels, homa - b, and ifg were generally consistent across all racial / ethnic groups.
idiopathic pulmonary hemosiderosis (iph) is a rare condition, which causes recurrent diffuse alveolar hemorrhage (dah). no definite treatment is available, however most case series and reports have reported some response to corticosteroids. it is sometimes associated with celiac disease (cd), which is a primarily a gut condition due to intolerance to gluten. it is important to recognize the association of cd and iph even in the absence of gastrointestinal symptoms as the treatment with gluten - free diet may lead to resolution of iph. dilated cardiomyopathy (dcm) is also reported in association with these two conditions, mainly in pediatric population. this is a case of an adult patient, presented to us with innocuous hemoptysis and found to have involvement of multiple organ systems that is iph, celiac disease and cardiomyopathy. a 19-year - old non - smoker gentleman presented to us in january 2011 with recurrent hemoptysis, about 10 ml per day for last 3 years. he also complained of generalized weakness and dyspnea, which progressed from medical research council grade i to grade ii. there was no history suggestive of recurrent chest infections, joint - pains, skin - rash, photo - sensitivity, alopecia, oral ulcers, raynaud 's phenomenon or dysuria. earlier, he was diagnosed to have iron - deficiency anemia and was treated with blood transfusions and hematinics. on examination investigations revealed low levels of hemoglobin (7.8 g / dl), mean corpuscular volume (mcv - 69.9 fl), mean corpuscular hemoglobin (mch - 19.3 pg), mean corpuscular hemoglobin concentration (mchc - 27.5 gms / dl) and the red blood cells in peripheral smear were microcytic, hypochromic with anisopoikilocytosis suggestive of iron - deficiency anemia. ecg showed sinus tachycardia, non - specific interventricular conduction delay and t - wave inversion (lead 1, avl, v4-v6). echocardiography revealed dcm and left ventricular dysfunction with ejection fraction (lvef) of 25%. contrast - enhanced computed tomography (cect) showed bilateral diffuse ground glass opacities suggestive of dah [figure 1 ]. pulmonary function tests (pft) showed restrictive pattern with forced vital capacity (fvc)- 3.05l, 68% of predicted) and increased diffusion capacity for carbon monoxide (dlco). blood eosinophil counts, serum angiotensin - converting enzyme and 1- antitrypsin enzyme levels were normal. serum markers of vasculitides and auto - immune disorders including rheumatoid factor, anti - nuclear antibodies (ana), anti - neutrophil cytoplasmic antibodies (anca), anti - cardiolipin antibody and anti - glomerular basement membrane antibodies were negative. bronchoalveolar lavage and transbronchial lung biopsy revealed type ii pneumocyte hyperplasia, chronic inflammatory cell infiltrate in interalveolar spaces and hemosiderin - laden macrophages in alveolar spaces. [figure 2 ] after excluding all other possibilities, patient was diagnosed as iph. his serum anti - tissue transglutamase antibodies were raised (291.66 iu / ml, normal < 30 iu / ml). duodenal biopsy showed subtotal villous atrophy and crypt hyperplasia (marsh grade iiib) consistent with cd. he was started on gluten - free diet, beta - blockers and diuretics. at 2-year follow - up, cect scan of chest showing bilateral diffuse ground glass opacities photomicrograph shows type ii pneumocyte hyperplasia, mild chronic inflammatory infiltrate in interalveolar septa and hemosiderin laden macrophages in alveolar spaces. dah refers to bleeding from the pulmonary microcirculation leading to blood leakage into the alveolar spaces. it has numerous causes which can be immune (19 - 42%) or non - immune (58 - 81%). iph is an uncommon disease and was originally described by virchow in 1864 as brown induaration. a comprehensive portrayal of iph was given by ceelen in 1931, also known as ceelen - gellerstedt syndrome. adult - onset iph (20%) is mostly reported in < 30 years of age with a male preponderance. usually it presents as a triad of hemoptysis, pulmonary infiltrates and iron deficiency anemia. it may also present as chronic disease with weight loss, growth failure, iron - deficiency anemia and respiratory impairment. diagnosis is made by exclusion of other causes, as was the case with our patient in whom no other cause of dah could be found. this was first described by lane and hamilton in 1971 and is recognized as the lane - hamilton syndrome. in a study by khemiri., that screened patients with iph for cd, an association was found in 44.45%. two other studies by kiper., and both cd and iph are believed to be immunologically mediated ; however, the pathogenetic link between the two conditions is not clear. several hypotheses were put forth, which include the deposition of circulating immune complexes from food allergens on the basal membrane of alveolar capillaries. in one of the case reports, although evidence of circulating immune complexes was found but evidence of damage to alveolar capillaries was not observed. the cross - reaction between the anti - reticulin antibodies and the alveolar basal membrane antigens, and the effect of adenovirus 12 were other possible mechanisms. more than 50% of patients show remittance of respiratory symptoms only with gluten - free diet. cardiomyopathy associated with cd is rare and till date eight studies have reported this association. a recent study by de bem., in which 12.18% cases of cardiomyopathy were reported to have cd have recommended screening for cd in all patients with dcm., studied patients (238) and the relatives of patients (418) with idiopathic cardiomyopathy and found that cd seems to be associated but not co - segregated within familial cases. frustaci., showed high prevalence of cd among patients with idiopathic congestive heart failure and biopsy proven myocarditis. mechanisms for pathogenesis of dcm in cd include nutritional deficiencies, infections and immune - mediated insults., suggested that co - morbidities like iron - deficiency anemia in patients with dcm should arouse suspicion of cd. cardiac functions improve with gluten - free diet as was the case with our patient. if a patient has iph, cd should be investigated even in the absence of gastrointestinal symptoms. similarly, all patients with idiopathic dcm should be investigated for cd with anti - tissue transglutamase antibodies and if positive, a biopsy may be performed ; though there is a general preference for a non - invasive approach to the diagnosis.	idiopathic pulmonary hemosiderosis (iph) is a rare cause of recurrent diffuse alveolar hemorrhage (dah) with no specific treatment. herein, we discuss a case of hemoptysis, who had iph and other rare associations. a 19-year - old man presented with recurrent hemoptysis, generalized weakness and progressive dyspnea for 3 years. earlier, he was diagnosed with anemia and was treated with blood transfusions and hematinics. on examination he had pallor, tachycardia and was underweight. investigations revealed low level of hemoglobin (7.8 g / dl) and iron deficiency. an electrocardiography (ecg) showed sinus tachycardia, interventricular conduction delay and t - wave inversion. echocardiography revealed dilated cardiomyopathy with left ventricular dysfunction. computed tomography of the chest demonstrated bilateral diffuse ground glass opacity suggestive of pulmonary hemorrhage. pulmonary function tests showed restrictive pattern with increased carbon monoxide diffusion. bronchoalveolar lavage and transbronchial lung biopsy showed hemosiderin - laden macrophages. patient could recall recurrent episodes of diarrhea in childhood. serum antitissue transglutamase antibodies were raised (291.66 iu / ml, normal < 30 iu / ml). duodenal biopsy showed subtotal villous atrophy consistent with celiac disease. he was started on gluten - free diet, beta blockers and diuretics. after two years of treatment, he has been showing consistent improvement. screening for cd is important in patients with iph. cardiomyopathy forms rare third association. all three show improvement with gluten - free diet.
the online version of this article (doi:10.1007/s10552 - 014 - 0386 - 2) contains supplementary material, which is available to authorized users. parity and age at first birth consistently have been shown to be associated with breast cancer risk in women worldwide (e.g.,). the mechanisms underlying the protective effect of pregnancy include a reduction in the number of mammary stem cells, an alteration in the responsiveness of the breast to estrogens, differentiation of mammary epithelial cells, and a change in the levels of circulating hormones (as reviewed in). in addition to the difference in breast cancer risk between nulliparous and parous women, there is evidence that the characteristics of pregnancy, particularly first birth, affect breast cancer risk. an increased risk of breast cancer has been associated with having had a pregnancy that resulted in a preterm birth, higher birthweight (e.g.,), or multifetal gestation, and a decreased risk has been found with having had a pregnancy in which the woman developed hypertension or preeclampsia [3, 5 ], had a smaller placental weight, or had nausea or vomiting [7, 8 ]. investigators have found that pregnancy characteristics may be particularly important in the first birth, and with a late age at first birth (e.g.,). research has suggested that this relationship is causal rather than correlational (e.g.,) thus providing a potentially important surrogate target for prevention studies. while research has consistently shown a negative association between increasing parity and breast density (e.g., [10, 11 ]), findings have been less consistent for an association between breast density and older age at first birth (as reviewed in). a few studies have demonstrated that some of the same birth characteristics shown to be associated with increased breast cancer risk were associated with high breast density, specifically, preterm birth, nulliparity / low parity, older age at first birth, and high birth weight [10, 11, 13, 14 ]. lope. found that duration of breastfeeding was positively associated with breast density, while butler a better understanding of the association between first pregnancy and breast density could inform breast cancer prevention efforts. if first birth characteristics are found to be associated with breast density in the same direction as their association with breast cancer, this would indicate that these pregnancy events affect breast cancer through their impact on breast density. this study therefore aimed to examine the association between first pregnancy and breast density (study 1). we additionally endeavored to further explore whether the association between first pregnancy and breast density varied by candidate germline gene variants, specifically single nucleotide polymorphisms (snps) known to be related to breast cancer, breast density, or pregnancy characteristics (study 2). this study was conducted using data from women enrolled in the marin women s study (mws). the mws is a cross - sectional study conducted at mammography centers in marin county, california associated with kaiser permanente, sutter health (novato community hospital), and marin general hospital. it is estimated that 80 % of mammograms conducted in marin residents are conducted at centers affiliated with these health care facilities, representing approximately 38,000 women annually. these mammography sites are also included in the san francisco mammography registry (sfmr), one of seven registries included in the national cancer institute breast cancer surveillance consortium. risk factors and saliva specimens collected in the mws are linked with breast density, measured as percent fibroglandular volume (% fgv), and breast cancer outcome. at the time of analysis, there were 11,361 women enrolled in the mws ; for purposes of this analysis (study 1), women were excluded if they were missing data on age (n = 80), if they reported ever having been diagnosed with breast cancer (n = 751), if they reported taking antiestrogens (n = 107), if they had never had a live birth (n = 2,814), if they did not have a measure of breast density assessed as % fgv (n = 4,326), if they had missing data on any of the model variables (n = 639), if they reported having had a hysterectomy with ovarectomy (n = 160), or if their first birth was not a singleton birth (n = 44), leaving an analysis population of 2,440. women are asked to report in - depth information on their reproductive history, including information on life course socioeconomic status, alcohol use, and use of exogenous hormones, and data on other established breast cancer risk factors, including family history. reproductive history includes parity and age at first birth, and for each pregnancy women are asked to report multifetal gestation, duration of breast - feeding, low and high birth weight (4,000 g), preterm birth, pregnancy weight gain (pounds), and high blood pressure during pregnancy [or pregnancy - induced hypertension (pih) ] (yes / no). self - reported height and weight are collected from the sfmr. at the time of entry into the mws those who consent to donate a saliva sample (89 % of population) are sent a kit in the mail and asked to return the tube of donated saliva in a preaddressed and postage - paid envelope to the buck institute for research on aging where the saliva is logged, stored, and processed. the specimens are processed by separation into supernatant available for steroid hormone analysis, and a cellular component from which dna is isolated using invitrogen s purelink genomic dna kits. samples are stored in the mws biorepository housed at the buck institute in novato, california. for study 2, participants were also selected from the mws population and were drawn from among 2,400 participants for whom snp data were available from donated saliva samples. twenty - five preselected candidate snps were multiplexed and analyzed in the mws dna samples by intergenetics, inc (oklahoma city, ok) in parallel with analysis of their panel of 22 oncovue snps. all snp assays were performed and evaluated as previously described, and the clia - approved 22 snp oncovue assay and algorithm were previously validated as a new individualized breast cancer risk estimator using buccal dna samples from an earlier (19971999) marin county case control study cohort. selection of the novel 25 snp candidates evaluated in the present study followed a detailed literature search of snps reported in gwas studies to be significantly associated (in at least one published report) to specific exposures, outcomes, and hypothesized breast cancer associated pathways of relevance to the current mws. in cases where there were more than one snp from a given gene, weight of literature, linkage characteristics, and potential functionality based on specific location of the snp within the gene for purposes of the analysis examining interactions with pih reported in this paper, we evaluated snps in the following seven genes : enos (nos3), esr2, vegf, edn1, il-10, hcfxi (klkb1), igfr1. one of the novel features of this study is the measure of % fgv assessed by the method single - energy x - ray absorptiometry (sxa) [1720 ]. this method uses a calibration phantom of the same thickness as the compressed breast, circumventing some of the problems associated with other breast density measures including their subjectivity and lack of absolute reference standards. the first generation calibration phantom (gamma) used for this method has provided preliminary data on 8,600 women to show that % fgv is reproducible to approximately 2 % between successive measures and accurate to known standards of breast composition using reference phantoms. investigators have demonstrated that % fgv is inversely correlated with age, body mass index (bmi), and menopausal status, as expected for a measurement of breast density, and is positively associated with breast cancer risk. % fgv data are collected by the sfmr at all sites in marin county which employ digital mammography and are obtained from the sfmr through a cooperative agreement. the mws was approved by the participating institutions, and participants provided written informed consent for participation in the study. multivariable linear regression analyses were conducted to examine the associations between % fgv and reproductive factors, controlling for relevant confounders. robust regression techniques were used to minimize the effects of outliers and influential observations (i.e., observations that have a large impact on the regression analysis). all models controlled for a base set of confounding variables, including current age, bmi, race, education, smoking, family history of breast cancer (whether a first degree relative has been diagnosed with breast cancer), hysterectomy status, menopausal status, and exogenous hormone use at the time of the mammogram. all genotyping data were checked for compliance of single - gene allelic frequencies with hardy weinberg frequency expectations using goodness - of - fit test. multivariable linear regression models were constructed adjusting for potential confounders including current bmi, age, race, and age of first live birth, and interaction terms were generated for the genotype of each snp compared to a baseline genotype. statistical tests were conducted to examine the interaction term for each level of the snp compared to the baseline level of the snp, as well as for the joint interaction effect of each snp overall and pih, and p values examined for statistical significance. this study was conducted using data from women enrolled in the marin women s study (mws). the mws is a cross - sectional study conducted at mammography centers in marin county, california associated with kaiser permanente, sutter health (novato community hospital), and marin general hospital. it is estimated that 80 % of mammograms conducted in marin residents are conducted at centers affiliated with these health care facilities, representing approximately 38,000 women annually. these mammography sites are also included in the san francisco mammography registry (sfmr), one of seven registries included in the national cancer institute breast cancer surveillance consortium. risk factors and saliva specimens collected in the mws are linked with breast density, measured as percent fibroglandular volume (% fgv), and breast cancer outcome. at the time of analysis, there were 11,361 women enrolled in the mws ; for purposes of this analysis (study 1), women were excluded if they were missing data on age (n = 80), if they reported ever having been diagnosed with breast cancer (n = 751), if they reported taking antiestrogens (n = 107), if they had never had a live birth (n = 2,814), if they did not have a measure of breast density assessed as % fgv (n = 4,326), if they had missing data on any of the model variables (n = 639), if they reported having had a hysterectomy with ovarectomy (n = 160), or if their first birth was not a singleton birth (n = 44), leaving an analysis population of 2,440. women are asked to report in - depth information on their reproductive history, including information on life course socioeconomic status, alcohol use, and use of exogenous hormones, and data on other established breast cancer risk factors, including family history. reproductive history includes parity and age at first birth, and for each pregnancy women are asked to report multifetal gestation, duration of breast - feeding, low and high birth weight (4,000 g), preterm birth, pregnancy weight gain (pounds), and high blood pressure during pregnancy [or pregnancy - induced hypertension (pih) ] (yes / no). self - reported height and weight are collected from the sfmr. at the time of entry into the mws those who consent to donate a saliva sample (89 % of population) are sent a kit in the mail and asked to return the tube of donated saliva in a preaddressed and postage - paid envelope to the buck institute for research on aging where the saliva is logged, stored, and processed. the specimens are processed by separation into supernatant available for steroid hormone analysis, and a cellular component from which dna is isolated using invitrogen s purelink genomic dna kits. samples are stored in the mws biorepository housed at the buck institute in novato, california. for study 2, participants were also selected from the mws population and were drawn from among 2,400 participants for whom snp data were available from donated saliva samples. twenty - five preselected candidate snps were multiplexed and analyzed in the mws dna samples by intergenetics, inc (oklahoma city, ok) in parallel with analysis of their panel of 22 oncovue snps. all snp assays were performed and evaluated as previously described, and the clia - approved 22 snp oncovue assay and algorithm were previously validated as a new individualized breast cancer risk estimator using buccal dna samples from an earlier (19971999) marin county case control study cohort. selection of the novel 25 snp candidates evaluated in the present study followed a detailed literature search of snps reported in gwas studies to be significantly associated (in at least one published report) to specific exposures, outcomes, and hypothesized breast cancer associated pathways of relevance to the current mws. in cases where there were more than one snp from a given gene, weight of literature, linkage characteristics, and potential functionality based on specific location of the snp within the gene gene frequency was also a major selection criterion to ensure adequate power for analysis. for purposes of the analysis examining interactions with pih reported in this paper, we evaluated snps in the following seven genes : enos (nos3), esr2, vegf, edn1, il-10, hcfxi (klkb1), igfr1. one of the novel features of this study is the measure of % fgv assessed by the method single - energy x - ray absorptiometry (sxa) [1720 ]. this method uses a calibration phantom of the same thickness as the compressed breast, circumventing some of the problems associated with other breast density measures including their subjectivity and lack of absolute reference standards. the first generation calibration phantom (gamma) used for this method has provided preliminary data on 8,600 women to show that % fgv is reproducible to approximately 2 % between successive measures and accurate to known standards of breast composition using reference phantoms. investigators have demonstrated that % fgv is inversely correlated with age, body mass index (bmi), and menopausal status, as expected for a measurement of breast density, and is positively associated with breast cancer risk. % fgv data are collected by the sfmr at all sites in marin county which employ digital mammography and are obtained from the sfmr through a cooperative agreement. the mws was approved by the participating institutions, and participants provided written informed consent for participation in the study. multivariable linear regression analyses were conducted to examine the associations between % fgv and reproductive factors, controlling for relevant confounders. robust regression techniques were used to minimize the effects of outliers and influential observations (i.e., observations that have a large impact on the regression analysis). all models controlled for a base set of confounding variables, including current age, bmi, race, education, smoking, family history of breast cancer (whether a first degree relative has been diagnosed with breast cancer), hysterectomy status, menopausal status, and exogenous hormone use at the time of the mammogram. prior to regression, all genotyping data were checked for compliance of single - gene allelic frequencies with hardy weinberg frequency expectations using goodness - of - fit test. multivariable linear regression models were constructed adjusting for potential confounders including current bmi, age, race, and age of first live birth, and interaction terms were generated for the genotype of each snp compared to a baseline genotype. statistical tests were conducted to examine the interaction term for each level of the snp compared to the baseline level of the snp, as well as for the joint interaction effect of each snp overall and pih, and p values examined for statistical significance. the distribution of covariates and the mean % fgv for each level of the covariate are presented in table 1. the vast majority of the women were white, had a college degree, and were postmenopausal. there were significant associations between % fgv and age, menopausal status, bmi, race, education, smoking status, hysterectomy status, and age at menarche ; all associations were in the expected directions.table 1mean percent fibroglandular volume (% fgv) by study population characteristics (study 1)characteristicparous women (n = 2,440) n mean % fgv (sd)all women2,44035.3 (21.0)age 4548149.4 (22.5) 465575639.1 (22.00) 566568028.4 (16.0) > 6552325.8 (14.2) f statistic, p value169.71, p 1233342.9 (24.1) f, p 37.72, p 4040935.8 (21.5) f, p 0.65, p = 0.62 mean percent fibroglandular volume (% fgv) by first pregnancy risk factors (study 1) table 3 presents the results of multivariable models examining the effect of first birth characteristics on breast density overall and stratified by age at first birth. because the results are presented in units corresponding to the square root transformation of % fgv, the point estimates are not interpretable on a % fgv scale, but can be interpreted in terms of the magnitude of the effect.table 3linear regression model : percent fibroglandular volume (% fgv) in parous mws women with % fgv measurement and model variables (study 1)overall (n = 2,440)age at first birth<30 (n = 1,271)30 + (n = 1,169)parity (number : 15)0.02 (0.04, 0.08)0.01 (0.06, 0.08)0.05 (0.16, 0.07)high blood pressure (vs. no)0.31 (0.52, 0.11)0.24 (0.53, 0.06)0.42 (0.71, 0.13)breast - feeding (months)0.01 (0.003, 0.02)0.01 (0.0002, 0.03) 0.01 (0.001, 0.02)gestational weight gain (lbs)0.002 (0.01, 0.002)0.0001 (0.01, 0.01)0.002 (0.01, 0.01)birthweight (vs. normal) low0.04 (0.26, 0.18)0.29 (0.57, 0.002) 0.05 (0.31, 0.40) high0.04 (0.21, 0.12)0.01 (0.21, 0.22)0.02 (0.28, 0.23)weeks gestation (versus 38 + weeks) 3637 weeks0.08 (0.14, 0.29)0.24 (0.05, 0.54)0.03 (0.28, 0.35) < 35 weeks0.02 (0.40, 0.35)0.23 (0.29, 0.76)0.002 (0.55, 0.54)menarche (vs. < 10) 11140.07 (0.19, 0.33)0.07 (0.24, 0.39)0.08 (0.52, 0.37) 15 + 0.26 (0.04, 0.56) 0.32 (0.05, 0.69) 0.04 (0.54, 0.47)age at first birth (vs. < 20) 20290.01 (0.26, 0.24)nana 30340.01 (0.26, 0.28) 35 + 0.13 (0.15, 0.41) r 0.490.380.44controlled for current age, bmi, race, education, smoking, first degree relative with breast cancer, hysterectomy status, menopausal status (except in models stratified by hysterectomy status), and hormone use at the time of the mammogram differences significant at the p < 0.05 level differences significant at the p < 0.10 level linear regression model : percent fibroglandular volume (% fgv) in parous mws women with % fgv measurement and model variables (study 1) controlled for current age, bmi, race, education, smoking, first degree relative with breast cancer, hysterectomy status, menopausal status (except in models stratified by hysterectomy status), and hormone use at the time of the mammogram differences significant at the p < 0.05 level differences significant at the p < 0.10 level overall, having experienced high blood pressure during the first pregnancy was associated with a significantly lower % fgv (0.31 ; 95 % ci 0.52, 0.11). in addition, each month of breast - feeding was associated with a significantly increased % fgv (0.01 ; 95 % ci 0.003, 0.02). late menarche (age 15 + vs. before age 10) was associated with a borderline significant increase in % fgv (0.26 ; 95 % ci 0.04, 0.56). models were stratified by whether the first birth occurred before age 30 or at age 30 or later to examine whether these associations varied by age at first birth (table 3). among women with a first birth before age 30, each month of breast - feeding was borderline significantly associated with an increase in breast density (0.01 ; 95 % ci 0.0002, 0.03). in addition, having had a low birthweight infant was associated with a borderline significantly decrease in % fgv (0.29 ; 95 % ci 0.57, 0.002), and having had a late menarche was associated with a borderline significant increase in % fgv (0.32 ; 95 % ci 0.05, 0.69). in women whose first birth occurred at age 30 or later, having experienced high blood pressure during the first pregnancy was associated with a significant decrease in % fgv (0.42 ; 95 % ci 0.71, 0.13), and months of breast - feeding were associated with a significant increase in % fgv (0.01 ; 95 % ci 0.001, 0.02). while the model explained 44 % of the variance in % fgv in women whose first birth was age 30 or greater, it explained 38 % of the variability in women whose first birth was age < 30 years. because of the significant protective associations between pih and % fgv, and the consistency of this finding with those in the literature indicating a significantly protective effect of pih on breast cancer, we analyzed salivary dna samples to determine whether interactions existed for the associations between pih and % fgv based on seven snps previously shown to be associated with pih (online resource 1). for this analysis, participants were excluded if they had a history of breast cancer or use of antiestrogens, if they did not have a valid % fgv measurement, or if they had never had a live birth, leaving an analysis sample size of 1,240, which was further reduced in multivariable models because of missing data on one or more of the variables included in the model, including snp results when genotyping was not successful for that snp. in line with the study 1 analysis that was limited to reproductive characteristics in the first pregnancy, our study 2 definition for the exposure all of the seven snps were found to be in compliance with hardy weinberg frequency expectations. in this study 2 subset, % fgv was lower in women with a history of pih, although this finding did not reach statistical significance. none of the bivariate associations between the genotypes of the seven selected snps and % fgv were statistically significant. multivariable analyses indicated no significant interactions between pih and five of the seven snps tested (table 4) ; however, two of the seven snps were associated with pih. a borderline significant interaction was found between the ct genotype of the vegf snp and pih on % fgv (p = 0.063) (compared to the cc genotype). the tt genotype, which has the lowest frequency, did not occur in any of the women with pih in the first pregnancy and thus does not appear in the results. statistically significant interactions were found between pih and the gt genotype of the igfr1 snp (p = 0.01) (compared to the gg genotype) ; those with pih and the gt genotype had significantly lower % fgv than those with gg genotype of the igfr1 snp (joint interaction term p value = 0.03). a borderline significant interaction was found between the tt genotype of the igfr1 snp and % fgv (compared to the gg genotype) (p = 0.07). this group was only half the size of the gt group, which may explain why, despite the apparent larger effect seen in the graphic, this did not reach statistical significance. the main effects terms for the gt and tt genotypes of the igfr1 snp were both 0.18 in this multivariable model that included the interaction terms. both of these sets of interactions are presented visually in figs. 1 and 2, below.table 4multivariate associations between genotypes of the 7 pih snps and percent fibroglandular volume (% fgv) (study 2)snpbaseline genotypeinteraction termestimate p valuejoint test p values for interaction n for each genotypeedngg471edn_gtpih0.1780.58266edn_ttpih0.3000.700.7738hcfxcc374hcfx_ctpih0.0040.99323hcfz_ttpih0.2630.570.8475nos3cc124nos3_ctpih0.0750.85342nos3_ttpih0.1760.680.91305il10cc186il10_ctpih0.0200.95365il10_ttpih0.1160.760.95214vegfccvegf_ctpih0.6210.06na557200igfr1gg174igfr1_gtpih0.9060.01389igfr1_ttpih0.7340.070.03195esr2cc131esr2_ctpih0.4030.31379esr2_ttpih0.0530.900.60251fig. 2interaction of pih and igfr1 multivariate associations between genotypes of the 7 pih snps and percent fibroglandular volume (% fgv) (study 2) interaction of pih and vegf interaction of pih and igfr1 in this case, it can be seen that having pih and the cc genotype resulted in no change in % fgv, while having pih with the ct genotype resulted in a decrease in % fgv. in this cross - sectional study, we found that pih during first pregnancy was associated with significantly reduced breast density measured by % fgv in later life, the effect of which was greatest in those women whose first birth occurred at age 30 or later, where preeclampsia and gestational hypertension (or pih) are more prevalent. to our knowledge, this is the first study to identify an association between experience of a pregnancy affected by pih / gestational hypertension and % fgv. pih has been fairly consistently associated with a reduction in breast cancer risk (e.g., [5, 21 ]), though some studies have found no (e.g., [6, 22 ]) or even increased risk [23, 24 ]. pih is a multifactorial disease with genetic and environmental factors known to be involved in its etiology although full understanding of its pathogenesis remains elusive despite decades of research. likewise, the mechanism by which pih affects breast cancer risk is unknown although potentially via the same etiologic basis as pih, with theories focusing on placental dysfunction and a subsequent lowering of circulating estrogens, increase in serum insulin - like growth factor, and/or angiogenic factors. in support of the theory of an altered hormonal milieu who found non - significantly reduced breast density in women born from a pregnancy in which their mothers experienced eclampsia / preeclampsia, and similar findings reported in a systematic review by xue and michels who found significantly decreased risk of breast cancer if a woman s mother had experienced preeclampsia or eclampsia. in this study, pih was shown to significantly interact with specific allelic variants of insulin growth factor receptor-1 (igfr1 ; rs2016347), and borderline significantly with a specific allelic variant of vascular endothelial growth factor - a (vegf - a ; rs3025039) genes, indicating that the protective effects of first pregnancy pih on later life breast density and breast cancer risk may depend on inherited functional variants in these two growth factor receptor and angiogenic factor genes. prior studies involving these allelic variants support this conclusion, since the vegf - a snp investigated (rs3025039) has been repeatedly linked to decreased plasma levels of vegf and reduced breast cancer risk, [3133 ] while the igfr1 snp investigated (rs2016347) has not only been previously associated with breast density but also found to be an independent prognostic marker for breast cancer recurrence [34, 35 ]). while these various sources of evidence make it unlikely that these two snps found to modulate the significant association between pih and % fgv were simply false positive discoveries, these novel observations require additional validation in larger population - based studies given the strong inheritance pattern underlying breast density. duration of breast - feeding in first pregnancy was associated with increased % fgv later in life in our overall study population and in women regardless of age at first birth (though these findings were of borderline significance). our finding of a positive association between duration of breast - feeding and % fgv is in agreement with that of lope., who found a positive association between breast density (using the cumulus method) and duration of lactation, but conflicts with those of others who found no association (e.g., [10, 25 ]) or an inverse association between breast - feeding and breast density (e.g.,). differences in the timing and measurement of breast density between these diverse studies are the most likely explanation for their disparate associations, though this could also be due to basic differences in the study populations. although breast - feeding has been thought to produce an overall protective effect against breast cancer (e.g.,), many studies find no association (e.g.,) and recent systematic reviews of the topic fail to support an association (e.g., [38, 39 ]). more research is needed in the area of breast - feeding and breast health to better elucidate whether a protective effect exists, and if so, when and how breast - feeding might affect breast density. a number of other first pregnancy factors were less consistently associated with % fgv, but were suggestive in certain sub - analyses, including gestational weight gain, infant birthweight, and preterm birth. other authors have found associations between breast density and birthweight [11, 14 ] and preterm birth and breast density. while our data were suggestive of relationships between % fgv and these birth characteristics, our findings were neither consistent nor strong. the differences may be due to differences in the breast density measure or characteristics of the study populations, including differences in the age structure (only 55 and younger in el - bastawissi. and 4568 in lope.). this study has a number of important strengths, including a large sample size, a contemporary sample, a novel measure of breast density, and the availability of a wide variety of reproductive characteristics for study. the primary limitation in this study is the use of self - reported data for reproductive history. though it is possible that women may not accurately recall information about their first pregnancy, particularly if it occurred in the distant past, we would expect that they would accurately recall the major events including their age, an experience of pih, and breast - feeding. to the extent that recall bias is present, it would be nondifferential (i.e., not associated with % fgv), and would thus bias the results toward the null. another limitation is that, despite the fact that the overall sample size in this study was large, the sample size was small for specific subgroup analyses. studies with larger populations may be better able to detect significant associations between birth characteristics and breast density where they exist. selection bias may be present in the sample of patients providing saliva samples for the snp analyses ; women who consented to donate saliva were significantly more likely to be of white non - hispanic race and to be of higher socioeconomic status based on education and income, but were not significantly different in terms of family history of breast cancer or current age. while this may limit the generalizability of the findings indicating an interaction between pih and vegf and igfr1 on % fgv, any selection bias present should not limit the validity of the findings. finally, this study was intentionally restricted to first births, but it will be important to determine whether the findings for first birth characteristics hold for all births or whether they are unique to the first birth (e.g., whether total duration of breast - feeding has the same association with breast density as duration of breast - feeding after the first birth). in summary, we found associations between first birth characteristics and breast density measured as % fgv that confirmed and extended the few published findings on birth characteristics and breast density. pih was associated with a decrease in breast density and breast - feeding a increase in breast density, which may help elucidate the pathway by which they operate to affect breast cancer. variation in the association between pih and % fgv by genotype of igfr1 and vegf suggest that the protective effect of pih on breast density may vary between women depending on genotype.	purpose pregnancy characteristics have been associated with breast cancer risk, but information is limited on their relationship with breast density. our objective was to examine the relationship between first pregnancy characteristics and later life breast density, and whether the association is modified by genotype.methods the marin women s study was initiated to examine breast cancer in a high - incidence mammography population (marin county, ca). reproductive characteristics and pregnancy information including pregnancy - induced hypertension (pih) were self - reported at the time of mammography. forty - seven candidate single nucleotide polymorphisms were obtained from saliva samples ; seven were assessed in relation to pih and percent fibroglandular volume (% fgv). breast density assessed as % fgv was measured on full - field digital mammograms by the san francisco mammography registry.resultsa multivariable regression model including 2,440 parous women showed that pih during first pregnancy was associated with a statistically significant decrease in % fgv (b = 0.31, 95 % ci 0.52, 0.11), while each month of breast - feeding after first birth was associated with a statistically significant increase in % fgv (b = 0.01, 95 % ci 0.003, 0.02). pih and breast - feeding associations with % fgv were modified by age at first birth. in a subsample of 1,240 women, there was evidence of modification in the association between pih and % fgv by specific vascular endothelial growth factor (vegf) (rs3025039) and insulin growth factor receptor-1 (igfr1) (rs2016347) gene variants.conclusionthese findings suggest that first pregnancy characteristics may exert an influence on extent of breast density later in life and that this influence may vary depending on inherited igfr1 and vegf genotypes.electronic supplementary materialthe online version of this article (doi:10.1007/s10552 - 014 - 0386 - 2) contains supplementary material, which is available to authorized users.
life expectancy has increased during the past century, and this has led to a dramatic increase in the aging population. the number of americans over 65 is expected to double from the years 2000 to 2030 : in 2000, there were 34.8 million over the age of 65 (12% of the population), and in 2030, it is predicted that there will be 70.3 million in this age group (representing 20% of the population). this change in population dynamics presents substantial medical issues, as aging is a primary independent risk factor for development of cardiovascular disorders. sir william osler (18491919), one of the founders of johns hopkins university hospital, stated in his textbook that, longevity is a vascular question a man is only as old as his arteries. from a strictly aging / vascular perspective, numerous recent articles have described changes in anatomical and histological properties with age (e.g., see) as well as biophysical alterations, such as with changes in pulse wave velocity with age (as described in). however, one possibility to consider is that these aforementioned changes are likely due to aging - mediated alterations in the molecular and biochemical factors that determine vascular tone. vascular tone is regulated by both the intimal (endothelial) and medial (vascular smooth muscle) layers as well as through interlayer interactions. age - related changes in the structure and function of each layer is well documented [4, 5 ]. smooth muscle cells represent the major arterial cell population, and these cells highly express adrenergic receptors that mediate smooth muscle tone. although all three beta adrenergic receptor (-ar) subtypes, 1, 2, and 3, are found in vascular smooth muscle cells, the 2-ar subtype is by far the most highly expressed. of specific relevance to this paper is that the vascular 2-ar exhibits an age - related decline in signaling with advancing age that leads to impaired vasorelaxation. in contrast, the intrinsic ability for vascular muscle contraction is generally maintained throughout the aging process. this change is important, because it may allow for multiple age - associated clinical conditions such as hypertension, arterial insufficiency, orthostatic hypotension, and arteriosclerosis. the underlying change is hypothesized to be a decrease in -ar - stimulated camp production., the -ar is a target for many medications prescribed to the elderly and are used to manage hypertension, angina, postmyocardial infarction risk, congestive heart failure, glaucoma, tremor, arrhythmias, and chronic obstructive pulmonary disorders. beta - ar - mediated signal transduction pathways are well described, but new discoveries continue to impart complexity (see and following sections). at present, no change in any one factor in the -ar signal cascade has been identified to fully explain the impaired -ar vascular function observed with aging. instead, the cause of the change is likely multifactorial. this notion is supported due to intricate nature of -ar signaling and two biochemical findings : first is that expression of 2-ar does not change with age, and secondly, although drugs that activate -ars do not elicit complete vasorelaxation with advancing age, drugs that act on proteins postreceptor in the signaling cascade do. that is, the physiologic factors that mediate vasorelaxation can not completely dilate blood vessels with advancing age ; however, the molecular and cellular / anatomic machinery postreceptor vascular tone is physically established in the medial layer of blood vessels, which is almost entirely composed of vascular smooth muscle cells. numerous agents (epinephrine, norepinephrine, acetylcholine, angiotensin ii, nitric oxide, etc.) function through their cognate receptors localized at vascular smooth muscle, and/or endothelial cells and influence an elaborate network of signal transduction pathways that yields homeostatic control. the molecular mechanisms regulating smooth muscle contraction and relaxation are beyond the scope of this paper ; however, excellent reviews are found elsewhere [14, 15 ]. in blood vessels, the -ar signal transduction cascade mediates smooth muscle vasorelaxation. activation of the -ar stimulates the dissociation of the g protein, gs, from the subunit. the g protein subunit can also affect various membrane and/or organelle channels whose action can rapidly alter the ionic milieu of the cell. two molecules of this second messenger bind one regulatory subunit of protein kinase a (pka). structurally, pka is a tetrameric kinase made up of regulatory and catalytic subunit dimers. functionally, pka is a multipurpose kinase that controls numerous cellular events by phosphorylating protein targets. pka is distributed to multiple discrete intracellular compartments via the function of a - kinase anchoring proteins (akap). phosphorylated -ars are targets for still another group of proteins, the -arrestins that desensitize -ars and mediate internalization, which leads to receptor recycling and/or degradation. in addition, -arrestins can serve as scaffolds and adaptors for other kinases such as extracellular signal - regulated kinase (erk), src, and raf that regulate several cellular pathways that result in the activation of map kinases. identifying a possible locus for the age - related decline in -ar function three subtypes of the -ar (1-ar, 2-ar, and 3-ar) are found in vascular smooth muscle cells. also, at least nine different isoforms of adenylyl cyclase have been identified [21, 22 ], along with at least six grk, three arrestin, and multiple types of akap isoforms are known to exist. therefore, to understand the mechanism(s) of impaired -ar function with age, recent research has focused on proteins that interact with the -ar, directly or indirectly, that may be critical for optimal receptor signaling. investigations on age - related changes in various modifications to the -ar, such as phosphorylation - mediated desensitization, scaffolding proteins that form postreceptor signalosomes, or proteins that directly interact and affect -ar function may provide insight to explain the change. the initial observation of an aging effect on vasorelaxation was made in the early 1970s involving analysis of vascular smooth muscle pharmacodynamics in general. blood vessels from 6-month - old animals relaxed 90% less to isoproterenol as compared to blood vessels from 1-month - old animals [27, 28 ]. to explain this physiologic change, biochemical analysis found that both basal- and isoproterenol - mediated camp synthesis significantly declined in isolated aorta with advancing age. this was in contrast to adenylyl cyclase and phosphodiesterase activity that was essentially unaffected by age. from these results, it was concluded that the decreased ability of isoproterenol to elevate intracellular camp concentration, and thus relax aortas from older rats was predicated by an upstream change in the -ar itself, rather than a downstream change specific to adenylyl cyclase. from these initial observations, numerous researchers have evaluated the aging vasculature in an attempt to uncover the mechanism of this change in -ar function with age. as described above, the -ar signaling cascade is multifaceted, including numerous protein factors, each of which exists with multiple subtypes. the following is a discussion of what is currently known regarding the mechanism of the age - related change in -ar function, organized in a sequential manner, starting upstream with the -ar itself and continuing through the cascade as we currently understand it. presently, three distinct subtypes of the -ar have been identified in mammals, (1-ar, 2-ar, and 3-ar : crystalline structures (including affect of agonist occupancy) have recently been described. most of the literature demonstrates that the primary -ar subtype in the vasculature is the 2-ar, but both 1-ar and 3-ar are present and mediate vasorelaxation. the overall distribution, and relative proportion, of each -ar subtype varies across vascular beds [33, 34 ]. these three -ar subtypes work in concert to alter vascular tone in a complementary manner, as they all couple to gs and promote camp production. both 1-ar and 2-ar desensitize with activation due to the function of grks, pka, as well as other kinases and/or factors [36, 37 ]. interestingly, agonist exposure to the 2-ar also initiates the transformation of its g protein - coupling selectivity from gs to gi. when the 2-ar is linked to gi, one cellular event that occurs is an inhibition of adenylyl cyclase activity that is manifested as a reduction of intracellular camp concentration. there are no data to suggest that 1-ar changes its g protein subtype coupling preference. in contrast, the 3-ar does not appear to undergo desensitization, as it lacks regulatory phosphorylation sites for grks, pka, or other kinases. this characteristic may allow for prolonged signaling compared to 1-ar- and 2-ar - mediated effects. although expression levels of the three adrenergic receptor types is differential, with 2 being the most predominant subtype, and 3 being the least expressed, patterns of function could be altered with pathology. for instance, under maintained stimulation, 1-ar is predicted to desensitize, 2-ar is predicted to desensitize and further inhibit camp production through its linkage to gi, and 3-ar is predicted to possibly represent a functional alternative for camp production. we have evaluated whether an agonist - mediated change in 2-ar / g protein coupling observed in the heart could explain the age - related change in vasorelaxation. using pertussis toxin (pertussis toxin irreversibly adp ribosylates and inactivates gi) to block the coupling of activated 2-ar to gi in aortae isolated from fischer 344 rats of increasing age, it was found that this treatment did not alter the age - related decline in relaxation. however, a population of 2-ar coupled to gi was found, as pertussis toxin treatment improved 2-ar - mediated vasorelaxation in aortae for all ages in equal proportion. however, left ventricular function and age - related heart failure were highly correlated with 3-ar expression in rats. these findings suggest that further investigation is warranted to characterize ar subtype expression patterns and 3-ar function throughout the vasculature. because the 2-ar is the most highly expressed subtype in the vasculature, much interest is the finding that 2-ar sensitivity substantially declines with age. results found that in aortic preparations from 1-month - old animals, 64% of the -ars were in the high affinity state. this compared to 40%, and 0% high affinity -ars for 6- and 24-month - old animals, respectively. to explain these results, age - related changes in the content of 2-ar bound to gs was examined : 2 ar : gs complexes were found only in aortic preparations from 1-month - old animals. these data strongly suggest that there is a substantial decline in high - affinity 2-ar with advancing age. this change did not appear to be related to a decline in the presence of -ars at the membrane (further confirmed) or caused by a switch in g protein coupling as occurs in cardiac tissue. another interesting possibility to explain the age - related decline in 2-ar signaling is the possibility that -ar can form hetero- and homodimers, and this could alter signaling fidelity. mercier. showed that -ars likely exist as either 1-ar : 1-ar homodimers, or 1-ar : 2-ar heterodimers, and they suggested that changes in the overall cellular configuration of monomers, heterodimers, and homodimers could be altered by agonist as well as disease state, and this finding was supported by lavoie.. also, 1-ar : 2-ar heterodimers have been shown to exhibit distinct functional and pharmacological properties, resulting in enhanced signaling efficiency in response to agonist stimulation and optimizing -adrenergic modulation of contractility in cardiomyocytes. however, controversy exists as to whether -ar dimerization occurs in cardiovascular tissue. a report by ianoul. that used higher - fidelity imaging techniques suggested that 1-ar and 2-ar may be localized in two different populations of microdomains in cardiomyocytes, an observation inconsistent with the existence dimers. of additional interest is a recent report by larocca. that showed, also in cardiomyocytes, that 2-ar form heterodimers with the chemokine receptor type 4 (cxcr4) and this interaction negatively regulated isoproterenol - mediated 2-ar signaling by altering 2-ar sensitivity. following from these studies, we evaluated whether cxcr4 activity modulation could alter -ar - meditated vasorelaxation. using aortae isolated from 2-month - old fischer 344 rats, it appeared that cxcr4 activation inhibited, and cxcr4 blockade improved the vasorelaxant effect of isoproterenol (figure 1). determination of age - related changes in cxcr4-mediated alterations in -ar - stimulated vasoreactivity, cxcr4 expression, and the interaction between 2-ar and cxcr4 are underway. the age - related change in 2-ar signaling appears to caused by changes in receptor sensitivity, and changes in g protein expression or function could manifest this physiology. however, its function appears to be age impaired, as direct gs activation - mediated camp production by was reduced in aortae isolated from old rats. our lab further characterized this observation by finding a marked decline in cholera toxin catalyzed adp ribosylation labeling of gs without a decline in the expression, suggesting some age - related alteration in g protein structure / function. we have also found that cholera toxin - mediated relaxation in aortic rings decreased with advancing age. further evidence that gs function is altered with advancing age in the vasculature is from a study where it was attempted to reverse age - related declines in -ar - mediated vasorelaxation by expressing a constitutively activated mutant of gs (gs - q227l) into aorta from 6-month - old fisher 344 rats. in that study, aorta that expressed gs - q227l exhibited enhanced isoproterenol - stimulated vasorelaxation, and both basal- and isoproterenol - stimulated camp production was increased. therefore, gs may undergo some age - related change that inhibits its ability to become activated via the action of an agonist - occupied receptor. in support of a functional change in gs with age is the identification of a regulator of g protein signaling (rgs)/gtpase activating protein (gap) that functions on gs called rgs - px1. age - related enhanced rgs - px1 activity could impair -ar signaling without changes in -ar or gs expression as agonist exposure would not initiate vasorelaxation as gs signaling would be quenched due to the high rgs / gap activity of rgs - px1. at present, rgs - px1 has not been evaluated within an aging paradigm. in total, the decline in -ar - mediated vasorelaxation and camp accumulation observed in old vessels could be caused by a change in the function but not expression of gs. another possible explanation for the decline in -ar - mediated signaling could be an increase in gi function, as this g protein subunit inhibits adenylyl cyclase activity and thus camp production. also, the 2-ar, the predominant receptor species in vascular smooth muscle, has been shown in cardiomyocytes to rapidly link to gi after agonist activation, and it is pka - phosphorylated. we actually found a slight decline in pertussis toxin labeling of gi with age. also, a 30% decrease in gi1&2 protein content between 6- and 24-month - old aortic preparations has been documented. g has been shown to either stimulate or inhibit adenylyl cyclase activity in the presence of activated gs. g also affects numerous plasma and organelle membrane - localized ion channels, thereby affecting the net polarity and potential for tonal changes of vascular smooth muscle. however, we have found no age - related changes in the expression of g subunit. as discussed, the fundamental change in blood vessels from older animals is a pronounced inability to relax to -ar stimulation. however, old vessels do maintain the ability to relax entirely, as acetylcholine-, forskolin-, and nitrate - mediated vasorelaxation is complete. therefore, a probable protein candidate for the impairment is gs (as discussed previous) or g protein receptor kinase (see following). the classical effector of gs is adenylyl cyclase. because forskolin stimulates blood vessels from young and old animals to relax completely, and to accumulate camp equally, it is generally thought that adenylyl cyclase function does not change with advancing age. however, the adenylyl cyclase family contains nine different isoforms, each with discrete tissue distribution. for instance, calcium (at relevant intracellular concentrations) stimulates adenylyl cyclase subtype-1, and subtype-8 but inhibits subtype-3 and subtype-9. interestingly, zhang. have demonstrated that the predominant adenylyl cyclase isoforms in vascular smooth muscle are of the calcium - sensitive variety. therefore, a possible explanation for impaired receptor - mediated - camp production is not with the cyclase itself, but rather the interaction between cyclase and another, critical and age - affected, cellular factor that could regulate intracellular calcium sequestration. another line of reasoning would be that adenylyl cyclase activity is unchanged across age, but camp processing is altered. therefore, changes in physiology could be due to age - related changes in the processing and degradation of camp through phosphodiesterases. we have determined that there are no age - related changes in general phosphodiesterase inhibitor - mediated vasorelaxation using 3-isobutyl-1-methylxanthine (ibmx), a nonspecific phosphodiesterase subtype inhibitory agent. however, others found that using a low dose of ibmx caused impaired camp accumulation in blood vessel preparation from older rats. the role of phosphodiesterases in mediating age - related changes in camp concentration is presently underevaluated. also, only recently have drugs specific to individual isoforms have become available. to that end, it has recently been shown that the vasodilator pathway associated with phosphodiesterase iii is likely unchanged with aging in humans. -ar desensitization is initiated by phosphorylation of the receptor, which is followed by its uncoupling from its signaling cascade. phosphorylated -ars are targets for another family of proteins that mediate uncoupling / desensitization, the arrestins. therefore, arrestins function in concert with grks to attenuate intracellular signaling [66, 67 ]. to date, six different grks have been identified. of interest, grk-2, grk-3, and grk-5 (grk-2 and grk-3 are also known as -ar kinases : -ark-1 and -ark-2, resp.) target -ars and significant increases in grk activity and expression have been observed in ventricles of failing human hearts. the progression of alzheimer 's disease has also been associated with enhanced grk function in fibroblasts taken from human skin. relating specifically to grk function and the vasculature, cultured vascular smooth muscle cells have been shown to express grk-2 both within the cytoplasm and at the membrane, and a transgenic mouse that overexpressed grk-2 in a vascular - specific manner has been developed. also, overexpression caused elevations in resting blood pressure and was accompanied by an increase in vascular thickening, suggesting a decline in camp generation. an age - related change in grk activity or expression in vascular tissue would implicate grks in the age - related decline in -ar mediated vasorelaxation. only a few studies have been performed to assess age - related changes in grks. no changes in grk activity or grk-2 and 5 expression were observed in lymphocytes of aged humans. however, expression of soluble grk-2 increased with maturation in thoracic aortic preparations from fischer 344/brown norway rats. we have also examined age - related changes in grks. in aorta from aged fischer 344 rats, total grk activity increased nearly 2.1-fold. in the soluble (cytosolic) fraction, grk-2 expression increased nearly 3.6-fold, grk-3 expression increased approximately 3.8-fold, and -arrestin expression increased approximately 1.6-fold. in the membrane fraction, grk-2 expression increased approximately 1.5-fold, grk-3 expression increased nearly 2.1-fold, while there was not an age - related change in the expression of grk-5. these data suggest that a critical feature of age - related impaired -ar signaling may be imparted through an increase in total pool of grk that could be explained by either increased expression, or decreased degradation. this increased pool could allow for enhanced targeting of these receptor kinases to the membrane, and hence the -ar. whether -ars from aged vessels have increased phosphorylated residues, and thus enhanced desensitization is yet to be established. also, the mechanism for the enhanced expression of grks with advancing age is likewise yet to be explained but is an active interest of our lab., -ar - mediated function requires appropriate localization (cytoplasmic versus membrane) and organization (to allow efficient and rapid interaction with one another) of these proteins. therefore, recent research has focused on scaffolding proteins, which are intracellular proteins that compartmentalize multiple related signaling molecules to specific intracellular domains. this theory has replaced the classical random collision - coupling theory of signal transduction. the -ar signaling cascade is anchored within the plasma membrane by the scaffolding protein caveolin. 2-ars [77, 78 ], numerous g - proteins (including gs) [79, 80 ], adenylyl cyclase (numerous isoforms) [8082 ], and grks 2, 3, and 5 all localize in caveolin - rich domains of the cell membrane. age - related changes in caveolin have recently been demonstrated revealing tissue - specific changes in expression. also, our results with fischer 344 rat aortic tissue show that the expression of caveolin-1 decreases with advancing age. an age - related change in caveolin expression could easily alter the milieu of proteins within a -ar signaling pocket, and thereby alter signaling. indeed, carman. our lab has shown that with advancing age, the interaction between caveolin-1 and grk2 substantially declines. therefore, both the scaffolding and protein activity - modulating functions of caveolin may be compromised with advancing age in the vasculature. produced caveolin-1 null mice and found that these animals exhibited impaired aortic steady - state maximal tension induced by phenylephrine (an -ar agonist). also, acetylcholine - mediated (nitric oxide - dependent) vasorelaxation was similarly altered in null animals. finally, caveolin-1 null mice displayed hyperproliferation in certain cell types, suggesting (but not documented) a decline in camp production. changes in -ar-, camp-, or age - mediated effects in the caveolin-1-null mice have yet to be evaluated. however, these data from transgenic animals clearly indicate that caveolin is an important modulator of vascular function. a potentially interesting phenomenon observed in molecular signaling is receptor cross - talk events. activation of pkc by gq - linked - agonists directed grks to the membrane, enhancing -ar phosphorylation and desensitization. also, it has been shown that grk-2 is more effective at desensitizing -ars after its activation by pkc. finally, antisense technologies have been used to knockout pkc expression and function ; these studies determined that this manipulation produced enhanced -ar agonist - induced desensitization rather than the expected attenuation result. in addition, these authors subsequently found that this effect was linked to phosphatase activity. these findings suggest that pkc might also be involved with -ar resensitization through interaction with a phosphatase. therefore, phosphorylation / dephosphorylation and desensitization / resensitization of -ars can be induced from a number of stimuli including angiotensin ii. in terms of vascular -ar being altered by receptor cross - talk are in vitro studies showing that angiotensin ii enhanced -ar - mediated camp production in cultured aortic vascular smooth muscle cells [61, 92, 93 ] as well as in preglomerular microvascular smooth muscle cells [94, 95 ]. in terms of vasorelaxation being affected was a study that found that angiotensin ii can enhance camp - mediated vasorelaxation via angiotensin ii - type 1-receptors (at1). we examined the interaction among aging, -ar - mediated vasorelaxation, and angiotensin ii. our results showed that this effect of angiotensin ii on agonist - mediated vasorelaxation was limited to young (6-week - old) or adult (6-month - old) rats, was absent in aged (12- and 24-month - old) animals, and was mediated by angiotensin ii - type 1 receptors. angiotensin ii appeared to amplify vasorelaxation in aorta from 6-week and 6-month - old animals via enhanced production of camp. the mechanisms involved with angiotensin ii enhanced, -ar - mediated signaling are unknown but may involve adenylyl cyclase, gs, or calcineurin. further study may show that aging may effect a factor common to both angiotensin ii and -ar signaling pathways or that aging may impair cross - talk between these two receptor pathways. a final interesting aspect of age - related changes in -ar - mediated signaling is understanding the role of various ion channels ; it is well understood that the function of numerous ion channels is responsible for determining membrane potential. the effect of isoproterenol on the ionic milieu of aortic vascular smooth muscle cells was characterized. results determined that isoproterenol functioned by inducing hyperpolarization via activating atp - sensitive potassium channels (katp). they also determined that the isoproterenol / katp - mediated hyperpolarization was impaired in smooth muscle cells from older rats. however, the effect of direct activation of katp was unchanged between young and old groups. therefore, their data fit well with what has been previously known about the age - related changes in -ar signaling the alteration appears to be localized proximal to adenylyl cyclase and may involve changes in the -ar itself or in its ability to couple to other regulatory molecules. a controversial topic is the endothelium - mediated effect on the age - related change in -ar function. it is clear that age - related changes in the endothelium occur, and there are endothelium - localized -ar. it is also well accepted that removal of endothelium reduces the effect of isoproterenol on vasorelaxation in a variety of isolated arteries and veins from different species, including humans. data show that endothelial cells have binding sites for -ar ligands and that isoproterenol increases nitric oxide synthase activity in these cells. compatible with these findings is that inhibition of nitric oxide synthase modestly decreased relaxation to -ar agonists. therefore, endothelium- and vascular smooth muscle - mediated function may be additive in that -ar - mediated vasorelaxation appears to be induced via both nitric oxide - mediated pathways (endothelial), and camp - mediated pathways (vascular smooth muscle). in terms of aging, however, found that the endothelial component did not change with age, whereas the vascular smooth muscle component did. they did identify an age - specific, endothelium - dependent effect in that vascular tone appeared to be mediated through an endothelium - derived hyperpolarizing factor (tetraethylammonium - sensitive k+ channels) that was increased with advancing age. age - related changes in membrane polarization are also discussed previously where katp - mediated hyperpolarization was found to be impaired in vascular smooth muscle cells from older rats. our lab has also produced data in support of a role for changes in polarization in mediating the age - related change in -ar - mediated vasorelaxation. when comparing the effect of age on isoproterenol - mediated vasorelaxation on phenylephrine- versus kcl - contracted aorta, vessels contracted with phenylephrine relaxed to a substantially higher degree that those contracted with kcl, although the age - effect was maintained. one interpretation of this result is that with advancing age, isoproterenol - mediated signaling pathways involve an increased role for k+ channels. further support that the aging change in -ar signaling is vascular smooth muscle-, rather than endothelium - dependent is that other agents that initiate vasorelaxation through vascular smooth muscle localized g protein - coupled receptors (adenosine, parathyroid hormone) also show impaired vasorelaxation with age [105, 106 ]. therefore, the age - related change is likely due to a factor common to all vascular smooth muscle - localized g protein - coupled receptors, such as grk (see above discussion), while a nonage - related endothelial dependent component contributes to -ar - stimulated vasorelaxation in general. hypertension, orthostatic hypotension, arterial insufficiency, atherosclerosis, and restenosis are common disorders in the elderly that lead to significant morbidity and mortality. these clinically significant conditions all may have a common feature in that they are associated with and age - related change in -ar signaling. impaired -ar - mediated vasorelaxation with age is observed throughout species and arterial beds, and in aged vascular tissue, -ars are desensitized. one cellular process that changes with age that modulates -ar sensitivity is phosphorylation by grk-2. but, there are multiple other protein factors that may modulate -ar function with aging. therefore, the overall complexity of the molecular pathways creates difficulties in isolating a single therapeutic target. regardless, a large and growing segment of the general population are age 65 or older, and this percentage will continue to rise. optimal care of this population is a priority for clinicians, and better understanding of this age - related change in the vasculature will allow for innovative strategies for the management of multiple disorders. findings will be applicable to other tissues and disease states where -ar signaling is altered (such as in the kidney and liver, heart, lung, and brain.	hypertension, orthostatic hypotension, arterial insufficiency, and atherosclerosis are common disorders in the elderly that lead to significant morbidity and mortality. one common factor to these conditions is an age - related decline in vascular beta - adrenergic receptor - mediated function and subsequent camp generation. presently, there is no single cellular factor that can explain this age - related decline, and thus, the primary cause of this homeostatic imbalance is yet to be identified. however, the etiology is clearly associated with an age - related change in the ability of beta - adrenergic receptor to respond to agonist at the cellular level in the vasculature. this paper will review what is presently understood regarding the molecular and biochemical basis of age - impaired beta - adrenergic receptor - mediated signaling. a fundamental understanding of why -ar - mediated vasorelaxation is impaired with age will provide new insights and innovative strategies for the management of multiple clinical disorders.
iron deficiency (i d) is the most common nutritional disorder among children and lead toxicity, especially in developing countries, is the most common environmental health threat, because of rapid urbanisation and uses of leaded fuels, dyes and glazed household pottery and so on.12 children especially infants are at high risk for i d because of high demand for iron and low diet iron during a period of rapid growth. both i d and lead poisoning are harmful for early development and growth in children and may cause profound neurologic and developmental effects such as cognitive and behaviour problems.345 during 1976 - 1980, blood lead concentration was more than 10 g / dl in 78% of people in the united state, but it was reduced to 20% in 1998.3 however, in developing countries such as iran lead pollution is a serious problem6 and previous studies in children have shown a blood lead concentration > 10 g / dl in 41 - 75% of children in large crowded cities.17 the us environmental protection agency has suggested a threshold level of 20 - 40 g / dl for anaemia in children. although some studies especially in children have showed an association between i d and high blood lead concentration,389101112131415 some other studies have not confirmed this.41016 it have been speculated that iron and lead compete for absorption in the small intestine and in children with inadequate amounts of iron consumption, lead absorption will be increased.1718 even some other study such as wolf a., showed that iron supplement therapy in children with i d could reduce blood lead level.18 there has been controversy on the association between i d and blood lead concentration in children and due to high frequency of i d and lead toxicity in this age group especially in underdeveloped and developing countries like iran, we evaluated this association. in addition, if this association is causative, then preventing i d persons in target high risk group might prevent lead poisoning.418 this cross sectional study was approved by local ethic committee and performed on 230 children aged 1 - 10 years, in imam reza teaching hospital of mashhad (a big city located in northeast iran) in 2010. after obtaining a informed consent and a short history from children referred to laboratory for evaluation of i d, 2 ml anticoagulated blood with ethylene diamine tetraacetic acid (edta)-k2 for measurement of complete blood count (cbc) and blood lead concentration and 4 ml blood without anticoagulant for determination of iron, total iron binding capacity (tibc) and ferritin was taken. because ferritin is an acute phase reactant, children with any evidence of acute or chronic infection in medical history and clinical examination were excluded from this study. blood lead concentration was measured by an atomic absorption spectrophotometer using heated graphite atomisation technique (perkin elemer, model 3030). quality control for this analysis was done with two normal and high levels of commercial control material (seronorm, sweden) everyday. according to the u.s. centres for disease control and world health organisation (who) standards, blood lead concentration 10 g / dl was considered as lead intoxication.4719 cbc, haemoglobin, haematocrit and red blood cells (rbc) indices were measured by a cell counter (sysmex k-21, japan). serum ferritin was determined by radioimmune assay (kavoshyar reagent - iran) and serum iron and tibc by colorimetric methods. iron deficiency anaemia (ida) in this age range group was defined as haemoglobin 350 g / dl (transferrin saturation 40 g / dl was observed in 80.6% of children in control group and 82.6% of children in patient group with no significant difference (p value = 0.117). table 1 also shows mean differences between ferritin, iron, tibc, haemoglobin, mcv, mch and mchc in two groups [table 1 ]. we also did not find any correlation between blood lead concentration and haemoglobin (r = 0.09, p = 0.186), ferritin (r = 0.045, p = 0.632), iron (r = 0.105, p = 0.252), tibc (r = 0.064, p = 497), mcv (r = 0.051, p = 457), mch (r = 0.007, p = 0.913), mchc (r = 0.096, p = 0.156), wbc (r = 0.092, p = 0.174) and platelets (r = 0.02, p = 0.764). chronic lead intoxication may affect children because they have more hand - to - mouth activities and they absorb lead in small intestine more efficiently than adults.101922 all children evaluated in this study had lead intoxication. it shows children breath high concentration of lead in this polluted big city that can be associated with renal, cardiovascular, hematologic, and irreversible neurologic toxicity.7 this high prevalence of lead poisoning has to decrease with a preventive national program especially with reducing leaded fuels. initially, iron stores and ferritin are depleted and iron absorption increase but serum iron is sufficient, in the second stage, serum iron is decreased and eventually in third stage, ida is created.23 hence, serum ferritin is a valuable marker of i d showing iron stores. children with high blood lead levels are more probably to show evidence of i d and children with i d probably have higher blood lead even after controlling for age, sex and socioeconomic status ; however, these pathways are different.18 according to the results of this study, we did nt observe any relation between blood lead concentration and i d and/or ida [table 1 ]. many studies especially in paediatric group have revealed that i d and/ or ida can elevate blood lead concentration with increase blood lead absorption in gastrointestinal tract.34911222425 there is a hypothesis for this increase in lead absorption. recent research have shown an iron transport channel in the intestine named the divalent metal transporter (dmt), which is regulated by iron regulatory proteins 1 and 2.2326 dmt1 is not specific for iron ; it can transport many of divalent metal ions, such as copper, zinc and lead. therefore, absorption of some other ions such as lead increase in low iron diet or in the high exposure of body to lead in the situation like pica observed specially in infants.3101127 however, bannon., (2003) in a study on cell lines in a knockdown model confirmed that although dmt is main way of lead absorption, it can also enter the cells by other mechanism,28 willows., (2002) in a study for evaluating of association between blood lead concentration and ida on infants characterised that infants with ida had a significantly higher blood lead concentration than infants without anaemia. they also found a significant negative correlation between blood lead and haemoglobin (r = 0.203, p = 0.006) and blood lead and ferritin concentrations (r = 0.245, p = 0.003). in another study, shah., (2010) evaluated environmental exposure of lead and ida in children 1 - 5 years and observed similar results.2 even some researchers have shown that iron fortification of food to children with high lead - exposed may decrease blood lead levels.1027 wright., (2003) in a longitudinal study on 1,275 children evaluated blood lead concentration, haematologic parameter and iron status in a 3-year period with two consecutive visits. logistic regression analysis showed i d was associated with subsequent lead intoxication.4 relation between blood lead concentration and ida in some other studies especially in adults is inconsistent.11116 they believed, initially, that environmental lead exposure is as aerosols that absorb from the respiratory tract not gastrointestinal tract. secondly, both diseases are more common in the lower socioeconomic class and, therefore, this relation might be secondary to common environmental risk factors, and i d may merely be a marker of high environmental blood lead.19 some other studies have shown the relationship of iron status and blood lead concentration varied within ethnic groups. they assumed genetic polymorphism in lead binding proteins can affect blood lead concentration independently of iron status.11 as we mentioned, our finding also did nt show any association between blood lead concentration and i d or ida. we speculate various reason for this result : it may be due to genetic polymorphism in proteins transmitting the iron and lead;11 this finding also can be cause by chance alone ; in addition, the studies have shown that haematopoiesis affects in blood lead concentration more than 25 g / dl, and anaemia is seen with blood lead concentration more than 50 g / dl.19 in this study, all children had lead intoxication and mean blood lead concentration was more than 50 g / dl in each group. therefore, the high blood lead concentration in the two groups, could affect the haematologic parameter evenly between them and it may be a reason that mean differences in haematologic parameter between two groups were not significant. in addition, it is possible that at this high level of lead in environment / blood, all children, regardless of whether i d or ida or not, will have similar blood lead concentration. it can be for this reason that lead will still find another ways of getting into the body, indeed absorption in intestine by dmt, when it is very abundant, and in the event of over abundance, it may not be very dependent on or limited by iron status. we suggest performing a similar study that also includes evaluation of children with low blood lead concentration such as children living in the rural area. rbcs indices were lower and platelets were higher in patients compared with control group [table 1 ] ; i d decrease rbc indices and can cause increase in platelets.23 we did nt determine any significant difference between blood lead and white blood cells (wbc). however, choi., showed that blood lead elevation can be with increase in leukocyte counts.13 we did nt find any association between i d and blood lead concentration in children. further studies in highly polluted and also unpolluted area are necessary to evaluate this association. indeed, because of high prevalence of lead toxicity in children in this area	background : iron deficiency anaemia is the most common nutritional anaemia among children. lead toxicity is a serious health threat, especially in developing countries due to environmental pollution. it was thus aimed to investigate correlation between blood lead concentration and iron deficiency in children of mashhad, iran.materials and methods : this cross sectional study was performed on children between 1 year and 10 years, in imam reza teaching hospital of mashhad, iran, in 2010. indeed during complete blood count (cbc), we measured iron and total iron binding capacity (tibc) by colorimetric methods, ferritin by radioimmune assay and blood lead concentration by atomic absorption method. results were analysed by statistical package for social sciences (spss) (version 11.5), using statistical tests including independent sample t - test, mann - whitney u test, spearman 's test and analysis of variance (anova) and pearson 's or spearman 's correlation coefficient. p value 0.05 was considered as a significant level.results:we studied 223 cases including 98 control children and 125 patients. all children had lead intoxication. mean (sd) blood lead concentration in the control group was 57.1 25.3 (ranged 20 - 212) g / dl and in the patient group was 57 20.4 (ranged 10.9 - 159) g / dl with no significant difference (p value = 0.713). we also did not find any correlation between blood lead concentration and haemoglobin, ferritin, iron, tibc, mean corpuscular volume (mcv), mean corpuscular haemoglobin (mch), mean corpuscular haemoglobin concentration (mchc), white blood cells (wbc) and platelets.conclusion:based on these results, no correlation was found between blood lead concentration and iron deficiency in the children. because all children had lead intoxication, further studies in highly polluted and a comparison with a low polluted area are necessary to make a general conclusion.
the calcineurin (can) inhibitor, tacrolimus, is widely used in patients undergoing allogeneic organ transplantation and in those with certain allergic diseases. recently, several reports have suggested that can is also associated with schizophrenia. however, little data are currently available on the direct effect of tacrolimus on the human brain. a 23-year - old japanese female experienced severe delusion of persecution, delusional mood, suspiciousness, aggression, and excitement. when she was 27 years old, she had severe general fatigue, persistent fever, systemic joint pain, gingival bleeding, and breathlessness and was diagnosed with acute myelomonocytic leukemia. later she underwent bone marrow transplantation (bmt), she was administered methotrexate and cyclosporin a to prevent graft versus host disease (gvhd). three weeks after bmt, she showed initial symptoms of gvhd and was prescribed tacrolimus instead of cyclosporin a. seven months after bmt at the age of 31 years, she died of progression of gvhd. pathological anatomy was examined after her death, including immunohistochemical analysis of her brain using anti - can antibodies. for comparison, we used our previous data from both a schizophrenia group and a healthy control group. no significant differences were observed in the percentage of can - immunoreactive neurons among the schizophrenia group, healthy control group, and the tacrolimus case (all p>0.5, analysis of covariance). compared with the healthy control group and schizophrenia group, the percentages of can - immunoreactive neurons in layers iii vi of the ba46 and the putamen tended to be lower in the tacrolimus case. in addition, we also found that the effect of tacrolimus on can immunore - activity in human brain was stronger than the effect of schizophrenia. the calcineurin (can) inhibitor, tacrolimus, is a nonsteroidal, anti - inflammatory immunosuppressive drug mainly used to treat patients undergoing allogeneic organ transplantation and those with atopic dermatitis.1 can is a heterodimeric calcium - dependent serine / threonine protein phosphatase, consisting of a catalytic (can a) and a regulatory (can b) subunits, and plays a critical role in cellular responses and calcium signaling.2 tacrolimus induces not only mild neurological side effects such as tremor but also psychiatric side effects such as manic - like psychosis and relapse of schizophrenia.3,4 on the other hand, several reports suggested that can dysfunction may be a risk factor for schizophrenia. forebrain - specific ppp3cc (encodes the can a -catalytic subunit) knockout mice display multiple abnormal activities related to schizophrenia, such as increased locomotor activity, decreased social interaction, impairments in prepulse inhibition, and latent inhibition.5,6 another report showed decreased can a protein levels and decreased mrna of the three subunits of can a in the hippocampus of schizophrenia patients.7 chang reported on the b - cell lymphoma-2 (bcl-2)can dopamine- and camp - regulated phosphoprotein of 32 kda (darpp-32) feedback mechanism in regulating serine 1755 phosphorylation and apoptosis in primary human chronic lymphocytic leukemia cells. however, there were no previous reports that acute myelomonocytic leukemia (aml) itself has direct influence on can in the brain. to investigate the effect of tacrolimus in the brain, we examined the immunoreactivity of can using immunohistochemistry in the dorsolateral prefrontal cortex, hippocampus, caudate, and putamen of a postmortem brain of a patient with schizophrenia who had been treated with tacrolimus. as healthy and disease controls, we used our previously reported data.9 there were no previous reports of aml itself having a direct influence on can in the brain. if we only aimed to investigate the effect of tacrolimus on the brain, we would have focused on characterizing the effect of tacrolimus in aml patients without prior history of schizophrenia. we aimed to investigate whether tacrolimus treatment or acquiring schizophrenia had a stronger effect on the can immunoreactivity in the brain. a 23-year - old japanese female visited a local psychiatric clinic in may 1990 due to nonspecific physical symptoms. several months after she first visited that clinic, she experienced severe delusion of persecution, delusional mood, suspiciousness, aggression, and excitement. because they shared the same delusion of persecution, they moved together many times to various places based on delusions. in november 1992, she began to take haloperidol (4.5 mg / d) and chlorpromazine (200 mg / d) for her psychotic symptoms. five months after starting antipsychotics, her psychotic symptoms had improved. in june 1996, she experienced severe general fatigue, persistent fever (38c), systemic joint pain, gingival bleeding, and breathlessness. her blood tests showed pancytopenia (white blood cells, 3.110/mm ; red blood cells, 2.610/l ; hemoglobin, 7.5 g / dl ; and platelets, 8.810/l) and the presence of blast cells. we consulted a hematologist, and she was diagnosed with aml. in september 1996, she underwent bone marrow transplantation (bmt). after bmt, she was administered methotrexate and cyclosporin a to prevent graft versus host disease (gvhd). at the same time we judged that she had initial symptoms of gvhd, and the hematologist prescribed tacrolimus instead of cyclosporine a. in december 1996, her auditory hallucination, delusions, and excitement recurred. her general condition gradually worsened. in february 1997, she experienced disturbance of consciousness, hypotension, and respiratory failure. in april 1997, she died of progression of gvhd at the age of 31 years. the use of postmortem human brain tissues for the present study was approved by the ethics committee of fukushima medical university (fmu) and complied with the declaration of helsinki. all procedures were carried out with the informed written consent of the next of kin. brain tissue blocks were obtained from the right hemisphere of the dorsolateral prefrontal cortex (ba46), hippocampus, caudate, and putamen and fixed in 10% formalin, embedded in paraffin, and sliced into 5 m sections. immunohistochemistry was performed in accordance with our previous protocol.911 the sections were deparaffinized in xylene and rehydrated through a decreasing alcohol series (100%, 90%, 80%, and 70% ethanol). endogenous peroxidase activity was quenched by incubating in 0.3% hydrogen peroxide in methanol at room temperature for 20 minutes. the sections were washed with phosphate - buffered saline (pbs : 0.01 m sodium phosphate, ph 7.3), microwaved for 15 minutes in 10 mm citrate buffer for antigen recovery, and washed with pbs. the sections were incubated in 5% skim milk in pbs for 30 minutes at room temperature to reduce nonspecific binding, incubated overnight at 4c in rabbit polyclonal antibodies against human can (1717 - 0909 ; abd serotec, oxford, uk, 1:50), which recognizes the 60 kda mammalian can catalytic subunit (can a), in pbs, and washed in pbs three times for 5 minutes each. the sections were incubated with the second antibody (biotinylated anti - rabbit igg ; nichirei corporation, tokyo, japan) for 20 minutes at room temperature and washed in pbs three times for 5 minutes each. subsequently, the sections were incubated with peroxidase - labeled streptavidin for 15 minutes at room temperature and washed in pbs three times for 5 minutes each. osaka, japan) for 5 minutes at room temperature to visualize the immunoreaction products and then washed with pbs to terminate the reaction. for nuclear counterstaining, the sections were treated with carracci s hematoxylin for 20 seconds. can - immunoreactive (can - ir) cells were examined under a microscope equipped with a digital camera (olympus bx51 ; olympus corporation, tokyo, japan) at 100 magnification, and parenchymal cell numbers were counted using winroof version 5.5 (mitani corporation, tokyo, japan). neurons and neuroglial cells were distinguished by morphology, nuclear size, nuclear membrane thickness, and chromatin homogeneity.1012 we defined neurons in which staining was more intense than background as can - ir. image acquisition was performed to show the representative staining pattern of each region and to include approximately the same number of total neurons. in the ba46 the mean percentage of can - ir neurons (number of can - ir neurons / number of total neurons) was calculated for each layer. in the hippocampus, the ca1, ca2, ca3, and ca4 subfields and dentate gyrus were investigated by randomly selecting three, one, one, three, and two images, respectively. a single researcher blinded to the tissue source was responsible for all cell counting and data analyses. the percentages of can - ir neurons in the ba46, hippocampus, caudate nucleus, and putamen are shown in figure 1a c. to compare this patient with a schizophrenia group and healthy control group, we used previously reported data.9 no significant differences were observed in the percentage of can - ir neurons among the schizophrenia group, healthy control group, and the tacrolimus case (all p>0.5, analysis of covariance). compared with the healthy control group and schizophrenia group, the percentages of can - ir neurons in layers iii vi of the ba46 and the putamen tended to be lower in the tacrolimus case (figure 1a and c). representative images of can - ir neurons in the prefrontal cortex (ba46, layers v vi) are shown in figure 2. first, we considered the possibility of an association between our patient s psychiatric symptoms and tacrolimus. the percentage of can - ir neurons in layers iii vi of the ba46 tended to be lower in patients with schizophrenia compared with the healthy control group. furthermore, the percentage of can - ir neurons was even lower in the tacrolimus patient compared with the schizophrenia alone group. cognitive dysfunction due to hypofunction of the frontal lobe is well known in schizophrenia.13 in addition, a side effect of tacrolimus is delirium, and this form of cognitive dysfunction is more severe than schizophrenia without delirium. thus, the decrease in can immunoreactivity in the frontal lobe may reflect cognitive dysfunction. the percentage of can - ir neurons in the putamen tended to be higher in patients with schizophrenia compared with healthy subjects, but the percentage of can - ir neurons was lower in the tacrolimus case than in the schizophrenia group. tacrolimus may cause side effects of tremor and dysarthria, which occur when a patient has dysfunction of the striatum. lower immunoreactivity of can induced by tacrolimus in the striatum may mediate these extrapyramidal symptoms. lee reported that tacrolimus reduces nuclear expression of can in a renal ischemia reperfusion injury mouse model. they suggested that tacrolimus decreases can expression both in the cytoplasm and in the nucleus. our data indicated that the percentages of can - ir neurons in layers iii vi of the ba46 and putamen tended to be lower in the tacrolimus case compared with the schizophrenia group. as in the report by lee, tacrolimus may decrease the expression of can in some regions of the human brain. overall, when we considered the results from the frontal, caudate, and putamen together, we found that the effect of tacrolimus in the human brain may be stronger than the effect of schizophrenia. eastwood reported decreased can a protein levels and decreased mrna of the three subunits of can a in the hippocampus of schizophrenia patients. the effect of tacrolimus on the human brain might be subject to intersubject differences in can immunoreactivity and thus generalization of these findings might be difficult. in addition, the lack of a control group (patients with leukemia but without tacrolimus treatment and schizophrenia, patients with leukemia treated using tacrolimus but without schizophrenia) makes drawing firm conclusion regarding efficacy difficult. it is also possible that the aml might have directly influenced can immunoreactivity in the brain. further studies using a larger sample size are warranted to investigate can immunoreactivity in the postmortem brains of leukemia patients without tacrolimus treatment and schizophrenia, leukemia patients treated with tacrolimus but without schizophrenia, and schizophrenia patients with tacrolimus - treated aml. third, can - ir neurons were defined as having greater staining intensity compared to background. this method allows for ambiguity because it is dependent on human assessment. in this context, the use of in situ hybridization for future studies is warranted. finally, if the patient carried the ppp3cc snp, the level of can might have already been low, instead of attributable to the tacrolimus treatment. however, we could not obtain the snps data because we were unable to use frozen brain samples in the tacrolimus case. we performed immunohistochemical analysis using human brain tissue from a patient with schizophrenia and aml treated with tacrolimus. our data suggested that tacrolimus may decrease the immunoreactivity of can in some regions of the human brain. in addition, the percentage of can - ir neurons in layers iii vi of the ba46, caudate, and putamen tended to be lower in patients with schizophrenia complicated by tacrolimus - treated aml compared to the schizophrenia group. these data also suggested that the effect of tacrolimus in the human brain was stronger than the effect of schizophrenia.	backgroundthe calcineurin (can) inhibitor, tacrolimus, is widely used in patients undergoing allogeneic organ transplantation and in those with certain allergic diseases. recently, several reports have suggested that can is also associated with schizophrenia. however, little data are currently available on the direct effect of tacrolimus on the human brain.casea 23-year - old japanese female experienced severe delusion of persecution, delusional mood, suspiciousness, aggression, and excitement. she visited our hospital and was diagnosed with schizophrenia. when she was 27 years old, she had severe general fatigue, persistent fever, systemic joint pain, gingival bleeding, and breathlessness and was diagnosed with acute myelomonocytic leukemia. later she underwent bone marrow transplantation (bmt), she was administered methotrexate and cyclosporin a to prevent graft versus host disease (gvhd). three weeks after bmt, she showed initial symptoms of gvhd and was prescribed tacrolimus instead of cyclosporin a. seven months after bmt at the age of 31 years, she died of progression of gvhd. pathological anatomy was examined after her death, including immunohistochemical analysis of her brain using anti - can antibodies. for comparison, we used our previous data from both a schizophrenia group and a healthy control group. no significant differences were observed in the percentage of can - immunoreactive neurons among the schizophrenia group, healthy control group, and the tacrolimus case (all p>0.5, analysis of covariance). compared with the healthy control group and schizophrenia group, the percentages of can - immunoreactive neurons in layers iii vi of the ba46 and the putamen tended to be lower in the tacrolimus case.conclusiontacrolimus may decrease can immunoreactivity in some regions of the human brain. thus, tacrolimus may introduce side effects such as cognitive dysfunction and extrapyramidal symptoms. in addition, we also found that the effect of tacrolimus on can immunore - activity in human brain was stronger than the effect of schizophrenia.
several studies have investigated the efficacy of early interventions for prevention of the transition to psychosis, including schizophrenia. patients with possible onset are prospectively identified as being in an at - risk mental state (arms)1 and early interventions have been carried out for those identified as ultra - high risk (uhr) for development of schizophrenia or other psychotic disorders.2,3 early intervention is supported by results for the duration of untreated psychosis in schizophrenia. thus, patients have a better prognosis with a shorter period from the onset of illness to intervention,4,5 and early detection before disease progression may have a good influence on the prognosis. uhr is diagnosed using the comprehensive assessment of at - risk mental states (caarms) or the structured interview for prodromal syndromes (sips).1,6 yearly rates of 13% to 50% for progression to psychosis have been reported.7 interventions for uhr patients mainly involve pharmacotherapy and cognitive behavior therapy (cbt).8 second - generation antipsychotics (sgas) and antidepressants are prescribed for uhr patients,9,10 but their use is restricted as first - line pharmacotherapy.8 antipsychotics are useful for urgent improvement in a crisis, such as threatened self - mutilation and suicide, and sgas are effective for preventing or delaying progression to psychosis.9,1114 ethical considerations regarding false positive identification of suspected prodromal patients and adverse reactions related to pharmacotherapy are of concern.9,11,15 it is difficult to distinguish a false positive case at the start of drug treatment using current diagnostic tests, and this prevents a consensus on the appropriate medication for the uhr patient population, in which false positive cases are intermingled. therefore, the aim of this study was to examine the speed of response, doses, and safety of treatment with sgas in patients at uhr compared to those with schizophrenia. the subjects were inpatients and outpatients aged 1430 years old who visited zikei hospital, okayama, japan from december 1, 2006 to december 1, 2011. the uhr group was diagnosed using the caarms, and the first - episode schizophrenia (fes) and multi - episode schizophrenia (mes) groups were diagnosed based on the international classification of diseases (10th revision).16 exclusion criteria were : cases in which no consent was obtained ; pregnancy ; iq under 70 ; neurological disorder and drug dependence ; mood disorders ; pervasive developmental disorders ; conduct disorders ; and personality disorders. patients with uhr and fes had not been prescribed antipsychotics for more than 2 weeks before the start of the study. for matching with the other two groups, patients with mes with a global assessment of functioning (gaf) score under 70 before relapse and with treatment - resistant disease in the last episode were excluded from the study. the uhr group was prescribed sgas, antidepressants, or benzodiazepines as the main agent, and the fes and mes groups were prescribed sgas as the main agent. other antipsychotics used for augmentation were used less than the main agent and were decreased or stopped when possible during the study. anticholinergic agents were reduced to minimum dosages during the study period, but preventive prescription was allowed. supportive psychotherapy and occupational therapy were also performed as standard medical treatment during the study. clinical evaluation was performed by raters other than physicians at baseline and after 4, 8, and 12 weeks of treatment. if treatment was ended before 12 weeks, an evaluation was performed at the end point. the clinical rating scales used included the positive and negative syndrome scale (panss),17 gaf,18 clinical global impression - severity scale (cgi - s),19 simpson - angus scale (sas),20 barnes akathisia rating scale (bars),21 and abnormal involuntary movement scale (aims).22 adverse events were documented and switching of sgas was evaluated based on reasons of efficacy and tolerability. the investigation was carried out in accordance with the latest version of the declaration of helsinki. informed consent was obtained from each patient. for patients who were minors, the consent of one or more guardians differences in ratios among the three groups were evaluated by and fisher s exact test. differences in data over time within groups were evaluated by friedman test and differences among the three groups at each time point were evaluated by kruskal wallis test. if significant differences were found by kruskal wallis test, comparison among the three groups at each time point was performed by steel dwass test. differences in the doses of antipsychotic drugs at individual time points within groups were evaluated by mann the subjects were inpatients and outpatients aged 1430 years old who visited zikei hospital, okayama, japan from december 1, 2006 to december 1, 2011. the uhr group was diagnosed using the caarms, and the first - episode schizophrenia (fes) and multi - episode schizophrenia (mes) groups were diagnosed based on the international classification of diseases (10th revision).16 exclusion criteria were : cases in which no consent was obtained ; pregnancy ; iq under 70 ; neurological disorder and drug dependence ; mood disorders ; pervasive developmental disorders ; conduct disorders ; and personality disorders. patients with uhr and fes had not been prescribed antipsychotics for more than 2 weeks before the start of the study. for matching with the other two groups, patients with mes with a global assessment of functioning (gaf) score under 70 before relapse and with treatment - resistant disease in the last episode were excluded from the study. the uhr group was prescribed sgas, antidepressants, or benzodiazepines as the main agent, and the fes and mes groups were prescribed sgas as the main agent. other antipsychotics used for augmentation were used less than the main agent and were decreased or stopped when possible during the study. anticholinergic agents were reduced to minimum dosages during the study period, but preventive prescription was allowed. supportive psychotherapy and occupational therapy were also performed as standard medical treatment during the study. clinical evaluation was performed by raters other than physicians at baseline and after 4, 8, and 12 weeks of treatment. if treatment was ended before 12 weeks, an evaluation was performed at the end point. the clinical rating scales used included the positive and negative syndrome scale (panss),17 gaf,18 clinical global impression - severity scale (cgi - s),19 simpson - angus scale (sas),20 barnes akathisia rating scale (bars),21 and abnormal involuntary movement scale (aims).22 adverse events were documented and switching of sgas was evaluated based on reasons of efficacy and tolerability. the investigation was carried out in accordance with the latest version of the declaration of helsinki. informed consent was obtained from each patient. for patients who were minors, the consent of one or more guardians differences in ratios among the three groups were evaluated by and fisher s exact test. differences in data over time within groups were evaluated by friedman test and differences among the three groups at each time point were evaluated by kruskal wallis test. if significant differences were found by kruskal wallis test, comparison among the three groups at each time point was performed by steel dwass test. differences in the doses of antipsychotic drugs at individual time points within groups were evaluated by mann a total of 72 patients were registered at zikei hospital, but only 61 (uhr = 17, fes = 23, mes = 21) were included as subjects because only cases treated with an antipsychotic drug as the main agent were eligible for this study. the numbers of males and females were almost equal, and 48 subjects were inpatients. the mes group was older than the fes group based on age at the time of entry into the study, but there were no significant differences in age at onset among the three groups. the gaf score was significantly lower in the fes group compared to the mes group, but did not differ significantly between the uhr and fes groups. the panss total score was significantly higher in the fes group compared with the uhr group. the positive symptoms score in the uhr group was significantly lower than those in the two schizophrenia groups, while the negative symptoms score in the uhr group was significantly lower than that in the fes group. the general symptoms scores in the uhr and mes groups were significantly lower than that in the fes group. cgi - s scores were significantly higher in the fes group compared with the uhr group. gaf scores improved significantly in the uhr, fes, and mes groups after 12 weeks (table 2). there was no significant difference in the baseline gaf score between the uhr group and the two schizophrenia groups, but the gaf scores at 4, 8, and 12 weeks were higher in the uhr group. therefore, uhr patients showed significantly faster and greater improvement in gaf compared to those with fes and mes (table 2 and figure 1). uhr patients showed rapid improvement from an average score of 29.7 at baseline to 64.1 at 4 weeks after initiation of therapy. in contrast, the gaf score in the fes group only improved from 24.3 at baseline to 43.5 at 4 weeks, at which time there was already a significant difference between the uhr and fes groups. similarly, the gaf score in the mes group only improved from 35.7 at baseline to 47.1 at 4 weeks, again with a significant difference between the uhr and mes groups at 4 weeks. there were significant differences in the panss score until 8 weeks between the uhr and fes groups, but this difference was lost at 12 weeks. this result does not indicate that the uhr group showed less improvement than the fes group in the later part of the study ; rather, the uhr group had already shown a major improvement on panss by an average of 50 points after 4 weeks and further improvement was unlikely. the early improvement of the uhr group was also shown by the significant improvement in panss scores after 4 weeks for this group compared to the mes group, given that the panss scores in these groups did not differ significantly at baseline. the tendency for early improvement in uhr patients was also seen on the cgi - s (table 2). in the uhr group, all scores were 3 points, although these scores decreased over 12 weeks. thus, uhr patients clearly had significantly better early improvement compared with patients with schizophrenia, based on the results for the gaf, panss, and cgi - s. there was no significant difference in the doses of sgas given as chlorpromazine equivalents in the uhr group between the start and other points (table 2). in contrast, the doses were significantly higher at weeks 4 and 8 in the fes group, compared to those at the start of the study. there was no significant difference in the doses between the start and other points in the mes group. the means of the maximum doses of sgas were 274.3 (239.9) (standard deviation [sd ]) mg in the uhr group, 527.8 (240) mg in the fes group, and 745.3 (287.8) mg in the mes group, with significant differences among the three groups (p 3 points, although these scores decreased over 12 weeks. thus, uhr patients clearly had significantly better early improvement compared with patients with schizophrenia, based on the results for the gaf, panss, and cgi - s. there was no significant difference in the doses of sgas given as chlorpromazine equivalents in the uhr group between the start and other points (table 2). in contrast, the doses were significantly higher at weeks 4 and 8 in the fes group, compared to those at the start of the study. there was no significant difference in the doses between the start and other points in the mes group. the means of the maximum doses of sgas were 274.3 (239.9) (standard deviation [sd ]) mg in the uhr group, 527.8 (240) mg in the fes group, and 745.3 (287.8) mg in the mes group, with significant differences among the three groups (p < 0.01 kruskal the maximum doses in the uhr group were significantly lower than those in the fes and mes groups, and the doses in the mes group were significantly the highest among the three groups. the means of modal doses of sgas were 183 (201.1) mg in the uhr group, 382.3 (191.9) mg in the fes group, and 574 (289.6) mg in the mes group (figure 2), again with significant differences among the three groups. the modal doses in the uhr group required for improvement was significantly lower than that in the other two groups, while the mes group received significantly the highest doses among three groups. switching of sgas was performed at a rate of almost 50% in all patients (table 3). the rates for switching due to poor efficacy were 44%, 70%, and 70% in the uhr, fes, and mes groups, respectively, and the rates for switching due to adverse events were 67%, 50%, and 70% in the same respective groups. each group was prescribed anticholinergic agents and the frequencies of extrapyramidal symptoms (epss) were 6%, 65%, and 29% in the uhr, fes, and mes groups, respectively, over the whole study period. at the end of the study, these rates were 6%, 39%, and 10%, respectively. the fes group had a significantly higher frequency of epss compared with the other two groups. adverse events other than epss included somnolence and fatigue in all three groups. the type and number of sgas prescribed to the three groups are shown in table 4. during the study period, we examined the speed of response, doses, and safety of treatment with sgas in patients at uhr compared to those with schizophrenia. on the gaf, uhr patients showed significantly faster and greater improvement compared to those with fes and mes. in the caarms, therefore, the panss score in the uhr group had a tendency to be lower than those in the fes and mes groups. although panss scores showed significant improvements in all three groups, those did not clarify about the speed of response to treatment in this study. gaf scores do not necessarily correlate with symptoms and give a low value in crisis patients that are acting out through self - mutilation and threatened suicide, or those with social withdrawal. treatment of uhr patients is often performed in outpatient settings, but there were more inpatients than outpatients in this study, indicating that many cases were in a crisis. this study suggests that the gaf is a very sensitive rating scale for mild symptoms such as those in uhr patients. mcglashan have also suggested that panss is not sensitive enough to show improvement of illness in uhr cases.9 the cgi - s is a rating scale that can be used after evaluation with gaf and panss. the cgi - s results showed that uhr patients had already reached a good level after 4 weeks, with greater improvement at this time point compared with the other two groups. in the uhr group, ceiling effects for improvement of symptoms and functions in contrast, patients with schizophrenia tended to show more gradual improvement over the duration of the study. a comparative study of the treatment response of patients with fes and recurrent schizophrenia has been reported,23,2528 but there has been no previous comparison of uhr and schizophrenia patients. thus, this is the first study to show faster improvement of functions and global impressions in uhr patients compared to those with fes and mes after treatment with sgas, based on the gaf and cgi - s. all the uhr patients were treated with sgas as the main agent in this study. the doses of sgas in the fes and mes groups were within the limits of the recommended doses for acute phase or recurrent acute - phase schizophrenia,24 whereas the uhr group was treated at a dose close to the minimum recommended dose. clinical studies on antipsychotics for uhr or arms patients9,1114 have used doses (chlorpromazine equivalents) of approximately 100375 mg / day. in the current study, the modal dose of sgas prescribed in the uhr group was 183 (201.1) mg / day and the results were similar to those found in previous studies. these results show that uhr patients in various crises can be treated effectively with sgas at a dose close to the minimum recommended for fes.30 significant differences in the modal dose were also found among the groups, similarly to the maximum dose. thus, the results suggest that uhr patients have a faster response to lower doses of sgas compared to patients with schizophrenia. switching of sgas was performed at a rate of almost 50% in all patients. in the uhr group, the rate of switching for adverse events was 67%, but that for poor response was only 44%. this reflects the good response of uhr patients to sgas, and thus switching was based more on adverse events. in contrast, both a poor response and adverse events led to switching in the fes and mes groups. however, serious adverse events did not occur in any of the groups over 12 weeks. anticholinergic agents were permitted in the study, including preventive prescription before appearance of epss. anticholinergic agents were prescribed at rates of 24%, 43%, and 29% over the study period in the uhr, fes, and mes groups, respectively, but these rates had fallen to 6%, 22%, and 24%, respectively, by the end of the study. epss occurred at a rate of only 6% (one patient) in the uhr group, but at 65% and 29% in the fes and mes groups, respectively, over the whole study. in the fes and mes groups, these rates had fallen to 39% and 10%, respectively, by the end of the study. thus, the uhr group showed the most improvement of functions and global impressions and the lowest incidence of epss, and our findings suggest that sgas can be administered safely in uhr patients by proper choice of the dosage and use of anticholinergic agents. epss occurred in one subject (6%) in the uhr group during the 12-week study period, although there were no serious side effects. therefore, if uhr patients are prescribed sgas at higher doses than necessary to improve symptoms, safety may be compromised and adverse events may reach levels observed in the fes group. since sufficient doses of sgas were already being prescribed upon commencement of the study in the uhr group, no large - scale titration of the dose of sgas was required until completion of the study (table 2). pharmacotherapy in uhr may be possible with doses of sgas lower than those shown in figure 2 and a further detailed titration may be necessary in a shorter period of time. moreover, sgas caused less epss compared with first - generation antipsychotics (fgas), and fgas may increase the incidence of epss in uhr patients. there was a required increase in the doses of sgas in the fes group because the symptoms at baseline were serious and there was insufficient improvement at weeks 4 and 8. age at baseline in the mes group tended to be higher than that in the fes group, although there was no significant difference in age of onset among the three groups. the slightly higher age in the mes group all sgas have some inhibitory effects on receptors related to sedation, such as the histamine or 1 receptor. a meta - analysis of placebo - controlled trials for bipolar disorder showed that risperidone, quetiapine, olanzapine, and aripiprazole frequently caused somnolence as an adverse event.29 thus, it is important to keep in mind that even sgas have a tendency to cause sedation as an adverse event in young patients such as those involved in this study. in a double - blind comparative study of olanzapine and placebo in uhr patients, mcglashan found a significant improvement in those treated with olanzapine from 8 weeks and a faster response to olanzapine compared to placebo.9 similarly, sgas have been shown to be significantly more effective than placebo in comparative studies in uhr patients from 8 to 12 weeks of treatment, with no difference in safety.9,12,13 thus, the short - term advantage of sgas in uhr patients is clear. similar results were found in this study and we suggest that antipsychotics are appropriate for use if immediate improvement is needed. however, the efficacy and safety of sgas for false positive cases remains uncertain. therefore, it is important to eliminate adverse events where possible and improve efficacy in the use of sgas in uhr patients ; however, in terms of prescribing sgas to uhr cases, prescription periods and methods must be studied further. analysis using the gaf, panss, and cgi - s showed that uhr patients had higher sensitivity and a faster response to a lower dose of sgas, compared to patients with fes or mes. we suggest that sgas can be safely prescribed to uhr patients with extremely mild positive symptoms but with serious acting out, if an appropriate dose is determined and anticholinergic agents are used. however, the efficacy and safety of sgas in these patients was only shown for 12 weeks in this study. longer - term effects require further study in a larger number of cases and with different evaluation scales.	aimthe aim of this study was to examine the speed of response, doses, and safety of treatment with second - generation antipsychotics (sgas) in patients at ultra - high risk (uhr) compared to those with schizophrenia.methodsa 12-week open - label, prospective study of sgas was performed in uhr patients and those with first - episode schizophrenia (fes) and multi - episode schizophrenia (mes). the subjects were 1430 years old and were recruited at zikei hospital, okayama, japan from december 1, 2006 to december 1, 2011. treatment was carried out in a natural setting in an open - label format, but clinical evaluation was performed blind. the clinical rating scales include the global assessment of functioning (gaf), the positive and negative syndrome scale (panss), and the clinical global impression - severity scale (cgi - s).resultsuhr (n = 17), fes (n = 23), and mes (n = 21) patients all showed significant improvements on the gaf, panss, and cgi - s. however, the uhr patients showed significantly greater improvement on the gaf at weeks 4, 8, and 12 compared to the other groups, and a significantly lower modal dose of sgas (chlorpromazine equivalent : 183 [201.1 ] mg / day, mean [sd ]) was needed for improvement in the uhr group. each group was also prescribed anticholinergic agents during the study period and the uhr group had significantly fewer extrapyramidal symptoms (only 6%) compared with the fes group.conclusionour findings suggest that uhr patients have a better response to sgas compared to patients with schizophrenia, and that these drugs can be given safely by minimizing the dosage of sgas and using anticholinergic agents.
the global burden of cancer continues to increase mostly because of increasing adoption of cancer - causing behaviors, particularly smoking and smokeless tobacco forms in economically developing countries. globally, about 5,00,000 new oral and pharyngeal cancers are diagnosed annually and three quarters of these are seen in the developing world, including about 65,000 cases reported in india. the world health organization (who) estimated that the proportion of deaths that result from tobacco - related diseases will rise in india from 1.4% of all deaths in 1990 to 13.3% of all deaths in 2020. the number of persons consuming tobacco is also likely to rise, according to the models presented in the 2002 report of the economic and social council (ecosoc) of the united nations. oral carcinomas are characterized by complex karyotypes that involve many chromosomal deletions, translocations, and structural abnormalities. cells often have errors in chromosome segregation that lead to the formation of a lagging chromosome or chromosome parts that become lost during the anaphase stage of cell separation and are excluded from the reforming nuclei. these are induced in oral exfoliated cells by a variety of substances, including genotoxic agents and carcinogenic compounds in tobacco, betel nut, and alcohol. the induction of micronucleated (mn) cells by carcinogens and mutagens is a sign of the genotoxic effect of such substances. these are also observed in exfoliated buccal cells, from people who are exposed to organic solvents, antineoplastic agents, diesel derivatives, polycyclic aromatic hydrocarbons, lead - containing paints and solvents, and drinking water, which is contaminated with arsenic. the assessment of micronuclei in exfoliated cells is a promising tool for the study of epithelial carcinogens and can be used to detect chromosome breakage or mitotic interference, thought to be relevant to carcinogenesis. due to its association with chromosomal aberrations, micronuclei have been used since 1937 as an indicator of genotoxic exposure based on the radiation studies conducted by brenneke and mather. the direct correlation between the micronuclei formation and genomic damage make the micronuclei assay an efficient alteration to the metaphase analysis. the present study aimed to detect micronuclei in exfoliated buccal mucosal cells in healthy tobacco users and healthy nontobacco users. the present study aimed to detect micronuclei in exfoliated buccal mucosal cells in healthy tobacco users and healthy nontobacco users. a total of 75 healthy male subjects (25 smokeless tobacco users, 25 smokers, and 25 non tobacco users) who attended the outpatient department of dental institute, were examined and were selected for the study. the study group comprised smokeless tobacco chewers who chewed five or more packets daily for at least 5 years. the smokers included smoked every day for at least 5 years and consumed 20 or more bidis / cigarettes in a day. individuals who were alcoholics, had a recent viral infection, had undergone radiation therapy, or those who had been under medication were excluded from the study. before sampling, each individual rinsed his mouth thoroughly with tap water. the exfoliated cells were obtained by scraping the buccal mucosa with a moistened wooden spatula and the scraped cells were placed on the clean glass slides and smears were prepared. all the cytologic smears were stained by papanicolaou technique using a commercially available staining kit rapidpap. from each slide, ~ 1000 cells were examined under the 400 magnification and where mn cells were located, they were examined under the 1000 magnification. the criterion which was developed by tolbert was used for counting the micronuclei. tolbert criteria parameters for identifying micronucleus are as follows : rounded smooth perimeter suggestive of a membrane.less than a third the diameter of associated nucleus, but large enough to discern shape and color.staining intensity similar to nucleus.texture similar to nucleus.same focal plane as nucleus.absence of overlap with or bridge to nucleus. less than a third the diameter of associated nucleus, but large enough to discern shape and color. dead or degenerating cells (karyolysis, karyorrhexis, nuclear fragmentation) were excluded from evaluation. nuclear blebbings (micronucleus - like structure connected with the main nucleus with a bridge) were also not considered. the exfoliated cells were obtained by scraping the buccal mucosa with a moistened wooden spatula and the scraped cells were placed on the clean glass slides and smears were prepared. all the cytologic smears were stained by papanicolaou technique using a commercially available staining kit rapidpap. from each slide, ~ 1000 cells were examined under the 400 magnification and where mn cells were located, they were examined under the 1000 magnification. the criterion which was developed by tolbert was used for counting the micronuclei. tolbert criteria parameters for identifying micronucleus are as follows : rounded smooth perimeter suggestive of a membrane.less than a third the diameter of associated nucleus, but large enough to discern shape and color.staining intensity similar to nucleus.texture similar to nucleus.same focal plane as nucleus.absence of overlap with or bridge to nucleus. less than a third the diameter of associated nucleus, but large enough to discern shape and color. dead or degenerating cells (karyolysis, karyorrhexis, nuclear fragmentation) were excluded from evaluation. nuclear blebbings (micronucleus - like structure connected with the main nucleus with a bridge) were also not considered. the mn cells observed are shown in figures 13. cell with a micronucleus pap (1000) (a) cell with two micronuclei pap (1000) ; (b) cell with two micronuclei pap (1000) (a) cell with three micronuclei pap (1000) ; (b) cell with three micronuclei pap (1000) the frequency of distribution of micronuclei in the three study groups number of micronuclei/1000 cells in smokeless tobacco users number of micronuclei/1000 cells in smokers the mean number of micronuclei observed in the three groups are presented in table 1. the mean number of micronuclei in smokeless tobacco chewers, smokers, and controls were 24.13 10.68, 11.96 4.23, and 4.17 2.99, respectively. the mean number of micronuclei found were more in smokeless tobacco chewers as compared with smokers and controls [table 1 ]. mean number of micronuclei in three study groups the mean values and the mean differences, which were obtained from the smokeless tobacco chewers, the smokers, and the controls were compared and are shown in table 2. in comparison, the mean difference between the number of micronuclei in smokeless tobacco chewers and smokers was 12.17 and was statistically significant (p 0.05). the incidence of micronuclei has been analyzed by various studies in normal patients, oral premalignancies, and osccs. casartelli observed micronuclei frequencies in exfoliated buccal cells in normal oral mucosa, precancerous lesions, and squamous cell carcinoma. they concluded that the gradual increase in micronucleus counts from normal mucosal to precancerous lesions to carcinoma suggested a link of this biomarker with neoplastic progression. a significant rise was also observed in the percentage of mn cells and micronuclei from control to precancer patients, and from precancer to cancer patients in a study by saran. in the present study, the mean number of micronuclei was higher in smokeless tobacco users indicating more genotoxic effect of smokeless tobacco as compared with smoking. from the present study, increase in the number of micronuclei provides the evidence that smokeless tobacco chewers and smokers may be at a high risk for developing oral cancer. in comparison, the cellular changes associated with smokeless tobacco use were more than that in smokers, thus indicating more carcinogenic potential of smokeless tobacco. micronucleus assay can be used as a biomarker of genotoxicity and epithelial carcinogenic progression. however, more research is required to establish it as a potential biomarker for oral carcinogenesis.	introduction : the assessment of micronuclei in exfoliated cells is a promising tool for the study of epithelial carcinogens and can be used to detect chromosome breakage or mitotic interference, thought to be relevant to carcinogenesis.aim:the present study aimed to detect micronuclei in exfoliated oral mucosal cells in individuals using various tobacco forms from the last 5 years.materials and methods : a total of 75 healthy male subjects (25 smokeless tobacco users, 25 smokers, and 25 non tobacco users) were selected for the study. smears were obtained with moistened wooden spatula from buccal mucosa and fixed with 95% alcohol. all the cytologic smears were stained by papanicolaou technique. from each slide, ~1000 cells were examined under the 400 magnification and where micronucleated (mn) cells were located, they were examined under the 1000 magnification.result:mn cells were found to be significantly higher in smokeless tobacco users than in smokers and controls.conclusion:a positive correlation is found between increased micronucleus frequency and tobacco - using habits. so micronucleus assay can be used as a biomarker of genotoxicity.
soft tissue sarcoma (sts) is a heterogeneous group of malignant tumors of mesodermal origin that constitute between 1.8 and 5.0 cases per 100,000 people per year [4 - 9 ]. however, these studies were based on western populations and focused on the incidence of sts. moreover, these studies typically included sts of all anatomical sites, whereas the treatment and prognoses of sts differ according to the anatomical location (e.g., head and neck, viscera, retroperitoneum, and extremity). thus, a large population - based study on the incidence and treatment patterns of extremity sts in korea is needed. south korea operates two mandatory nationwide databases for medical records that cover the entire population. these are the korea national cancer incidence (knci) database from the korean central cancer registry (kccr) and the korean health insurance review and assessment service (hira) database from hira. the knci database has covered the whole population under the population - based regional cancer registry program since 1999, and it has high diagnostic accuracy but lacks treatment information. conversely, the hira database includes the health insurance claim records under the korean national health insurance program, and thus provides detailed information on patient treatment, but has less diagnostic accuracy. in this study, we combined and utilized the knci and hira databases to estimate the nationwide incidence and treatment pattern of extremity sts in the korean population. kccr, initiated as a nationwide hospital - based cancer registry by the ministry of health and welfare in 1980, was expanded in 1999 to cover the entire population under the population - based regional cancer registry program. since 2002, kccr has constructed knci, a nationwide cancer registry database, and presented the annual incidence of cancer based on the whole korean population. cancer cases were classified in accordance to the international classification of disease for oncology (icd - o) third edition, and subsequently re - classified according to the 10th revised version of the international classification of disease (icd-10). the korean national health insurance program covers the entire korean population (97% of the population have health insurance, and 3% are covered by medical aid). hira, operated under the korean national health insurance program, contains the adjusted medical and pharmacy claims for all koreans, and its database has been used for many epidemiological studies. all clinics and hospitals submit claims data for inpatient and outpatient care, including diagnoses, procedures, prescription records, demographic information, and direct medical costs, to hira to obtain reimbursement of medical costs from the government. the hira database was established based on the sixth edition of the korean classification of disease (kcd-6), which is a modified version of the icd-10 for the korean health care system. for patients with serious diseases or cancer, if patients are confirmed to have a malignant disease, they are registered by kccr and paid only 5% of their total medical costs. the remaining 95% of costs are paid by the national health insurance corporation, and the associated review data are saved in the hira database. thus, virtually all patients with malignant disease are registered by both kccr and hira. all korean residents receive a unique identification number (korean resident registration number, krrn) at birth, allowing easy identification of every citizen. this is widely used in government - operated databases, including the knci and hira databases. these databases can therefore be used to obtain the health care records and demographic characteristics of specific patients without any duplications or omissions. public access to both databases is not allowed ; however, access can be granted with permission from the deliberative committee if they believe that the requested study will ultimately be for the greater good. after receiving approval for the study from the deliberative committee of hira and permission to access the requested data from kccr, the de - identified individual information from the hira database were accessed and the total annual incident cases of extremity sts from the knci database were obtained. the access to the data was allowed on september 2013 and february 2014 from hira and kccr, respectively. each de - identified individual s (1) diagnostic code and claim date, and referral level of the hospital where the first confirmed diagnosis of sts was registered and regional distribution of those hospitals ; and (2) the procedure code and its claim date. although each database uses krrn for data processing, the authors are prohibited to access the identified information. therefore, informed consent from each patient was not acquired, and the study was exempted by the seoul national university hospital institutional review board (irb no. 1311 - 054 - 533) because the authors could access only de - identified data. to identify patients with newly diagnosed extremity sts from the knci database, the following icd-10 diagnostic codes were searched : malignant neoplasm of the peripheral nerves and autonomic nervous system (icd-10 diagnostic code : c47.1 for upper extremity and c47.2 for lower extremity) or malignant neoplasm of other connective and soft tissues (c49.1 for upper extremity and c49.2 for lower extremity). as a result, kaposi sarcoma and malignant neoplasms of the skin, such as malignant melanoma, were excluded from this study. the most recent data available are for 2011, and thus, we obtained data for cases between 2009 and 2011 from the knci database. to identify patients who were newly diagnosed and treated for extremity sts from the hira database, with the following icd-10 diagnostic codes were searched : c47.1, c47.2, c49.1, and c49.2. because hira only provides data from the previous 5 yearsfor example if the data were requested in 2013, hira would provide data for 2008 - 2012 we assessed patients with extremity sts in the entire korean population between 2008 and 2011 from the hira database (to match the knci database search period). all claims records of outpatient visits or hospital admissions of patients containing diagnosis of sts between january 1, 2008 and december 31, 2011 were identified in the database. for cases involving more than one claim record that satisfied the inclusion criteria, only the claim record with the earliest date there were 3,155 patients, who were initially diagnosed with sts between 2008 and 2011 : 1,132 in 2008, 671 in 2009, 685 in 2010, and 667 in 2011. orthopedic oncologists in korea are advised to treat patients in adherence to the national comprehensive cancer network clinical practice guidelines in oncology (nccn guidelines). the nccn guidelines for sts recommended that stage i tumors are routinely followed up every 3 - 6 months for 2 - 3 years and then annually ; and stage ii, iii, or iv disease is followed up every 3 - 6 months for 2 - 3 years, then every 6 months for the next 2 years, and then annually. actually, in our clinical experience, most of the follow - up intervals for sts are ; and stage ii, iii, or iv disease is followed up every 3 - 6 months for 2 - 3 years, then every 6 months for the next 2 years, and then annually. actually, in our clinical experience, most of the follow - up intervals for sts are 50 years. as the incidence of sts tends to increase with age (fig. 2), the relatively young population in korea might contribute to the low incidence of sts. if this is true, as the population grows older, the incidence of sts is likely to increase in the future due to its higher incidence rate in older people. although its incidence is lower than that of other cancer types, the changes in the incidence of extremity sts should be carefully monitored because of the high mortality associated with this disease. of the extremity there was a minor tendency for older people to not receive treatment, with patients aged 70 - 89 years least likely to receive treatment (table 3). among patients who were treated for extremity sts, this usually involved surgical excision in approximately 85% of cases. according to one hospital - based study, only 6% of sts patients did not undergo surgery, but a greater proportion of patients (15%) did not undergo surgical excision in our study ; overall 26% and 28% of patients in our study received chemotherapy and radiotherapy, respectively. this is rather higher than the results reported in a previous study, and it is unclear whether these differences reflect the fact that the previous study was hospital - based, while ours was population - based, or whether it actually reflects the differences in treatment patterns between countries. some demographic characteristics, such as sex, also varied among different countries and studies. on contrary to many other studies, our study, together with many previous studies, found that sts was more common in men. most previous studies in korea regarding sts also found that men had sts more frequently, although none of these were population - based. overall, no clear tendency with respect to sex bias in sts development can be discerned. approximately 75%-85% of stss are reported in adults. in our study, only 7.0% (87 of 1,236) of cases involved patients aged 80 years. this is the first nationwide study of the incidence and treatment patterns of extremity sts in korea using two national databases that include the whole population (knci and hira).	purposewe conducted a nationwide study to assess the incidence and treatment patterns of extremity soft tissue sarcoma (sts) in south korea.materials and methodsthe nationwide incidence and treatment patterns of extremity sts were assessed using two nationwide databases, the korea national cancer incidence (knci) database and the health insurance review and assessment service (hira) database.resultsa total of 1,236 patients were newly diagnosed with extremity sts during the 3-year study period, from 2009 to 2011. the annual incidence of extremity sts in the korean population was approximately 0.9 per 100,000 people with a male bias that increased with age and was especially pronounced amongst individuals aged > 80 years. approximately 7% of patients did not receive any treatment, and surgical excision was performed for 85% of those who were treated.conclusionthis is the first nationwide study of the incidence and treatment patterns of extremity sts in korea using two national databases (knci and hira), which include the entire korean population. the results of this study may be useful for future planning and management of sts, at the national level.
litigation against tobacco companies plays an important role in reinforcing and advancing tobacco control policies. through lawsuits, as hidden strategies of tobacco companies were revealed to the public, the truth about tobacco products, smoking behavior, and the industry was also exposed. in short, through this revelation, everyone can reach a consensus on the need to reinforce tobacco control policies. the history of us tobacco litigation shows that such lawsuits have been public teaching programs. " throughout the process, classified documents of tobacco companies have been disclosed to the public, and the immoral and unethical nature of the industry has become known to the world. the importance of tobacco litigation has been emphasized in the world health organization (who) framework convention on tobacco control (fctc) as well. article 19 of the fctc recommends that all parties need to strengthen municipal law and build litigation support systems for facilitating tobacco lawsuits. for example, establishing municipal law, which enables seeking compensation against tobacco industry for financial losses due to smoking through litigation, is recommended. facilitating tobacco lawsuits is essential for strengthening and advancing tobacco control policies. in korea, several lawsuits have also been filed against the tobacco industry. in september 1999, a patient with lung cancer who had a long history of smoking and his family filed a compensation suit against the korean government and korea tobacco & ginseng corp. in december of the same year, six lung cancer victims and their families also filed a suit against the korean government and korea tobacco & ginseng corp. furthermore, although different from disease - related compensation claims caused by smoking, in january 2009, gyeonggi province sued kt&g for compensation for damages caused by a fire started from a cigarette butt. against this backdrop, the korea national health insurance service (nhis), a state health insurer, filed 53.7 billion korean won worth of compensation suits against three tobacco companies unlike the previous cases where individuals sued tobacco companies, this time, the nhis, a government organization, did, which is noteworthy and raises hopes for successful results. however, if the nhis were to lose, the ramifications would include serious obstruction to the advancement of national tobacco control policies as well as fatal adverse effects to public health. that is why careful attention by the government, academia, and the public is much needed in the current suit brought by the nhis. according to the cases of other countries, tobacco companies steer litigation toward outcomes favorable to themselves by adopting various strategies during suits. a case in point is misuse of scientific evidence submitted as supporting evidence on the issues of the lawsuit. the companies also win over scientists who can provide evidence or testimonies favorable to them, so as to secure an advantage in the suits. in this present study, we aim to analyze strategies employed by tobacco companies during the nhis s ongoing litigation and to identify what activities the companies are carrying out in implementing the key strategies misuse of scientific evidence and recruitment of scientists. in order to identify and analyze tobacco companies strategies in the litigation between the nhis and three tobacco companies (2014 gahap 525 054 compensation claim), content analysis was performed on the petition submitted by the nhis to the seoul central district court and preparatory documents submitted by the three companies to the court against the petition. documents submitted as supporting evidence by the korea tobacco & ginseng corp. to the court in the compensation claim lawsuit (99 gahap 104 973 compensation claim [miscellaneous ]) brought by six lung cancer patients and their families against korea tobacco & ginseng corp. were also analyzed. content analysis is a quantitative method of information communicated mainly via documents, tv, or radio. in content analysis, words, topics, or sentences can be a unit of analysis, and the subjects of the analysis are classified by frequency or category. in this present study, content analysis was performed in an attempt to identify the claims and intentions of the tobacco companies of the case, focusing on the words and topics of the petition and preparatory documents. the petition and preparatory documents of the nhis litigation were obtained by the author in the process of consulting for the current litigation as a member of the nhis normalization promotion committee. in addition, the list of the supporting evidence that the korea tobacco & ginseng corp. submitted to the court was obtained through the legal agent of the plaintiff of the case. later, to access the original copies of the supporting evidence on the list, the legacy tobacco documents library (https://industrydocuments.library.ucsf.edu/tobacco/), where internal documents of multinational tobacco companies are kept, was searched. for research reports or academic papers among the supporting evidence submitted by the tobacco companies, the possibility of any conflicts of interest or connections among the authors, participating researchers, and the tobacco companies was investigated by checking for the nhis to win the current compensation claim suit, what is most needed is to prove that smoking causes diseases. the defendants, tobacco companies, are claiming that epidemiological approaches and results that the nhis uses to prove the causality are far - fetched. therefore, proving the causal link is the paramount issue in this case, as it has been in past cases. besides demonstrating the causal relationship between smoking and disease, another key issue in this case is product liability : tobacco industry is responsible for their products. if they deliberately manipulate the product and harm their consumers, they, as manufacturers, become liable for the damage caused to the consumers. with regard to product liability, the nhis is aggressively making an issue of the tobacco companies use of various additives. the nhis claims that additives included in tobacco reinforce the addictive nature of nicotine and encourage smoking behavior. in other words, the nhis points out that including additives itself generates new smokers and worsens nicotine s addiction. about this issue, the defendants argue that the additives to tobacco are harmless. to back up their claim, philip morris korea cited reports of the who and american cancer society (acs) in opposition to the argument of the nhis. below is how kim & chang, of the law firm representing philip morris korea, cited the reports of the who and acs (figure 1). for instance, the who stated that cigarettes claimed to be without additives and made of organic tobacco have never been demonstrated to be less dangerous or addictive than conventional cigarettes smoke from all cigarettes, natural or otherwise, has many chemicals that can cause cancer (carcinogens) and toxins that come from burning the tobacco itself, including tar and carbon monoxide. the defendant has not used additives to increase the harmful or addictive nature of tobacco as the plaintiff argues. in citing or explaining the reports of the who and acs, the tobacco companies misused scientific evidence. the reason why both reports compared the danger of tobacco with and without additives was that tobacco companies such as american spirit were being irresponsibly promoted as additive - free tobacco at the time (figure 2). therefore, the two organizations wrote and published the reports to inform the public that the tobacco products labeled as additive - free were no different from common tobacco products with additives, in terms of danger. nonetheless, tobacco companies misinterpreted the reports to be communicating exactly the opposite idea in order to dodge the criticism by the nhis. in other words, the companies interpreted the reports warning of the danger of the tobacco advertised as additive - free in a way that tobacco products with additives were just as harmless as the ones without additives ; thus, adding additives to tobacco is harmless. in fact, the who and acs reports explained that tobacco products labeled as additive - free were also found to contain additives through component analysis. therefore, tobacco products putatively with and without additives are both dangerous. what is more interesting in philip morris korea s citation of the reports from the who and acs can be found in the reference of the preparatory documents, marked in the footnotes. while the other international references are all in english, the report from the who was translated into korean, although it was originally written in english. in other words, the acs report was presented in english in the footnote, and the who report was presented in korean in the footnote. the original title of the who report is tobacco : deadly in any form or disguise. however, in order to hide the word deadly, which means fatal, or leading to death, they translated the title into korean and footnoted the reference as tobacco, harmful in any form. the author reviewed the supporting evidence submitted by kt&g (the defendant of the current litigation and of other individual lawsuits that have been ongoing for more than ten years) to the court responsible for the individual lawsuits then, claiming that tobacco additives were harmless : eulna exhibit no.188, entitled a safety assessment of ingredients added to tobacco in the manufacture of cigarettes (march 1994), a research paper written by john doull, john p. frawley, william george, ted loomis, robert a. squire, and stephen l. taylor. the key message of the paper was that the ingredients of tobacco additives were not dangerous. john doull was a long - term consultant (mainly in the dangers of flavours) to tobacco companies. john doull, professor at the university of kansas medical school, was appointed by senate minority leader robert dole (r - kansas). both dole and doull were long - term friends of the tobacco industry. to examine how those researchers conducting such studies were linked with tobacco companies, the author performed investigations by accessing the science - for - sale website (www.sciencecorruption.com) and investigating john doull, the lead author of the paper. the results revealed that he had worked as a consultant of tobacco companies for a long time. in short, a person who had long consulted for the tobacco industry published a research paper, and tobacco companies used it as evidence to support their claim that tobacco additives were safe. science - for - sale introduces john doull as follows : john doull was a long - term consultant (mainly in the dangers of flavours) to tobacco companies. john doull, professor at the university of kansas medical school, who was appointed by senate minority leader robert dole (r - kansas). both dole and doull were long - term friends of the tobacco industry. tobacco industry has responded to the claims of plaintiffs, using various strategies during litigation. a key tactic is to provide scientific evidence to support their positions and to recruit scientists who can endorse the tobacco industry, by giving testimonies favorable to themselves in court. in addition, targeting the lack of expertise of the department of justice, they have sometimes submitted a vast number of expert research reports. in the process, the researchers selectively submitted the research reports that could be advantageous to tobacco companies or they sometimes misused scientific evidence. it is likely that the tobacco companies have been using or will use these two strategies in response to the current litigation that the nhis has filed. the misuse of scientific evidence has already been attempted as we have reviewed in this study. the recruitment of scientists although it was revealed through the analysis of the materials used in the prior individual lawsuits is highly likely to be done in the present case. when it comes to tobacco litigation, tobacco companies worldwide have allied among themselves as partners, not rivals. a prior study analyzing the internal documents of tobacco companies introduced a letter that korea tobacco & ginseng corp. sent to its rival philip morris korea, asking for support for litigation at the point when a tobacco lawsuit was first filed. this means that it is highly possible that materials or evidence to be used in litigation are shared inside the tobacco industry. to address such an issue, thorough examination of all supporting evidence submitted by tobacco companies in the current lawsuit brought by the nhis is imperative : experts on tobacco control should actively participate in reviewing the supporting evidence. for research reports or academic papers, in particular, investigations need to be performed on various aspects, including their research design, analysis methods, results, conflicts of interest among the researchers, and research funding sources. the experts also need to be well aware of the litigation strategies that the tobacco industry has adopted and is likely to adopt in the future, such as asking researchers to conduct studies to gain supporting evidence or recruiting scientists across diverse disciplines to request testimonies in the suits [9 - 12 ]. article 5.3 of the who fctc has set various recommendations to protect tobacco control policies from the vested interests of the tobacco industry. among them, the recommendation to refuse to partner with tobacco companies is particularly noteworthy for healthcare professionals, a likely target of such recruitment. acknowledging that working with the tobacco industry undermines tobacco control polices and negatively affects public health policies, health professionals and researchers the current nhis litigation is gaining worldwide attention. as the number of cases in which tobacco companies lose increases, more tobacco lawsuits will be filed globally. furthermore, such a spread of tobacco lawsuits may lead to reinforcement of tobacco control policies, and ultimately, even the end of the tobacco industry. the nhis s position and strategies for preparing for the current tobacco lawsuit will be the key to determining the outcome of the suit. the key message of the present study to the nhis is that the organization should make every effort to thoroughly review the supporting evidence that tobacco companies submit during the case, regardless of the time and budget required. even though the plaintiff lost in the first case, the experience provided the groundwork for developing strategies for the next cases. with more litigation experience,, the nhis and experts in tobacco control policy should be more proactive in informing the public of the hidden truth the misuse of scientific evidence and recruitment of scientists by the tobacco industry which was not carefully examined in prior lawsuits and yet was identified in the present study. if the nhis wins the current case, it would have a positive impact on not only the tobacco control policies in korea, but also similar tobacco litigation in other parts of the world. considering the implications of the suit, the nhis and related experts should prepare for the case meticulously with a great sense of responsibility. finally, the scope of this study was limited to the analysis of only a portion of the materials from all ongoing tobacco litigation. therefore, further analysis of the documents that remain to be submitted by tobacco companies should also be performed promptly and comprehensively.	south korea s state health insurer, the national health insurance service (nhis), is in the process of a compensation suit against tobacco industry. the tobacco companies have habitually endeavored to ensure favorable outcomes in litigation by misusing scientific evidence or recruiting scientists to support its interests. this study analyzed strategies that tobacco companies have used during the nhis litigation, which has been receiving world - wide attention. to understand the litigation strategies of tobacco companies, the present study reviewed the existing literature and carried out content analysis of petitions, preparatory documents, and supporting evidence submitted to the court by the nhis and the tobacco companies during the suit. tobacco companies misrepresented the world health organization (who) report s argument and misused scientific evidence, and removed the word deadly from the title of the citation. tobacco companies submitted the research results of scientists who had worked as a consultant for the tobacco industry as evidence. such litigation strategies employed by the tobacco companies internationally were applied similarly in korean lawsuits. results of tobacco litigation have a huge influence on tobacco control policies. for desirable outcomes of the suits, healthcare professionals need to pay a great deal of attention to the enormous volume of written opinions and supporting evidence that tobacco companies submit. they also need to face the fact that the companies engage in recruitment of scientists. healthcare professionals should refuse to partner with tobacco industry, as recommended by article 5.3 of the who framework convention on tobacco control.
to further evaluate the risk for cholera transfer through ballast water under existing management approaches, we applied the imo ballast water exchange depth and distance criteria to the caribbean region. buffers of 50 and 200 nautical miles were generated on the basis of the global self - consistent, hierarchical, high - resolution shoreline database (version 2.1 ; www.ngdc.noaa.gov/mgg/shorelines/gshhs.html) and overlaid on bathymetric data from the etopo1 global relief model (www.ngdc.noaa.gov/mgg/global/global.html) (10,11). we acquired these datasets through the national oceanic and atmospheric administration s national geophysical data center (www.ngdc.noaa.gov). mapping indicates that waters around haiti where the imo guidelines can be followed are extremely limited (figure). to exchange ballast > 200 nautical miles from shore in water 200 m deep, ships must travel 280 nautical miles northeast of haiti (figure, a) or to the gulf of mexico (figure, b). to exchange ballast at the minimum 50 nautical miles from shore in water > 200 m deep, ships must travel > 90 nautical miles northeast (figure, c) or 50 nautical miles south (figure, d) of haiti or conduct the exchange in an area 200 nautical miles from shore in water 200 m deep, ships must travel 280 nautical miles northeast of haiti (a) or to the gulf of mexico (b). to exchange ballast at the minimum 50 nautical miles from shore in water > 200 m deep, ships must travel > 90 nautical miles northeast (c) or 50 nautical miles south (d) of haiti or conduct the exchange in an area 50 nautical miles but 200 m. dark blue shading indicates distance from land is > 200 nautical miles and seawater depth is > 200 m. after discussions with staff at cdc and the pan american health organization, the director general of the haitian ministry of health and population issued 2 memoranda addressing ballast water management. the first, issued november 10, 2011, asked ship captains not to exchange ballast water in haitian harbors. the second, issued november 15, 2011, reminded ship captains to adhere to imo guidelines for ballast water exchange. however, ships operating in the caribbean sea face practical difficulties in conducting ballast water exchange at the recommended distances without making large deviations from usual routes. the bwm convention recommends that designated ballast water exchange areas should be on or close to existing maritime routes and does not require that ships deviate course or delay voyages to comply with ballast water exchange guidelines (6). ballast water exchange at sea also presents operational and safety challenges to ships and might not be completely effective in preventing spread of aquatic organisms (12). as an alternative management measure, ballast water treatment systems are being developed to meet the bwm convention s numeric discharge standards, and several systems are available (13). these systems combine filtration with nonchemical (e.g., uv light, shear, heat) and chemical biocides to remove or kill organisms (14). ballast water treatment systems are designed to achieve the bwm convention efficacy levels, and their future use in the caribbean region would likely be a more effective management approach than ballast water exchange. the haiti cholera outbreak spread to the neighboring dominican republic, and cholera cases associated with travel to haiti were recognized in the united states (7), but there is no evidence that v. cholerae was transferred by ship ballast water in these instances. the wider caribbean region comprises 28 island and continental countries with coasts on the caribbean sea, the gulf of mexico, and adjacent waters of the atlantic ocean. this area is at elevated risk for transfer of contaminated ballast water because of the high volume of cargo trade in the region and has been prioritized by the global ballast water management program (http://globallast.imo.org/index.asp), a program administered by imo, the global environment facility, and the united nations development program to assist developing countries implement ballast water management measures (15). a comprehensive regional strategic plan has been developed to promote ratification of the bwm convention and to facilitate its implementation within the wider caribbean region through regional cooperation, training, communication, compliance monitoring and enforcement, and promotion of national - level legislation, task forces, action plans, and sustainable resources to support activities (15). implementation of this plan will help protect public health by reducing the likelihood that v. cholerae and other pathogens will be transferred by ballast water.	organisms, including vibrio cholerae, can be transferred between harbors in the ballast water of ships. zones in the caribbean region where distance from shore and water depth meet international maritime organization guidelines for ballast water exchange are extremely limited. use of ballast water treatment systems could mitigate the risk for organism transfer.
wheat grains (triticum aestivum) and wheat flour (extraction rate 82%) were supplied from lantmnnen, jrna, sweden. grains were harvested in the year 2008 and derived from an unknown mix of varieties commonly grown in sweden. dried yeast (kronjst, jstbolaget, sollentuna, sweden) was purchased in a food store in uppsala. germination and drying experiments and subsequent milling for the preparation of wheat flour for pita bread baking were carried out as depicted in fig. germination was carried out in a leavening cupboard (elektro helios, stockholm, sweden) during 48 hr at 20, 25, 30, and 35c. the following drying conditions for wheat germ (germinated at 30c) were tested using a drying oven (heraeus, d-63450, hannover, germany) : 35c for 6 hr ; 50c for 4 hr ; 70c for 2 hr ; and 90c for 1 preparation of germinated wheat flour (left) and pita bread baking (right). drying conditions were 35c for 6 hr, 50c for 4 hr, 70c for 2 hr, and 90c for 1 hr. ; 300 g flour, 1.5 g dried yeast, 3 g nacl, 3 g sugar, 174 ml water), using flour (extraction rate 82%) substituted with 0, 25, 50, or 100% sieved (219 m) germinated (30c for 48 hr, fig. dm in cereal, dough, and bread samples was determined according to aoac (18). rheological properties of the dough were determined by farinograph (brabender ohg, duisburg, germany) according to aacc (19). data are expressed as mean and standard error of mean (sem) from duplicate trials and duplicate analysis. the sum of quantified folate forms (tetrahydrofolate, 5-methyl - tetrahydrofolate, and 10-formyl - folic acid) was after correction for molecular weights expressed as folic acid in g/100 g dm (20). effects of the different conditions within each treatment were analysed using tukey 's pairwise comparison (sas, version 9.1, sas institute inc. wheat grains (triticum aestivum) and wheat flour (extraction rate 82%) were supplied from lantmnnen, jrna, sweden. grains were harvested in the year 2008 and derived from an unknown mix of varieties commonly grown in sweden. dried yeast (kronjst, jstbolaget, sollentuna, sweden) was purchased in a food store in uppsala. germination and drying experiments and subsequent milling for the preparation of wheat flour for pita bread baking were carried out as depicted in fig. 2. germination was carried out in a leavening cupboard (elektro helios, stockholm, sweden) during 48 hr at 20, 25, 30, and 35c. the following drying conditions for wheat germ (germinated at 30c) were tested using a drying oven (heraeus, d-63450, hannover, germany) : 35c for 6 hr ; 50c for 4 hr ; 70c for 2 hr ; and 90c for 1 preparation of germinated wheat flour (left) and pita bread baking (right). hr, 70c for 2 hr, and 90c for 1 hr. rh = relative humidity, dm = dry matter. pita bread was baked using a sponge dough method (fig. 2 ; 300 g flour, 1.5 g dried yeast, 3 g nacl, 3 g sugar, 174 ml water), using flour (extraction rate 82%) substituted with 0, 25, 50, or 100% sieved (219 m) germinated (30c for 48 hr, fig. dm in cereal, dough, and bread samples was determined according to aoac (18). rheological properties of the dough were determined by farinograph (brabender ohg, duisburg, germany) according to aacc (19). data are expressed as mean and standard error of mean (sem) from duplicate trials and duplicate analysis. the sum of quantified folate forms (tetrahydrofolate, 5-methyl - tetrahydrofolate, and 10-formyl - folic acid) was after correction for molecular weights expressed as folic acid in g/100 g dm (20). effects of the different conditions within each treatment were analysed using tukey 's pairwise comparison (sas, version 9.1, sas institute inc. germination of wheat grains resulted, in accordance to findings by others (16, 17), in a 3- to 4-fold higher folate content as compared to the initial 14 g/100 g (fig. the folate content of unsieved and sieved germinated wheat flour (gwf) (after germination at 30c) was 72 + 3 g/100 g and 56 + 3 g/100 g dm. drying of germinated wheat grains did not significantly (p=0.61) affect the folate content as compared to fresh germinated grains, resulting in a folate content of 5672 g/100 g dm. folate content (g/100 g dmsem) in native and germinated wheat grains at different temperatures. folate values (means from duplicate trials and duplicate analyses) are the sum of quantified folate forms (tetrahydrofolate, 5-methyl - tetrahydrofolate, and 10-formyl - folic acid) expressed as folic acid after correction for molecular weights. the addition of increasing amounts of sieved germinated wheat flour resulted in enhanced folate content in dough, but rheological properties were affected adversely (fig. 4). baking of pita bread after addition of various amounts of sieved germinated wheat flour resulted in a net increase of folate content in both flour and bread (fig. pita bread baked with 50% sieved gwf was acceptable with respect to taste, colour, and layer separation (data not shown ; 20), and had a folate content of 50 g/100 g dm compared with 30 g/100 g dm in conventional bread (0% gwf). 6) ; similar observations were reported for other types of bread (13, 14). folate content (g/100 g dm) and dough stability time (min) in dough samples prepared after substitution with sieved germinated wheat flour (gwf). folate values (means from duplicate trials and duplicate analyses) are the sum of quantified folate forms (tetrahydrofolate, 5-methyl - tetrahydrofolate, and 10-formyl - folic acid) expressed as folic acid after correction for molecular weights. folate content (g/100 g dm, sem) in flour (left, green bars) and bread (right, maroon bars) after substitution with sieved gwf. folate values (mean from duplicate trials and duplicate analyses) are the sum of quantified folate forms (tetrahydrofolate, 5-methyl - tetrahydrofolate, and 10-formyl - folic acid) expressed as folic acid after correction for molecular weights. bars with different letters within the same group are significantly different (p<0.05). egyptian pita bread with enhanced folate content of 50 g/100 g (compared to 30 g/100 g using native wheat flour) could be produced by adding 50% sieved gwf. consumption of this bread would increase the average daily folate intake by approximately 75 g. net increase of folate content (g/100 g dm, sem) in pita bread with 50% sieved gwf during baking. folate values (mean from duplicate trials and duplicate analyses) are the sum of quantified folate forms (tetrahydrofolate, 5-methyl - tetrahydrofolate, and 10-formyl - folic acid) expressed as folic acid after correction for molecular weights. bars with different letters are significantly different (p<0.05). a phd grant (mh) from the egyptian ministry of higher education is gratefully acknowledged. we would like to thank lantmnnen r&d, jrna, sweden, for supplying the wheat grains and flour and lantmnnen mills ab, uppsala, sweden, for the milling.	introductionegypt has a high incidence of neural tube defects related to folate deficiency. one major food source for folate is pita (baladi) bread, which is consumed daily. bioprocessing (e.g. germination) has been reported to increase the folate content in cereals. the aim was to produce pita bread with increased folate content using germinated wheat flour (gwf).methodsprior to milling the effects of germination and drying conditions on folate content in wheat grains were studied. pita bread was baked from wheat flour substituted with different levels of gwf. the folate content in dough and bread and rheological properties of dough were determined.resultsgermination of wheat grains resulted in, depending on temperature, 3- to 4-fold higher folate content with a maximum of 61 g/100 g dm (dry matter). the folate content in both flour and bread increased 1.5 to 4-fold depending on the level of flour replacement with gwf. pita bread baked with 50% sieved gwf was acceptable with respect to colour and layer separation, and had a folate content of 50 g/100 g dm compared with 30 g/100 g dm in conventional pita bread (0% gwf).conclusionusing 50% gwf, pita bread with increased folate content, acceptable for the egyptian consumer, was produced. consumption of this bread would increase the average daily folate intake by 75 g.
the study cohort was composed of 225 patients with organ - confined, muscle - invasive (pt2n0m0), extravesical (pt3 - 4an0m0), and node - positive (ptanyn1 - 3m0) ucb who underwent radical cystectomy at the university of southern california between 1998 and 2004 (2). each patient had a minimum two - year follow - up postcystectomy unless they recurred prior to that date. patients receiving neoadjuvant chemotherapy, and those with clinical evidence of lymphadenopathy or distant metastasis at diagnosis were excluded. ninety - eight (43.6%) patients received adjuvant chemotherapy per physician and patient preference. postoperative follow - up was at four - month intervals in year one, six - month intervals in year two, and annually thereafter (4). with bioinformaticians who generated the prognostic classifiers remaining blinded to clinical data, two - thirds of the cohort was assigned to a discovery set and one - third to a validation set, with clinicopathologic characteristics balanced between both sets. clinical endpoint for biomarker discovery was cancer recurrence. recurrence - free survival (rfs) duration was defined as time from cystectomy to cancer recurrence at local or distant soft tissue sites ; patients who were recurrence - free at end of the study were censored at death or last follow - up. de novo urothelial carcinoma in upper tract or urethra was not considered as recurrence. rfs was preferred as the endpoint over cancer - specific survival because most patients who die of ucb have documentation of disease recurrence (3) ; this also avoided censoring patients with aggressive disease who recurred but died of other comorbidities. formalin - fixed paraffin - embedded (ffpe) primary tumor specimens underwent histopathological rereview, rna extraction and profiling using genechip human exon 1.0 st microarrays (affymetrix, santa clara, ca). these arrays profile coding and noncoding regions of the entire human transcriptome using probe selection regions, hereinafter referred to as features. samples from 199 (88.4%) patients passed microarray quality control ; relative patient proportions in discovery (n = 133) and validation (n = 66) sets were maintained (table 1). the university of southern california institutional review board approved this study and all patients consented to analysis of their tumor tissues. clinicopathologic characteristics of patients in the discovery and validation sets p values based on two - sided fisher s exact test. features most predictive of rfs were identified and combined as a signature to produce a genomic classifier (gc) (see the supplementary methods, available online). prognostic ability of gc was benchmarked against two clinical nomograms for predicting postcystectomy recurrence risk : the international bladder cancer nomogram consortium (ibcnc) (8), and an in - house clinical - only classifier (cc). the latter incorporated age, gender, pathological stage, and lymphovascular invasion status into a logistic regression model. additionally, to evaluate the joint prognostic value of genomic and clinicopathologic variables, gc was integrated with ibcnc and cc into g - ibcnc and g - cc, respectively (collectively referred to as genomic - clinicopathologic classifiers) by logistic regression. all classifiers output a continuous score between 0 and 1, with higher scores indicating higher probability of recurrence. gc performance was compared with prior prognostic genomic signatures for muscle - invasive or node - positive ucb (13 - 15,21,22). prognostic ability of gc was also independently validated on four external ucb datasets (see the supplementary methods, available online). discrimination and prognostic abilities of classifiers were compared using area under receiver - operating characteristic (roc) curves (aucs) for binary and time - to - event outcomes, and other methods. cumulative incidence curves for rfs were constructed using fine - gray competing risk analyses (23). decision curve and reclassification analyses were used to determine the clinical value of classifiers. where categorized, classifier scores of greater than or equal to 0.5 and less than 0.5 were grouped as high - risk and low - risk, respectively. biological interactions of gc components with their first - degree partners were extracted using human signaling network v5 (2427). processes were grouped using enrichment map, with nodes and links representing gene ontology (go) terms and degree of overlap between them, respectively (28). the study cohort was composed of 225 patients with organ - confined, muscle - invasive (pt2n0m0), extravesical (pt3 - 4an0m0), and node - positive (ptanyn1 - 3m0) ucb who underwent radical cystectomy at the university of southern california between 1998 and 2004 (2). each patient had a minimum two - year follow - up postcystectomy unless they recurred prior to that date. patients receiving neoadjuvant chemotherapy, and those with clinical evidence of lymphadenopathy or distant metastasis at diagnosis were excluded. ninety - eight (43.6%) patients received adjuvant chemotherapy per physician and patient preference. postoperative follow - up was at four - month intervals in year one, six - month intervals in year two, and annually thereafter (4). with bioinformaticians who generated the prognostic classifiers remaining blinded to clinical data, two - thirds of the cohort was assigned to a discovery set and one - third to a validation set, with clinicopathologic characteristics balanced between both sets. clinical endpoint for biomarker discovery was cancer recurrence. recurrence - free survival (rfs) duration was defined as time from cystectomy to cancer recurrence at local or distant soft tissue sites ; patients who were recurrence - free at end of the study were censored at death or last follow - up. de novo urothelial carcinoma in upper tract or urethra was not considered as recurrence. rfs was preferred as the endpoint over cancer - specific survival because most patients who die of ucb have documentation of disease recurrence (3) ; this also avoided censoring patients with aggressive disease who recurred but died of other comorbidities. formalin - fixed paraffin - embedded (ffpe) primary tumor specimens underwent histopathological rereview, rna extraction and profiling using genechip human exon 1.0 st microarrays (affymetrix, santa clara, ca). these arrays profile coding and noncoding regions of the entire human transcriptome using probe selection regions, hereinafter referred to as features. samples from 199 (88.4%) patients passed microarray quality control ; relative patient proportions in discovery (n = 133) and validation (n = 66) sets were maintained (table 1). the university of southern california institutional review board approved this study and all patients consented to analysis of their tumor tissues. clinicopathologic characteristics of patients in the discovery and validation sets p values based on two - sided fisher s exact test. features most predictive of rfs were identified and combined as a signature to produce a genomic classifier (gc) (see the supplementary methods, available online). prognostic ability of gc was benchmarked against two clinical nomograms for predicting postcystectomy recurrence risk : the international bladder cancer nomogram consortium (ibcnc) (8), and an in - house clinical - only classifier (cc). the latter incorporated age, gender, pathological stage, and lymphovascular invasion status into a logistic regression model. additionally, to evaluate the joint prognostic value of genomic and clinicopathologic variables, gc was integrated with ibcnc and cc into g - ibcnc and g - cc, respectively (collectively referred to as genomic - clinicopathologic classifiers) by logistic regression. all classifiers output a continuous score between 0 and 1, with higher scores indicating higher probability of recurrence. gc performance was compared with prior prognostic genomic signatures for muscle - invasive or node - positive ucb (13 - 15,21,22). prognostic ability of gc was also independently validated on four external ucb datasets (see the supplementary methods, available online). discrimination and prognostic abilities of classifiers were compared using area under receiver - operating characteristic (roc) curves (aucs) for binary and time - to - event outcomes, and other methods. cumulative incidence curves for rfs were constructed using fine - gray competing risk analyses (23). decision curve and reclassification analyses were used to determine the clinical value of classifiers. where categorized, classifier scores of greater than or equal to 0.5 and less than 0.5 were grouped as high - risk and low - risk, respectively. biological interactions of gc components with their first - degree partners were extracted using human signaling network v5 (2427). processes were grouped using enrichment map, with nodes and links representing gene ontology (go) terms and degree of overlap between them, respectively (28). to test the hypothesis that risk - prediction models incorporating genomic signatures can better characterize the biology that determines propensity of ucb to recur postcystectomy than clinical variables alone, a genomic classifier (gc) was developed and tested on the discovery set. median follow - up was 9.3 years ; 68 (51.1%) patients recurred, and 77 (57.9%) patients were dead at last follow - up. expression of nearly 1.410 rna features in tumors of patients who recurred was compared with those who remained recurrence - free at last follow - up (supplementary figure 2, available online). following normalization and feature selection, 15 markers were identified corresponding to rnas from protein - coding and noncoding regions of the genome that were differentially expressed based on recurrence (supplementary table 1, available online). a random forest machine - learning algorithm assembled these markers into a gc, and this locked classifier assigned a score to each patient. the feature associated with mecom was determined to be the most important variable (supplementary figure 3, available online). gc performance was compared with individual clinicopathologic variables, the ibcnc postoperative nomogram (8), and a clinical - only classifier (cc) that represented an optimized clinicopathologic prognostic model developed on the discovery set. gc had an auc of 0.88 (95% confidence interval [ci ] = 0.81 to 0.93) in the discovery set (figure 1). as gc was optimized in the discovery set, it could not be compared with individual clinicopathologic variables. however, we could assess the prognostic value of adding genomic information to clinical risk prediction models. when gc was added to ibcnc (g - ibcnc), the auc increased from 0.73 to 0.89 (95% ci = 0.84 to 0.95). when gc was added to cc (g - cc), the auc increased from 0.81 to 0.93 (95% ci = 0.88 to 0.97). this suggested that combined genomic - clinicopathologic classifiers could achieve superior prognostic performance than either alone. performance of classifiers and individual clinicopathologic variables as assessed by standard - roc analysis in the discovery and validation sets for predicting postcystectomy recurrence. roc = receiver - operating characteristic ; auc = area under roc curve ; gc = genomic classifier ; ibcnc = postcystectomy recurrence nomogram from the international bladder cancer nomogram consortium ; g - ibcnc = integrated genomic - ibcnc classifier ; cc = clinical - only classifier ; g - cc = integrated genomic - cc classifier. prognostic performance of the locked classifiers was then assessed (with bioinformaticians blinded to clinical variables) on an independent validation set of 66 ucb patients. median follow - up was 10.8 years ; 33 (50%) patients recurred, and 45 (68.2%) patients were dead at last follow - up. by univariate analysis, gc (p =.003), nodal status (p =.006) and lymphovascular invasion (p =.005) were prognostic for rfs, with gc having the highest hazard ratio (1.23 for every 10% score increment) (table 2). gc performance as measured by standard roc analysis was superior to single clinicopathologic variables (figure 1). this was confirmed by survival roc analysis where auc for gc (0.77 ; 95% ci = 0.65 to 0.91) was higher than any individual clinicopathologic variable (figure 2a). univariate associations of genomic and clinicopathologic features with risk of postcystectomy bladder cancer recurrence in the validation set ci = confidence interval ; gc = genomic classifier. performance of individual clinicopathologic variables and classifiers in the validation set for predicting cancer recurrence. a) survival roc curves show that gc outperforms individual clinicopathologic variables for predicting postcystectomy recurrence. in addition, b) g - ibcnc had higher auc compared to ibcnc, and c) g - cc had higher auc compared to cc by survival - roc analysis. aucs and associated 95% confidence intervals are shown at the bottom right of each roc curve panel. d) cumulative incidence plot for recurrence - free survival comparing patients with high versus low g - cc scores as determined by majority rule (cutoff = 0.5) indicate a statistically significantly elevated recurrence probability for patients with high g - cc scores (log - rank p <.001). roc = receiver - operating characteristic ; auc = area under roc curve ; gc = genomic classifier ; ibcnc = post - cystectomy recurrence nomogram from the international bladder cancer nomogram consortium ; g - ibcnc = integrated genomic - ibcnc classifier ; cc = clinical - only classifier ; g - cc = integrated genomic - cc classifier. prognostic performance of ibcnc and cc were comparable (standard - roc auc = 0.74 and 0.77, respectively) with gc in the validation set. again, performance of the combined genomic - clinicopathologic classifiers measured the highest of all models tested (figure 1). furthermore, aucs by survival roc analysis that consider time to recurrence showed marked boost in performance for combined models (ibcnc vs g - ibcnc, 0.73 vs 0.82 ; cc vs g - cc, 0.78 vs 0.86). combined genomic - clinicopathologic classifiers had the highest specificity and sensitivity across the widest range of cutoffs (figure 2, b and c). g - cc had the highest auc among the combined genomic - clinicopathologic classifiers tested. multivariable analyses showed the independent prognostic significance of combined genomic - clinicopathologic classifiers compared with clinical models (supplementary table 2, available online). decision curve analysis showed a higher overall net benefit for genomic - based classifiers, thereby highlighting their value as better tools for planning patient management compared to clinical - only risk models based on risk for recurrence (supplementary figure 4, available online). discrimination plots confirmed that while gc identified patients who recurred better than clinical models alone, the combined g - ibcnc and g - cc models were superior, with g - cc performing the best (p = 5.5510) (supplementary figure 5, available online). g - cc was therefore considered the benchmark genomic - clinicopathologic classifier for further evaluation. when survival - roc aucs were measured across a range of postcystectomy intervals, g - cc showed consistently superior performance compared with cc and gc alone, indicating that the former was most prognostic for rfs at any point in time following cystectomy (supplementary figure 6a, available online). validation set patients were then categorized as high - risk (g - cc score 0.5) or low - risk (g - cc score < 0.5). accounting for competing risk, high - risk patients had elevated cumulative incidence of recurrence compared with low - risk patients (four - year probability : 81.5% vs 20.6%, respectively ; p < patients were similarly risk - stratified by ibcnc and g - ibcnc scores, and categorization by the clinical - only ibcnc nomogram was compared with genomic - clinicopathologic classifiers (figure 3). addition of genomic features to the ibcnc nomogram (as g - ibcnc) reclassified 18 validation set patients into different predicted risk categories than those classified by ibcnc, of which 12 (66.7%) were reclassified correctly based on outcome. similarly, g - cc reclassified 12 patients originally risk stratified by ibcnc, of which 10 (83.3%) were reclassified correctly. reclassification of ibcnc score categories by genomic - clinicopathologic classifier scores for patients in the validation set. after categorizing based on their ibcnc scores, patients were reclassified based on their a) g - ibcnc and b) g - cc scores. individual patients are represented as dots colored by recurrence event ; sizes of dots represent pathological stage as indicated. gray quadrants represent situations where the genomic - clinicopathologic classifier reclassifies patients compared to ibcnc. patients who did not recur (blue dots) in the top - left quadrant and patients who recurred (red dots) in the bottom - right quadrant are reclassified correctly by the genomic - clinicopathologic classifier. of patients who were reclassified, a majority were done so correctly. ibcnc = postcystectomy recurrence nomogram from the international bladder cancer nomogram consortium ; g - ibcnc = integrated genomic - ibcnc classifier ; g - cc = integrated genomic - clinical classifier. when categorized by pathological stage, median gc scores were consistently higher in validation set patients who recurred (supplementary figure 7, available online). by multivariable analysis, after adjusting for demographic, clinicopathologic and treatment covariates, gc remained prognostic for recurrence (p =.005) (table 3). in this analysis, a statistically significant interaction was noted between gc and nodal status (p =.030) that prompted further exploration of gc score distribution based on nodal stage. when categorized by nodal status, gc scores were able to better discriminate validation set patients based on recurrence status as evidenced by nonoverlapping 95% cis of medians compared with cc (supplementary figure 8, available online) (29). multivariable associations of genomic and clinicopathologic features with risk of postcystectomy bladder cancer recurrence in the validation set ci = confidence interval ; gc = genomic classifier. these represent subjects who may not routinely receive adjuvant therapy. assuming that higher - risk patients may gain more from therapy intensification, improved identification of such candidates using more accurate risk - prediction models cc showed a trend towards significance in separating node - negative patients into risk groups (p =.051). however, gc statistically significantly stratified these patients based on recurrence (four - year probabilities : high 56.2% vs low 13% ; p =.004) (figure 4). when measured across time, aucs for genomic - based classifiers were consistently higher compared with cc in node - negative patients (supplementary figure 6b, available online). cumulative incidence plots for recurrence - free survival for node - negative patients in the validation set. patients were stratified based on their a) cc and b) gc scores into high - risk and low - risk groups as determined by majority rule (cutoff = 0.5). risk stratification based on gc scores was statistically significant (p =.004) while that based on cc scores showed a trend towards statistical significance (p =.051). p values were determined by the log - rank test and are two - sided. cc = clinical - only classifier ; gc = genomic classifier. to analyze the biological relevance of features within gc, a network - based enrichment analysis of go terms and biological pathways related to the markers and their 86 first - degree partners was performed (supplementary figure 9, available online). functional analysis revealed enrichment of pathways associated with cellular signaling including mapk, wnt, and other cancer - associated processes. additional go clusters associated with cell movement and adhesion suggested roles in cell - cell interaction and tumor cell migration. this indicated that gc components likely reflect the pathobiology behind ucb development and progression beyond mere statistical associations. comparison with genes associated with ucb revealed that four markers within gc were identified in prior studies (supplementary results, available online). the transcriptome - wide coverage of human exon arrays also allowed evaluation of previously reported prognostic signatures for muscle - invasive or node - positive ucb. performances of seven genomic signatures were optimized in the discovery set and compared with gc (supplementary table 3) (13 - 15,21,22). as expected, nearly all prior signatures demonstrated high survival - roc aucs in the discovery set, as observed with gc. however, when these locked signatures were applied to the validation set with bioinformaticians being blinded to clinical variables, the survival - roc auc for each model decreased, with gc having the best validated performance (supplementary figure 10, available online). a previously reported 61-feature panel for postcystectomy survival had the next - best performance in the validation set (auc = 0.72 ; 95% ci = 0.60 to 0.87) (21). performance of the 15-feature gc was independently validated on 341 ucb patients from four external datasets sourced through tcga and ncbi - geo (13,21,30,31). when stratified based on the combined expression of gc markers, high - risk patients had statistically significantly poorer survival probabilities in all datasets (p.016) (figure 5). concordance indices ranged from 0.65 to 0.80, and hazard ratios ranged from 2.23 to 7.57 across all available endpoints (supplementary table 4, available online). bladder cancer patients from four external datasets including a) tcga bladder urothelial carcinoma database (p =.016), b) ncbi geo gse31684 (p =.013) were stratified into low - risk (green) and high - risk (red) groups as determined by median split rule. stratification was based on the prognostic index, which was calculated using all available markers related to the 15-feature gc. p values were determined by the log - rank test and are two - sided. to test the hypothesis that risk - prediction models incorporating genomic signatures can better characterize the biology that determines propensity of ucb to recur postcystectomy than clinical variables alone, a genomic classifier (gc) was developed and tested on the discovery set. median follow - up was 9.3 years ; 68 (51.1%) patients recurred, and 77 (57.9%) patients were dead at last follow - up. expression of nearly 1.410 rna features in tumors of patients who recurred was compared with those who remained recurrence - free at last follow - up (supplementary figure 2, available online). following normalization and feature selection, 15 markers were identified corresponding to rnas from protein - coding and noncoding regions of the genome that were differentially expressed based on recurrence (supplementary table 1, available online). a random forest machine - learning algorithm assembled these markers into a gc, and this locked classifier assigned a score to each patient. the feature associated with mecom was determined to be the most important variable (supplementary figure 3, available online). gc performance was compared with individual clinicopathologic variables, the ibcnc postoperative nomogram (8), and a clinical - only classifier (cc) that represented an optimized clinicopathologic prognostic model developed on the discovery set. gc had an auc of 0.88 (95% confidence interval [ci ] = 0.81 to 0.93) in the discovery set (figure 1). as gc was optimized in the discovery set, it could not be compared with individual clinicopathologic variables. however, we could assess the prognostic value of adding genomic information to clinical risk prediction models. when gc was added to ibcnc (g - ibcnc), the auc increased from 0.73 to 0.89 (95% ci = 0.84 to 0.95). when gc was added to cc (g - cc), the auc increased from 0.81 to 0.93 (95% ci = 0.88 to 0.97). this suggested that combined genomic - clinicopathologic classifiers could achieve superior prognostic performance than either alone. performance of classifiers and individual clinicopathologic variables as assessed by standard - roc analysis in the discovery and validation sets for predicting postcystectomy recurrence. roc = receiver - operating characteristic ; auc = area under roc curve ; gc = genomic classifier ; ibcnc = postcystectomy recurrence nomogram from the international bladder cancer nomogram consortium ; g - ibcnc = integrated genomic - ibcnc classifier ; cc = clinical - only classifier ; g - cc = integrated genomic - cc classifier. prognostic performance of the locked classifiers was then assessed (with bioinformaticians blinded to clinical variables) on an independent validation set of 66 ucb patients. median follow - up was 10.8 years ; 33 (50%) patients recurred, and 45 (68.2%) patients were dead at last follow - up. by univariate analysis, gc (p =.003), nodal status (p =.006) and lymphovascular invasion (p =.005) were prognostic for rfs, with gc having the highest hazard ratio (1.23 for every 10% score increment) (table 2). gc performance as measured by standard roc analysis was superior to single clinicopathologic variables (figure 1). this was confirmed by survival roc analysis where auc for gc (0.77 ; 95% ci = 0.65 to 0.91) was higher than any individual clinicopathologic variable (figure 2a). univariate associations of genomic and clinicopathologic features with risk of postcystectomy bladder cancer recurrence in the validation set ci = confidence interval ; gc = genomic classifier. performance of individual clinicopathologic variables and classifiers in the validation set for predicting cancer recurrence. a) survival roc curves show that gc outperforms individual clinicopathologic variables for predicting postcystectomy recurrence. in addition, b) g - ibcnc had higher auc compared to ibcnc, and c) g - cc had higher auc compared to cc by survival - roc analysis. aucs and associated 95% confidence intervals are shown at the bottom right of each roc curve panel. d) cumulative incidence plot for recurrence - free survival comparing patients with high versus low g - cc scores as determined by majority rule (cutoff = 0.5) indicate a statistically significantly elevated recurrence probability for patients with high g - cc scores (log - rank p <.001). roc = receiver - operating characteristic ; auc = area under roc curve ; gc = genomic classifier ; ibcnc = post - cystectomy recurrence nomogram from the international bladder cancer nomogram consortium ; g - ibcnc = integrated genomic - ibcnc classifier ; cc = clinical - only classifier ; g - cc = integrated genomic - cc classifier. prognostic performance of ibcnc and cc were comparable (standard - roc auc = 0.74 and 0.77, respectively) with gc in the validation set. again, performance of the combined genomic - clinicopathologic classifiers measured the highest of all models tested (figure 1). furthermore, aucs by survival roc analysis that consider time to recurrence showed marked boost in performance for combined models (ibcnc vs g - ibcnc, 0.73 vs 0.82 ; cc vs g - cc, 0.78 vs 0.86). combined genomic - clinicopathologic classifiers had the highest specificity and sensitivity across the widest range of cutoffs (figure 2, b and c). g - cc had the highest auc among the combined genomic - clinicopathologic classifiers tested. multivariable analyses showed the independent prognostic significance of combined genomic - clinicopathologic classifiers compared with clinical models (supplementary table 2, available online). decision curve analysis showed a higher overall net benefit for genomic - based classifiers, thereby highlighting their value as better tools for planning patient management compared to clinical - only risk models based on risk for recurrence (supplementary figure 4, available online). discrimination plots confirmed that while gc identified patients who recurred better than clinical models alone, the combined g - ibcnc and g - cc models were superior, with g - cc performing the best (p = 5.5510) (supplementary figure 5, available online). g - cc was therefore considered the benchmark genomic - clinicopathologic classifier for further evaluation. when survival - roc aucs were measured across a range of postcystectomy intervals, g - cc showed consistently superior performance compared with cc and gc alone, indicating that the former was most prognostic for rfs at any point in time following cystectomy (supplementary figure 6a, available online). validation set patients were then categorized as high - risk (g - cc score 0.5) or low - risk (g - cc score < 0.5). accounting for competing risk, high - risk patients had elevated cumulative incidence of recurrence compared with low - risk patients (four - year probability : 81.5% vs 20.6%, respectively ; p < patients were similarly risk - stratified by ibcnc and g - ibcnc scores, and categorization by the clinical - only ibcnc nomogram was compared with genomic - clinicopathologic classifiers (figure 3). addition of genomic features to the ibcnc nomogram (as g - ibcnc) reclassified 18 validation set patients into different predicted risk categories than those classified by ibcnc, of which 12 (66.7%) were reclassified correctly based on outcome. similarly, g - cc reclassified 12 patients originally risk stratified by ibcnc, of which 10 (83.3%) were reclassified correctly. reclassification of ibcnc score categories by genomic - clinicopathologic classifier scores for patients in the validation set. after categorizing based on their ibcnc scores, patients were reclassified based on their a) g - ibcnc and b) g - cc scores. individual patients are represented as dots colored by recurrence event ; sizes of dots represent pathological stage as indicated. gray quadrants represent situations where the genomic - clinicopathologic classifier reclassifies patients compared to ibcnc. patients who did not recur (blue dots) in the top - left quadrant and patients who recurred (red dots) in the bottom - right quadrant are reclassified correctly by the genomic - clinicopathologic classifier. of patients who were reclassified, a majority were done so correctly. ibcnc = postcystectomy recurrence nomogram from the international bladder cancer nomogram consortium ; g - ibcnc = integrated genomic - ibcnc classifier ; g - cc = integrated genomic - clinical classifier. when categorized by pathological stage, median gc scores were consistently higher in validation set patients who recurred (supplementary figure 7, available online). by multivariable analysis, after adjusting for demographic, clinicopathologic and treatment covariates, gc remained prognostic for recurrence (p =.005) (table 3). in this analysis, a statistically significant interaction was noted between gc and nodal status (p =.030) that prompted further exploration of gc score distribution based on nodal stage. when categorized by nodal status, gc scores were able to better discriminate validation set patients based on recurrence status as evidenced by nonoverlapping 95% cis of medians compared with cc (supplementary figure 8, available online) (29). multivariable associations of genomic and clinicopathologic features with risk of postcystectomy bladder cancer recurrence in the validation set ci = confidence interval ; gc = genomic classifier. these represent subjects who may not routinely receive adjuvant therapy. assuming that higher - risk patients may gain more from therapy intensification, improved identification of such candidates using more accurate risk - prediction models cc showed a trend towards significance in separating node - negative patients into risk groups (p =.051). however, gc statistically significantly stratified these patients based on recurrence (four - year probabilities : high 56.2% vs low 13% ; p =.004) (figure 4). when measured across time, aucs for genomic - based classifiers were consistently higher compared with cc in node - negative patients (supplementary figure 6b, available online). cumulative incidence plots for recurrence - free survival for node - negative patients in the validation set. patients were stratified based on their a) cc and b) gc scores into high - risk and low - risk groups as determined by majority rule (cutoff = 0.5). risk stratification based on gc scores was statistically significant (p =.004) while that based on cc scores showed a trend towards statistical significance (p =.051). p values were determined by the log - rank test and are two - sided. to analyze the biological relevance of features within gc, a network - based enrichment analysis of go terms and biological pathways related to the markers and their 86 first - degree partners was performed (supplementary figure 9, available online). functional analysis revealed enrichment of pathways associated with cellular signaling including mapk, wnt, and other cancer - associated processes. additional go clusters associated with cell movement and adhesion suggested roles in cell - cell interaction and tumor cell migration. this indicated that gc components likely reflect the pathobiology behind ucb development and progression beyond mere statistical associations. comparison with genes associated with ucb revealed that four markers within gc were identified in prior studies (supplementary results, available online). the transcriptome - wide coverage of human exon arrays also allowed evaluation of previously reported prognostic signatures for muscle - invasive or node - positive ucb. performances of seven genomic signatures were optimized in the discovery set and compared with gc (supplementary table 3) (13 - 15,21,22). as expected, nearly all prior signatures demonstrated high survival - roc aucs in the discovery set, as observed with gc. however, when these locked signatures were applied to the validation set with bioinformaticians being blinded to clinical variables, the survival - roc auc for each model decreased, with gc having the best validated performance (supplementary figure 10, available online). a previously reported 61-feature panel for postcystectomy survival had the next - best performance in the validation set (auc = 0.72 ; 95% ci = 0.60 to 0.87) (21). performance of the 15-feature gc was independently validated on 341 ucb patients from four external datasets sourced through tcga and ncbi - geo (13,21,30,31). when stratified based on the combined expression of gc markers, high - risk patients had statistically significantly poorer survival probabilities in all datasets (p.016) (figure 5). concordance indices ranged from 0.65 to 0.80, and hazard ratios ranged from 2.23 to 7.57 across all available endpoints (supplementary table 4, available online). bladder cancer patients from four external datasets including a) tcga bladder urothelial carcinoma database (p =.016), b) ncbi geo gse31684 (p =.013) were stratified into low - risk (green) and high - risk (red) groups as determined by median split rule. stratification was based on the prognostic index, which was calculated using all available markers related to the 15-feature gc. p values were determined by the log - rank test and are two - sided. patients with muscle - invasive or node - positive ucb at cystectomy are at higher risk for recurrence, although a substantial proportion remains recurrence - free after meticulous surgery and lymphadenectomy (2,32). identification of candidates at highest risk for recurrence who may need adjuvant therapy is currently based on clinical criteria that may not reflect the entire biology of the disease (8). we hypothesized that an unbiased transcriptome - wide profiling approach of primary ucbs from clinically high - risk patients could be employed to accurately stratify the risk of postcystectomy recurrence. to our knowledge, this represents the largest effort to discover and validate a prognostic genomic signature for clinically high - risk ucb to date. the 15-feature gc surpassed the prognostic potential of standard clinical variables and previously reported genomic signatures for muscle - invasive ucb. such a tool can improve patient counseling after cystectomy and can better identify candidates who need more aggressive management. although patients with muscle - invasive ucb at cystectomy are considered to be at higher risk for recurrence clinically, most prior prognostic genomic profiling investigations have analyzed all ucb stages as part of their discovery effort, including non - muscle - invasive disease (13 - 15,21). clinically high - risk patients in prior studies did not always receive consistent surgical management, and prognostic signatures were often generated towards non - disease - specific outcomes such as overall survival (supplementary table 3, available online). further, prior efforts were limited to scarce frozen tumor specimens and employed low - density platforms that would only profile select protein - coding transcripts. the current study focused exclusively on identifying patients with muscle - invasive and/or node - positive ucb on final pathology who, based on expression profiles of their tumors, need intensified management to prevent recurrence. patients were neoadjuvant chemotherapy - nave, received consistent surgical management and had long follow - up. performance of genomic signatures and clinical nomograms were measured against rfs, a highly disease - specific endpoint in this group of patients. profiles were generated from ffpe tumors that represent an abundant and practical tissue source using technologies that reliably and reproducibly assess differential expression from archival specimens similar to unfixed tissues (33). the high - density profiling arrays in this investigation provide comprehensive whole - genome - scale coverage, including empirically supported and predicted transcribed sequences, enabling the discovery of previously unidentified events (34). this helped leverage the added value of noncoding transcripts that represent majority of the transcriptome, promote neoplastic transformation, and are potentially prognostic (3537). addition of clinical information in the form of established (ibcnc) or novel (cc) nomograms to gc improved its performance, thereby suggesting that clinical and genomic variables provide independent and complementary prognostic information. genomic - clinicopathologic classifiers had the highest net clinical benefit by decision curve analysis across a range of recurrence probabilities. when applied to node - negative patients, the subgroup where postcystectomy risk stratification is most critical (38), gc identified patients at risk for recurrence better than clinicopathologic metrics and remained statistically significant after adjusting for adjuvant chemotherapy administration. gc components include transcripts involved in cell proliferation and differentiation, apoptosis, cell - cycle and transcriptional regulation, and signal transduction when benchmarked against previously reported gene signatures for clinically high - risk ucb, gc performance was superior when applied in a blinded manner on the validation set. this comparison was possible because of the transcriptome - wide profiling platform that allowed prior signatures to be modeled on our cohort. of prior signatures aucs in the validation set, lower bounds of 95% cis of 5/7 signatures were greater than 0.50, suggesting a statistically significant ability to predict recurrence beyond chance. this indicated that while some prior signatures were prognostic for clinically high - risk ucb, gc provides the highest relative performance that improved when combined with clinicopathologic metrics. study limitations include the inability to further evaluate gc because of the absence of public, clinically - annotated human exon array - profiled ucb datasets. however, its constituent markers could be mapped to other profiling platforms for assessment of performance. this exercise across four external datasets showed that gc could statistically significantly predict outcomes, thereby further validating its prognostic potential. patients at elevated risk for recurrence may benefit from adjuvant therapy, although the efficacy of such therapy in patients with high gc or g - cc scores needs further investigation. in conclusion, we document the discovery and validation of a 15-feature genomic classifier composed of biologically - relevant rna sequences that can stratify patients with pathologically - confirmed muscle - invasive and/or node - positive ucb postcystectomy based on their risk for recurrence, especially when combined with clinicopathologic variables. classifier performance was superior to clinical metrics and prior genomic signatures for such patients, and was validated in external datasets. this may be attributable to the use of a large cohort, clinically relevant endpoint, and high - density transcriptome - wide expression profiling approach. the standardized patient management and use of ffpe tissues for profiling also make this signature more clinically applicable. while additional analysis is warranted to better characterize the classifiers prognostic potential, our results suggest that the combination of these markers and clinicopathologic parameters may be prognostic for clinically high - risk ucb. this work was supported by a proof - of - concept program grant from genome british columbia.	backgroundnearly half of muscle - invasive bladder cancer patients succumb to their disease following cystectomy. selecting candidates for adjuvant therapy is currently based on clinical parameters with limited predictive power. this study aimed to develop and validate genomic - based signatures that can better identify patients at risk for recurrence than clinical models alone.methodstranscriptome-wide expression profiles were generated using 1.4 million feature - arrays on archival tumors from 225 patients who underwent radical cystectomy and had muscle - invasive and/or node - positive bladder cancer. genomic (gc) and clinical (cc) classifiers for predicting recurrence were developed on a discovery set (n = 133). performances of gc, cc, an independent clinical nomogram (ibcnc), and genomic - clinicopathologic classifiers (g - cc, g - ibcnc) were assessed in the discovery and independent validation (n = 66) sets. gc was further validated on four external datasets (n = 341). discrimination and prognostic abilities of classifiers were compared using area under receiver - operating characteristic curves (aucs). all statistical tests were two-sided.resultsa 15-feature gc was developed on the discovery set with area under curve (auc) of 0.77 in the validation set. this was higher than individual clinical variables, ibcnc (auc = 0.73), and comparable to cc (auc = 0.78). performance was improved upon combining gc with clinical nomograms (g - ibcnc, auc = 0.82 ; g - cc, auc = 0.86). g - cc high - risk patients had elevated recurrence probabilities (p <.001), with gc being the best predictor by multivariable analysis (p =.005). genomic - clinicopathologic classifiers outperformed clinical nomograms by decision curve and reclassification analyses. gc performed the best in validation compared with seven prior signatures. gc markers remained prognostic across four independent datasets.conclusionsthe validated genomic - based classifiers outperform clinical models for predicting postcystectomy bladder cancer recurrence. this may be used to better identify patients who need more aggressive management.
an invitation was extended to 26 health care institutes (hcis) across the country which were either regional centres of amrsrn or part of amsp steering committee representing different regions of the country, from both government and private sectors to reply to a questionnaire on amsp practices being followed by them for the period from january 1, 2013 to december 31, 2013. the questionnaire carried the following information : (i)general information : location, population type seeking care, sponsor, level of health care, bed strength [including intensive care units (icus) ], specialties and super specialty services offered (including transplant, oncology programmes, etc.), accreditations of hospitals and diagnostic laboratories.(ii)amsp, hic : written documents, teams, frequency of meetings, communication system of minutes of meetings, hic guidelines & audit of compliance, record of health care associated infections (hcai), investigation of outbreaks of hcai.(iii)amr data analysis : frequency, as out patient department (opd), in patient department (ipd), icu, community acquired infections (cai), hcai, as per site of infection, as per pathogen, antimicrobial susceptibility testing (amst) guidelines, communication system.(iv)ama usage data analysis : frequency, ama, units.(v)amsp strategies : formulary restriction, empirical prescription approval, automatic stop, controlling authority.(vi)ama prescription guidelines : icu, hcai, cai, infections post - transplant, infections in oncology and immunocompromised.(vii)ama usage audit and feedback : frequency, audit team, prescription appropriateness.(viii)amsp implementation outcome studies : ama usage, appropriateness of ama usage, amr, hcai incidence, clinical markers, trends in amsp outcomes.(ix)usage of computer assisted programmes in amsp.(x)amsp implementation leading to perception of loss of prescription autonomy. general information : location, population type seeking care, sponsor, level of health care, bed strength [including intensive care units (icus) ], specialties and super specialty services offered (including transplant, oncology programmes, etc.), accreditations of hospitals and diagnostic laboratories. amsp, hic : written documents, teams, frequency of meetings, communication system of minutes of meetings, hic guidelines & audit of compliance, record of health care associated infections (hcai), investigation of outbreaks of hcai. amr data analysis : frequency, as out patient department (opd), in patient department (ipd), icu, community acquired infections (cai), hcai, as per site of infection, as per pathogen, antimicrobial susceptibility testing (amst) guidelines, communication system. amsp strategies : formulary restriction, empirical prescription approval, automatic stop, controlling authority. ama prescription guidelines : icu, hcai, cai, infections post - transplant, infections in oncology and immunocompromised. amsp implementation outcome studies : ama usage, appropriateness of ama usage, amr, hcai incidence, clinical markers, trends in amsp outcomes. usage of computer assisted programmes in amsp. completed questionnaires were received from 20 hci [12 government, i.e.(ghci), and 8 private, i.e. (phci) ] from various states and union territories such as jammu and kashmir, national capital territory of delhi, haryana, uttar pradesh, west bengal, chandigarh, maharashtra, andhra pradesh, karnataka, tamil nadu and puducherry. in addition, all offered most specialities and super - speciality training, including programmes in transplantations and oncology. the authorized bed strengths varied between 182 to 3000 (icu 11 - 200), with an average bed occupancy of 65 - 100 per cent. accreditation was certified for nine hospitals and seven laboratories, the majority being in phci (7 & 5) rather than ghci (2 & 2), respectively. amsp written documents were available with eight of 20 (40%) hci (ghci 2, phci 6) though amsp teams were formed in 50 per cent hci (table i). infectious disease (i d) physicians were part of these teams in 30 per cent hci (ghci nil, phci 3). physicians, surgeons, paediatricians, clinical microbiologists and hospital administrators were well represented on these teams. clinical pharmacists were available in 50 per cent of teams (ghci 1, phci 4). only one amsp team had a hospital epidemiologist and two hospitals had information technology (it) personnel as members of amsp teams - all in phci. antimicrobial stewardship programme (amsp) and hospital infection control (hic) features in government and private health care institutions (ghci and phci). written documents were available in 15 of 26 (75%) hcis (ghci 7, phci 8) and hic teams in 18 (90%) (ghci 10, phci 8). i d physicians were available in four (22.2%) (ghci 1, phci 3). physicians, surgeons, paediatricians, clinical microbiologists, hospital administrators and staff nurses were well represented. regular meetings were held at most hcis, though at variable frequencies (table i). hospital infection control (hic) guidelines (table ii) were available in 75 per cent (n=15) of hci (ghci 7, phci 8). house keeping, laundry and catering guidelines were available in 13 (65%) hcis. hand sanitation guidelines were available in 15 (75%) and guidelines on personal protective clothing, urine catheterisation, central and peripheral venous lines, operation theatre, mechanical ventilation, endoscopes and icu in 60 - 70 per cent and in lower percentages of 40 - 55 per cent for bedside minor procedures, lumbar puncture, dialysis and phlebotomy. availability of hospital infection control (hic) and prescription guidelines in health care institutions (hcis). guidelines were available in 12 hci (60%) for isolation of patients (ghci 4, phci 8). in decreasing order of availability, these pertained to methicillin resistant staphylococcus aureus (mrsa), respiratory infections, carbapenem resistant enterobacteriaceae (cre), vancomycin resistant enterococci (vre), carbapenem resistant non - fermentative gram - negative bacilli (crnfgnb) and esbl enterobacteriaceae. some level of audit of implementation of hic guidelines was seen in 60 per cent of hci (ghci 4, phci 8). hcai record (table iii) was being maintained in 12 hcis (60%) (ghci 4, phci 8), in 12 (60%) each for ventilator associated pneumonia (vap) and central line associated blood stream infections (clabsi), 11 (55%) for catheter associated urinary tract infections (cauti), 10 (50%) for surgical site - infections (ssi) early (nil for ssi late), in three (15%) for health care associated pneumonia (hcap) and in two (10%) for health care associated septicaemia (hcas). incidence of hcai was not compared because of paucity of comparable figures and for keeping analysis anonymous. guidelines for hcai outbreak investigations were available in 13 (65%) hci (ghci 5, phci 8). half of the hcis reported investigating hcai outbreaks (n=16) (ghci 9, phci 7). reports were available online in five phci and in printed form in three phci and all ghci. number of health care institutions (hcis) - both government and private hcis maintaining record of health care associated infections (hcais). amr data analysis (table iv) was regularly performed by 16 of 20 (80%) hci (ghci 8, phci 8) at varied frequencies (monthly 20%, quarterly 20%, half yearly 35%, yearly 25%). analysis as cai was performed by only one phci, and as hcai by one each ghci and phci. overall figures for phci (87.5 - 100%) were higher than for ghci (41.7 - 58.3%). analysis of specific bacterial data (55%) was less preferred than for aerobic gram - positive and gram - negative bacteria (70% each). online reporting of data analysis was performed by 12 (60%) hci and printed in 50 per cent. antimicrobial resistance (amr) data analysis patterns in government and private health care institutions (hcis). ama usage data (table v) were regularly analysed only by five (25%) hcis. the preferred units for data collection were daily defined doses (ddd) and days of therapy (dot). amsp strategies (table v) in some form were utilized by only six (30%) hcis. antimicrobial agents (ama) usage data and amsp strategies utilization analysis in health care institutions (hcis). ama prescription guidelines (table ii) were available with 13 (65%) hcis (ghci 5, phci 8). guidelines for icu for septicaemia were available with 50 per cent, and vap 45 per cent. guidelines for treatment of hcai were available in descending order for pneumonia and mrsa (40%), extended spectrum beta lactamase (esbl) enterobacteriaceae and carbapenem resistant enterobacteriaceae (35%), septicaemia and vre (30%), urinary tract infections (uti), antibiotic associated diarrhoea and crnfgnb (25%). treatment guidelines for cai in descending order were available for upper respiratory tract infections (urti) (50%), lower respiratory tract infections (lrti), diarrhoea and meningitis (45%), uti (40%), gynaecological infections (35%), septicaemia, tuberculosis and paediatric infections (30%), sexually transmitted infections (20%), infections in transplantation and hiv (15%) and infections in oncology, diabetes mellitus and autoimmune diseases (5%). ama prescription guidelines for surgical prophylaxis were available in 11 (55%) hci (ghci 3, phci 8). in descending order, guidelines were for clean contaminated surgery (50%), clean surgery (45%), contaminated and orthopedic surgeries (40%), orthopedic implants (35%), cardiac catheterization (30%), minimal access surgery, cardiac surgery, neurosurgery, gynaecological surgery and joint replacement surgery (25%). ama prescription audit and feedback was practiced by only six (30%) hcis (ghci 2, phci 4), with variable frequencies. all prescriptions were audited in four hci and icu prescriptions in two (table vi). amsp implementation outcome was analysed by only seven (35%) hci (ghci 2, phci 5). features analysed were total cost of ama used, reduction in ama usage, amr, incidence of hcai, length of hospital stay, and all cause mortality. none of the hcis analysed increase of appropriateness of ama usage, infection related mortality, readmission rates, clinical cure. computer assisted programmes were utilized by only six (30 %) hcis (ghci 1, phci 5). phci had ama prescription guidelines online in six centres, online ama prescriptions in three and autoreview of ama prescriptions in two. antimicrobial agent (ama) prescription audit and feedback data analysis in health care institutions (hcis). universally, the health care providers are increasingly recognizing the importance of amsp in hci as a major contributor to sustaining usefulness of ama in the treatment of infections. despite being few in number, compiled evidence from meta - analyses suggests that clinical outcomes are better or at least similar for patients when antimicrobial stewardship is performed9. other components like discovery and development of newer classes of ama, restricting use of ama (at least those used for human infections) in animal farming and agriculture are equally important101112. the major inputs for amsp are appropriateness of usage of ama in individual patients, amr data of infecting pathogens (indicates usefulness of specific ama for specific pathogen) and control and reduction of incidence of infections, especially hcai. with a view to understand various nuances in practice of amsp in india, a detailed questionnaire was prepared, vetted by the amsp committee of the icmr and sent to hcis, both government hci (ghci) and private hci (phci), across the country. these belonged both to ghci and phci, were tertiary care institutes with multiple specialties and were engaged in research and academics of some degree with bed strengths ranging between 182 to 3000. the data collected showed that phci had better accreditations in place, possibly out of commercial necessity, but the same was not true for ghci. it is now universally accepted that accreditation of hospitals and diagnostic laboratories is an important step towards standardization of health care delivery. this will automatically make amsp practices in ghci at par with phci, in addition to providing many other benefits. although the physicians recognize the challenges of antimicrobial resistance, this figures at the bottom of the list of factors influencing their choice of antibiotics, as the antibiotics are chosen for individual benefit13. having a treatment guidelines document which takes into account the regional data on antimicrobial resistance will thus help physicians in making relevant choices. it was noted that only 50 per cent of hci had amsp teams functioning, and only 40 per cent had written amsp document to follow, leaving much to the choice of team members. the emphasis should be to reach figures of 100 per cent for both amsp written documents and amsp teams formation. the i d physician is regarded as essential to the efficient functioning of amsp, diagnosis and treatment of difficult infections, as also control of hcai4. in our survey, only three phci had departments of i d and none were present in ghci. other poorly represented member of the team was the clinical pharmacist, having an undisputed role in determining appropriateness of prescriptions, dosing, drug combinations and drug interactions, etc. which can interfere with the efficiency of ama prescribed as also other drugs4. the situation concerning hic was encouraging as written documents were available in 75 per cent hci and hic teams were in place in 90 per cent of the participating institutions. the reason for this could be the early recognition of importance of hic by the medical fraternity. however, the aim should be to achieve figures of 100 per cent for written documents and formation of teams. these should include both infrastructure guidelines like house keeping, laundry and catering, as well as for the functioning of health care worker, the most important component being hand sanitation and personal protective clothing. guidelines on clinical procedures are important and make health care workers more confident of their functioning and help in reducing hcai. it is important to perform regular audits of implementation of hic guideline to keep health care workers attentive and alert to impending risks. the final parameter of efficiency of hic guideline implementation is the incidence of hcai, which must be recorded truthfully and accurately to be of any value. in this survey, it is equally important to have guidelines for investigation of hcai outbreaks, carry out investigations rapidly and communicate results without loss of time for facilitating corrective actions14. the amr data describe both need and outcome of amsp measures and is fundamental to amsp1315. the methods, components and ensuing analysis of amr will determine its usefulness for the purpose of laying down policies of amsp, especially treatment guidelines. the frequency of analysis should be monthly and also cumulative. it should be communicated to stakeholders at half yearly or yearly intervals, unless monthly data have some significant abnormal deviation which needs to be urgently reported. regular analysis need to be raised to 100 per cent from the reported 80 per cent in the current survey. even though it is ideal to analyse amr data as cai and hcai, but almost all prefer to analyse it as opd, ipd and icu.. it will be more meaningful to analyse data as per specific pathogen rather than as gram - negative, gram - positive, total yeasts or moulds. similarly, it would be more prudent to analyse data as per site of infection and additionally as subsites. the most controversial aspect of amsp is the implementation strategies457 to be applied, which were being done by only 30 per cent hci. controlled or restricted ama prescription guidelines must be easily available to all clinicians, and should be updated, preferably, on yearly basis, in line with amr data changes. guidelines on icu infections were better covered than other hcai. important guidelines which were not well covered were the management of infections in transplantation, hiv, oncology, diabetes mellitus and autoimmune diseases. the survey results indicated that minimal access surgery, cardiac surgery, neurosurgery, gynaecological surgery and joint replacement surgery were less likely to be covered under the available guidelines. since inappropriate choice of ama and duration and timing of ama are common when given for surgical prophylaxis, this lacunae should be addressed in the guidelines. important features should include appropriateness, ama combinations, ama antimicrobial susceptibility testing (amst) mismatch, dosing, duration, intravenous to oral switch and drug interactions. this important strategy is yet underutilized in india as in this survey only 30 per cent hcis were found to utilize this facility of prescription audit and feedback. carling analysed results from three years pre - intervention and seven years post - intervention, and documented a reduction in use of third - generation cephalosporins and aztreonam and a stable rate of use of fluoroquinolones and imipenem when i d physician and clinical pharmacist monitored prescription of broad - spectrum antimicrobials and gave feedback to prescribers. this also led to a reduction in clostridium difficile infections and infections with drug resistant enterobacteriaceae, when compared to pre - intervention rates and remained stable thereafter. only 35 per cent of hci were undertaking amsp outcome analysis and this must be improved. all hic and ama guidelines as well as reports can be put up online for ease and speed of access. availability of it personnel is essential for extensive and meaningful utilization of computer assisted programmes in amsp4. the increasing use of computer assisted programmes in different spheres of hospital functioning offers new opportunities to educate physicians and can be utilized for optimizing antimicrobial use. this can be achieved by introducing online prescription order entry systems in hospitals, providing a link to the institution 's guidelines for therapy to promote their wider usage, or for devising customized therapeutic regimens for patients using patient - specific laboratory and microbiology data. one of the cardinal requirements for the success of amsp is its acceptance and implementation by physicians without a feeling of loss of prescription autonomy45. a major finding of this study was better performance by phci than ghci on almost all aspects of amsp. one of the obvious factors was higher level of accreditation in phci, on account of basic commercial requirement. the very process of accreditation requires extensive preparation of guidelines for almost each and every aspect of their functioning. it is time the government makes accreditation of its hci mandatory to have this advantage. it is also important to strengthen amsp in our hci and also continue research in all controversial aspects of amsp so that better and more effective strategies may be evolved and followed and those found less useful amended or even discarded171819. (i) for the standardization of health care (including amsp practices) in the country, the government must make accreditation of all hospitals and their diagnostic laboratories mandatory. (ii) i d physicians must be available in all hci providing tertiary and secondary health care. (iii) clinical pharmacists must be available in all tertiary and secondary hci for better control and use of therapeutics especially ama. (iv) all hci must have written documents on amsp and perform frequent audits to ascertain how well guidelines are being followed. (v) a comprehensive record of hcai must be kept and trends analyzed regularly. (vi) amr data must be regularly analyzed specific to acquisition of infection, site of infection and the pathogen. (viii) regular education, easy availability of guidelines and audit of ama prescription practices and their feedback are essential. (ix) major stakeholders, physicians and surgeons must be involved in and even be leaders in all aspects of amsp, guidelines and audits. (x) continuous research is warranted in all aspects of amsp to obtain the best programme for local needs.	a survey was conducted to ascertain practice of antimicrobial stewardship programme (amsp) in india for 2013. a total of 20 health care institutions (hci) responded to a detailed questionnaire. all the institutions contacted were tertiary care hci, of which 12 were funded by government (ghci) and 8 were corporate / private hci (phci). further, all catered to both rural and urban populations and were spread across the country. written documents were available with 40 per cent for amsp, 75 per cent for hospital infection control (hic) and hic guidelines and 65 per cent for antimicrobial agents (ama) prescription guidelines. records were maintained for health care associated infections (hcai) by 60 per cent hci. antimicrobial resistance (amr) data were being analysed by 80 per cent hci. ama usage data were analysed by only 25 per cent hci and ama prescription audit and feedback by 30 per cent. phci performed better than ghci across all fields of amsp. the main contributory factor was possibly the much higher level of accreditation of phci hospitals and their diagnostic laboratories. the absence of infectious diseases physicians and clinical pharmacists is worrying and demands careful attention.
dengue is a disease caused by four antigenically distinct but genetically related virus serotypes that cause dengue : dengue virus (denv 14). it is transmitted by the aedes aegypti mosquito with clinical presentation of the disease ranging from mild forms, such as dengue fever (df), to serious and even fatal forms. in the mild form of the disease, clinical manifestations include fever, headache, prostration, arthralgia, retroorbital pain, nausea, rash, itchy skin, and others. the main severe form of dengue is dengue hemorrhagic fever (dhf), characterized by bleeding tendency, thrombocytopenia, and plasmatic effusion, which can progress to circulatory failure, characterizing dengue shock syndrome (dss), and death [26 ]. a recent study estimates there to be 390 million dengue infections every year, of which 96 million manifest any level of clinical or subclinical severity. in 2007, there were over 890,000 dengue cases in america, of which 26,000 were dhf. the disease is endemic in over 100 countries in africa, americas, eastern mediterranean, southeast asia, and eastern pacific., only nine countries had experienced dhf epidemics, a number that has increased more than four times in 1995. brazil has experienced several epidemics of dengue. from 2001 to october 2013, there were 13,955 confirmed cases of dengue in divinpolis, midwest of minas gerais (mg), brazil, of which 4,350 were in 2010. since the first case reported of dengue, eight deaths were registered. during this period, viral isolation detected the circulation of denv-1 and denv-2 serotypes in 2001 ; denv-3 's emergence in 2002 ; and denv-1 and denv-3 in 2003. the viral typing for the years 2010, 2011, 2012, and 2013 was performed in this work. the increase of dengue cases accompanied by severe forms of disease and the detection of three dengue serotypes in the city show that it is necessary to study the clinical, molecular, and epidemiological characteristics of dengue cases during a major dengue outbreak in divinopolis, mg. the information system for notifiable diseases and the local epidemiological surveillance database were used to access the data of patients with df. dengue fever cases were confirmed by epidemiological criteria or serological tests by different laboratories for igm / igg detection using an igm antibody - capture enzyme - linked immunosorbent assay (mac - elisa) (panbio, brisbane, australia) or rapid chromatographic immunoassay kit for detection of dengue igg / igm antivirus (wama diagnstica, sp, brasil). forms from the information system for notifiable diseases consist of general data (city, date of notification, and health care institution), individual data (identification, gender, race, and education), residence (address, urban, or rural), clinical manifestations (fever, headache, prostration, arthralgia, myalgia, exanthema, vomiting, diarrhea, bleeding, retroocular, and abdominal pain), and laboratory to qualify the case. the data analyzed in this study include gender, age, clinical manifestations, days of the symptoms, results of serological tests, and data about hospital admission. cases without fever but with many other symptoms described above were considered suspicious once atypical dengue cases were previously described in the country. the research data were entered in duplicate, validated, and processed in microsoft office excel 2007. statistical package for the social sciences (spss) 14.0 was used for statistical analysis. univariate analyses between categorical variables, sex and hospital admission and bleeding and hospital admission, were performed. univariate analyses between days with symptoms and hospitalization and days with symptoms and bleeding were performed as well. the chi - square, odds rates, and mann whitney test were used to estimate the odds ratio and median with a significance level of 5%. there were 5,032 reported cases of dengue in 2010 in the municipality, but only cases confirmed by laboratory test or epidemiological criteria were included. of the total, 699 forms were discarded without confirmation of the disease and some others discarded by incomplete filling or by typographical errors. the collection was performed from january to march of each year at the hospital so judas tadeu or at the centro municipal de apoio sade (cemas), both in divinopolis, mg. the samples were collected from patients with less than 7 days of symptoms after the consent on the research project. this study was approved by the research 's ethics committee of the universidade federal de so joo del rei, under the identification 012/2010. plasma for molecular analyses was obtained after centrifugation of 3 to 5 ml of whole blood and stored at 80c until rna extraction. the rna extraction was performed with the qiaamp viral rna minikit (qiagen, usa) and the rna fractionated and stored at 80c. for the detection and typing of the denv serotypes, primers described by lanciotti. were used. after linearization with reverse primer, the rna was subjected to rt - pcr using the following conditions : 1 h at 42c and 15 min at 70c using buffer rt enzyme (5x), dntp (1 mm), and rt enzyme (100 u). denv were detected using 5 ul of cdna, 1.25 ul of taq dna polymerase, taq buffer (5x), 2 mm mgcl2, 10 pmoles of each primer, and 100 um of dntp. nested pcr was performed for viral typing using 5 l from the dilution of the amplified product (1 : 10 in sterile water). the pcrs for detection and denv typing were performed under the same conditions : 5 min at 94c, 30 cycles of 94c for 30 s, 55c for 30 s, and 72c for 30 s. fragments were visualized on 8% polyacrylamide gels stained with silver nitrate. the most frequent symptoms present in the notification form from the information system for notifiable diseases are described in figure 1. prostration, arthralgia, exanthema, vomiting, diarrhea, bleeding, retroocular and abdominal pain, headache, and fever may be observed. in this analysis, the most common symptoms were fever (82.8%), followed by headache (82.7%), myalgia (78.6%), and pain retroocular (73.4%). of the total subjects studied, 206 (5.0%) reported the presence of some other symptoms besides those described in the information system for notifiable diseases form. the most frequent were petechiae at 44.7% (92/206) and itching at 22.8% (47/206). in this study, epistaxis (50.7%), gingival bleeding (25.3%), and metrorrhagia (19.4%) were the most frequent types. even among individuals who reported bleeding, six of them (8.9%) had more than one type. of the 4,110 individuals analyzed, 40.2% (1,651) were male and among them 2.7% (44/1,651) were hospitalized. among the women (59.8% of the sample), 3.0% however, chi - squared testing was used to verify that there was no association between gender of the patient and hospital admission (p = 0.517). individuals who had been admitted to hospital showed on average five days of symptoms compared with three days of symptoms of those not admitted. in the statistical analyses, the difference between the median number of the days of symptoms in patients who were or were not admitted to hospital was significant (p < 0.01). in the association analysis of the hospital admission and bleeding, data were available for 110 individuals that were admitted to hospital and for 3,222 individuals that were not admitted. so, the correlation analysis between hospital admission and bleeding was restricted to 3,432 patients. 9.7% had any kind of bleeding (334/3,432), and, among these, 10.5% (35/334) were hospitalized. on the other hand, from 90.3% with no bleeding (3,098/3,432), 2.4% were admitted to hospital (75/3,098) but other warning signs as vomiting and abdominal pain were present in 63% of these individuals. the median number of days with symptoms was four in people who had bleeding and three in people without bleeding. only 23% (946) of cases were confirmed by serological tests and the other 70,1% (2,880) were confirmed by epidemiological criteria. of the 190 blood samples collected from patients with suspected dengue from 2010 to 2013, 23% were positive for denv by molecular tests. in 2010, of the 82 blood samples collected, 22 were positive in the molecular test. just one sample was positive for denv-3. among 22 positive cases detected by molecular diagnosis,, the typing of denv has continued with denv-3 detected in the single positive sample in 2011 from 32 samples collected and denv-1 in one positive sample from 6 suspected cases collected in 2012. the years 2011 and 2012 had just 35 and 27 confirmed dengue cases by serological test. in 2013, 5,998 cases were confirmed until october of this year with denv-1 detected in 20 positive samples from 70 suspected cases collected. molecular diagnosis allowed the detection of denv in samples with only one day of symptoms. positive cases in the years analyzed were distributed as follows : five cases with one day of symptoms, 13 cases with two days of symptoms, 16 cases with three days of symptoms, five cases with four days of symptoms, one case with five days of symptoms, and one case with 6 days of symptoms. one patient had a negative serology but a positive molecular diagnosis and three other patients had positive serology but negative molecular diagnoses. dengue fever, which is a public health problem, is part of a scenario that consists of two factors of concern, since the presentation clinic is nonspecific : (i) most of the dengue cases go unreported ; (ii) during epidemic periods, erroneous notifications may occur in some individuals with similar clinical manifestations caused by other viral agents. the scarcity of financial resources and specific laboratory for dengue diagnosis contribute to these problems [2, 12, 13 ]. thus, clinical, molecular, and epidemiological studies of dengue cases during epidemic periods are important to review the proposed criteria for classification of dengue frames of the health ministry, to emphasize the need for surveillance and the need for caution of health professionals in evaluating suspected cases. the symptoms observed in this study are consistent with the protocol of the department of health surveillance, but not all patients had fever and this data was confirmed by epidemiological and molecular evaluation. according to the health ministry, dengue infection ranges from asymptomatic forms to severe cases, but fever has been used together with other symptoms to classify the disease. furthermore, in this present study, rt - pcr identified nine positive cases without fever among the samples collected in 2010. our results corroborate other published article, which reported in their study about higher accuracy of a predictive model for early dengue diagnosis, including clinical and laboratory features better than the current who guidelines for suspected dengue. in the association analysis of hospital admission and bleeding (table 1), the chance of hospital admission was 1.55 (1.13 to 1.97) higher in patients with any kind of bleeding than those without bleeding. regarding admissions for dengue infection, it is important to note that there are criteria recommended by the brazilian ministry of health, as the presence of warning signs, refusal in food intake and liquids, respiratory compromise, chest pain, difficulty breathing, decreased breath sounds or other signs of severity, and platelets < 20.000/ul regardless of hemorrhagic manifestations. of the 334 patients with bleeding, 90% were not hospitalized. in 2010, according to the recommendations of the ministry health, inexpressive bleeding did not require hospitalization which may explain the small number of hospitalized individuals even with bleeding. any bleeding in mucosa was included as a warning sign only after that year. among the 75 (2.4%) individuals who were admitted in the hospital without bleeding, at least 63% presented other warning signs as vomiting, but records about other criteria described above were not assessed in this study and probably justify this clinical practice. in spite of the large number of individuals hospitalized in 2010, 37 cases were classified by the department of health surveillance of divinopolis as dengue with complications, with two deaths and four cases of dengue hemorrhagic fever. the results found in this study are consistent with the literature showing patients with an average of 5 days of symptoms being admitted more to hospital compared with 3 days of symptoms of those not admitted. moreover, the number of days of symptoms on average observed for those individuals with bleeding was also higher. usually the critical phase can be observed on days 37 of the illness after the febrile phase that is marked mainly by dehydration and high fever. the critical phase can include shock from plasma leakage, severe hemorrhage, and organ impairment [2, 15 ]. therefore, monitoring for warning signs and other clinical parameters is crucial to recognizing progression to the critical phase. according to data released by the department of health surveillance, reported cases of dengue in brazil in 2008 by sex are higher in women than in men. this fact is also confirmed in this study and is easily understood since the vector is commonly residential. however, when evaluating statistically the number of the hospital admission in relation to sex, there was no significant association (p = 0.517). contrastingly, a study reported that male sex was predominant (65.2%) among dengue cases but was not among nondengue patients (47.9%). identification of virus, viral rna, and viral antigen and the detection of an antibody response are preferable for the diagnosis of dengue. if samples are collected after day 5 of illness, reliable serological tests can be performed in a reference laboratory. serological assays may be used to determine the extent of outbreaks. however, during major epidemics most cases are epidemiologically confirmed, since it would be impossible to test all of them in a reference laboratory. in this study, this fact accounts for the low percentage at 23% of serological tests performed in 2010. in 2010, to identify the serotype of virus circulating in the municipality, molecular diagnosis was performed for 82 patients with suspected dengue. denv-2 and the epidemic of 2010 can be due to the introduction of denv-2, which had not circulated in the municipality for many years. denv-1 and denv-2 were isolated in samples collected in 2001 in divinopolis, and denv-3 was the major serotype found in the 2002 epidemic in brazil, which resulted in a higher incidence of hemorrhagic cases leading to death. according to the epidemiological surveillance of divinopolis, serological identifications during 2002 confirmed the cocirculation of denv-3 and denv-1. the prevalence rates decreased from 2004 onwards, possibly due to the induction of partial group immunity. the number of reported cases increased gradually from 2004 to 2009 years in which studies identifying viruses were not performed. in 2010, probably with the reintroduction of denv-2, a new epidemic has occurred. in 2011 and 2012, the number of cases has decreased sharply with 35 and 27 confirmed cases, respectively. denv-3 was identified in 2011 and denv-1 in 2012 in only positive samples by rt - pcr. in 2013, 1,476,917 suspected dengue cases were reported in brazil. this number is almost three times higher than the same period in the last year. the increase in the number of cases is primarily due to proliferation of denv-4 ; a new strain of the virus reached a population still immunologically unprotected which led to an epidemic. in this same year, the largest epidemic in the history of the divinopolis took place with 6,015 cases notified and denv-1 and denv-4 detected in samples analyzed by molecular test. denv-1 was isolated in divinopolis 12 years ago and in only a positive sample in 2012, and denv-4 was isolated for the first time in 2013, which could explain the epidemic in the municipality. molecular diagnosis allowed the detection of denv in samples with only one and two days of symptoms. the serological test requires the production of antibodies, which takes about seven days of symptoms. the sensitivity of molecular diagnosis falls as the days of symptoms increase because the antibodies begin to act against the virus and there is a decrease in viral load. this relationship was also observed in this present study where one patient had a negative serology but a positive molecular diagnosis. this patient had two days of symptoms and most likely serological testing was not done at the right time accounting for the discordant results. the discordant results may be due to the viral load reduction as the days of symptoms increase and the bias that may be incorporated by reports that do not correspond to the days of exact symptoms of the patient. the results of this study confirm the need to review the criteria proposed by the health ministry to classify cases of dengue since fever is not present in all of cases and is classified as the first manifestation of the infection. the health professionals should be cautious in evaluating suspected cases since the presentation clinic of the disease is nonspecific. the typing viral in these last years in divinopolis reflects what takes place in the rest of the country being the denv-4, for the first time, confirmed in clinical samples from 2013 in the municipality. future sequencing and phylogenetic analysis of the rt - pcr amplified virus will be performed in positive samples that were found in divinopolis, mainly to increase the knowledge about the denv-4 once a distinct genotype was described recently in brazil, increasing the potential of outbreaks in the country.	this study aims to perform the first molecular and clinical - epidemiological analysis of dengue cases in divinopolis, mg, brazil. data from 4,110 cases of dengue were accessed and 190 clinical samples were collected for molecular analyses. in this study, 2.7% of the men and 3.0% of the women were admitted to hospital. there was no association between gender and hospital admission. the symptoms observed in this study are according to the health ministry, but fever was present in 82.2% and not in 100% of cases. the chance of hospital admission was 1.55 higher in patients with any kind of bleeding (334) and 2.4% of individuals without bleeding were also hospitalized due to other warning signs. in the molecular analyses, 23% of the samples were positive for denv. denv-2 and denv-3 were identified in 2010, denv-3 in 2011, denv-1 in 2012, and denv-1 and denv-4 in 2013. denv detection was possible in samples with only one day of symptoms. this first report of dengue data in divinpolis provided more insight into the viral types and effects of disease in the city, confirming the need for caution in assessing cases of suspected dengue and for revision of the criteria proposed by the health ministry to classify cases of the disease.
initially, a 28-year - old man had visited our hospital complaining of poor vision and a white pupil in his right eye in august, 2001. he had been diagnosed as having a hypermature cataract, presumed to be congenital, and an exotropia of 40 prism diopters in his right eye. b - scan ultrasonography showed a flat retina without vitreous floaters in the right eye. uncomplicated irrigation and aspiration without intraocular lens implantation and strabismus surgery had been performed on his right eye in september and october, 2001, respectively. the postoperative best - corrected visual acuity (bcva) was 0.02 in the right eye because of profound amblyopia and was 1.0 in the left eye. postoperatively, the anterior segment was clear and the retina was flat with a long axial length of 30.45 mm in the right eye. the subject was lost to follow - up for eight years, and revisited our clinic presenting with a two - month history of a painful, blind right eye in august, 2009. the bcva was no light perception in the right eye and 1.0 in the left eye. ophthalmologic examination revealed numerous polychromatic crystals in the anterior chamber and vitreous cavity, unilaterally elevated iop (39 mmhg), mild rubeosis iridis, and surgical aphakia in his right eye. a huge quantity of crystals was floating in the anterior chamber according to eye movements, and some of the crystals were adhered to the iris surface or sedimented at the inferior angle, resembling a pseudohypopyon (fig., the patient 's trabecular meshwork was obscured by dense precipitates of crystals all over the angle and scattered peripheral anterior synechiae (fig. a mild rubeosis iridis was evident near the pupil margin, but the neovascularization of the anterior chamber angle could not be clearly confirmed due to the presence of so many crystals near the angle. a funnel - shaped total rd was noted on the b - scan ultrasonograph (fig. our diagnoses were as follows : 1) cholesterosis bulbi accompanied by long - standing rd and 2) presumed neovascular glaucoma or secondary glaucoma associated with trabecular obstruction by crystals. anti - glaucoma medication failed to lower the iop to a comfortable level in the patient 's right eye. pars plana vitrectomy and anterior chamber irrigation were performed to remove abundant crystal clumps, in conjunction with intravitreal injection of bevacizumab (avastin, 2.5 mg / 0.1 ml) to diminish the neovascularization of the iris on the right eye. during the pars plana vitrectomy although there was total rd, we could not find any retinal breaks due to the hazy media and proliferative vitreoretinopathy changes in the retina. despite our efforts, we could not remove the cholesterol crystals completely due to a continuous influx of the crystals. microscopic analysis of the unprocessed aqueous humor aspirate disclosed transparent rhomboidal crystals with notched corners that are typical of cholesterol (fig. a week after the surgery, the iris neovascularization started to regress, but the amount of crystals in the anterior chamber did not decrease markedly as compared with the preoperative status, due to the continuous influx of cholesterol crystals from the vitreous cavity (fig. the iop was still as high as 40 mmhg despite maximal medical treatment. a month after the surgery, the rubeosis iridis had completely disappeared and the iop had decreased to normal range so that the patient could discontinue the administration of all of the iop - lowering agents in the right eye. during the four months post - operative, the iop in the right eye has been maintained at an adequate level. intraocular cholesterosis is a rare phenomenon that can occur in eyes that have undergone severe trauma, intraocular hemorrhage, severe inflammation, or chronic retinal detachment. forsius reported on a typical microscopic photograph of cholesterol crystals in the aqueous humor of patients with cholesterosis bulbi. also, a microscopic and chemical study by kumar suggested that the crystals in the anterior chamber were cholesterol. andrews. confirmed that the crystalline material observed in cases of cholesterosis bulbi was indeed cholesterol by thin - layer chromatography. the concentration of cholesterol in normal vitreous was reported to be much lower than that in normal blood. pathological changes in the blood - aqueous barrier may allow cholesterol to enter the vitreous or the anterior chamber, or it may enter via intraocular hemorrhages. kennedy suggested that anterior chamber cholesterosis may develop in eyes with chronic retinal detachments in the absence of detectable intraocular hemorrhage. in such cases, it is likely that the crystals diffuse into the anterior chamber through retinal breaks from the subretinal fluid, which is rich in cholesterol. in the same manner, coats ' disease with exudative retinal detachment can also be a cause of anterior chamber cholesterosis. finally, phacolysis is reported to be another source of the anterior chamber cholesterol crystals. hubbersty and gourlay described three cases of the anterior chamber crystals with lenticular origins that were associated with hypermature cataracts and lens capsular dehiscence. yu. first reported a 16-year - old male patient who had anterior chamber cholesterosis in the high myopic and exotropic eye with a visual acuity of 0.1. a 25-year - old male patient, who had experienced a penetrating injury by a piece of metallic foreign body at the age of 13, underwent an intraocular foreign body removal and a lens extraction due to traumatic cataract ; and a 21-year - old female patient had a long - standing cataract in the affected eye. the latter two patients had bcvas of no light perception, and they had no evidence of retinal detachment. in all three cases, iops were normal and the typical cholesterol crystals in the aqueous aspirates were confirmed by wet field light microscopy. among the korean cases, our patient is the only one that had cholesterosis bulbi following long - standing rd, which accompanied an elevated iop. it is not certain which factors contributed to iop elevation in our patient ; the abundant cholesterol crystals that settled down at the anterior chamber angle could have caused an inflammatory reaction and disturbed the aqueous outflow, or the concurrent neovascularization of the iris and probably the angle by long - standing rd might have raised the iop. after the pars plana vitrectomy, anterior chamber irrigation, and intravitreal injection of bevacizumab were performed in the affected eye, abundant crystals still existed in the anterior chamber, although their quantity was reduced as compared with the preoperative status. therefore, we could not confirm the angle status or the regression of angle neovascularization by gonioscopy. however, iris neovascularization regressed completely, and iop returned to the normal range. this suggests that the predominant mechanism responsible for the acute rise of iop was the obstruction of the trabecular outflow by new vessels. historically, enucleation was mainly performed on patients with cholesterosis bulbi due to the absence of effective treatment, or out of concern for sympathetic inflammation of the other eye. reported a case that had uncontrolled secondary open - angle glaucoma from the anterior chamber cholesterosis associated with coats ' disease. they suggested that the removal of the cholesterol crystals and treatment of the anomalous vessels by pars plana vitrectomy and endolaser photocoagulation may be sufficient to control the iop. our case showed that the treatment of the new vessels of the iris and anterior chamber angle and possibly abnormal retinal vessels by the injection of bevacizumab, which is a well - known antagonist of vascular endothelial growth factor, was useful for the management of secondary iop elevation caused by new vessels. reducing the amount of crystal clumps in the anterior chamber by pars plana vitrectomy combined with pars plana vitrectomy and anterior chamber irrigation, may be a viable alternative for enucleation in the treatment of cholesterosis bulbi accompanied by long - standing rd and presumed neovascular glaucoma, although a longer follow - up period will be required.	we report on a case of cholesterosis bulbi concurrent with secondary glaucoma. a 36-year - old man, with a history of long - standing retinal detachment in his right eye after the irrigation and aspiration of a congenital cataract, presented with a clinical picture of elevated intraocular pressure and ocular pain. upon slit - lamp examination, we found a ciliary injection and a pseudohypopyon of polychromatic crystals. gonioscopic examination revealed a large amount of crystals deposited on the trabecular meshwork and mild rubeosis iridis, but the neovascularization of the angle could not be clearly confirmed due to the presence of so many crystals. pars plana vitrectomy was performed to remove clusters of crystals and bevacizumab was injected intravitreally to treat iris neovascularization. aqueous aspirate was examined by light microscopy and the typical highly refringent cholesterol crystals were identified. intraocular pressure returned to a normal level after the bevacizumab injection, although severe cholesterosis was still evident in the anterior chamber. to our knowledge, this would be the first korean case of cholesterosis bulbi combined with chronic retinal detachment and presumed neovascular glaucoma, which was treated by pars plana vitrectomy and intravitreal bevacizumab injection.
the clinical nurse consultant (cnc) role was established in new south wales (nsw), australia, in the late 1980s [1, 2 ]. it is equivalent to the clinical nurse specialist role in the united states of america [1, 3 ] and united kingdom. heals states that part of the cncs role is to improve clinical practice, facilitate change, disseminate evidence - based practice, and improve communication in and beyond the health team. there has been continued confusion about the cnc role [1, 2, 6 ]. in 2005, nsw health reaffirmed the five domains of practice for cncs in nsw as clinical service and consultancy, clinical leadership, research, education, and clinical service planning and management. cncs are required to dedicate their time evenly to each of these domains, however, a study by o'baugh. found that within the research domain less than 60% of cncs surveyed played a significant part in the development of clinical research. this trend was confirmed in a study in victoria, australia, by bloomer and cross where a similarly titled role existed. in both studies, the cncs cited lack of support and workload as reasons for not being able to enact all domains, (in particular, leadership and research), as health service management tends to place a greater emphasis on the clinical service and consultancy domains. this paper reports on a team and mentoring approach which was implemented by the authors, in 2012, to assist a group of generalist paediatric cncs practising in nsw to comply with the demands of the research domain. the first step in this research capacity building was to determine the research priorities of the group. a nursing research culture may involve an organisation constructing an environment that enables and supports creative work to generate new knowledge that provides researchers with opportunities to interact and grow. an enabling research culture is essential to building research capacity (ability to plan and conduct research) in nursing. this research culture is characterised by research productivity, positive collegial relationships, inclusiveness, noncompetiveness, and effective research processes and training. nurses need to develop their research capacity, that is their ability (or skill level) to undertake research projects [811 ]. the fundamental motivation for this capacity building is to optimise research performance with high - quality outcomes in the academic and clinical arenas [911 ]. an enabling research culture has, at its centre positive collegial relationships, productivity, inclusiveness, and effective research processes and training [8, 12, 13 ]. one step in establishing collegial relationships with common goals is for a group to determine their research priorities. the setting of research priorities is an established way to provide research direction for health services and groups [14, 15 ]. this may aid the development of cross - organisational and professional collaboration (depending on who the members of the group are) and ultimately may aid in establishing research culture. further, setting research priorities allows nurses to explore important issues in an era when the research dollar is shrinking [17, 18 ] and enables a direct link between nursing research and the development of healthcare practice. when setting research priorities not only do nurses take an interest in future research, but also their commitment will initiate knowledge development within clinical practice and hopefully fuel an enabling research culture among clinicians. another essential element for developing a research culture and producing research outputs is developing the capacity of the nurses who need to conduct the research [19, 20 ]. this capacity building can be initiated by the nurses becoming informed and keen consumers of research. unless nurses ' research capacity is directed and translated by nurse specialists such as cncs to build patient care, there will be a significant gap between knowledge generation and usage. this aspect of building research capacity will assist nurses to implement their research findings. systematic reviews of the literature are an essential part of this development so that policy is based on strong evidence [23, 24 ]. developing research capacity can be accomplished in a number of ways including formal education through postgraduate degrees in nursing, in - house informal research education programs, and involvement with successful research teams [12, 25, 26 ]. mentoring is considered to be an essential aspect of building the research capacity of clinical nurses [27, 28 ]. this mentoring involves a senior experienced professional working with less advanced nurses to aid them in developing research skills and knowledge. byrne and keefe conducted a literature review of medline and cinal databases between 1990 and 2001, and they found that a mentor working with a group of nurses to set research priorities and objectives for a given nursing setting was an optimal method to produce scholarly research. research priority setting in paediatric nursing, including specialties, has been a focus of studies since the 1990s [15, 3033 ]. in america, a delphi technique has been used by both schmidt., and broome. to determine the research priorities for paediatric nurses. the schmidt study identified the top five priorities as analgesic drip weaning, central line dressings, analgesic dosing, procedural pain, growth and development knowledge. study identified their top six priorities as interventions to prevent repeated child abuse, efficacy and quality of paediatric home care, postoperative pain relief, educating for effective parenting skills, and strategies to manage pain in infants. wilson. conducted a study in western australia in 2005 - 2006 using a randomised selection of registered nurses at a paediatric referral hospital. using a delphi technique their top five priorities were reducing medication errors, impact of pain assessment on pain management, exploring new health promotion strategies, impact on family and support required for children needing long - term care, school and antenatal health education programs for children and parents, and reasons for parent noncompliance with treatment. as part of a research mentoring program for paediatric cncs across nsw, it was decided that the group should determine their research priorities and establish a relationship with the team. the cncs felt that through conducting clinical research they would raise their profile in the paediatric health community and in so doing, improve child and adolescent health care in the state and internationally. this paper focuses on the process of establishing research priorities for the group and, in so doing developing a research relationship with each other and a mentor, who was a member of the team. the use of consensus methods, such as delphi surveys or nominal group techniques (ngt), is common when developing research priorities to guide the commissioning of health research [1517, 30, 31, 34, 35 ]. this study utilised a quantitative ngt [3638 ] with a group of paediatric cncs. an ngt provides an orderly procedure for obtaining relevant and reliable information from a group of experts within a focus group setting or a small group meeting. ngt is a method which promotes creative and meaningful interpersonal disclosures from the participants by gathering equally weighted responses that can offer valid representations of the group 's views. the collaborative nature of ngt increases the likelihood that the group will work together on problem identification, generate research questions, and develop solutions to change and enhance nursing practice and policy. the monthly meetings held by the cncs provided a forum in order to conduct the ngt. the study was conducted in a meeting room of the neonatal emergency transport service (nets) in sydney, nsw, australia. the population of children under 15 years of age was 1.36 million. in the nsw public health service and within the structure of the nsw child health networks (chn) there are a total of 23 cncs providing generalist paediatric nursing care, across urban, rural, and remote areas outside the two designated children 's tertiary hospitals. the tertiary hospital paediatric cncs were excluded as they are specialists in their clinical area and were not members of the nsw chn paediatric cnc group which the research team was invited to attend. the researchers conducted a two - hour workshop on setting research priorities using a ngt. a total of sixteen cncs attended the meeting and their demographic characteristics were collected (see table 1). a workshop was conducted using a nominal group technique (ngt) during a meeting of the nsw chn paediatric cnc group on 21st march 2012. the meeting participants were asked to think about the research question : what are the most important research priorities for paediatric nurses in nsw ? phase 1the researchers gave a forty - three item list of research topics for paediatric nurses identified from the literature to the ngt [15, 35 ]. this list was derived from a combination of the resulting top research priorities for paediatric nurses as identified by moreno - casbas., and wilson. each research topic was randomly assigned a number between 1 and 43 to determine their order on the combined list used in this study. the researchers gave a forty - three item list of research topics for paediatric nurses identified from the literature to the ngt [15, 35 ]. this list was derived from a combination of the resulting top research priorities for paediatric nurses as identified by moreno - casbas., and wilson. each research topic was randomly assigned a number between 1 and 43 to determine their order on the combined list used in this study. the participants were divided into small groups of four and asked to read the list and add any missing or additional items. a forty minute period was allowed for them to discuss the pros and cons of each item including their additions. the small groups then came back together, and using a round robin, each participant in turn stated one or more additional items. the items were written on a white board and discussion by the group was limited to clarification only. using a laptop, the additional items listed on the board were merged, simplified and organised with the agreement of the group (removing any overlap and duplication). the additional items were added to a master list containing the original 43 items. phase 2a new typed list of research topics was provided to each participant and they were asked to rank each item using the point scale provided (not important (n / a) = 0, important = 1 or most importance = 2). a new typed list of research topics was provided to each participant and they were asked to rank each item using the point scale provided (not important (n / a) = 0, important = 1 or most importance = 2). phase 3the results of phase 2 were provided to the participants at the meeting and a final consensus of agreement was achieved. before leaving the meeting, the participants were asked to complete a demographic survey with questions on gender, age, position title and highest paediatric / academic qualifications. the advantage of the method was that the initial data analysis was achieved at the same time as data collection and the participants had a sense of ownership in the results. following the meeting the responses were entered into excel and further analysed. the results of phase 2 were provided to the participants at the meeting and a final consensus of agreement was achieved. before leaving the meeting, the participants were asked to complete a demographic survey with questions on gender, age, position title and highest paediatric / academic qualifications. the advantage of the method was that the initial data analysis was achieved at the same time as data collection and the participants had a sense of ownership in the results. following the meeting the responses were entered into excel and further analysed. phase 4the mentor revisited the nsw chns paediatric cnc group at their next meeting in may 2012 to go over the findings from the ngt and to revisit the priorities with the group. the mentor explored with the members the research methods that could be utilised to commence research projects based on the ngt findings. the mentor revisited the nsw chns paediatric cnc group at their next meeting in may 2012 to go over the findings from the ngt and to revisit the priorities with the group. the mentor explored with the members the research methods that could be utilised to commence research projects based on the ngt findings. the university of western sydney human research ethic committee was consulted and advised that according to the national health research medical council guidelines formal ethics approval was not required. the committee advised that the researchers needed to have their attendance and activities recorded in the meeting minutes as the cnc group invited the researchers to conduct the research priority setting exercise. the workshop was listed on the meeting agenda and all paediatric nurses attending the meeting consented to participate in the workshop. the results from the nominal group discussion were recorded in the meeting minutes, and a copy was provided to the researchers with permission to publish the results. sixteen generalist paediatric cncs were members of the nsw chn paediatric cnc group. seven (46%) cncs had a hospital certificate, four (25%) had a bachelor of nursing, and one (6.3%) had a bachelor of nursing honours degree, as an initial nursing qualification. the participants ranged in age from 30 to 58 years, with a mean age of 44 years. fourteen of the cncs had a paediatric qualification, with nine (56.3%) at masters level and five (31.3%) at graduate certificate level. nineteen research priorities were added by the cncs to the original master list of 43 items. table 2 displays the top 30 research topics on the master list (62 items) which had a participant mean of 1 (important) or greater. they are listed in descending order of importance to the group according to the mean score. included in this list were 15 (50%) of the additional research topics added by the ngt participants. of the 30 research topics, five items (11, 46, 47, 15, and 60) were related to pain either its assessment or effectiveness of actions to manage it. item 11, encompassing a broad view of issues related to pain, was ranked as the top research priority. four items (55, 56, 59, and 32) were related to issues in the emergency department (ed). three of these were added by the participants to the master list and included reasons why children re - present to ed (55), parent expectations of the ed (56), and when observations are done in ed (59) ? item 32, explore families ' reasons for presenting to the emergency department, came from the wilson.. to determine the focus of the priority, the research topics were grouped by the researchers using wilson. 's categories : (1) research topics of greatest value to patients, (2) research topics of greatest value to the families, and (3) research topics that would most facilitate health in children and young people and reduced hospitalisation (see table 2). the grouped items presented in table 2 were emailed to the cnc group and confirmed at their next meeting. fifteen priorities have greatest value for the patient (i.e., clinical issues, psychosocial issues, safety issues, quality care, and role competence issues), the top three issues ranked by the ngt were 11, 46, and 47, and they all related to pain. two priorities have the greatest value for the family. these were parent expectations of the ed and paediatric units (56) and assessed parent understanding and usefulness of information provided (printed and other modes) regarding child 's care and discharge, including long - term outcomes (22). twelve priorities would most facilitate the health of the children and reduce hospital admission (clinical issues, health education issues, and models of care). the top three ranked by the nurses were the reasons children re - present at ed (55), non - compliance of clinical practice guidelines (cpg) by gps and visiting medical officers (53), and exploring the families ' reasons for presenting to ed (32). fifteen priorities have greatest value for the patient (i.e., clinical issues, psychosocial issues, safety issues, quality care, and role competence issues), the top three issues ranked by the ngt were 11, 46, and 47, and they all related to pain. two priorities have the greatest value for the family. these were parent expectations of the ed and paediatric units (56) and assessed parent understanding and usefulness of information provided (printed and other modes) regarding child 's care and discharge, including long - term outcomes (22). twelve priorities would most facilitate the health of the children and reduce hospital admission (clinical issues, health education issues, and models of care). the top three ranked by the nurses were the reasons children re - present at ed (55), non - compliance of clinical practice guidelines (cpg) by gps and visiting medical officers (53), and exploring the families ' reasons for presenting to ed (32). at the next meeting of the nsw chns paediatric cnc group in may 2012 research methods that could be utilised to commence research projects based on the ngt findings were discussed, including conducting systematic reviews of the literature. the cncs decided to pursue the two priorities : reasons for children re - presenting to ed and a comparison of the use of high - flow and low - flow nasal prongs in children with bronchiolitis. while pain - related topics were the top priorities, the group felt they wanted to pursue the two nominated topics as they were currently an issue in their fields of practice. another priority highlighted was medical clinician adherence to clinical standard guidelines, however, this was already being pursued by one of the cncs in a local multidisciplinary project. the group established two subgroups of four to five nurses to conduct systematic reviews of the literature on both topics. the mentor was available by phone or face - to - face meetings which were followed up by one of the subgroups. eight months after the research priority setting exercise, group one continued with their literature review on readmission to emergency, while group two, examining high and low oxygen, prepared an ethics application under the guidance of the mentor. the priorities set by the cnc group reflect results from other studies [15, 35 ] with pain management being the top priority research area. ongoing poor management of pain continues to be an issue. the top priority identified by wilson. was related to medication errors but was ranked at 10 by the nurses in the current study, therefore, reflecting a need to examine research in this area. it is clear that the research priorities considered of greatest value to improving practice were those related to patient care and reduction of readmissions to hospital according to wilson one issue not addressed in this process of ranking research needs in paediatrics is what consumers need or want. as suggested by gillies, multistakeholder groups including the children, parents, and practitioners need to be used in setting research priorities. this project has started the journey to engage cncs in the research process and to establish relationships which can enable their further development in the skills and knowledge required for research in paediatric nursing. the group has worked together with a common focus, thus, the beginnings of a research culture have been established. in establishing these groups of nurse researchers, the nurses can become motivated and supported in fulfilling the research domain of their cnc role. kajermo. argue that cncs are ideally placed to promote research - based nursing practice if prepared and supported. cncs, however, need support and funds from health services management to achieve this. as suggested by corchon. it could be useful to have management involved with specialist nurses in setting research priorities. this will integrate management and clinical priorities and may assist the motivation for management to support clinical research the cncs need to be educated to the masters level and as seen in our sample only 56.3%. (n = 9) had completed education to this level, although it is recommended by nsw health. management needs to find ways to support the cncs, in relation to time release and financial support, for example, paid study leave. while in - house research education continues in hospitals across nsw, when a speciality is so small it makes it difficult for specialities to collaborate. working together on the systematic review and establishing collaborative projects across organisational and geographical boundaries, with ongoing mentoring, will help develop and establish this research culture. as suggested by bishop and freshwater participating in a research culture however, the research culture will not flourish if the organisations where the nurses are employed do not value research and provide an environment where it is encouraged [47, 48 ]. as indicated in the findings of this study the cncs held similar priorities as paediatric nurses in other studies. however, the process has opened new paths to mentor these cncs and develop research collaborations across organisational and geographical boundaries. as shown by gagliardi., mentoring can facilitate the development of research capacity in nurses and encourage knowledge transfer into clinical practice. this capacity is stressed in the study by mannix. as necessary for nurses to become clinical scholars which should be the aim of cncs. leadership development is essential for cncs as they appear to not address this domain, as well as they could [1, 6 ]. initiating knowledge transfer to practice and especially to policy development in order to enhance patient care is one bridge to this development. however, resources and personal support from management are essential for any change to occur. a limitation of this study was that only one group of expert paediatric nurses was used for the ngt, however, 16 is an ideal number of participants for an ngt. the benefit of this one - off process is that a substantial amount of information can be gathered in a relatively short time. this study has opened the way for a group of cncs in paediatric nursing to combine their research capacity and influence clinical knowledge. as recommended by mccance. the first step in research capacity building is encouraging nurses to value research and legitimise it as an essential activity to improve their professional practice.. it could be useful to survey cncs in a tertiary paediatric hospital in nsw using the research priorities developed in this study. this study provides a model others can use to encourage research culture development in groups of cncs.	this paper reports a research capacity building exercise with a group of cncs practicing in the speciality of paediatrics in new south wales (nsw), australia. it explores the first step in building a research culture, through identifying the research priorities of members of the nsw child health networks paediatric clinical nurse consultant group, and this forms the major focus of this paper. a nominal group technique (ngt) was utilised with sixteen members to identify research topics for investigation which were considered a priority for improving children 's health care. the group reviewed and prioritised 43 research topics in children 's health which were identified in the literature. as a result of conducting this research prioritisation exercise, the group chose two research topics to investigate : reasons for children representing to the emergency department and a comparison of the use of high - flow and low - flow nasal prongs in children with bronchiolitis. the research team will continue to mentor the nurses throughout their research projects which resulted from the ngt. one bridge to leadership development in enhancing patient care is translating knowledge to practice and policy development. this study leads the way for a group of cncs in paediatric nursing to combine their research capacity and influence clinical knowledge.
calcifying epithelial odontogenic tumor (ceot) (also known as pindborg tumor), which was first designated as a distinct disease entity by pindborg, is an uncommon benign odontogenic lesion that accounts for less than 1% of all odontogenic tumors. it most often occurs in the posterior mandible and is most frequently found in patients between 30 and 50 years of age, with no sex predilection. in addition to the intraosseous lesion, a number of extraosseous counterparts of cceot have also been documented. the classical histopathological characteristics of ceot comprise sheets and islands of polyhedral eosinophilic epithelial cells with calcifications as well as deposition of an amyloid - like substance ; however, occasionally, focal areas of clear cells can be observed in the clear - cell variant of ceot (ccceot). through a medline search for ccceot in the english - language literature (19672011), 14 cases were found ; however, this unusual lesion still needs continual documentation in order to have more information regarding clinical, microscopic features or behavior, particularly, the potential origins of the clear tumor cells. therefore, the aim of the current report was to describe the clinical, radiographic, and histological findings in a case of mandibular ccceot. the clinical features as well as the potential origins of the clear tumor cells of previously reported cases of intraosseous ccceot are reviewed. a 59-year - old female was referred for evaluation of a painless swelling over the left retromolar area. the patient 's medical history was significant for the diagnosis of hypertension. intraoral examination showed a hard, non - tender 3 cm2 cm mass on the lingual aspect of the left retromolar area up to half of the mandibular ramus. a panoramic radiograph showed a well - defined unilocular radiolucence with a corticated margin extending from the distal root of tooth 38 up to half of the left ramus area, and from the left retromolar area down to the mandibular body, which measured about 3 cm2 cm in diameter (figure 1b). the specimen was sent to the oral pathology department of our institution for histological examination. microscopic examination of the incisional biopsy showed that a large portion of the tumor was arranged in a pseudoglandular pattern consisting of nests of pale, uniform, clear cells with dark - stained nuclei without abnormal mitotic figures and necrosis (figure 2a), whereas some areas were admixed with clusters of polyhedral epithelial cells (figure 2b). the cells were separated by thin bands of connective tissue in areas showing deposits of amorphous eosinophilic material. small foci of calcifications were also noted, but no liesegang rings were observed (figure 2c). staining was negative for periodic acid fast stain (pas) stains with and without diastase digestion (data not shown), as well as mucicarmine stain (data not shown), but positive for congo red stain throughout the intercellular eosinophilic material (figure 3a). with regard to immunohistochemical stainings, the tumor cells were positive for cytokeratin only (figure 3b), and negative for s-100 protein (figure 3c) and smooth muscle actin (figure 3d). the findings for ki-67 were positive in only a small number of scattered cells (figure 3e). similar microscopic findings to the incisional biopsy were observed for the surgical specimen (figure 2d). the postoperative course of the patient was uneventful, and there was no evidence of disease at the 2-year follow - up. the histopathology of ceot, in its classic pattern, comprises sheets of polyhedral epithelial cells with well - defined cell borders and distinct intercellular bridges ; these neoplastic cells may demonstrate pleomorphism, but only rarely typical mitoses. additionally, the other most characteristic findings are the presence of amyloid - like substances and calcified concentric liesegang rings. to date, five histopathologic patterns of ceot have been documented : (i) strands / sheets / islands of polyhedral cells with intracellular bridges ; (ii) a cribriform arrangement with many spaces containing an eosinophilic (amyloid - like) substance ; (iii) densely - populated neoplastic cells with interspersed multinucleated giant cells ; (iv) nests of epithelial cells similar to neoplasm of the salivary gland ; and (v) prominent clear - cell arranged in a pseudoglandular manner. the last pattern is referred to as the clear - cell variant of ceot, and the histopathological findings of the current case were consistent with this pattern, showing abundant clear cells arranged in a pseudoglandular pattern containing an amyloid - like material. in the current case, a relatively high proportion of the clear tumor cell components were observed. the diagnosis of ccceot in the present case was reached according to the positivity of cytokeratin staining, the absence of pas - positive staining, the presence of congo red - positive material between tumor islands (amyloid - like material) and the absence of mitotic figures. malignance of salivary gland origin was ruled out by the absence of actin and s-100 expression, and clear - cell odontogenic carcinoma was ruled out by the lack of overt cellular atypia, a well - circumscribed lesion, and the presence of amyloid - like material as well as the very low ki-67 labeling index. additionally, a lack of mitotic figures and the generally good circumscription of the lesion are not characteristics of metastatic diseases of any origin. it should be noted that clear cells may also occur in other epithelial odontogenic leisons such as ameloblastoma, and calcifying odontogenic cyst. it has been demonstrated that the clear cells of the ameloblastoma was clearly of odontogenic epithelial origin. moreover, it has also been of opinion that the clear cells of the calcifying odontogenic cyst are possibly odontogenic epithelial cells, which have undergone aberrant degeneration. for ccceot, it has been claimed that the clear cells represent a degenerative process, whereas another suggestion indicated that the clear tumor cells represent a feature of cytodifferentiation rather than the degenerative phenomenon. in order to obtain some valuable information, we hereby summarize the reported histochemical, immnohistochemical, and electron microscopic findings (including the current case) for the potential origins of clear tumor cells in ccceot in table 1. as observed from table 1, in two cases, the clear tumor cells have been proved to be langerhans cells, as evidenced by the presence of birbeck 's granules using electron microscopy. indeed, some authors have regarded ceot containing langerhans cells as non - calcifying ceot (pindborg tumor) with langerhans cells. on the other hand, it has been shown that the clear cells of ccceot contain glycogen in four cases by pas stain and in two cases by electron microscopy. an odontogenic epithelial origin has also been demonstrated for the clear tumor cells in three cases by positive immunohistochemical stainings (chiefly cytokeratin) together with negative stainings for pas and mucicarmine stains and the present case, and in one case by electron microscopy. both pas and cytokeratin positivity have been simultaneously reported in one case, in which transition between clear cells and ceot tumor cells was evident throughout the tumor tissues. taken together, it may be speculated that the clear - cell change might be derived from the cytokeratin - positive ceot tumor cells ; these tumor cells initially showed a congregation of initially pas - positive substance, but when the clear - cell phenomenon enhances, the pas positivity is consistently absent. consequently, the potential origin and function of the clear tumor cells in ccceot would be more complex as compared to other epithelial odontogenic lesions, and the clear - cells in ccceot may in fact, represent a more diversity of cell origins encompassing langerhans cells, degenerated cells containing glycogen granules, and cells of overt odontogenic epithelial origin. additionally, one may also question whether the presence of clear cells is of clinical relevance. in 1994, hicks. suggested that the existence of clear tumor cells in ccceot may imply a more aggressive performance. however, other authors considered that too few cases of ccceot have been described to date to attain a confirmative conclusion concerning the impact of the clear - cell population on the biologic activity of ccceot (ref. 2). today, the choice of surgical management of the ceot depends on the site, size, and amount of bone destruction of the lesion. for mandiblular lesions, the suggested surgical approach is enucleation with vigorous curettage ; however, for lesions with more advanced bone infiltration, resection of the tumor should be considered. the treatment of choice in the current mandibular case is total excision of the tumor. on the other hand, hemimaxillectomy is suggested as the treatment of choice for lesions of the maxilla, because maxilla tumors could easily intrude on vital structures. finally, anavi. after almost ten years, as shown in table 1, we have updated the data of anavi. by adding three more cases, including the present case. with only three additional cases, it is unsurprising to observe that the main clinical data shown in table 1 are largely compatible with the report of anavi. we report an uncommon case of ccceot arising in the mandible, and additionally, a review of pertinent literature as well as discussion of the potential origins of the clear tumor cells have been presented.	we present an uncommon case (female patient aged 59 years) of the clear - cell variant of calcifying epithelial odontogenic tumor (ceot) (also known as pindborg tumor) in the mandible. the clinical characteristics and probable origins of the clear tumor cells of previously reported cases of clear - cell variant of intraosseous ceot are also summarized and discussed.
substance abuse is a common cause of visit to the hospital and cocaine is among the most commonly encountered drug. the abdominal complications related to cocaine are less often recognized as they are less often considered. however a patient with cocaine use presenting with acute abdominal pain could be harboring a life - threatening emergency that warrants prompt recognition and treatment. the important differentials that need to be considered are bowel ischemia, bowel perforation and splenic hematomas or rupture. we discuss a case of spontaneous splenic rupture causing hemoperitoneum in a patient with cocaine use. a 58-year - old male with history of polysubstance abuse and hiv infection on antiretroviral therapy presented to the emergency department with complaints of shortness of breath and coughing for 2 days. he also reported a vague pain in the left chest and upper abdomen that was aggravated by deep inspiration. though he initially denied recent substance use, after specific questioning he admitted to smoking crack cocaine two days prior. on examination, he had sinus tachycardia with a heart rate of 115 per min, a respiratory rate of 22 per min and blood pressure of 115/66 mm hg. chest examination revealed equal breath sounds and he exhibited vague tenderness in the epigastric region and left upper quadrant of the abdomen. laboratory testing showed a total leukocyte count of 16,200 with 88% neutrophils, hemoglobin of 13.9 g / dl, creatinine of 4.1 mg / dl, lactate of 2.2 mmol / l and total ck of 41,000 u / l. he was diagnosed with sepsis from pneumonia and acute kidney injury from rhabdomyolysis, likely due to cocaine use. he was given broad - spectrum antibiotics, started on aggressive intravenous hydration and transferred to the medical step - down unit. in a few hours after his arrival, his abdominal pain acutely worsened and was also radiating to his back. on repeat examination, the abdomen was tender diffusely, with diffuse guarding and rigidity. sinus tachycardia persisted and there was a drop in his blood pressures to 86/62 mm hg. there was a drop in the hemoglobin to 10.7 g / dl, leukocyte count increased to 25,400 and lactate to 7.8 mmol / l. his clinical condition continued to deteriorate, as his blood pressures were further dropping and not responding to fluid resuscitation. his respirations were getting irregular, the pain in his abdomen was worsening and he was developing confusion. given the clinical and laboratory picture, a possibility of bleeding in the abdomen was considered. following initial stabilization he underwent a computerized tomography (ct) of the abdomen and pelvis that revealed a perisplenic hemorrhage, moderate hemoperitoneum in the abdomen tracking down to the pelvis (figure 1) and ill - defined hypodense splenic lesions (figure 2). following this, he was taken for splenic angiography that demonstrated multiple foci of active extravasation in the mid and lower region of the spleen (figure 3). successful occlusion of the distal splenic artery was carried out with gelfoam and embolization coils, with follow up angiography not showing any further extravasation (figure 4). repeat ct imaging in four days showed decrease in hemoperitoneum and residual splenic lesions, which were most likely hemorrhages. cocaine is an addictive stimulant drug extract from the leaves of erythroxylon coca plant native to south america. one is cocaine hydrochloride that is in powder or granule form and can be taken orally, intravenously or intranasally (snorting). the other form, widely known as crack cocaine or the free - base cocaine is heat - stable and hence it can be smoked. crack cocaine is the most addictive formulation as it has a rapid onset of euphoria (3 - 5 s) and an easy administration route. cocaine is a triple reuptake inhibitor that acts by inhibiting the reuptake of three neurotransmitters - dopamine, norepinephrine and serotonin. blocking the reuptake of dopamine and norepinephrine increases the availability of these transmitters in the synaptic cleft, thereby making cocaine a powerful sympathomimetic agent. cocaine s nervous system stimulant effects and abuse potential are considered primarily secondary to its enhancement of brain dopamine activity, particularly in the mesolimbic dopamine reward pathway. majority of cocaine is metabolized by hydrolysis of its ester bonds in the liver and plasma to benzoylecgonine (be) and ecgonine methylester (eme), which are excreted in the urine along with some minor metabolites. since be, eme and cocaine are the major urinary analytes after cocaine use, they are recommended as screening tests for urine toxicology. cocaine is generally detectable in the urine only for 8 h, be can be detected from 48 to 144 h depending on the assay used. due to its long elimination half - life, eme has the longest detection time for up to 164 h. the adverse effects of cocaine have been described and studied in various organ systems. cardiopulmonary symptoms are the most common reason for cocaine users seeking medical help, with chest pain being the most frequent presentation. cocaine acutely increases heart rate, blood pressure and systemic vascular resistance, mainly by increasing adrenergic activity in the heart. it has been shown to increase heart rate by 30 beats per minute and blood pressure by 20/10 mm of hg. acute cocaine intoxication can present as coronary ischemia (including acute myocardial infarction), elevated blood pressures, dysrhythmias and sudden cardiac death. central nervous system effects include seizures, ischemic and hemorrhagic strokes. in the respiratory system acute pulmonary edema, pulmonary hemorrhage and pneumothorax have all been described, but less commonly. crack lung is an acute pulmonary syndrome of unknown pathogenesis that manifests with fever, hypoxia, hemoptysis and alveolar infiltrates after smoking cocaine. in the kidneys, it can cause acute kidney injury from rhabdomyolysis and contribute to progression of chronic kidney disease, especially in patients with hypertension. the effects of acute cocaine intoxication in the gastro - intestinal system are not as common as the cardiac and nervous system ones, however they are potentially life threatening. the major ones are gastro - duodenal perforations and bowel ischemia, and the others include delayed gastric emptying and peptic ulceration. perforations are more common with crack cocaine and are mostly in the juxtapyloric region and the first part of duodenum. they can present anywhere between an hour to three days after crack cocaine use. hence if the clinical suspicion for perforation is high in a patient with cocaine use, immediate exploration should be considered. a retrospective study observed that most of these gastric perforations are treated with omental patch repair, though anti - ulcer surgery had a slightly lower recurrence rate. the pathogenesis of perforation is vasoconstriction causing tissue ischemia and necrosis, predisposing to perforation. first is the direct vasoconstrictive effect mediated via increased influx of calcium across the vascular endothelium as has been proven in animal studies. the other is vasospasm and poor blood flow in the mucosal vessels due to the sympathomimetic effects of cocaine in the gut vasculature. apart from vasoconstriction, cocaine can cause ischemia by promoting intramural thrombus formation. increased platelet activation and aggregation and elevated concentrations of plasminogen - activator inhibitor contribute to the thrombogenicity of cocaine. intestinal ischemia after cocaine use is also well known and presents with acute abdominal pain with or without bloody stools. induced ischemic colitis, the time interval between drug use and onset of symptoms varies from one hour to two days. some of these cases are self - resolving with conservative management, while others need surgical intervention especially if complicated by perforation or peritonitis. the underlying pathology is vasoconstriction causing ischemia or direct toxic effect of cocaine on the gut mucosa. another intra - abdominal organ that is affected by cocaine but is less commonly recognized is the spleen. there are two case reports that have described splenic infarction following cocaine use in sickle cell trait patients presenting as acute onset abdominal pain., there is another report of autopsy findings in a patient with cocaine abuse that describes multiple splenic infarcts, some with necrosis and abscess formation. homler and colleagues described a patient with cocaine snorting who presented with left upper quadrant pain and was found to have a subcapsular splenic hematoma on ct imaging. azar and colleagues reported a case of atraumatic splenic rupture and hemoperitoneum following intranasal cocaine use where the patient was managed supportively and improved. there is one other report of hemoperitoneum following intravenous cocaine use, in which an exploratory laparotomy did not reveal the source of bleeding. none of the cases had a splenic angiogram performed that would have better demonstrated the bleeding in the spleen. though vasoconstriction and thrombus formation can explain the cases of splenic infarct, there is no clear explanation for the splenic rupture, hematomas or bleeding. cocaine has been shown to transiently reduce splenic volume by approximately 20% in human subjects, which is believed to be from splenic constriction. it is possible that the initial insult on the spleen could be ischemic ; with hemorrhage occurring in the ischemic area later after the vasoconstriction is resolved. despite the evidence that cocaine promotes platelet aggregation, some studies have proven that cocaine inhibits platelet aggregation and interferes with the process of thrombus formation by activated platelets. it has also been shown to induce expression of platelet - derived growth factor in endothelial cells, thereby increasing vascular permeability. hence inhibition of platelet aggregation and increased vascular permeability could also be contributing to the bleeding. another possible mechanism could be splenic arterial or arteriolar rupture from hypertension caused by cocaine. it is unclear whether anatomical factors like a pseudoaneurysm can predispose to splenic hematoma and bleeding. in the case described, the patient may have had a splenic hematoma or ischemia at the time of presentation and the rapid change in clinical status was due to splenic rupture resulting in hemoperitoneum. given the increasing abuse of cocaine and its varied presentations, it is important that providers be aware of the various abdominal complications of the same. it can be a challenging task to elicit the history of drug use and perform a good abdominal examination in these patients. gastroduodenal perforations, bowel ischemia and splenic rupture are all acute and potentially life - threatening conditions that can be associated with cocaine use. splenic rupture in particular can be easily missed unless an astute clinician looks for it. it should be considered in any patient presenting with acute abdominal pain or surgical abdomen without any other obvious cause. a knowledge of these conditions and their association with cocaine can be vital as timely recognition of these emergencies can be life - saving.	cocaine abuse is frequent in patients visiting the emergency department. the knowledge of the cardiovascular complications of cocaine is excellent among physicians. however the awareness regarding its abdominal complications, the most important of which include gastroduodenal perforation, bowel ischemia and splenic rupture is less adequate. we report a 58-year - old with cocaine use who presents with upper abdominal pain and a rapidly worsening clinical status. he was found to have atraumatic splenic rupture causing a hemoperitoneum that was managed by intervention radiology guided splenic artery embolization. splenic hemorrhage and rupture need timely recognition, as they are difficult to diagnose clinically and can be potentially fatal. in the encounter of patients with cocaine use who present with chest or upper abdominal pain, clinicians should consider imaging to look for splenic rupture as it is often masked or overlooked due to the complicated clinical picture.
the optimal care of open, high - velocity, lower limb injury requires surgical skills in debridement, different modes of skeletal stabilization, and in providing appropriate soft tissue cover. when faced with a near epidemic of these injuries in developing countries, existing approaches to this problem may have to be modified. it has been shown that 90% of disability - adjusted life years lost from road traffic injury occurs in developing countries. in india alone, it is expected that there will be three million hospitalizations due to road traffic accidents in 2010. an orthopedic surgeon in the developing world sees at least 20 times the number of gustilo iii b open tibial fractures than his counterpart in developed world. the numbers of open ankle and foot injuries are likely to be exponentially higher in a barefoot population. despite these statistics, trauma care in the developing world tends to follow the developed world model with multiple teams caring for the different facets of open injuries. this segment of society can scarce afford to pay for highly specialized services, which do exist in urban centers. although the results of single - stage reconstruction are good, timely coordination between orthopedic and plastic surgeons is often difficult.[357 ] the question of who does soft tissue cover ? it is clear that the workforce and availability of flap surgeons has become a problem. furthermore, levin hinted that the days of godina will never again be reached and to be more practical. it is even more imperative in developing countries ; with emerging economies and with greater magnitude of open injuries, a different solution is sought. although attempts have been made toward this, there is reluctance by those who influence opinion, to address this problem, as it may be radically different to conventional thinking. we performed these flaps in our attempt to provide a cost - effective solution, keeping in mind the best interests of the patient. the same constraints are likely to be present in many parts of the developing world. in these parts of the world, the paradox remains that despite the increasing number of open injuries, they remain a low priority for resource managers. in this scenario, the orthopedic community is more aware than others of this problem, has more resources, and is also keen to treat these injuries, especially as they often present at untimely hours. this paper intends to promulgate the fact that some orthopedic surgeons have been able to accomplish soft tissue coverage in the lower limb. in our center, soft tissue cover of this magnitude is provided by orthopedic surgeons only in the recent past, though there has been a tradition of involvement in performing soft tissue cover. the senior author undertook a short fellowship in plastic surgery, and his encouragement and teaching has resulted in the other authors performing flap surgeries under supervision initially and then independently. we reviewed our results of the flaps performed for open limb injuries at and below the level of knee. we retrospectively reviewed the results of flaps at and below the level of knee done in the orthopedic department of our 2 200 bed, multispeciality institution in a developing country between january 2005 and december 2006. flap cover was done in 64 patients (62%) for type iiib gustilo and anderson fractures of both bones of leg and in 39 patients (38%) for isolated soft tissue injuries resulting in exposure of bone or vital structures like tendons and neurovascular bundle. in 45 patients (44%), either tibia or fibula was exposed ; in 11 patients (10%), major tendons were exposed ; in 12 patients (12%), either knee or ankle joint was exposed ; and in 35 patients (34%), a combination of the above three was exposed. the flaps done were 51 reverse sural artery, 35 gastrocnemius, 25 local fasciocutaneous, and 7 foot flaps (lateral calcaneal artery - based flap 5, extensor digitorum brevis muscle rotation flap 1, abductor hallucis muscle rotation flap 1). in our series, gastrocnemius flaps were used to cover defects in the knee and proximal third of tibia [figures 1 and 2 ]. we used the gastrocnemius myocutaneous flaps and fasciocutaneous flaps for middle third cover, since soleus is usually traumatized in middle third leg injuries [figures 35 ]. the sural artery flap was the mainstay of cover for the distal leg and foot [figures 68 ]. photograph of patient with open tibial plateau fracture after plating and loss of soft tissue over proximal third of tibia photograph showing exposed plate covered by medial gastrocnemius flap and distally by fasciocutaneous flap photograph of 14-year - old girl with type iiib open fracture of mid leg photograph showing distally based fasciocutaneous flap cover and external fixation of fracture photograph of 14-year - old girl with open ankle and loss of soft tissue of dorsum of foot photograph after debridement, skeletal stabilization, and reverse sural flap cover photograph at follow - up at 2 months twenty - five flaps (21%) had delayed healing [table 1 ]. there was complete necrosis of three reverse sural artery flaps. one patient had bilateral reverse sural artery flaps for bilateral heel pad injury, after a fall from a height of 15 feet. he occasionally has breakdown of heel split skin graft that heals with total contact cast. outcome of flaps with delayed healing another 18-year - old boy presented with open fracture dislocation of talus. he underwent debridement, open reduction of talus, and reverse sural flap cover for the exposed ankle joint. his wound healed after 60 days. at a follow - up of one year, complete necrosis of reverse sural artery flap healed with secondary split skin graft at follow - up after 14 months nine (36%) of the 25 flaps which had necrosis healed without any surgical intervention [table 2 ]. one reverse sural artery flap (4%) required secondary suturing after debridement of the necrosed tip and another (4%) with 20% necrosis was managed with fasciocutaneous flap to cover the necrosed area. there are two options, the traditional approach being bony injuries treated by orthopedic surgeons and soft tissue by plastic surgeons. this approach, requiring excellent cooperation and coordination between two busy surgical specialties in a large institution, is difficult to achieve. in addition, in our situation, because of the absence of protective clothing in patients with these injuries, they invariably arrive with gross contamination. frequently, there is established infection when patients present late. in this situation, masquelet described this as a holistic approach to open fractures rather than subjecting the patient to multiple surgical procedures by different teams at different intervals. as far as we know, few centers in the world have near perfect understanding and coordination of three different disciplines, i.e., orthopedics, plastic surgery, and anesthesia. this is possible only in centers with few specialties and in centers that treat only trauma. the advantage in our set up is that a single surgical team is involved in all stages of treatment, from debridement, skeletal stabilization, to soft tissue cover and for secondary procedures to accelerate fracture healing. the whole treatment is planned at the time of presentation in accident and emergency department, taking into consideration the associated systemic injuries and the general condition of the patient. it is imperative that the orthopedic surgeons, who are involved in emergency debridement and who are already comfortable with limb anatomy, familiarize themselves with performing soft tissue cover also, to optimize treatment. in our region of practice, the orthopedic surgeon bears primary responsibility in treatment of limb injuries and hence it is vital that they be more involved in providing soft tissue cover. though these techniques may be new to an orthopedic surgeon, careful soft tissue handling and biological fixation have become an integral part of the orthopedic surgeon 's work. our results showed that the majority of these flaps, which were mainly in the difficult area of lower leg and foot, healed primarily. the results of this study are comparable with other large studies with the sural artery flap, though the failures are often the nature of the bed and the infected state at arrival rather than the flap itself, making strict comparison between studies difficult. the sural artery flap also remains a good alternative to free flaps in the lower third of the leg. the main limitation of this study is that it does not compare with the earlier results when more than one team of surgeons provided the same treatment. though plastic surgeons will always be involved in extensive reconstructions and free flaps, involvement of orthopedic surgeons in soft tissue cover of open limb injuries helps in improved and timely treatment of patients in developing countries. soft tissue cover of lower limb injuries by a single team involved in bony stabilization and reconstruction using local flaps is an alternate and simpler solution to a difficult problem, especially when resources are limited and cooperation between two different teams is not always possible.	background : optimal care of open, high - velocity, lower limb injury requires surgical skills in debridement, skeletal stabilization, and in providing appropriate soft tissue cover. timely coordination between orthopedic and plastic surgeons, though ideal, is often difficult. in our center, orthopedic surgeons undertake comprehensive treatment of open fractures including soft tissue cover. we reviewed the results of the local flaps of lower limb, done by orthopedic surgeons.materials and methods : we retrospectively reviewed the results of the lower limb flaps done between january 2005 and december 2006. all flaps done at and below the level of knee were included.results:there were 105 patients with 120 flaps during this period. two patients with two flaps were lost to follow - up. the average age was 32 years. sixty - four patients had type iiib gustilo and anderson injuries. thirty - nine patients had isolated soft tissue injuries. the indications for flaps were exposed bone, tendon, and joint in 45, 11, and 12, respectively, or a combination in 35 patients. the flaps done were 51 reverse sural artery, 35 gastrocnemius, 25 local fasciocutaneous, and seven foot flaps. the flap dimensions ranged from 2 2 to 30 15 cm. ninety - three flaps (79%) healed primarily. among 25 flaps (21%) with necrosis, 14 flaps required secondary split skin graft for healing, while the other nine flaps healed without further surgery.conclusion:appropriate soft tissue cover provided by orthopedic surgeons can help in providing independent, composite care of lower limb injuries.
the increasing use of methamphetamine (meth) in the last decades has made it the second - most abused drug in the world. it has been reported that nearly 0.7% of the global population (33.8 million people) aged 15 to 64 years - old, used amphetamine - type stimulants in 2010 (1). relative ease of availability and low cost of synthesis are among the reasons for rise in meth abuse (2). meth abusers utilize various routes of administration including smoking, injection, ingestion, nasal insufflation or snorting, sublingually or even rectally (1). meth abuse can adversely affect health, causing acute mental and behavioral disorders and long - term health complications following chronic use (3). every year, approximately 94000 cases of meth intoxication are admitted to the emergency wards of united states hospitals (4). the incidence of meth intoxication in all autopsies in san diego county has been 4.9% (5). patients with acute meth intoxication may present with a variety of clinical manifestations including : hypertension, tachycardia, dysrhythmias, myocardial ischemia, hyperthermia, agitation, seizures, intra- cerebral hemorrhage, headache, euphoria, choreo- athetoid movements, hyperreflexia, paranoid psychosis, rhabdomyolysis, muscle rigidity and acute lung injury. benzodiazepines are the recommended treatment for meth toxicity and can reduce agitation, seizures, delirium or hallucinations (6). however, at the present time, there is no available antidote for acute meth toxicity intravenous lipid emulsion (ile) is a lipid rich emulsion that has been used to provide calories and essential free fatty acid to patients in peripheral parenteral nutrition (7). recently, ile has been utilized for delivering lipid - soluble drugs, such as fentanyl, decotaxl and etopsides, intravenously, because of the biocompatible nature of the lipid - based delivery systems (8 - 10). advances in the area of ile have resulted in potential application of ile for treating poisoning. there is considerable evidence from animal studies and case reports to support the effectiveness of ile in alleviating drug or xenobiotic toxicity and reversing the hemodynamic or neurological toxicity (11 - 13). the present study aims to investigate the potential role of ile as an antidote in acute meth poisoning. two groups of six male wistar rats weighing 250 - 300 g ; were obtained from the animal center, school of pharmacy, mashhad university of medical sciences. animals were kept on a 12 hr light / dark cycle and temperature of 23 1 c (room temperature) with free access to standard laboratory food and tap water. all experimental procedures were carried out in accordance with the guidelines of the animal care committee of mashhad university of medical sciences. ile was obtained from fresenius kabi ab, spain and meth was donated by the department of medicinal chemistry, school of pharmacy, mashhad university of medical sciences (mashhad, iran). all animals were treated by meth at a dose of 45 mg / kg, intraperitoneally. five to seven min after meth injection, they received an infusion of 18.6 ml / kg ile 20% in the experimental group or normal saline (ns) in the control group in the same volume through the tail vein. the locomotors activity and stereotypical behavior of all animals were assessed in an open field at 0 (before meth injection), 0.5, 1, 2, 4, 8 and 24 hr after meth administration. briefly each rat was placed in the center of an open - field apparatus and its activity was recorded by digital video for 10 min. motor activity was measured by the number of peripheral, central and total square crossings ; freezing length and number of rearing. stereo - typed behavior was rated using the scoring system of sanberg (15) : asleep or stationary (0), active (1), predominantly active but with bursts (2), stereotyped activity such as sniffing along a fixed path in the cage (3), stereotyped sniffing or rearing maintained in one location (4), stereotyped behavior in one location with bursts of gnawing or licking (5). the body temperatures of the rats in both control and experimental groups were assessed by rectal thermometer during the same intervals. the mortality rate was calculated for each group after 24 hrs of drug administration. on the third day after drug administration, the brain and internal organs including the lung, kidney and pancreas of animals were removed after euthanasia and fixed by immersion in 10% buffered formalin. the tissues were then embedded into paraffin blocks and were cut at a thickness of 5 m tissue sections. for the histopathological examination, paraffin embedded sections were also scored for apoptosis by terminal deoxynucleotidyl transferase - mediated dutp nick end labeling (tunel). the assay was performed according to the manufacturer s instructions (roche / germany). cells with condensed dark nuclei and morphological apoptotic changes were scored as tunel positive (16). unpaired t test was used to evaluate differences in the body temperature in two groups and two - way repeated - measures anova for evaluating the differences among various times of intra - groups. kruskal - wallis anova and mann - whitney testing were used for evaluating open - filed variables, stereotyping motions, histopathological findings and tunel results. two groups of six male wistar rats weighing 250 - 300 g ; were obtained from the animal center, school of pharmacy, mashhad university of medical sciences. animals were kept on a 12 hr light / dark cycle and temperature of 23 1 c (room temperature) with free access to standard laboratory food and tap water. all experimental procedures were carried out in accordance with the guidelines of the animal care committee of mashhad university of medical sciences. ile was obtained from fresenius kabi ab, spain and meth was donated by the department of medicinal chemistry, school of pharmacy, mashhad university of medical sciences (mashhad, iran). all animals were treated by meth at a dose of 45 mg / kg, intraperitoneally. five to seven min after meth injection, they received an infusion of 18.6 ml / kg ile 20% in the experimental group or normal saline (ns) in the control group in the same volume through the tail vein. the locomotors activity and stereotypical behavior of all animals were assessed in an open field at 0 (before meth injection), 0.5, 1, 2, 4, 8 and 24 hr after meth administration. briefly each rat was placed in the center of an open - field apparatus and its activity was recorded by digital video for 10 min. motor activity was measured by the number of peripheral, central and total square crossings ; freezing length and number of rearing. stereo - typed behavior was rated using the scoring system of sanberg (15) : asleep or stationary (0), active (1), predominantly active but with bursts (2), stereotyped activity such as sniffing along a fixed path in the cage (3), stereotyped sniffing or rearing maintained in one location (4), stereotyped behavior in one location with bursts of gnawing or licking (5). the body temperatures of the rats in both control and experimental groups were assessed by rectal thermometer during the same intervals. on the third day after drug administration, the brain and internal organs including the lung, kidney and pancreas of animals were removed after euthanasia and fixed by immersion in 10% buffered formalin. the tissues were then embedded into paraffin blocks and were cut at a thickness of 5 m tissue sections. for the histopathological examination, paraffin embedded sections were also scored for apoptosis by terminal deoxynucleotidyl transferase - mediated dutp nick end labeling (tunel). the assay was performed according to the manufacturer s instructions (roche / germany). cells with condensed dark nuclei and morphological apoptotic changes were scored as tunel positive (16). unpaired t test was used to evaluate differences in the body temperature in two groups and two - way repeated - measures anova for evaluating the differences among various times of intra - groups. kruskal - wallis anova and mann - whitney testing were used for evaluating open - filed variables, stereotyping motions, histopathological findings and tunel results. fisher exact tests were used to evaluate differences in mortality rate. the alpha level was set at 0.05. ile therapy for poisoning by meth in rats could protect them from death and all of them were alive at the time of the last evaluation. nevertheless, only 50% of rats treated by meth+ns were survived (mean survival time of meth+ns = 133.7 hrs and meth+ile = 24 0 hr, p<0.05). all meth - treated rats became hyperthermic after 30 min ; however the mean rectal temperatures of ile treated group returned to normal condition more quickly than the other group (p<0.05) (figure 1). rectal temperature of rats received methamphetamine (45mg / kg) plus intravenous lipid emulsion 20% (18.6 ml / kg). comparison of data by values of zero time were analyzed by anova, posttest = tuky, n=6. # p<0.05, # # p<0.01 and$ (not significant) compared to control group. comparison means between groups were analyzed by non - paired t test, p<0.05 within - group comparisons the mean time spends for freezing for each group is presented in figure 2. meth in both groups significantly enhanced the freezing response until 2 hr after injection in comparison with zero time. in subsequent times, meth - induced freezing was significantly lessened by ile compared with ns (p<0.01 and p<0.05) and could reverse the effect to normal. effect of methamphetamine (45 mg / kg) administration plus intravenous lipid emulsion 20% (18.6 ml / kg) or normal saline on freezing and rearing in rats during different time intervals (hr). comparison of values of two groups with each other at each episode of time was analyzed by non - paired t test, p<0.05, p<0.01 meth administration also decreased the rearing responses during the first two hrs that was significantly increased in the next time interval in the ile receiving group (p<0.01 and p<0.05) (figure 2). the meth intoxicated rats lost the ability to cross the lines of open filed (figure 3). however, rats that were exposed to the meth+ile, especially during the last hrs of the experiment, crossed the central and peripheral squares significantly more than the other group (p<0.01) and returned to original condition (figure 3). indeed, the animals receiving meth engaged in vigorous stereotyped movements and ile could reduce stereotyped behavior eight hrs after meth administration (p<0.05) (figure 4). effect of methamphetamine (45 mg / kg) administration plus intravenous lipid emulsion 20% (18.6 ml / kg) or normal saline on locomotor activity in rats at different time intervals (hr). comparison of values of two groups with each other s on each time was analyzed by non - paired t test, p<0.05, p<0.01 stereotyped behavioral response to methamphetamine (45 mg / kg) administration plus intravenous lipid emulsion 20% (18.6 ml / kg) or normal saline every 10-min interval during 24 hr. comparison of values of two groups with each other s on each time was analyzed by non - paired t test, p<0.05 h&e staining of rat lungs following meth exposure showed the presence of emphysematous changes in the lungs, bronchiolitis and interstitial inflammation sections (figure 5). light photomicrograph of hematoxylin and eosin stained sections in the lung tissue of rats that were exposed to methamphetamine (45 mg / kg) plus intravenous lipid emulsion 20% (18.6 ml / kg) or normal saline. a) emphysema b) bronchiolitist c) interestitial inflamation d) normal lung the frequency of these findings was significantly lower in the ile - treated group in comparison with the control group (figure 6). effect of methamphetamine (45 mg / kg) plus intravenous lipid emulsion 20% (18.6 ml / kg) or normal saline on histopatological changes in the lung tissue of rats after 3 days. the tunel assays revealed an induction of apoptosis in lung and brain tissues in groups receiving meth (figure 7). however the percentages of tunel positive cells in the brain was significantly decreased only in groups that received meth+ ile, in comparison with the control group (p<0.05) (figure 8). immunohistochemical staining for tunel in the lung and brains tissue of rats that were exposed to methamphetamine (45 mg / kg) plus intravenous lipid emulsion 20% (18.6 ml / kg) or normal saline. the brain tissue from group received meth + ns (a), meth+ile (b), and lung tissue from rats received meth + ns (c) and meth+ile (d) effect of methamphetamine (meth) (45 mg / kg) plus intravenous lipid emulsion 20% (ile) (18.6 ml / kg) or normal saline on cells apoptosis in the lung and brain tissues as revealed by tunel assay. ile therapy for poisoning by meth in rats could protect them from death and all of them were alive at the time of the last evaluation. nevertheless, only 50% of rats treated by meth+ns were survived (mean survival time of meth+ns = 133.7 hrs and meth+ile = 24 0 hr, p<0.05). all meth - treated rats became hyperthermic after 30 min ; however the mean rectal temperatures of ile treated group returned to normal condition more quickly than the other group (p<0.05) (figure 1). rectal temperature of rats received methamphetamine (45mg / kg) plus intravenous lipid emulsion 20% (18.6 ml / kg). comparison of data by values of zero time were analyzed by anova, posttest = tuky, n=6. # p<0.05, # # p<0.01 and$ (not significant) compared to control group. comparison means between groups were analyzed by non - paired t test, p<0.05 within - group comparisons meth in both groups significantly enhanced the freezing response until 2 hr after injection in comparison with zero time. in subsequent times, meth - induced freezing was significantly lessened by ile compared with ns (p<0.01 and p<0.05) and could reverse the effect to normal. effect of methamphetamine (45 mg / kg) administration plus intravenous lipid emulsion 20% (18.6 ml / kg) or normal saline on freezing and rearing in rats during different time intervals (hr). comparison of values of two groups with each other at each episode of time was analyzed by non - paired t test, p<0.05, p<0.01 meth administration also decreased the rearing responses during the first two hrs that was significantly increased in the next time interval in the ile receiving group (p<0.01 and p<0.05) (figure 2). the meth intoxicated rats lost the ability to cross the lines of open filed (figure 3). however, rats that were exposed to the meth+ile, especially during the last hrs of the experiment, crossed the central and peripheral squares significantly more than the other group (p<0.01) and returned to original condition (figure 3). indeed, the animals receiving meth engaged in vigorous stereotyped movements and ile could reduce stereotyped behavior eight hrs after meth administration (p<0.05) (figure 4). effect of methamphetamine (45 mg / kg) administration plus intravenous lipid emulsion 20% (18.6 ml / kg) or normal saline on locomotor activity in rats at different time intervals (hr). comparison of values of two groups with each other s on each time was analyzed by non - paired t test, p<0.05, p<0.01 stereotyped behavioral response to methamphetamine (45 mg / kg) administration plus intravenous lipid emulsion 20% (18.6 ml / kg) or normal saline every 10-min interval during 24 hr. comparison of values of two groups with each other s on each time was analyzed by non - paired t test, p<0.05 h&e staining of rat lungs following meth exposure showed the presence of emphysematous changes in the lungs, bronchiolitis and interstitial inflammation sections (figure 5). light photomicrograph of hematoxylin and eosin stained sections in the lung tissue of rats that were exposed to methamphetamine (45 mg / kg) plus intravenous lipid emulsion 20% (18.6 ml / kg) or normal saline. d) normal lung the frequency of these findings was significantly lower in the ile - treated group in comparison with the control group (figure 6). effect of methamphetamine (45 mg / kg) plus intravenous lipid emulsion 20% (18.6 ml / kg) or normal saline on histopatological changes in the lung tissue of rats after 3 days. the tunel assays revealed an induction of apoptosis in lung and brain tissues in groups receiving meth (figure 7). however the percentages of tunel positive cells in the brain was significantly decreased only in groups that received meth+ ile, in comparison with the control group (p<0.05) (figure 8). immunohistochemical staining for tunel in the lung and brains tissue of rats that were exposed to methamphetamine (45 mg / kg) plus intravenous lipid emulsion 20% (18.6 ml / kg) or normal saline. the brain tissue from group received meth + ns (a), meth+ile (b), and lung tissue from rats received meth + ns (c) and meth+ile (d) effect of methamphetamine (meth) (45 mg / kg) plus intravenous lipid emulsion 20% (ile) (18.6 ml / kg) or normal saline on cells apoptosis in the lung and brain tissues as revealed by tunel assay. the first ile use for overdose / poisoning was reported in 2006 in the management of acute local anesthetic toxicity (17), especially restoring cardiac and hemodynamic stability. nowadays the resuscitative effects of ile has been evaluated against several classes of drugs and xenobiotic such as beta blockers (18), calcium channel blockers (19), organophosphates (20), tricyclic antidepressants (21), antipsychotics (22), cocaine(23), and others. although numerous mechanisms for ile antidote activity have been suggested, the lipid sink (partitioning) theory is predominant. based on this theory, the intravenous administration of lipid emulsion at high doses creates a new lipid compartment in the blood which causes fat - soluble drugs to move away from the target receptors. subsequently, the concentration of drug is reduced at the target site and reverse the intoxication (24). there is a positive relationship between the fat solubility of drug ; based on octanol / water partition coefficient and the volume of distribution ; and decreasing serum drug concentration treated by ile (25). on the other hand, meth is a highly lipophilic drug with a volume distribution of 3.2 - 4.2 l / kg and log p (octanol / water) of 2.1 (26, 27). thus it can cross the blood brain barrier more easily than other drugs (3). therefore, ile could redistribute meth from the action site, nervous system, and reverse intoxication in rats. the common manifestations of meth overdose are as follows : agitation, shivering, dyspnea, hyperactivity, hyperpyrexia and tremor (4). amphetamine administration to the rats can produce behavioral excitation including forward locomotion, exploratory behavior, and different kinds of stereotyped behavior (28). in this study, the severity of intoxication and high frequency of stereotypical movements make animals unable to move normally. however, after one or two half - lives and diminishing of the severity of toxicity, the rats showed normal activity. the rats treated by ile could return to normal behavior and body temperature faster than the control group. ile could also reduce the mortality rate of meth significantly in comparison with the control group. a similar effect has been reported for many other drugs such as haloperidol, tramadol, propranolol, bupivacaine and cocaine (13, 22, 29, 30). in most reports several studies confirm that ile has also been able to reduce the complications or mortality induced by neurotoxic agents (13, 22, 31, 32). there are some human case reports regarding the successful treatment by ile of poisoning when multiple drugs are used simultaneously, such as cocaine and amphetamine. a 28-year - old man was admitted to the hospital complaining of convulsion, hemodynamic ins - tability, tachycardia, supraventricular and ventricular arrhythmias, hyperthermia and loss of conciseness after overdosing on cocaine (33). arora (23), in a similar case, successfully treated cocaine poisoning with ile in a patient who did not respond to standard treatment for seizure and cardiac outcomes. pre - treatment on cocaine toxicity with ile could significantly reduce cocaine - induced mortality rate, cardiovascular collapse and blunted hypotension in rats. cocaine is a similar drug, in terms of mechanism and toxicity, to amphetamines. both of cocaine and amphetamine are cns stimulant and have similar action mechanisms ; however cocaine is more cardio - toxic than meth and meth is more neurotoxic than cocaine. the use of ile 70 min after an unsuccessful standard cardiopulmonary resuscitation in a 17-year - old girl, who developed seizures and cardiovascular collapse after an intentional ingesting of an overdose of bupropion, lamotrigine and amphetamine, improved her cardiovascular status and her neurologic function (34). recently a middle - aged man was admitted to the hospital because of swallowing an egg - sized portion of meth (35). he remained refractory to standard treatment, ile at an amount of 100 ml could reverse the symptoms of toxicity, 20 min after infusion. our pathological finding showed that meth administration can cause bronchiolitis, emphysema and interstitial inflammation. clinical studies have also revealed that meth intoxication induces acute lung injury, pulmonary edema and pulmonary congestion (36, 37). it should be mentioned that the administration of ile at high doses can induce dose dependent pulmonary fat emboli (22). acute respiratory distress syndrome (ards) has been reported as the most common pulmonary complication in resuscitated patients with ile (38). pulmonary complications associated with ile infusion are related to lipid load and underling pulmonary disease (22). ile infusion released a high concentration of free - fatty acids into the blood stream (39, 40) that can induce impaired endothelium - dependent vasorelaxation and vasoconstriction (41). pulmonary vasoconstriction and shunt have increased in patients with ards that received ile infusion (42, 43). however, these effects have not been reported in patients with normal lung function (43, 44). meth is also a known potent vasoconstrictor and co - administration of these two compounds, meth and ile, in theory, may have synergic effect on lung complications. but in our research, ile reduced the meth pulmonary inflammation and percentage of pulmonary cell apoptosis. the percentage of brain cell apoptosis was reduced in the ile - treated group in comparison with the control group. meth is a potent neurotoxin and induces neuronal apoptosis especially at high doses (45). ile could reduce the severity of meth- induced toxicity and mortality rate of animals by lipid sink theory or direct effect. although the possible adverse effects of ile and interferences with other rescue treatments should be considered, intravenous infusion of lipid emulsion may save the life of patients with acute meth intoxication who do not respond to initial treatment. further human and animal studies are needed to determine the exact indications and their possible mechanisms.	objective(s):the increasing use of methamphetamine (meth) in the last decades has made it the second most abused drug. advancs in the area of intravenous lipid emulsion (ile) have led to its potential application in the treatment of poisoning. the present study aims to investigate the potential role of ile as an antidote for acute meth poisoning.materials and methods : two groups of six male rats were treated by meth (45 mg / kg), intraperitoneally. five to seven min later, they received an infusion of 18.6 ml / kg ile 20% through the tail vein or normal saline (ns). locomotor and behavioral activity was assessed at different time after meth administration. body temperature and survival rates were also evaluated. brain and internal organs were then removed for histological examination and tunel assay.results:ile therapy for meth poisoning in rats could prevent rats mortalities and returned the meth - induced hyperthermia to normal rates (p<0.05). ile reduced freezing and stereotyped behaviors and increased rearing responses (p<0.05). locomotor activity also returned to control levels especially during the last hours of the experiment. ile administration decreased the prevalence of pulmonary emphysema in the lungs (p<0.05 and p<0.01) and percentages of tunel positive cells in the brain (p<0.05), in comparison with the control group.conclusion:ile could reduce the severity of meth- induced toxicity as well as mortality rate in the animals. intravenous infusion of lipid emulsion may save the life of patients with acute meth intoxication who do not respond to standard initial therapy.
atrial fibrillation (af) is the most common cardiac arrhythmia observed in clinical practice. it is associated with a heavy burden of morbidity and mortality, mainly due to an increased risk of cerebrovascular events and cardiac failure. the prevalence of af in the general population increases exponentially with age (approximately 0.4 % in the general population, 35 % in subjects aged > 65 years and 10 % in subjects aged > 80 years) [1, 2 ]. af is associated with a high risk of systemic thromboembolism, of which stroke is the most frequent manifestation [15 ]. recently, it has been hypothesized that af can be a threat to the brain not only because of the risk of stroke, but also because it leads to cognitive deterioration resulting in dementia, even without first having developed a stroke [6, 7 ]. the efficacy of oral anticoagulant (oac) treatment in the prevention of stroke and systemic thromboembolism has been demonstrated in randomized controlled trials and is strongly recommended in the guidelines from the european society of cardiology (esc). despite strong evidence of the efficacy of oac treatment, its use in clinical practice for prevention of thromboembolism in patients with af is still limited [9, 10 ]. although diagnostic and therapeutic strategies in af patients are usually initiated by cardiologists or other in - hospital specialists, management of co - morbidities and drug adherence also involves primary care physicians (pcps). in this context, prevention of thromboembolism associated with af is an essential component of the global cardiovascular prevention strategy in primary care. a canadian registry showed limited use of the chads2 (cardiac failure, hypertension, age, diabetes, stroke [doubled ]) score by family physicians for assessing the need for anticoagulant therapy for stroke prevention in af patients [4, 11 ]. a number of other observational studies in the primary care setting have also found that adherence to guidelines for stroke prevention in patients with af is low and that anticoagulation is underused [9, 12 ]. moreover, even when warfarin is prescribed, achievement of target international normalized ratio (inr) 2.03.0 is inadequate. the primary objective of this study was to estimate the prevalence and epidemiological features of af in this population with a focus on ischaemic and bleeding risk assessment. a secondary objective was to examine the pcps level of adherence to the guidelines for the prevention of thromboembolic risk in these patients. data were obtained from 128 pcps in naples who provided information to the health search (hs)/thales database. we performed an observational, retrospective analysis of patients with a diagnosis of af over a 2-year period (april 2009april 2011). institutional review board / ethics committee approval is not required for retrospective, observational studies based on database analysis in italy. the hs / thales database is an italian general practice registry that collects data from the electronic patient records of a selected group of italian pcps who voluntarily agreed to collect patient information and to attend specific training courses on data entry. in the hs / thales database patient demographic details are linked with a range of clinical parameters (e.g., diagnosis, diagnostic procedures, drug prescription information, hospital admissions) by the use of an encrypted patient code. the research validity of the hs / thales database has been confirmed by a number of published comparative studies [1416 ]. all subjects 18 years of age recorded in the database in april 2009 were included in the analysis if they persisted with the same pcp for at least 1 year after the index diagnosis. we identified patients with a diagnosis of af according to the international classification of diseases, 9th revision, clinical modification (icd-9-cm) [items 427.3 and 427.32 ]. however, diagnoses derived from electronic medical records did not distinguish between paroxysmal, persistent or permanent af, or patients whose af subsequently resolved. in order to reduce potential bias due to the missing diagnoses of af, use of amiodarone, dronedarone, flecainide, sotalol and warfarin was considered as potential markers of af and matched with diagnostic and electrocardiography (ecg) findings. demographic and clinical data (risk factors, co - morbidities) were collected for all the enrolled subjects, and diagnostic procedures and therapeutic management were also analysed for patients with a diagnosis of af.. risk stratification of patients was performed with the chads2 and cha2ds2-vasc (congestive heart failure, hypertension, age 75 years [doubled ], diabetes, stroke [doubled ], vascular disease, age 6574 years, sex category [female ]) scores for ischaemic risk and has - bled (hypertension, abnormal renal / liver function, stroke, bleeding history or predisposition, labile inr, elderly [> 65 years ], drugs / alcohol concomitantly) score for bleeding risk in accordance with esc guidelines. in addition, we compared the patients has - bled and atria (anticoagulation and risk factors in atrial fibrillation) scores. instrumental diagnostic procedures (ecg, echocardiography) were carried out at specialist centres and recorded in the database. since this was an observational study, no additional diagnostic or therapeutic interventions were performed. due to the observational nature of the study a formal calculation of the study sample size standardized definitions of all patient - related variables were used and clinical diagnoses were assessed according to the icd-9-cm system. categorical variables are presented as percentage and comparisons were performed using a chi - squared () test. the hs / thales database is an italian general practice registry that collects data from the electronic patient records of a selected group of italian pcps who voluntarily agreed to collect patient information and to attend specific training courses on data entry. in the hs / thales database patient demographic details are linked with a range of clinical parameters (e.g., diagnosis, diagnostic procedures, drug prescription information, hospital admissions) by the use of an encrypted patient code. the research validity of the hs / thales database has been confirmed by a number of published comparative studies [1416 ]. all subjects 18 years of age recorded in the database in april 2009 were included in the analysis if they persisted with the same pcp for at least 1 year after the index diagnosis. we identified patients with a diagnosis of af according to the international classification of diseases, 9th revision, clinical modification (icd-9-cm) [items 427.3 and 427.32 ]. however, diagnoses derived from electronic medical records did not distinguish between paroxysmal, persistent or permanent af, or patients whose af subsequently resolved. in order to reduce potential bias due to the missing diagnoses of af, use of amiodarone, dronedarone, flecainide, sotalol and warfarin was considered as potential markers of af and matched with diagnostic and electrocardiography (ecg) findings. demographic and clinical data (risk factors, co - morbidities) were collected for all the enrolled subjects, and diagnostic procedures and therapeutic management were also analysed for patients with a diagnosis of af. co - morbidities and risk factors for cardiovascular events were identified according to the icd-9-cm. risk stratification of patients was performed with the chads2 and cha2ds2-vasc (congestive heart failure, hypertension, age 75 years [doubled ], diabetes, stroke [doubled ], vascular disease, age 6574 years, sex category [female ]) scores for ischaemic risk and has - bled (hypertension, abnormal renal / liver function, stroke, bleeding history or predisposition, labile inr, elderly [> 65 years ], drugs / alcohol concomitantly) score for bleeding risk in accordance with esc guidelines. in addition, we compared the patients has - bled and atria (anticoagulation and risk factors in atrial fibrillation) scores. instrumental diagnostic procedures (ecg, echocardiography) were carried out at specialist centres and recorded in the database. since this was an observational study, no additional diagnostic or therapeutic interventions were performed. due to the observational nature of the study a formal calculation of the study sample size was not applicable and only descriptive analyses were performed. standardized definitions of all patient - related variables were used and clinical diagnoses were assessed according to the icd-9-cm system. categorical variables are presented as percentage and comparisons were performed using a chi - squared () test. a total of 2,173 patients (out of 167,056 eligible subjects enrolled in the hs / thales database) had a diagnosis of af (1.3 %) and details of their demographic and clinical characteristics at baseline are shown in table 1. the prevalence of af was highest in women (women 1.4 % vs men 1.2 %), and in the age group 75 years (6.9 % vs 3.5 % in patients aged 65 years), drugs / alcohol concomitantlyfig. 3risk of bleeding in patients with af based on atria score (n = 2,173). af atrial fibrillation ; atria anticoagulation and risk factors in atrial fibrillation stratification of ischaemic stroke risk in patients with atrial fibrillation based on chads2 and cha2ds2-vasc scores (n = 2,173). chads 2 cardiac failure, hypertension, age, diabetes, stroke (doubled) ; cha 2 ds 2-vasc congestive heart failure, hypertension, age 75 years (doubled), diabetes, stroke (doubled), vascular disease, age 6574 years, sex category (female) risk of bleeding in patients with atrial fibrillation based on has - bled score (n = 2,173). has - bled hypertension, abnormal renal / liver function, stroke, bleeding history or predisposition, labile international normalized ratio, elderly (> 65 years), drugs / alcohol concomitantly risk of bleeding in patients with af based on atria score (n = 2,173). af atrial fibrillation ; atria anticoagulation and risk factors in atrial fibrillation of the 2,173 patients with af, 84 % (n = 1,827) took oac treatment at the time of diagnosis. among patients on oac treatment, 91 % had a cha2ds2-vasc score 2, 7.3 % were at moderate risk and 1.7 % were at low risk ; similar rates were observed for antiplatelet drugs. however, at the 2-year follow - up, 64.8 % of patients (n = 1184) had discontinued oacs in favour of antiplatelet treatment and only 29.6 % (n = 643) were still being treated with oacs. inr values were available in the database for only 620 patients but the majority of them (63.2 % ; n = 392) were not within the target therapeutic range for af (inr 2.03.0) at the last recorded examination. af is the most common cardiac arrhythmia, conferring substantial mortality and morbidity from stroke, thromboembolism and heart failure, and significant impairment of quality of life [21, 22 ]. it has been suggested that a regular pulse measurement followed by ecg can detect a number of additional cases in comparison with the current clinical practice. the prevalence of af has been strongly associated with increasing age, affecting about 5 % of people over 65 years of age and 9 % of people aged more than 80 years [1, 24 ]. the results of an observational cohort study of 1,599 italian patients aged 65 years showed a 7.4 % prevalence for af. in our study in contrast to epidemiological data from the literature, which has indicated a higher rate of af in men, we observed a higher prevalence in women, which can probably be attributed to a higher number of visits to pcps by female patients. according to the esc guidelines, the chads2 score should be used as an initial, rapid and easy - to - remember means of assessing stroke risk. a limitation to the use of the chads2 score is that it only includes a few stroke risk factors ; as a result more moderate - risk patients (with a chads2 score of 1) could potentially derive greater benefit with oac therapy compared with aspirin (acetylsalicylic acid). in order to improve the choice of antithrombotic strategy in non - valvular af, some stroke risk modifier risk factors have been added to the chads2 score, thus deriving a more refined stroke risk stratification, named cha2ds2-vasc. truly low risk atrial fibrillation patients as those with a cha2ds2-vasc score of 0, who can probably be treated with no antithrombotic therapy. all others (i.e., cha2ds2-vasc score of 1), can be considered for oral anticoagulation. the esc guidelines suggest that the cha2ds2-vasc score should be assessed in all patients presenting with at least one stroke risk factor (resulting in stroke risk stratification according to chads2 showed that 20 % of af patients were at high risk but, when stratified by the more refined cha2ds2-vasc scores, the proportion at high risk of stroke increased up to 86 %. the patient s risk of bleeding should also be evaluated prior to the initiation of oac treatment, and the has - bled score should be used for this purpose. high bleeding risk and some caution and regular review of the patient is needed following the initiation of antithrombotic therapy, whether with oral anticoagulation or aspirin. the has - bled score was derived and validated in an af population and its predictability has been recently compared to the hemorr2hages (hepatic or renal disease, ethanol use, malignancy, older age [> 75 years ], reduced platelet count, re - bleeding [doubled ], hypertension uncontrolled, anaemia, genetic factors, elevated risk of fall, stroke) score. both of these schemes performed similarly in predicting major bleeding, but a substantial advantage of has - bled is its relative simplicity, allowing ease of use in everyday clinical practice [19, 26 ]. it has been suggested that the atria score (including five weighted risk factors : anaemia, severe renal disease, age 75 years and older, previous haemorrhage, and diagnosed hypertension) may be used to support the choice of antithrombotic strategy in patients at moderate ischaemic risk, especially in view of the introduction of the novel oacs. we compared the results from has - bled to atria and found that the latter identified a lower number of high risk patients. a debate has recently followed the atria score publication, as some concerns have been raised with regard to the derivation of the new schema and its applicability. in particular, inclusion criteria and risk factors from which the model was derived warfarin - experienced cohort in the study, atria might not be applicable in warfarin - nave patients, notoriously at higher risk of bleeding. thus, to date, the use of the atria score does not seem to be considered as an alternative to the has - bled score for stratification of bleeding risk. many observational studies have shown that in clinical practice oacs are frequently underutilized, with reported percentages of prescriptions between 30 and 60 % despite the recognized clinical benefits of prevention of af - related strokes [2830 ]. a previous italian primary care registry has shown a significantly lower proportion of af patients treated with oacs (persistence with oac was < 50 % at 1 year and reduced to approximately 25 % by 2 years), when compared with the euro heart survey of af patients or other european countries. in our analysis, despite a higher percentage of oac prescriptions following the diagnosis of af, levels of adherence among the pcps patients were very low with less than one - third of patients maintained on warfarin at the end of the 2-year follow - up. notably, inr values were recorded in a minority of cases and were not within the target therapeutic range in most of them. the underutilization of oac therapy may also be explained by a lack of awareness of patient risk stratification criteria or poor appreciation of the risk benefit ratio of oacs, with an overestimation of their bleeding risks [9, 13 ]. moreover, this may reflect the difficulty of managing patients on conventional oac treatment regimens. inr monitoring is important for management of oacs currently used in clinical practice but is not easy to arrange for all patients due to the need for regular blood tests and frequent dose adjustments. furthermore, the established oacs have a narrow therapeutic range and exhibit a wide variability of patient response, which leads many patients to spend significant amounts of time outside the therapeutic inr range and may favour drug discontinuation. thus, thromboembolic risk stratification only partially influences the choice of the oac therapy for stroke prophylaxis and is underutilized, even in high - risk patients. an italian survey of antithrombotic therapy in patients with af showed that treatment was discontinued in one - third of patients due to fear of bleeding and difficulty in performing adequate monitoring. the lack of use of oac therapy is particularly evident in elderly patients who were also at highest risk of stroke. even when there is evidence that age and af independently increase stroke risk, elderly people with af are less likely to receive oac therapy. recently, olesen. demonstrated in a nationwide cohort analysis in denmark that the net clinical benefit was more positive in patients at high bleeding risk, and the absolute benefit in reducing stroke with warfarin would outweigh the small increase in intracranial haemorrhage with warfarin. bafta (birmingham atrial fibrillation treatment of the aged study) showed that warfarin significantly reduces stroke risk in the elderly. in the atria cohort, singer. also showed a reduction in absolute stroke risk with warfarin : the benefit was greater in very elderly patients (age 85 years) and among those at high stroke risk. the bleeding risk with antiplatelet therapy is similar to that with warfarin, especially in the elderly who can benefit most, in terms of risk of bleeding, from the accurate use of oac treatment, as compared with the use of antiplatelet treatments [9, 35, 37 ]. the recent european position document on bleeding risk assessment and management in af states that bleeding risk is almost inevitably lower than stroke in patients with af but can be minimized by refining antithrombotic therapy in patients identified to be at high risk. firstly, data were collected retrospectively and the analysis was based on the diagnoses recorded by the pcps in their database, which may affect the accuracy in reporting anamnestic data. nevertheless, since it was our intent to describe the epidemiology and management of af patients referred to pcps, these data represent a picture of a second issue is that in italy there are some local directives and restrictions affecting the prescription of drugs and diagnostic procedures that could potentially influence physicians behaviour, which may limit the generalizability of these results to a non - italian population. this survey does not provide information on outcomes and the potential relationship with management strategies, and can not be considered a study on the appropriateness of diagnostic or therapeutic approaches in patients with af as the aim was to provide epidemiological data in the pcp setting. this study provides confirmation of previous epidemiological data on af prevalence in italy. despite the high stroke risk in this population, anticoagulant therapy is underutilized, although, in comparison with other observational studies, we found a higher rate of oac prescription at the time of the diagnosis. in contrast, we detected a very high rate of drug discontinuation and poor attention to monitoring of the effectiveness of oac therapy by measurement of patients inr values. in real world practice, evaluation of ischaemic and bleeding risks, which should direct therapeutic decisions concerning the prevention of thromboembolism in af patients, are not balanced and often the choice to avoid oac therapy is led by an overestimation of the haemorrhagic risk. in addition, the management of chronic oac therapy using inr monitoring appears to represent a real challenge in the pcp setting. the utilization of simple risk scores could provide more sensitive instruments for a real risk profile definition in these patients, and support physicians therapeutic decisions in the primary care setting. our results strengthen the importance of promoting educational programmes amongst pcps, focusing on risk stratification and adequate monitoring of patients on oac therapy. how much the availability of novel oacs will change prevention strategies in this setting is still unknown. however, even in this new landscape, net clinical benefit in stroke prophylaxis will remain bound to balancing ischaemic and bleeding risk. this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.	backgroundatrial fibrillation (af) is the most common cardiac arrhythmia and is associated with a heavy burden of morbidity and mortality, mainly due to an increased risk of cerebrovascular events and cardiac failure. oral anticoagulant (oac) treatment prevents stroke and systemic thromboembolism in patients with af and its use is strongly recommended in guidelines. however, its use in this patient group remains limited. primary care physicians (pcps) have an important role to play in this context.objectivethe primary objective was to estimate prevalence and epidemiological features of af in the primary care setting, focusing on ischaemic and bleeding risk assessment. a secondary objective was to examine the pcps level of adherence to the guidelines for the prevention of thromboembolic risk in these patients.methodsthis retrospective, observational study was based on data entered by 128 pcps into the health search (hs) thales database, identifying patients with a diagnosis of af at the time of the analysis.resultsout of 167,056 patients analysed, 2,173 (1.3 %) were diagnosed with af, with 86 % at high risk for ischaemic stroke, according to cha2ds2-vasc (congestive heart failure, hypertension, age 75 years [doubled ], diabetes, stroke [doubled ], vascular disease, age 6574 years, sex category [female ]) stratification. after the diagnosis of af, 84 % of patients were prescribed oac treatment. however, at 2 years follow - up, only 29.6 % were still being treated with oacs.conclusionthe prevalence of af in this analysis was consistent with previously reported italian national epidemiological data. adherence to the european society of cardiology af guidelines by pcps was low, despite the high levels of stroke risk. at the end of the observation period less than one - third of patients were still on oac therapy. awareness of the benefits of oacs in stroke prevention in af patients needs to be improved.
study protocols were approved by the american university of armenia institutional review board. given the lack of data on tb among migrant workers, we contacted tb physicians working in all 72 outpatient tb centers in armenia. the attending tb physicians were familiar with the migrant worker status of their patients, and a list of their patients formed a census study frame for all migrant workers with tb. a study participant had to be a migrant worker outside armenia for 3 months during 20082011, be 18 years of age, have armenian citizenship, and speak armenian. we collected data from eligible study participants in the census study time frame through face - to - face interviews and abstraction of data from medical records. after considering national data on tb prevalence in armenia and the number of migrant workers with a tb diagnosis provided by tb physicians and confirmed during the study, we estimated that 7.0% of all tb patients treated during 20082011 in armenia were migrant workers. we attempted to contact all 126 eligible migrant workers in armenia at the time of the survey (january march 2012) and recruited 95 study participants (75.0% response rate). approximately 91.0% of study participants had either a high school or professional technical education, 85.0% were from low - income households, and 33.0% were registered in a social support program for persons living in poverty. median ages of surveyed persons and the population with tb in armenia in 2012 were similar (46 and 45 years, respectively). the 95 study participants reported 166 visits for seasonal work to other countries during 20082011 ; the russian federation was the most common destination (147 visits) (figure). the type of work in which study participants were involved outside armenia was construction (65.0%), driving / transportation (11.0%), commerce (8.0%), production (7.0%), services (5.0%), food industry (4.0%), and farming (1.0%). for 63.0% of all host country work visits, work was in confined spaces, of which 23.0% had > 6 persons working in the same enclosed space. for 7.0% of all visits percentage of work visits by migrant workers to host countries - of - work and tuberculosis (tb) prevalence/100,000 persons in host countries compared with that in armenia, 20082011 (7). medical record reviews of 95 study participants indicated that 28.3% (26/92) of respondents had mdr tb and 5.3% (5/94) had hiv / tb co - infection compared with 5.6% and 3.1%, respectively, for all tb patients in armenia reported by the national tb control program in 2011 (table 1). all study participants were treated for tb at least once, 73.6% (70/95) were reportedly treated only once, 13.7% (13/95) were treated twice, 12.6% (12/95) were treated 3 times, and 44.2% (42/95) of migrant workers were reportedly first given a diagnosis of tb in the host country of work. mdr tb, multidrug - resistant tuberculosis ; ss+, sputum smear positive. the period between first diagnosis and first treatment was reportedly 5 times longer (95% ci 1.934.8, p = 0.06) for those who were given a diagnosis in the host country of work than for those given a diagnosis in armenia (table 2). approximately 92.5% (86/93) workers reported that they received inpatient hospital care during their first tb treatment, of whom 5 reportedly did not complete the full course of inpatient treatment. the mean duration for the first inpatient tb treatment for participants who received tb treatment in armenia was 78 days (range 40425 days). for those who received their first inpatient treatment in the host country of work, the mean duration was 164 days (range 20912 days). those workers who received treatment in the host country of work were 3.9 times more likely to have a failed or defaulted treatment outcome than those who received treatment in armenia (95% ci 11.474.1, p = 0.001). of those reporting, 9.3% (8/86) experienced an interruption of prescribed tb treatment for 1 day during the inpatient phase of their first treatment (mean duration of interruption 7 days, range 130 days). marginally significant difference (0.1p0.05). during the ambulatory phase of their first treatment, 20.4% (19/93) of respondents reported treatment interruptions (mean duration of longest interruption 7.8 days, range 160 days). approximately 80.8% (55/68) of persons who completed their first treatment for tb reported that they completed the treatment (cured or completed treatment with no treatment failure), 11.7% (8/68) reported defaulting (lost - to - follow - up), and 7.4% (5/68) reported as being sputum - smear - positive at the end of treatment (treatment failed) (8). bivariate analysis showed that patients who had incomplete ambulatory treatment had greater odds of having mdr tb than having drug - sensitive tb than did those who completed ambulatory treatment (odds ratio [or ] 4.0, 95% ci 1.610.1, p = 0.003). multivariate logistic regression showed that persons who did not receive standardized tb treatment with supervision during the ambulatory phase of treatment had 4 times greater odds (or 4.4, 95% ci 1.314.9, p = 0.02) of having 2 tb treatments (versus only 1 tb treatment) than persons who received standardized treatment with supervision during the ambulatory phase. after adjusting for confounding, we found that persons who had mdr tb had 2 times greater odds (or 2.3, 95% ci 1.15.0, p = 0.04) of having 2 tb treatments (versus only 1 tb treatment) than persons who had tb (table 3). dot, directly observed therapy ; na, not applicable ; mdr tb, multidrug - resistant tuberculosis ; ni, not included. we found that among migrant workers with a diagnosis of tb, migratory work was associated with higher rates of mdr tb and hiv co - infection, which suggested that migratory work may provide impetus for spread of hiv infection and tb. treatment for tb that started in the host country of work was usually interrupted because migrant workers wanted to return to armenia. those migrant workers with tb who experienced treatment delays, dropped out of therapy, or had therapy interrupted were likely to increase the period of infectivity and spread tb to other persons. there is no official referral system between the armenian national tb program and their counterparts in host countries of work. on the basis of our study findings, we recommend establishing closer collaboration between health systems of countries supplying migratory workers and host countries for work at governmental and nongovernmental levels to improve treatment completion rates and reduce adverse outcomes. this study could become a basis for further efforts in understanding the mechanisms that impede access of immigrants to treatment and planning target strategies for early detection of tb and treatment in this group of patients. it could also become an example for further large - scale projects worldwide, especially in the commonwealth of independent states region to explore the correlation between migrant labor and tb.	to understand use of tuberculosis (tb) services for migrant workers, we conducted a cross - sectional census of 95 migrant workers with tb from armenia by using medical record reviews and face - to - face interviews. prolonged time between diagnosis and treatment, treatment interruption, and treatment defaults caused by migrant work might increase the risk for multidrug - resistant tb.
in carrying out their professional work, dentists are exposed to a number of occupational hazards. these cause the appearance of various ailments specific to the profession, which develop and intensify with years. in many cases they result in diseases and disease complexes, these hazards can be broadly categorized as : physical, chemical, biological, mechanical and psychological. physical and mechanical hazards include eye injuries occurring from projectiles, cuts from sharp instruments, or puncture wounds from needles or other sharps. such injuries can result in the transmission of serious infectious diseases to the dental worker. harmful radiation like non - ionizing radiation (visible and uv light) and ionizing radiation (x - rays) can cause damage to various body cells. musculoskeletal problems like wrist ache, lower backache, and neck ache can occur due to the need to work in specific working positions using a continuous repetitive motion. chemical hazards can be inorganic (mercury toxicity), organic (solvents, resins, gases), caustic (formaldehyde, hydrogen peroxide) and latex glove allergy (contact dermatitis). biological hazards can occur due to allergens of biological origin, infections, dental material toxicity, and cross contamination. lastly, psychological problems can arise due to stress / excess work load, lack of job satisfaction / insecurity, professional burnout, and medico - legal problems. therefore, there is a need to educate and make the dentists aware of these hazards and the methods of their prevention. before carrying out such an educational program we must have a baseline data of the prevalence and awareness among the study group regarding the topic. majority of studies on occupational hazards among dentists are conducted in the developed world however, the minimal data is available from the developing countries. hence, this study was aimed at finding the status of occupational hazards among dentists working in private clinics in chandigarh city. the other objective of the study was to know whether the dentists were adopting proper measures for their prevention. the present study was conducted among the dentists practicing in private clinics in chandigarh city. since, the data on the total number of registered dental clinics were not available, it was decided to cover maximum number of dental clinics in the survey. in total 130 dentists were requested to fill up the questionnaire, out of which 113 returned the filled forms. verbal as well as written informed consent was obtained from the study participants. a 22 questions closed - ended questionnaire was prepared to assess the occurrence of occupational hazards and the necessary steps taken for their prevention. subjects were questioned about their age, gender, qualification, years of practice, daily working hours, and average number of patients attended in a day. further, questions assessed whether they have suffered from any of the various occupational hazards like injury from sharps and what precaution they had taken after the incident. other hazards included were burns, hearing problems, musculoskeletal problems (back pain, cervical spondylitis, and carpel tunnel syndrome), allergies (allergy from dental materials like acrylic, latex etc.), and stress and eye injuries. the next part of questionnaire included questions on administration of hepatitis b vaccination and its booster dose, and the following of various cross infection control measures in the clinic. next, questions were asked on use of amalgam in the clinic and its method of disposal, use of x - ray and measures available in the clinic for protection of staff from it. lastly, subjects were asked questions related to their physical activity, job satisfaction, stress and its management through vacation, hobbies etc. spss software version-17 was used for analysis and level of significance was set at p 0.05). this questionnaire based survey was conducted on private dental practitioners in chandigarh city, india to assess the status of occupational hazards and their prevention among them. total 113 dentists completed the questionnaire ; response rate was 86.9%, which can be considered satisfactory. most common reason for not participating in the study (n = 17) was busy schedule of the dentists in their clinics. mean age of participants was 38.2 years (range 24 - 70 years). qualification of participants showed 55 graduates and 58 post - graduates, this clearly indicates that different categories of dentists were represented appropriately in this study. injury from sharps is a major concern for the profession as it is a source of transmission of various infectious agents like different types of hepatitis viruses and hiv. in our study 77% dentists gave a positive history of such injuries, which is higher compared to study done by 27.7% in queensland, australia, 36% among the nigerian dentists and 50% among dentists in thailand. most common cause of such injury was from the needle (49.5%), which is in agreement with other studies conducted in different countries. stress due to job / business is present in all professions and dentistry is no exception to it as 43.3% of our study participants were a victim of this. these self - reported values are considerably less when compared to studies carried out by leggat. in thailand, kay and lowe and myers and myers in england and gijbels. in belgium, they reported prevalence of 96.1%, 86%, 60% and 54% respectively. the main reasons for stress among dentists are related to working hours, job satisfaction, staff / patient interactions and medico legal problems. musculoskeletal disorders (msd) like lower backache, wrist ache, and neck, and shoulder pain are common hazards of our profession. a total of 39.8% participants complain one or more msd in this study. msd is one of the major factor for premature retirement among dentists along with stress and cardiovascular disease. around quarter (23.8%) of the dentists were suffering from allergies due to dental materials, gloves etc. this percentage is quite similar to as reported in the studies by chowanadisai. immunization against hepatitis b virus was received by 88.4% of participants and 70% had even got the booster dose administered, these figures similar to reported in other parts of the world. protection from hepatitis b virus is essential for the dentists as they are exposed to a variety of body fluids of patients. majority of dentists were following proper infection control (73.4%) and hospital waste disposal management (96.4%) in the their clinics, other authors have also reported the same, but these values are self - reported and so can have some degree of bias when responding to such questions as most dentists are aware that they have to follow the above procedures. by far the most dangerous material used in dentistry is mercury in dental amalgam which poses a serious threat to dental as well as general population if not handled and disposed - off properly. in our study, 66% dentists who were carrying out amalgam restoration were storing excess amalgam in water ; these figures are more than as reported by leggat in thailand. only 11% were storing excess amalgam in radiographic fixer, which is considered to be the proper method. most of the participants (85.7%) were using an appropriate protection barrier while taking radiographs, but 72.5% were not using any devices to measure the amount of radiation exposure, this can be a cause of concern especially, considering that 38% of them were having conical positioning device in their x - ray machine. overall, majority of them were satisfied with their job and working hours and were taking regular breaks from their routine. 70% of them were also involved in some kind of physical activity like sports ; this percentage is comparable to a study done by kay on uk dentists. as with any questionnaire study, the present study has limitations especially of false reporting as dentists are aware of what corrective measures are required to be taken for prevention of various occupational hazards which they must have studied in dental school. no physical inspection was carried out of the clinics by the investigators so such false reporting is possible. although, pilot study was conducted to check feasibility of the study and to do necessary modifications of questions, validity, and reliability of questionnaire was not assessed statistically. as the results of the present study demonstrates that there is a need to make dental practitioners of chandigarh more aware about the prevention of different occupational hazards in dentistry. areas, which require special attentions are radiation protection, proper storage of excess amalgam, post - exposure prophylaxis after sharps injury and stress management. this can be carried out by carrying various informative programs like continuing dental education programs, seminars, and workshops on prevention of occupational hazards in the region. future research can include medical examination of the participants as well as physical examination to verify the answers provided by dentists.	background : to assess the status of occupational hazards and their prevention among the practicing dentists in chandigarh city, india.materials and methods : a closed - ended questionnaire was prepared to record demographic status, types of occupational hazards encountered, and status of measures used for their prevention. a total 113 out of 130 dentists completed the questionnaire and the response rate was 86.9%.frequency tables were prepared and coefficient of correlation was computed to check correlation between different variables. statistical significance was set at p < 0.05.results:the most common occupational hazard reported was injury from sharps (77%), out of which needle prick injury was the most frequent. of the other occupational problems job related stress (43.3%), musculoskeletal problems (39.8%), and allergies (23.8%) from things used in dental clinics were most common. a reasonably high percentage of dentists were immunized against hepatitis - b virus (88.4%) and were following proper infection control measures and hospital waste disposal methods. very few dentists were following the correct method of disposal of excess amalgam (11%) and measurement of radiation exposure (27.5%) within their clinic. most of them (90.2%) were satisfied with their current working hours and job.conclusion:prevalence of occupational hazards among the studied group was high and certain preventive measures were not being followed properly. therefore, there is a need to improve the knowledge of dentists regarding these hazards and their prevention.
this inner city 307-bed community hospital is also the home of the first chest pain emergency center in the world. interventional cardiology services are available, but the hospital does not provide open heart surgery. upon review of lab ordering data, the testing volumes of both ck - mb and myoglobin totaled over 10,000 a year despite increased evidence of their low specificity and sensitivity in acs workup verses troponins (3). examination of the cardiac enzyme order set in newly implemented cpoe revealed a possible explanation. the laboratory tests in the cardiac enzyme order set included ck - mb, myoglobin, ck, sgot, and sgpt, and all were pre - checked. as a result, providers were more likely to order all these tests due to convenience. based on these findings and pursuant discussion, the leadership in departments of internal medicine, cardiology, and pathology and laboratory reached an agreement that, given the high sensitivity, specificity, and easy availability of cardiac troponin testing, there was no need for physicians to routinely order ck - mb and myoglobin along with ck, sgot, and sgpt as part of workup for acs in the vast majority of patients. therefore, those tests would be removed from the cardiac enzyme order set. after the above agreement was approved by the hospital administration, provider education to de - emphasize the use of ck - mb and myoglobin in diagnosing acs was conducted through grand rounds (held on november 7, 2013) on high - value care and conferences to the staff. in addition, this information was sent via an email notice (was sent on november 22, 2013) co - signed by the chiefs of cardiology, pathology and internal medicine to all licensed medical providers in internal medicine and emergency medicine departments at the institution informing of the change and the evidence behind the change. the other tests remained available as stand - alone assays to be used in conditions such as re - infarction or infarction extension. numbers of tests ordered (based on lab procedure charges) from february 2013 through january 2014 (year 1) and march 2014 through february 2015 (year 2) were provided by reimbursement & compliance department. student 's t - test was used to compare the mean difference in utilization of these tests before and after the intervention. numbers of tests ordered (based on lab procedure charges) from february 2013 through january 2014 (year 1) and march 2014 through february 2015 (year 2) were provided by reimbursement & compliance department. student 's t - test was used to compare the mean difference in utilization of these tests before and after the intervention. as can be seen from fig. 1, before ck, ck - mb, myoglobin, sgot, and sgpt were removed from the routine acs order set, the yearly testing volumes of the above tests were 36,435 ; 11,768 ; 11,420 ; 4,403 ; and 4,032, respectively (does not include numbers of sgot and sgpt as part of a liver function panel or comprehensive metabolic panel). since the change was implemented in february 2014, the annual volumes of ck, ck - mb, myoglobin, sgot, and sgpt dropped to 4,121 ; 2,063 ; 1,561 ; 4,043 ; and 4,032. notably, there was a decrease in the volumes of ck, ck - mb, and myoglobin before the cpoe change, which was most likely the result of grand rounds and email notification in november. the acs order set test volume from february 2013 to february 2015. as shown in table 1, the mean tests volume of ck, ck - mb, myoglobin, sgot, and sgpt was significantly decreased due to the intervention (table 2), while the mean volume of troponin test remained the same (p=0.283). the mean monthly volume of ck decreased from 3,036.3 to 343.4 (88.7% of reduction) ; the mean monthly volume of ck - mb dropped from 980.7 to 171.9 (82.5% of reduction) ; the mean monthly volume of myoglobin decreased from 951.7 to 130.1 (86.3% of reduction) ; and the mean monthly volume of sgot and sgpt dropped from 336.9 to 100.3 (70.0%) and 336.0 to 100.2 (70.2%), respectively. cost savings analysis ts, total savings ; lft, liver function test ; ck, creatinine kinase mean monthly tests volume before and after the intervention y1 : february 2013 through january 2014 (before cpoe intervention). it has been noted (9) that there is a large variability in healthcare costs based on geography, which can not all be explained by varying charges or population differences, and there is a general recognition of stylistic differences in medical practice within or amongst institutions as well. one of the possible explanations for the substantial variation in testing practices could be that a large fraction of tests may be unnecessary (9). identification of appropriate and necessary tests with elimination of those not proven essential or useful is crucial to providing high - value health care. the choosing wisely campaign by abim and its partners has been invaluable in highlighting areas for change. sgot), and ldh were some of the first biomarkers used in the diagnosis of acs, but in comparison with troponins, their utility has waned (10). a recent study suggested that a positive myoglobin result did not offer an additional diagnostic value, but rather led to an additional confirmatory testing compared with troponin i testing (4). when reviewing the utility of ck - mb, the slight lag in troponin rise compared to ck - mb could be the reason for its continued use in acs protocols due to the fear of missing early acs (5). however, volz reviewed 11,092 ed patient encounters and concluded that ck - mb is not necessary in the initial screening for ami (acute myocardial infarction) and may safely be excluded in patients with negative troponins (6). the only potential indication for the ck - mb assay is in the case of re - infarction or an extension of recent infarction in a patient with acs. however, according to a recent study, even in this scenario, ck - mb could be safely substituted by serial troponin testing (7). university of missouri - kansas city reported an exciting study at their institution involving removal of these older biomarkers, facilitated by close collaboration between cardiology and pathology departments (7). it also highlighted the importance of early involvement of the ed and medical leadership, which was consistent with our experience as well (7). our study, which was conducted in a community hospital setting, re - emphasizes the paramount importance of joint efforts among different specialties and departments. this collaborative initiative has clearly led to significant yearly cost savings (based on billable charges) of $ 463,744.7. in the era of nearly ubiquitous electronic medical records and cpoe, the process of changing physician practice with clinical decision support or by developing order sets is a rapidly evolving area of health care (7). given the presumed variability in resources and practice between community hospitals and university hospitals, one may argue that what can be done in a university program may not be applicable at a community hospital. our ability to successfully launch the project and maintain continued support with minimal resources may motivate other institutions to explore opportunities to assist all providers in practicing high - value care. high - value cost - conscious care and stewardship of resources have been proposed as the seventh general competency for all physicians (11). many recent studies provide successful examples of involving physician trainees in quality improvement at an institutional level, leading to significant cost savings both to the healthcare system and to the medical center (12). engaging residents and medical students not only provides them the learning opportunities in principles of quality improvement and high - value care, but also teaches them the practical tools for process change (12). this is an essential component in changing the overall culture of overconsumption and improving the value of healthcare delivery (13). one limitation of our study was that education interventions, including grand rounds and email notifications, were held around 3 and 2 months prior to actual cpoe change respectively, which makes statistical analysis challenging. however, this is practically acceptable (since it is difficult to implement all changes at the same time), and both the education and the cpoe changes helped care providers to change their behavior. further, despite the pre - cpoe implementation decline in test ordering, the number of tests ordered in year 2 was significantly reduced from year 1. no change in the overall morbidity or mortality across the entire hospital patient population was noted during the 2, 12-month periods of comparison (year 1 vs. year 2). exploring these comparisons may certainly be an important next step in rationalizing value versus quality of care.	objectivesnearly one - third of healthcare costs are potentially avoidable and would not compromise medical care if eliminated. therefore, we sought to evaluate the financial impact of reduction in use of creatinine kinase (ck)-mb and myoglobin tests after removing them from the cardiac enzyme order set at a community hospital.methodsgrand rounds were held, and an email notification was sent to de - emphasize the use of ck, ck - mb, myoglobin, sgot (glutamic - oxaloacetic transaminase), and sgpt (serum glutamic - pyruvic transaminase) in acute coronary syndrome (acs) work up. the above tests were removed from the pre - checked cardiac enzyme order set in the computerized physician order entry on february 13, 2014. the tests continued to be available, but needed to be ordered individually. the mean monthly volume of cardiac enzyme tests for 12 months after this intervention was compared with the mean monthly volume of 12 months before the change. total cost savings were calculated.resultsafter the intervention, the number of ck, ck - mb, myoglobin, sgot, and sgpt tests utilized for acs workup decreased dramatically (p<0.001). the volume of troponin testing remained the same (p=0.283). the total annual savings of billable charges to healthcare payers was $ 463,744.7.conclusionsremoval of ck - mb, myoglobin, ck, sgot, and sgpt tests from cardiac enzyme order sets can successfully reduce unnecessary laboratory testing for acs workup, leading to significant cost savings to the healthcare system.
avian systemic salmonellosis has three distinct phases and during each phase there is significant interaction with the immune system. the second phase is the establishment of systemic infection mainly as an intracellular infection of macrophages. finally infection may be cleared by the immune response, the bird may succumb to the infection, or a subclinical carrier state may develop. it has been established that attenuated salmonella enterica serovar typhi (s. typhi) strains can serve as safe and effective oral vaccines to prevent typhoid fever. in addition, live attenuated s. typhimurium strains have been evaluated for use as live vaccines to express or deliver a variety of pathogen 's antigen or dna, respectively, to mucosal lymphoid tissue including the ha of avian influenza virus (aiv), polyprotein of infectious bursal disease virus (ibdv), vapa antigen of rhodococcus equi, and 5401 gene of eimeria tenella. in all the above studies, oral administration of the attenuated salmonella the pathogenicity of the bacteria such as s. typhimurium can be reduced significantly by various attenuation methods while still retaining their invasion capacity and thus deliver the heterologous genes into mammal cells. s. typhimurium sv4089 is a double mutant (dam and phop), derived from wild - type s. typhimurium sl1344. this mutant of salmonella did not show pathogenicity to chickens at a dose level as high as 10 cfu / ml delivered orally [4, 6 ]. the oral ld50 determined for chicken, of wild - type sl1344, is about 10 cfu. studies have shown that dam methylation regulates other virulence - related loci besides spv genes in the virulence plasmid such as lps modification genes, salmonella pathogenicity island 1, and the std fimbrial operon are under dam methylation control [8, 9 ], whilst phop - phoq is regulatory system that controls expression of several virulence properties in s. typhimurium. in addition, the virulence possessions that the phop phoq system controls include the ability to survive inside macrophages, withstanding acidic ph, invasion of epithelial cells, confrontation of killing by antimicrobial peptides, formation of spacious vacuoles, and the ability to alter antigen presentation. although s. typhimurium has successfully been used as a carrier for vaccine, characteristics of the bacteria and its in vitro and in vivo stability in chickens are poorly studied. hence, the purpose of this study is to characterize the attenuated s. typhimurium sv4089 transfected with eukaryotic expression plasmid encoding aiv genes. attenuated salmonella enterica sv. typhimurium (s. typhimurium) sv4089 was kindly provided by dr. the bacteria is a double mutant (dam228::mudj (dam) phop7953::tn10 (phop)), derived from wild - type s. typhimurium sl1344 [4, 6 ]. the eukaryotic expression plasmids, pcdna3.1 (invitrogen, usa) expressing aiv subtype h5n1 a / ck / malaysia/5858/04, ha, na, and np genes (pcdna3.1/ha, na, and np) were constructed by jalilian.. the nucleotide sequences of the full length of ha, na, and np are available at genbank under accession number dq320934, dq321066, and dq321132, respectively. a single colony of attenuated s. typhimurium sv4089 was grown in lb broth to an optimal density od600 0.60.8 and resuspended in ice - cold nuclease - free water. the plasmids pcdna3.1/ha, na, np and control plasmid (pcdna3.1) were purified (qiagen, germany) and transformed into s. typhimurium competent cells by electroporation set at 2.5 kv, 25 f, and 200400 (gene pulser, bio - rad, usa). the transformed culture (100 l) was suspended into lb plates supplemented with 50 g / ml ampicillin [4, 6 ]. resistant colonies harboring the plasmid were cultured and stored after confirmation by pcr and fish to the targeted aiv ha, na, and np. to determine the in vitro stability of the transfected plasmid in a population of attenuated s. typhimurium, bacterial cultures were passaged for approximately 100 generations (25 days) without antibiotic selection. the percentage stability of the plasmid was estimated by calculating the number of cells containing the plasmid at each passage divided by the number of salmonella. the human breast cancer cell lines, mcf-7 and mcf-10a, were maintained in dulbecco 's modified eagles medium (dmem) with 10% (v / v) fetal calf serum and 10% (v / v) antibiotic and antimycotic (gibco, invitrogen, usa) then infected with s. typhimurium as described by cano. with modification. briefly, 10 cfu of bacteria were added to the cell culture at 60 to 70% confluency in 24-well plates with each well containing 10 cell, multiplicity of 10 : 1 infection (bacterium / eukaryotic cell ratio). after incubation for 1 h, extracellular bacteria were killed by incubation for 2 h at 37c in dmem containing 100 g of gentamicin / ml. cells were washed three times in phosphate - buffered saline (pbs), ph 7.4, and lysed with 1% a set of primers were designed based on the consensus sequences for amplification of ha, na, and np genes of a / ck / malaysia/5858/04 (h5n1) (table 1). amplification was facilitated by an additional 6 bp to the 5 end of the primer. gene fragments of interest were amplified by gradient pcr using a pcr thermal cycler (mj research, ptc-225). briefly, in a 50 l volume, 25 l dh2o was mixed with 1.5 l of 10x reaction pcr buffer, 0.6 l of 50 mm mgcl2, 0.6 l of dntp mix (10 mm each), 10 l each of 100 mm forward and reverse primers, 0.3 l taq dna polymerase (5 u/l) (vivantis, malaysia), and 2 l (50 ng) template. the amplification was performed in a pcr thermal cycler using the following parameters : one cycle of 94c for 5 min ; 30 cycles of 94c for 1 min, 56.9c for 1 min, 72c for 2 min, final extension step of 72c for 10 min, and a hold cycle at 4c. pcr assays were conducted based on the detection of random amplified polymorphic dna (rapd) as described by gurakan.. briefly, in a 50 l reaction, 27 l dh2o was mixed with 10 l (50 ng) template, 0.6 l of 50 mm mgcl2 ; 1.5 l of 10x reaction pcr buffer ; 0.6 l 0f 10 mm deoxynucleoside triphosphate ; 0.3 u of taq dna polymerase (vivantis, malaysia) ; 10 l of 10 mm primer 5-cgt gca cgc-3. the reaction mixture was denaturated at 90c for 5 min followed by 35 [89c, 1 min ; 32c, 1 min ; 72c, 1.5 min ], an extension cycle at 50c for 3 min, and a hold cycle at 4c. the pcr product and loading dye were mixed and separated on 1% agarose gel with 1x tae buffer (promega, germany) at 80 v for 40 min. the targeted ha, na, and np genes in transfected salmonella were detected using specifically designed and labeled ha, na, and np probes (table 2) by fish. meanwhile, a specific probe for the 23s rrna of salmonella was used, sal3 (5-aatcacttcacctacgtg-3) [15, 16 ] to detect the attenuated s. typhimurium. the probe was synthesized and labeled with cy5 at the 5 end (nextgene, germany). briefly, salmonella and salmonella / pcdna3.1/ha, na, and np were fixed and hybridized using the protocol described by tabatabaei. with some modification. the cells (2 ml) were fixed in 6 ml of 3% paraformaldehyde / phosphate buffer saline. the fixed cell suspensions were diluted with ethanol / pbs mixture (1 : 1) and were spotted on coated glass slides (cel - line, new hampshire, uk). bacterial smears were covered with 20 l of lysozyme, incubated for 20 min at 30c. the slides were thoroughly rinsed with double - distilled water followed by air - drying at room temperature for the termination of enzymatic digestion. samples in 8 l of hybridization buffer and 1 l of sal3, ha, na, and np probe were separately applied to the wells on the slides and incubated. unbound probes were removed by rinsing the slide in 1 ml of washing solution. the slides were then incubated at 48c for 20 min in 50 ml of washing solution, rinsed briefly with distilled water, air - dried, and mounted with slowfade antifading reagent (molecular probes, or, usa). fluorescence was observed using confocal laser microscopy (mrc 1024es, biorad, usa). the inocula were prepared from typical salmonella (clear with black center on xlt4) colonies of an overnight culture, which were transferred 3 times in lb medium. the bacterial cells were collected by centrifugation at 5000 g for 10 min and serially diluted in sterile buffered peptone water (bpw) (ph 7.2) to a concentration of approximately 10 cfu / ml. eighteen - day - old spf eggs were purchased from malaysian vaccines and pharmaceuticals and reared under salmonella - free environment. cloacal swabs from one - day - old spf chicks were directly preenriched in bpw at 37c for 24 h, after which the samples were enriched by the addition of 1 ml of this suspension to 9 ml of rappaport - vassiliadis 10 broth (aes lab, india). following the incubation at 42c for 20 h, 100 l of this suspension was inoculated on xlt4 (aes lab, india) plates with novobiocin (25 g / ml)/nalidixic acid (20 g / ml) (no - na) of culture media and incubated for 20 h at 37c. newly hatched chickens were subjected to oral inoculation with attenuated salmonella using an autoclaved gavage plastic tube and a 1 ml syringe. the inocula were drawn through the gavage plastic tube, which was then dipped in glycerol for a smoother passageway down the chicken esophagus. a dose of 500 l containing approximately 10 cells was administered orally to each chick. chickens were monitored daily for clinical changes and were provided free access to water and a balanced unmedicated corn - soybean - meal - based mash layer diet that met or exceeded the national research council (nrc) requirements for nutrients. the experimental trials were approved by the animal care and use committee at the faculty of veterinary medicine, universiti putra malaysia, upm / fpv / ps/3.2.1.551/aup - r72. during the period of clinical observation, four chickens at day 3, 5, 7, 10, 14, 21, 28, and 35 were euthanized for sample collection. cecum, liver, heart, and spleen were collected in sterile condition and homogenized, and 10-fold dilutions were made in bpw starting from 10 to 10,000. from each dilution, the number of cfu / g of tissue was determined by counting the bacterial colonies. freshly collected blood were directly preenriched and enriched with bpw and rappaport - vassiliadis 10 broth (aes lab, india). one milliliter of this culture was then plated on no - na xlt4 and incubated for 20 h at 37c, and examined for growth. blood was collected for serum slide agglutination of antibodies to salmonella o antiserum group d1 (factors 1, 9, 12) (difco, becton dickinson, usa). plating results of s. typhimurium sv4089 on xlt4 provide good presumptive morphologic evidence for the presence of salmonella species. colonies on xlt4 plate typically are large, black with a creamy margin (after 30 h) and circular, with convex to umbonate / nippled elevations and entire margins. both the nontransfected as well as pcdna3.1, pcdna3.1/ha, pcdna3.1/na, and pcdna3.1/np transfected attenuated s. typhimurium sv4089 are no - na resistant. the average invasion rate obtained in standard 60 min infection was 1.2% 0.09 in mcf-7 and 0.5% 0.08 in mcf-10a cells. s. typhimurium sv4089 transfected with pcdna3.1/ha, na, np and pcdna3.1 were grown without antibiotic and each point represents the percentage of bacterial population that has retained the plasmid. the plasmid was stably detected over 90% of the attenuated s. typhimurium population after 100 generations of growth in antibiotic - free media (figure 1). pcr screening of the transfected salmonella showed that pcdna3.1/ha, pcdna3.1/na and pcdna3.1/np from transfected salmonella were successfully transformed into attenuated s. typhimurium cells separately. the viral ha, na and np genes of a / ck / malaysia/5858/04 (h5n1) virus were amplified from the respective transfected salmonella by pcr. figures 2 and 3 showed the expected sizes of the amplified product for ha (1.7 kb), na (1.5 kb), np (1.6 kb) and circular pcdna3.1 (5.1 kb), pcdna3.1/ha (6.8 kb), pcdna3.1/na (6.6 kb), pcdna3.1/np (6.72 kb), respectively. no salmonella - positive chickens were found. to test the possibility of colonization of s. typhimurium sv4089 into organs, salmonella was found in the spleen, liver, and cecum of the infected birds after 3 days of infection. the systemic counts of salmonella - positive peaked at 7 days after infection in all the selected organs following which they declined at day 14, where no bacteria were detected in those organs although salmonella showed good stability in cecum until day 35 (data not shown). no salmonella growth was detected in the heart. as expected, the control group was salmonella - free throughout the experiment. antibody against salmonella was detected by adding one drop of salmonella o antisera group d1 with one drop of the collected serum on a slide. although, salmonella were not detected in blood cultures, sera from inoculated chickens from as early as 7 days of vaccination up to 35 days were detected positive for salmonella - specific antibodies. under optimal hybridization conditions, transfected ha, na and np genes into salmonella were specifically visualized and detected using the corresponding probes (figure 4). meanwhile, salmonella was specifically detected using genus - specific probe, sal3 from homogenized spleen, liver and cecum sections of infected chicken, whereby a distinct fluorescent signal from rod - shaped bacteria could be detected following hybridization (figure 5). salmonella was detected from infected organs (spleen, liver and cecum) 3 days after inoculation. after two weeks, the infected chickens showed good clearance of salmonella from spleen and liver, while salmonella were detected in cecum even at 35 days after inoculation. detection of salmonella using pcr was performed using genus - specific primers for rapid screening of pure culture and extracted salmonella from tissue. as shown in figure 6, the rapd patterns contained three different bands, in which the size varied between 300 and 1,000 bp. the presence of 700 bp amplification product was found as specific polymorphic region for s. typhimurium. a major disadvantage of dna vaccine is its low efficiency, which is inherent in the way the vaccine is currently being administered. in addition, parenteral administration of dna vaccine is not practical in livestock animals including poultry that requires mass vaccination. thus, it is essential to develop carrier systems that improve efficacy of genetic vaccines. live bacteria that contain recombinant plasmids encoding heterologous antigen genes from other pathogens have the potential as oral delivery vectors for dna vaccines. the pathogenicity of bacteria such as s. typhimurium could be decreased significantly by various attenuation methods while still retaining their invasion capacity to deliver the heterologous genes into mammalian cells. a few studies in chickens have shown that s. typhimurium harboring heterologous genes were capable of eliciting specific humoral and cellular immune responses locally and at the systemic level [4, 6 ]. however, characterization studies on attenuated salmonella as dna vaccine carrier in chicken is limited. in this study, the characteristics of the attenuated s. typhimurium sv4089/harboring pcdna3.1, pcdna3.1/ha, na, and np gene were investigated. first, the transfected bacteria were genetically stable in vitro when passing on lb agar with or without antibiotic selection as well as on xlt4 agar. secondly, pcr, fish, and culturing on xlt4 were able to detect salmonella from homogenized spleen, liver, and cecum. no chickens displayed any clinical aberrations, side effects, and abnormalities after inoculation with the attenuated salmonella at dose of 10 cfu within 5 weeks after oral inoculation. previous studies have shown that the attenuated bacteria are not pathogenic to chickens at dose levels as high as 10 cfu / ml delivered orally [4, 6 ]. however, the virulent strain of the salmonella sl1344 has an oral ld50 of 10. salmonella colonize the small intestine and invade normally nonphagocytic epithelial cells in order to gain access to the underlying lymph tissue. invasion is mediated by a type iii secretion system (t3ss) encoded on salmonella pathogenicity island 1 (spi1). the spi1 t3ss forms a needle - like complex that is responsible for the injection of bacterial effector proteins into the host cell cytosol. these mechanisms enable serovar typhimurium to efficiently go through the intestinal epithelial barrier and distribute to deeper tissues. the lack of phop - phoq two - component system has always been linked to major intramacrophage survival defects and marked attenuation in the murine typhoid model. hence, human cancer cell lines are commonly used to study invasion of salmonella to eukaryotic cells. the attenuated salmonella used in this study still maintained its ability to invade mcf-7 and mcf-10a mammalian cells, where 1.2% and 0.5% of the cells were infected, respectively. in addition, in vitro study of salmonella serovar enteritidis phage type 4, strain 76sa88 showed invasion into t84 human colonic adenocarcinoma cell line up to 5.75%. meanwhile, the average invasion rate of salmonella enterica serovar typhimurium strains into hela cells and nrk-49f rat fibroblasts was up to 1 - 2% and 0.20.5%, respectively. in order to enhance the specificity and sensitivity of s. typhimurium sv4089 detection among the other enterobacteriaceae in the gastrointestinal tract, xlt4 media supplemented with no - na could serve as a selective medium for s. typhimurium sv4089. in addition, we showed that initial resuscitation of salmonella in selective medium with no - na followed by other detection methods such as pcr and fish is effective to avoid any - false positive results. previously, grakan. have used rapd analysis to detect s. typhimurium from other selected enterobacteriaceae and showed specific band for the detection of s. typhimurium is 700 bp. using similar approach, a 700 bp band was detected in smear and infected organs of s. typhimurium sv4089. the safety of a live vaccine and stability of an expression plasmid in a vaccine vector may potentially affect the efficacy of the vaccine and alter the outcome of vaccination. one of the limitations of using bacteria especially salmonella as a carrier is monitoring the stability and availability of bacteria without any cultivation. our study demonstrated that 5 monolabeled dna oligonucleotide is able to detect plasmid dna in prokaryotes without signal amplification - based or target amplification by in situ rt - pcr (figure 4). a 23s rrna fish method, using sal3 probe was used as a rapid and direct screening tool on attenuated s. typhimurium sv4089 in pure culture and tissue homogenates from chickens inoculated with salmonella (figure 5). the method allowed us to detect the bacteria from tissues of the inoculated chickens within approximately 7 h prior to the culture of salmonella (necessary time for fixation, hybridization, and observation of the sample) [2224 ]. in this study, rod - shaped salmonella was detected from homogenized spleen, liver, and cecum as early as 3 days after inoculation. the salmonella count peaked at day 7 although at day 14 no bacteria were detected in those organs except from cecum. previous study has shown that s. enteritidis aroa invades phagocytes of the liver, spleen, and bone marrow. clearance of s. typhimurium from the gastrointestinal tract occurred considerably later than the clearance from the spleen and liver [26, 27 ]. the results presented here indicate that although attenuated s. typhimurium sv4089 is able to move through different organs of chicken, it is not able to cause septicemia. in addition, sera from the inoculated chickens react strongly with o antiserum group d1 indicating the attenuated salmonella is a suitable vaccine candidate for oral vaccination. salmonella has been used as live vehicles for inducing protective responses to a wide variety of diseases since due to its ability to improve vaccine efficacy by targeting vaccines in vivo inductive sites on mucosal surface or internal organs. in addition, salmonella naturally possesses lps that can function as an adjuvant to stimulate systemic immune responses. in conclusion, live attenuated salmonella serovar typhimurium provides a unique alternative in terms of in vitro stability of transfected plasmid, in vivo stability, relatively inexpensive to produce in large scale, and easy detection by acting as a carrier in chickens.	attenuated salmonella has been used as a carrier for dna vaccine. however, in vitro and in vivo studies on the bacteria following transfection of plasmid dna were poorly studied. in this paper, eukaryotic expression plasmids encoding avian influenza virus (aiv) subtype h5n1 genes, pcdna3.1/ha, na, and np, were transfected into an attenuated salmonella enteric typhimurium sv4089. in vitro stability of the transfected plasmids into salmonella were over 90% after 100 generations. the attenuated salmonella were able to invade mcf-7 (1.2%) and mcf-10a (0.5%) human breast cancer cells. newly hatched specific - pathogen - free (spf) chicks were inoculated once by oral gavage with 109 colony - forming unit (cfu) of the attenuated salmonella. no abnormal clinical signs or deaths were recorded after inoculation. viable bacteria were detected 3 days after inoculation by plating from spleen, liver, and cecum. fluorescent in situ hybridization (fish) and polymerase chain reaction (pcr) were carried out for confirmation. salmonella was not detected in blood cultures although serum antibody immune responses to salmonella o antiserum group d1 factor 1, 9, and 12 antigens were observed in all the inoculated chickens after 7 days up to 35 days. our results showed that live attenuated s. typhimurium sv4089 harboring pcdna3.1/ha, na, and np may provide a unique alternative as a carrier for dna oral vaccine in chickens.
giardia duodenalis is an important cause of diarrhea in humans worldwide, especially in children, and is responsible for an estimated 2.0 million cases per year. the prevalence of this infection is higher in developing countries as the poor sanitary conditions favour the contamination of water and food with cysts. this parasite has been included in the neglected diseases initiative of the who due to its diffusion among children in these regions of the world, and being a significant cause of diarrhea and nutritional disorders in institutional and community settings. approximately 200 million people have symptomatic giardiasis in asia, africa, and latin america and about 500,000 new cases are reported each year according to the world health organization (who). giardiasis exerts a significant public health impact because of the high prevalence and disease burden of the infection and its propensity in causing major outbreaks and emergency responses. an important aspect of the epidemiology of giardiasis is to understand the host range of different giardia species and strains / genotypes, the potential for cross - species transmission, and risk environmental factors involved in the exposure of the pathogen. this is particularly important in determining the human disease burden attributable to parasites of animal origin and in determining the zoonotic potential of giardia infections in domestic animals. g. duodenalis, which is found in humans and many other mammals, including pets and livestock, is now considered a multispecies complex that comprises at least eight distinct genetic groups, referred to as assemblages a to h. the analysis of more than a thousand human isolates from different geographical locations, examined by pcr amplification of dna extracted directly from stool, has demonstrated that, in almost all cases, only g. duodenalis assemblages a and b are associated with human infections. in the course of these studies, it has been observed that the analysis of more than one gene is needed to address the occurrence of mixed infection in humans and the correlation between clinical symptoms and the type of giardia assemblage. the spectrum of clinical manifestations in human giardiasis is relatively variable, ranging from the absence of symptoms to acute or chronic diarrhea, dehydration, abdominal pain, nausea, vomiting, and weight loss. children might also suffer more serious consequences, including retarded growth and development, poor cognitive function, and detrimental effects on nutritional status. with regard to that issue, studies on the correlation between the assemblages and clinical symptoms have reported controversial results. some studies have pointed out that symptoms are more associated with assemblage a, while others have found that assemblage b infections are more likely to be symptomatic. (2014) in a group of children from la habana have found that children harboring assemblage b of giardia were more likely to have symptomatic infections than children with isolates from assemblage a [9, 10 ]. giardiasis is an endemic intestinal parasitic infection in cuba with prevalence rates ranging from 10 to 55% in the most recent studies, mainly in the capital and the western provinces of the country [1113 ]. in the last national survey of intestinal parasitic infections (ipi) carried out in 2009, giardia duodenalis was the most frequently identified pathogenic protozoan. since the identification of the genetical assemblage is considered a primary element in the study of human giardiasis, it is important to develop and apply, in laboratories with basic molecular equipment, single step pcr methods that allow one to detect and distinguish assemblages a and b and mixed infections from human faecal specimens simply by gel electrophoresis of the amplification products. that is why the present study was conducted to explore whether there is an association between assemblages and clinical manifestations by the performance of two conventional pcr tests and to identify epidemiological risk factors associated with this infection. a descriptive cross - sectional study was conducted on 639 children from 4 primary schools located in urban settings belonging to the plaza de la revolucin, playa, and boyeros municipalities, in la habana. they were subjected to a routine examination as part of a program of intestinal parasitic surveillance, from january 2013 to december 2013. in this work, we studied the frequency of g. duodenalis in children from preschool up to 4th grade from each school. cases in which coinfection with other intestinal parasites was found were not included in the investigation between infecting assemblages and clinical signs. stool collection containers were handed to the parents who were instructed to bring a fresh sample from the child on the day of examination. three stool samples from each child involved in this study were examined for intestinal parasites. a standard questionnaire was used to collect information on each child based on the demographic (e.g., age, sex, and number of household members), environmental (e.g., availability and types of toilets in the household, types of water supply, garbage disposal, and presence of domestic animals), personal hygiene (e.g., washing hands before eating, after defecation, and after playing with animals, washing vegetables and fruits before consumption, boiling water before consumption, and bathing place), and general health status of the participants (i.e., symptoms related to intestinal parasitic infections such as diarrhoea (defined according to who (1998)), nausea, vomiting, and abdominal pain and a history of receiving anthelmintic treatment). symptomatic children were defined as those children who had at least one of the symptoms mentioned above and included in the questionnaire, while the asymptomatic children were referred to the complete absence of symptoms. a portion of stool was examined at field by a wet smear stained with lugol 's iodine and followed by formalin ethyl acetate concentration techniques under light microscope at 10x and 40x magnifications. all the diarrheal stool samples were stained by modified acid - fast for cryptosporidium spp., cyclospora, and cystoisospora. the kato - katz smear method was used for species specific identification of parasite eggs, including ascaris lumbricoides, trichuris trichiura, and hookworm. all giardia - positive samples were stored in potassium dichromate (2.5%) and stored at 20c for molecular characterization. giardia cysts were purified and concentrated from stool samples in a sucrose gradient with a specific gravity of 0.85 m and then washed with distilled water, following the protocol described by babaei.. the cyst wall was disrupted by 810 freeze - thaw cycles in liquid nitrogen alternated with a 95c water bath. after that, purified cysts were mixed with 300 l of buffer lysis (50 mm tris - hcl, ph 7.5 ; 25 mm edta, 25 mm nacl, and 1% of sodium dodecyl sulphate (sds)) and vortexed. after adding 100 g / ml of proteinase k, the suspension was incubated at 56c for 2 h. the dna lysate was then treated with phenol / chloroform / isoamyl alcohol (24 : 24 : 1), followed by chloroform / isoamyl alcohol (24 : 1) according to sambrook and russell protocol (2001). the dna was precipitated by the addition of 1 ml chilled ethanol and stored at 20c until use. the dried dna was resuspended in 30 l distilled water and used as a template for pcr. dna molecular analysis was carried out using published pcr - relevant protocols [15, 19 ]. in brief, the genotyping analysis was performed using assemblages - specific primers that amplify, in the case of the pcr - tpi, a 148-bp fragment of the assemblage a and 81-bp fragment of assemblage b. the other pcr (pcr - e1-hp) amplifies a 165-bp amplicon for assemblage a and 272-bp fragment for assemblage b. the pcr reaction mixture was done using a amplitaq dna polymerase with geneamp 10x pcr buffer kit (applied biosystems, usa) in a total volume of 25 l and comprised 10 l of 10x pcr buffer (applied biosystems, usa), 0.2 mm of each deoxynucleoside triphosphate (dntp) (applied biosystems, usa), 1 u of taq polymerase (applied biosystems, usa), 0.4 m of each primer, and 5 l of dna template, with ultrapure water used as a negative control. the dna was amplified using a thermocycler (gene amp pcr system 9700, applied biosystems, usa). the pcr products were analyzed by 2% agarose gel electrophoresis, stained with 0.5 g / ml of ethidium bromide, and then visualized on a uv transilluminator (syngene, u : genius, belgium). dna from axenic cultures of g. duodenalis strains wb - c6 (assemblage a) and gs (assemblage b) was used as positive controls, while ultrapure water was included in negative controls. chi square test and proportion tests were employed to assess the significance of the associations. a multivariate logistic regression using introduction tests was used to determine whether each independent variable was significantly related to the outcome variable. the odds ratio (or) with 95% confidence intervals (ci) was used to approximate the relative risk associated with exposure. the research protocol was approved by the ethics committee of the pedro kour institute. written informed consent was obtained from parents / guardians for children to participate in the study. from a total of 639 children examined, 76 were infected with g. duodenalis for an overall prevalence of 11.9%. in two cases, dispar were recorded. from the 76 giardia - infected children included in the study, 41 (53.9%) were symptomatic and 35 (46.1%) did not present symptoms. no significant association was found between g. duodenalis infection related to sex, residing area, age, or development of diarrhea between symptomatic and asymptomatic children (p > 0.05) (table 1). the 76 stool samples microscopically positive for giardia were analyzed by pcr using two loci (tpi and e1-hp) as targets. from these samples, 63 (82.9%) and 58 (76.3%) scored positive in the pcr - tpi (figure 1) and pcr - e1-hp (figure 2), respectively, providing cumulated positivity of 85.5% (table 2). in total, 43 positive samples (56.6%) revealed genotyping results that were confirmed by both pcrs. eleven samples (14.5%) scored negative in both pcrs, 10 of which showed very few cysts in the coprological examination. g. duodenalis assemblage b was most frequently detected by both pcrs : 47.6% by pcr - tpi, and 67.3% by pcr - e1-hp. we detected only 7 cases (8.6%) of assemblage a - type infection confirmed by pcr - tpi and 5 cases (11.1%) by pcr - e1-hp only. interestingly, mixed infections consisting of both assemblages were found in a relatively high proportion (41.3% by pcr - tpi and 24.1% by pcr - e1-hp, resp.). we also focused on the analysis of the clinical significance of g. duodenalis infection in children that harbored giardia genotypes consistently identified in both pcrs (table 3). due to the low number of exclusive assemblage a - type infections detected in the child population, no statistical correlation between this particular assemblage and any symptom typical for giardiasis was possible. diarrhea was the only clinical characteristic statistically associated with infection by assemblage b of g. duodenalis (p < 0.05) at least as compared to mixed a + b infections. for the other variables analyzed, no correlation was found between symptoms and infection with specific assemblages. table 4 shows that univariate analysis reveals that omission of washing hands before eating (or = 1.6, 95% ci : 1.002.64 ; p = 0.04) and keeping dogs at home (or = 2.6, 95% ci : 1.235.36 ; p = 0.004) were significant risk factors for giardia infection. multivariate analysis using introduction test logistic regression ratified the association of contacts with dogs (or = 2.1, 95% ci : 1.114.76 ; p = 0.01) with this intestinal infection. giardia duodenalis, originally regarded as a commensal microorganism until 1978, is the etiologic agent of giardiasis, a gastrointestinal disease of humans and animals. transmission is either direct, through the faecal - oral route, or indirect, through ingestion of contaminated water or food. in developing, giardia infection is acquired during early infancy and its prevalence peaks at up to 30% in children younger than 10 years of age. apart from diarrhea, giardia infection in children in these countries can result in faltering of long - term growth and impairment of cognitive function [1, 20 ]. that is why it is important to assess the prevalence of this parasite in the child population, as well as the infecting assemblage, in order to elucidate the relationship between developed symptomatology and genetic groups of giardia that could lead to changes and reduce the need for treatment of children. previous studies demonstrated giardia as the principal intestinal protozoan that infects children around the country with prevalence rates reported from 10 to 55% [1113 ]. in this investigation, a frequency of 11.9% was found in all children included. this result is lower when compared with a recent study conducted by puebla. (2014) in symptomatic and asymptomatic children from la habana, which showed an overall prevalence of giardiasis at 22% (n = 452). that confirms the endemism of this disease in younger children and the relatively high prevalence of giardia infection in cuban children. the molecular characterization of giardia from stool samples yielded the amplification of 63 of 76 samples (82.9%) by the tpi gene and 58 of 76 (76.3%) by the assemblage - specific pcr assay reported by vanni.. here, g. duodenalis assemblage b parasites were preferentially detected in both pcrs (47.6% (pcr - tpi) and 67.2% (pcr - e1-hp), resp.). this observation is in accordance with most large - scale studies where the distribution of assemblage b was more commonly identified in developing (58%) than in developed countries (55%) and at a higher prevalence than assemblage a (37% versus 40%). in the present study, we found a high portion of mixed infections (41.3% by pcr - tpi and 24.1% by pcr - e1-hp, resp. ; see table 2) among the children studied. in a previous investigation recent studies using either conventional pcr with assemblage - specific primers or real time pcr including sequencing of the resulting amplification products showed a much larger degree of mixed assemblage infections in humans as previously reported [8, 24, 25 ]. the occurrence of mixed infections was also noted lately in some investigations using assemblage - specific tpi primers [26, 27 ], which allow detection of a much higher number of mixed assemblages a and b infections than approaches based on the use of more general primers for pcr [8, 25 ]. (2012) can even detect individual assemblages when they are 9 times underrepresented in the sample in relation to assemblage b. the reliable detection of cases of mixed infections is influenced by several factors, including the proportion of each assemblage in the specimen and biased amplification efficiencies of one assemblage over the other. in the present study, the use of the tpi gene for pcr was very important because it allowed detection of a higher number of mixed infections as compared to the pcrs designed by vanni. furthermore, the high variability of the tpi sequence (geurden., 2008) makes the respective pcr an ideal molecular tool for a differential diagnosis of assemblage a - type versus assemblage b - type infections. in the present study, inconsistent genotyping results in the pcrs applied were observed in 22 isolates (28.9%), representing a much higher number of such cases than previously reported (10 and 30%) [28, 29 ]. this discrepancy might be due to a bias of the pcr - e1-h towards a detection of assemblage b (see table 2). molecular characterization of g. duodenalis is complicated by our incomplete understanding of the genetics of this organism. many studies were based on the analysis of a single locus, particularly the 18s rdna locus. nowadays, through the application of multilocus pcr analysis there is a higher identification of mixed infections caused by assemblages a and b, and then inconsistent results can arise for assemblage (or even species) determination of given giardia isolates. inconsistencies in genotyping results were observed in both human and animal (particularly dogs but also cattle) isolates of g. duodenalis and can be formally explained by two distinct phenomena : (i) the presence of genetically different cysts in a faecal sample in combination with preferential amplification of a particular assemblage - specific marker gene compared to another marker gene indicative for a second assemblage (i.e., a true mixed infection followed by biased pcr amplification) ; or (ii) previous occurrence of sexual recombination among two giardia assemblages that had led to a mixed genotype as far as the two pcr targets are concerned. evidence of mixed infections with particularly high prevalences in infected individuals living in developing countries has been frequently provided [8, 30 ]. however, the second possibility also should not be underestimated because sexual genetic exchange between giardia isolates has been previously (and is currently) discussed as a realistic scenario. differences in symptomatology of giardiasis with different assemblages were initially described for dutch patients. here, assemblage a was associated with mild intermittent disease but assemblage b was associated with severe persistent disease. since then, several studies have reported correlations between assemblages and symptoms, but there has been a lack of concordance in the data obtained [9, 32, 33 ]. however, these findings are contrasted by data that counterindicate a relationship between symptomatology and the infecting assemblage [33, 34 ]. herein, we did a correlation analysis between giardia assemblages and symptoms manifested in children. in the 43 children with identical genotyping results, we found that assemblage b was significantly more associated with diarrhea (p < 0.05) when compared with mixed infections by both assemblages. more data are needed to determine whether mixed infections could reduce symptoms induced by assemblage b. in previous studies done in our country by pelayo. (2014), a close association between assemblage b and symptomatic school children was found. however, the high proportion of mixed infections reported in both investigations further complicated the attribution of symptoms to infection with a specific assemblage. compared to our previous studies [9, 10 ], our present data indicate a decline in the prevalence of assemblage b in symptomatic children although those studies had different overall prevalence rates of giardia and the genetic characterization employed different markers. nonetheless, assemblage b still has to be considered as the predominant assemblage within the giardia - infected children in our country. conversely, in this survey only a statistically insignificant number of assemblage a - type infections were found, thus excluding an association of this assemblage with a certain symptomatology. the prevalence of giardia is strongly associated with a variety of risk factors related to the host, such as sociodemographic, environmental, and zoonotic conditions. since water plays a major role in giardia transmission, the quality of water is an essential parameter for risk factor assessment in giardiasis. apart from that, hygienic behaviour of children must be considered as a main risk factor of this disease. surprisingly, however, most studies on hand hygiene related to intestinal parasites undertaken so far have not placed special emphasis on giardiasis. referring to the clinical significance of g. duodenalis among children in our country, we decided to investigate the abovementioned risk factors as well as various other epidemiological parameters regarding their relevance for transmission of this disease. finally, omission of washing hands before eating and keeping dogs in - house turned out to be the only factors that were significantly associated with higher giardia infection rates. these results are in agreement with recent findings in school children populations from developing asian countries [37, 38 ]. the zoonotic transmission of g. duodenalis has gained increasing evidence, particularly as far as the role of domestic animals is concerned. furthermore, it has been reported that dogs can harbor either zoonotic or canine - specific giardia assemblages. there is clear evidence that cysts of zoonotic giardia do contaminate the environment in areas where the potential for zoonotic transmission exists. in the past, many epidemiological studies on giardiasis were able to identify a risk for zoonotic transmission of the disease. however, evidence of how frequently this phenomenon occurs requires intense focal studies in defined endemic areas where transmission dynamics and range of hosts involved in giardia infections are known. considering the interest in unraveling the complex questions about the epidemiology of g. duodenalis, studies have focused on application of molecular methods to gain insight into genetic diversity of isolates and the public health significance of this, mainly in populations living in developing countries. the present study provides information on the giardia assemblages associated with human infection and, more specifically, the association of epidemiological parameters with the infection of giardia in school children. further studies on the epidemiology of giardiasis, especially investigations of risk factors associated with giardia assemblages, subassemblages, and genotypes, would help to better understand the characteristics of this disease. such information will certainly be highly beneficial for the development of prevention and control strategies in giardiasis. in conclusion, giardiasis is one of the most frequent intestinal parasitic infections in cuban children. determination of g. duodenalis assemblages is a valuable approach to understanding the complex dynamics underlying transmission of giardia infections. in our study, assemblage b turned out to be the predominant genetical group in cuban school children, but also a high frequency of mixed infections was found. omission of washing hands before eating and keeping dogs at home were identified as significant risk factors of acquiring a g. duodenalis infection. in order to verify the possibility of zoonotic transmission and the potential of household pets as reservoir hosts, further large - scale studies involving microscopical coprology and pcr - based, multilocus genotyping of giardia isolates in respective faecal samples are recommended and to get a few sequences combined with a phylogenetic analysis to see how the cuban isolates are related to giardia isolates identified in other places in the world. these investigations are expected to provide in future studies definite evidence for zoonotic transmission in giardiasis.	giardia duodenalis is considered the most common protozoan infecting humans worldwide. molecular characterization of g. duodenalis isolates has revealed the existence of eight groups (assemblages a to h) which differ in their host distribution. a cross - sectional study was conducted in 639 children from la habana between january and december 2013. two assemblage - specific pcrs were carried out for the molecular characterization. the overall prevalence of giardia infection was 11.9%. dna from 63 of 76 (82.9%) samples was successfully amplified by pcr - tpi, while 58 from 76 (76.3%) were detected by pcre1-hf. similar results by both pcrs were obtained in 54 from 76 samples (71%). according to these analyses, assemblage b and mixed assemblages a + b account for most of the giardia infections in the cohort of children tested. our current study identified assemblage b as predominant genotype in children infected with giardia. univariate analysis indicated that omission of washing hands before eating and keeping dogs at home were significant risk factors for a giardia infection. in the future, novel molecular tools for a better discrimination of assemblages at the subassemblages level are needed to verify possible correlations between giardia genotypes and symptomatology of giardiasis.
clinical improvement in the community acquired pneumonia (cap) should occur within 3 to 5 days. non - resolving pneumonia is defined as pneumonia with a slow resolution of radiologic infiltrates or clinical symptoms despite adequate antibiotic therapy. then we should exclude the associated factors causing the systemic or local immunodeficiency [intra - bronchial obstruction, smoking, diabetes, chronic obstructive pulmonary disease, malignancy, concomitant human immunodeficiency virus (hiv) infection, alcoholism or addictions, immunosuppressant therapy ] and complications responsible for the delayed resolution (empyema, necrotizing pneumonia, metastatic spread of infection and bacterial super - infection). however, we may rarely encounter some unexpected cause of a non - resolving pneumonia. histoplasmosis is a fungal disease caused by histoplasma capsulatum, named after darling who first described this clinical entity well in details. the endemic area includes the ohio and mississippi river valleys, central and south america, and microfoci in the eastern united states, southern europe, africa, and southeast asia. scattered cases of cutaneous, laryngeal and disseminated histoplasmosis in immunosuppressed persons have been reported from india. we herewith report, from eastern part of india, a geographically rare and unexpected case of progressive disseminated histoplasmosis (pdh) in a 17-year - old immunocompetent male patient that presented as non - resolving pneumonia. a 17-year - old male patient, student and nonsmoker, consulted a local physician at murshidabad, west bengal with high grade intermittent fever with chills but no rigors, cough with scanty mucoid expectoration, dull aching chest pain, and generalized weakness for 5 days. the patient 's chest x - ray postero - anterior (pa) view at that time showed middle lobe consolidation [figure 1a ]. the patient was treated as cap with oral amoxicillin + clavulanic acid (625 mg) 8 hourly and levofloxacin (500 mg) daily was added afterwards. as the patient 's condition deteriorated with documented weight loss of 5 kg in 2 weeks, his father brought him to our hospital. on examination, the patient was very toxic with blood pressure 90/60 mmhg, pulse rate 130/min regular, respiratory rate 32/min with moderate pallor. we treated the case as non - resolving pneumonia and did antibiotics adjustment with the intravenous cefoperazone - sulbactam (1.5 g) 8 hourly and oral clarithromycin (500 mg) twice daily along with the other supporting treatment. the investigations revealed hemoglobin 6.4 g%, total white cell count 15200/mm with 94% neutrophils ; fasting blood sugar 86 mg / dl, urea 45 mg / dl, creatinine 0.8 mg / dl, total bilirubin 0.96 mg / dl, alanine transaminase 78 u / l, aspartate transaminase 82 u / l and alkaline phosphatase 238 u / l. routine urine examination was normal and culture revealed no growth after 72 h of incubation. enzyme - linked immunosorbent assay (elisa) for hiv 1 and 2 was non - reactive. the patient 's condition deteriorated further with appearance of right - sided pleural effusion [figure 1b ] and hepatosplenomegaly. examination of pleural fluid showed cell count of 560/mm with 70% lymphocytes, protein 3.4 gm / dl, sugar 46 mg / dl, lactate dehydrogenase 600 u / l and adenosine deaminase 36 u / l. the patient 's contrast enhanced computed tomography (cect) of thorax showed organization of right middle lobe consolidation with enlargement of right paratracheal and subcarinal lymph nodes with central necrosis [figure 1c ]. fibre - optic bronchoscopy revealed pus coming from the right main bronchus and a gelatinous mass in the posterior wall of trachea and a widened carina with constricted right main bronchus [figure 2 ]. bronchoscopic biopsy from the mass showed inflammatory granulation tissue with proliferation of capillaries and infiltration of a large number of inflammatory cells. gomori 's methenamine silver stain showed fungal spores morphologically resembling histoplasma capsulatum [figure 3a ]. bronchoalveolar lavage (bal) and transbronchial needle aspiration (tbna) from subcarinal lymph node also showed the presence of capsulated round microorganism on a background of inflammatory exudates compatible with histoplasma capsulatum. the patient was treated with intravenous amphotericin b 50 mg / day for 1 week followed by oral itraconazole 200 mg twice daily. significant clinical improvement was noticed by 7 days and follow - up chest x - ray after one month showed a significant radiological resolution [figure 3b ]. chest x - ray pa view showing the right middle lobe consolidation (a) and the right sided pleural effusion with consolidation (b) ct scan thorax with contrast showing the necrotic lymph nodes in paratracheal and subcarinal sites (c) fibre - optic bronchoscopy showing the wide carina with constricted right main bronchus and pus coming out from the right main bronchus fungal stain of bronchial mucosal biopsy specimen showing the fungal elements suggestive of histoplasma capsulatum (a) and follow - up chest x - ray pa view (b) after 1 month of treatment showing improvement of the right lung opacity in respect to pretreatment chest x - ray incidence of non - resolving pneumonia was found to be 10% to 15% among hospitalized patients with cap and of them mortality of un - resolving pneumonia ranges from 27 to 49%. approximately, 20% of presumed non - responding cap had noninfectious etiology. old age, multilobar pneumonia, pneumonia severity index more than 90, legionella pneumonia, gram negative pneumonia and discordant antimicrobial therapy were the common responsible for non - resolving pneumonia. non - resolving pneumonia was found to be responsible for 15% of inpatient pulmonary consultations and 8% of bronchoscopies. in a recent study from south india tuberculosis (tb) was the cause of non - resolving pneumonia in 35.7% cases and malignancies were responsible for another 27% cases, whereas western literature has showed malignancies being responsible for up to 11% cases of non - resolving pneumonia. histoplasmosis is caused by inhalation of the fungus histoplama spp, and the degree of infection and clinical presentation are determined by the size of inoculum, immune status of the individual and presence of underlying lung disease. like tb primary histoplasmosis heals spontaneously in about 99% of cases and only a few progress to pdh. adrenal gland involvement, lesions of oral mucosa, gastrointestinal tract and skin may be seen in 5 to 10% of cases. chronic pulmonary and progressive disseminated histoplasmosis are often fatal with reported mortality 83 - 100% in untreated cases and 7 - 23% in amphotericin b treated patients. in case of appropriate clinical context, disseminated histoplasmosis should be considered in both immunocompromised and immunocompetent patients, in endemic or non - endemic areas. in our case the diagnosis of histoplasmosis was established by histopathological examination of tracheal lesion, bal fluid and tbna results, positive culture from sputum and therapeutic response to anti - fungal treatment. the case was more interesting because no factor responsible for immunosuppression could be demonstrated in the patient and pdh presenting as non - resolving pneumonia has not been reported in literature.	histoplasmosis, a fungal disease caused by histoplasma capsulatum, is endemic in north and south america. except few scattered cases, the disease is considered to be a non - entity in india. furthermore, disseminated histoplasmosis is rare in the immunocompetent individuals. we report an adolescent boy presenting as middle lobe consolidation which did not respond to antibiotics. his condition deteriorated with the development of mediastinal lymphadenopathy, pleural effusion and hepatosplenomegaly. a diagnosis of progressive disseminated histoplasmosis was established by his clinical findings as well as bronchoscopic biopsy, transbronchial needle aspiration cytology and bronchoalveolar lavage culture demonstrating histoplasma capsulatum. the case represents a unique example of progressive disseminated histoplasmosis in an immunocompetent individual in india.
congenital adrenal hyperplasia (cah) is an inherited disorder in which adrenal glands produce excessive amount of androgens.. however non - classical cah are chromosomal females (46xx) born with normal genitalia becoming symptomatic only during adolescence causing hirsutism, acne, menstrual irregularity and rarely gender dysphoria. the frequency of the classical disease has been estimated in europe and the united states, ranging from 1/5,000 to 1/115,000. it is postulated that a general psychiatrist is unlikely to see more than one or two cases in lifetime. a young stocky built adult patient appearing in early twenties dressed in shirt and pant with a thick moustache and beard accompanied with parents walked inside psychiatry out - patient department (opd) with a referral from plastic surgery opd concerning gender affirmation surgery. t, a 21-year old female patient and expressed desire to be named as mr. t, in further conversation. on interview, parents reported patient to be their eldest daughter born of a non - consanguineous marriage following a full term normal vaginal delivery. parents reported since early childhood, patient was tomboyish, more comfortable playing with boys, watching wrestling and never showed interest in dolls or playing with younger sister or girls at school. patient used to wear shirt and skirt to school, but at home used to prefer wearing t - shirt and jeans. from adolescence onwards, patient started to gain weight, developed hirsutism and never attained menarche. patient started to develop strong disliking towards self being called as a female, withdrew self from female friends and interest in studies declined ; but never verbalised these feelings to anyone. thereafter, with parents consent patient started to dress as males, stopped removing facial hairs. from 18 years difficulties faced were using common urinals, feeling ashamed of getting the anatomical sex being disclosed and inability to study further or procure a white collared job because of high school certificate mentioning sex as female. despite these problems, patient was more comfortable in male gender role and since past 2 years, started seeking help for gender affirmation surgery. patient appeared to be of average intelligence and had no persistent aggressive, violent or criminal tendencies. external body habitus was of male and external genitalia was of female [figures 1 - 3 ]. electrolytes, liver function test, lipid profile, thyroid function test, insulin, prolactin, follicle stimulating hormone, luteinizing hormone, estrogen, cortisol were within the normal limit. 17-oh progesterone and dehydro - epiandrosteine were elevated and showed complete suppression with low dose dexamethasone suppression test suggesting diagnosis of non - classical congenital adrenal hyperplasia. hirsutism, masculine body habitus and female external genitalia breast development : tanner stage iii female pattern pubic hairs : tanner stage iii ultrasound showing female internal genitalia gender identity describes psychological recognition of self as well as wish to be regarded by others as fitting into the social categories of male or female. these social categories generate expectations of gender roles that is how one is expected to behave in the society. gender dysphoria refers to persistent sense of one 's gender identity being in discord with anatomical sex and may be accompanied by gender discordant behaviour as was present in our case. without knowledge of ms. t 's intersexuality, presentation was most consistent with gender identity disorder of diagnostic and statistical manual - iv, text revision (dsm - iv - tr). she had strong and persistent cross - gender identification, persistent discomfort with her natally assigned sex and clinically significant distress or socio occupational impairment. ms t was diagnosed with non - classical cah, a mild variant of one of the common causes of female pseudohermaphroditism. non - classical cah is an autosomal recessive genetic disorder due to deficiency of 21-hydroxylase enzyme, being more common in ashkenazi jews, hispanics and italians. it is also called attenuated, late - onset, acquired and cryptic adrenal hyperplasia. the age of 13 years is taken as an arbitrary dividing line between early and late onset cases to discriminate pre and post pubertal onset. non classical forms have more commonly been associated with hirsutism, acne, menstrual irregularities, but rarely with gender identity crisis as in our case. available research on cah focuses more on sexual orientation than gender identity. in a sample of 62 swedish females with cah, frisn. reported more male - dominant occupations (30%), greater interest in rough sports (74%), non - heterosexual orientation (19%) whereas other psychosexual parameters assessed were comparable to controls. studied 82 women with non - classical cah and reported gender dysphoria in 2 and sex change in1, rest all were comparable to controls in terms of gender identity. berenbaum. did not find any difference in gender identity of the 43 women with cah studied when compared to controls. in adults presenting with gender dysphoria it requires (1) a thorough medical history and physical examination, (2) karyotyping and (3) a thorough psychiatric examination. only after ruling out t 's case was by virtue of male external body habitus, no hormonal treatment was required along with gender affirmation surgery. however, post - operatively, ms. t will continue to face legal issues of change in name and sex on birth certificates, identity cards. tista das is currently the only transsexual in india to have a valid voter identity and ration card. indian laws regarding marriage, adultery, sexual offences, adoptions, succession and industrial laws will require modifications when dealing with these individuals. a social, legal and legislative change is the need of hour.	gender identity is the sense of belonging that one feels for a particular sex psychologically and socially, independent of one 's biological sex. there is much less systematic data on gender identity in females with congenital adrenal hyperplasia (cah). we report a case of non - classical cah presenting as a case of gender identity disorder.
recent studies have identified assisted reproductive treatments and infertility as risk factors. however, neither a definite mechanism nor clear risk factors were identified and therefore prevention strategies are yet unavailable. in this article, we present a case of ovarian pregnancy occurring following in vitro fertilization treatment and a fresh embryo transfer. the couple was diagnosed with unexplained infertility and no identifiable risk factors for extrauterine pregnancy. the diagnosis of ovarian pregnancy was made during explorative laparoscopy performed due to suspected extrauterine pregnancy. ovarian pregnancy is an infrequent and a challenging diagnosis. yet, late diagnosis and lack of appropriate intervention may have long - term implications. several mechanisms and risk factors are proposed, and their acknowledgment may improve early diagnosis and prevention of complications. it is well established that fertility treatments including assisted reproductive technology (art) increase the risk of ectopic pregnancy.1 ovarian pregnancy is a very rare complication of both spontaneous and art - induced pregnancies ; the estimated incidence is 0.5%3% of all ectopic pregnancies.1,2 ovarian pregnancy can be explained by one of the following two mechanisms : direct fertilization of an anovulated mature egg inside the ovary or retrograde embryo migration back into the ovary via the fallopian tube. accordingly, ovarian pregnancy is classified as primary, follicular fertilization, or secondary, embryo reflux through the tube.3 in in vitro fertilization (ivf), embryos are transferred to the uterus with ultrasound guidance. therefore, ovarian pregnancy in ivf most likely occurs via retrograde migration of the embryo through the tube and implantation in the ovary.4 however, the remote possibility of fertilization of an intrafollicular anovulated egg during intercourse around the time of egg retrieval can not be ruled out. early detection of ovarian pregnancy is challenging, and in many cases, the diagnosis is made during an urgent surgical intervention due to acute abdominal pain or bleeding. the diagnosis is usually confirmed when chorionic villi are detected within the ovary. in this case report the couple, a 33-year - old female patient and her 33-year - old male partner both with unremarkable medical history, underwent fertility investigation for primary infertility for more than one year. her day 3 serum follicle stimulating hormone (fsh) was 11 iu, estradiol (e2) 177 pmol / l, anti - mllerian hormone 1.6 ng / ml, and the rest of the hormonal measurements were normal. the patient had a sonohysterogram that demonstrated an intramural fibroid with a diameter of 2 cm that did not interfere with uterine cavity and patent tubes with normal spillage. the couple had three cycles of ovarian stimulation with good response (letrozole followed by recombinant fsh) combined with intrauterine insemination ; however, no pregnancy was achieved. her ivf treatment cycle was done using an antagonist protocol. on day 3 of the cycle, the patient began injections of 300 iu of recombinant fsh daily (puregon ; merck canada inc.). was started on day 7 of stimulation and was combined with a 100 iu of human chorionic gonadotropins (hcgs ; pregnyl ; merck canada inc.). pmol / l on the day on which she was given 10,000 iu of hcg (pregnyl ; merck canada inc.) to trigger ovulation ; 13 eggs were retrieved, 10 of them were mature. the eggs were fertilized with intracytoplasmic sperm injection ; six blastocysts developed, of which five were frozen and a single embryo grade 4aa was transferred on day 5. first hcg, two weeks later, was 329 iu and increased to 941 iu in two days. two weeks later, the patient was admitted with acute left abdominal pain. upon admission, she had normal vital signs and a tender abdomen. a transvaginal ultrasound demonstrated a large amount of blood and blood clots in the pelvis. no intrauterine pregnancy was observed, and a suspicious mass was seen in the left adnexa. the tissue was removed from the ovary, and the edges of the ruptured follicle were confirmed to be clear. the patient was instructed to have serial measurements of serum hcg concentration until it declines to zero. in a follow - up visit in our clinic, the patient was feeling well and symptom free. a frozen embryo transfer cycle was planned ; it will be a medicated cycle with estrace and intramuscular injection of progesterone expecting that the latter will achieve a more quiescent uterus. the pathological report confirmed the presence of chorionic villi in the ovary, validating the diagnosis of an ovarian ectopic pregnancy. its incidence is reported to be 0.5%3% of all extrauterine pregnancies (1:30001:7000 of all pregnancies).2,46 some reports suggest that the apparent increase in the incidence of ovarian pregnancy is the result of improved diagnostic tools.6,7 presumably, some of the ovarian pregnancies are mistakenly diagnosed as tubal pregnancies and treated conservatively or medically without a final confirmation of the exact location.8 due to the scarcity of ovarian pregnancies, it is hard to demonstrate a causal relationship between art and the occurrence of ovarian pregnancy. one study among ivf patients showed a higher frequency of ovarian pregnancy comprising 4.5%6% of extrauterine pregnancies and 0.35% of all clinical pregnancies.1 however, there may be a detection bias since ivf pregnancies are usually monitored earlier and more carefully than natural pregnancies ; and therefore, short - lived ivf - induced ovarian pregnancies have a higher likelihood of being detected. the ovarian pregnancy case presented here is a result of fresh embryo transfer of single embryo. the most likely explanation to its occurrence is a retrograde migration of the blastocyst through the tube and implantation into the ovary, possibly through one of the aspiration needle puncture sites. ovarian pregnancy presents with similar symptoms to tubal pregnancy symptoms, namely, delayed menses, vaginal bleeding, and abdominal pain ; however, asymptomatic ovarian pregnancies were also described.9 as with other extrauterine pregnancies, an empty uterus and rising serum hcg should raise suspicion ; however, preoperative diagnosis is not common as the sonographic appearance might mimic a corpus luteum, a hemorrhagic ovarian cyst, or a tubal pregnancy. koo did, however, report about seven cases of confirmed preoperative diagnosis. in 1868, these criteria are based on intraoperative findings : (1) intact fallopian tube on the ipsilateral side, (2) the gestational sac is in the same position as the ovary, (3) the ovary is connected to the uterus by the utero - ovarian ligament, and (4) the ovarian tissue is present in the wall of the gestational sac.8 ovarian pregnancy is more common in fertile women;6 however, no other clear risk factors for ovarian pregnancy were reported. nonetheless, several publications reported an increased rate of ovarian pregnancies that was associated with certain clinical conditions. choi suggested that spontaneous ovarian pregnancies were related to the concurrent use of intrauterine device. a possible association was suggested between pelvic inflammation reaction secondary to a pelvic inflammatory disease or surgical intervention and ovarian pregnancy.11 it should be noted that a high concentration of estradiol, as reported in this case report, may promote pelvic inflammation. factors associated with extrauterine pregnancy in ivf are tubal infertility, fresh embryo transfer compared to frozen,1214 cleavage stage compared to blastocyst stage embryos, and large number of transferred embryos.15 interestingly, several studies had reported an increased ectopic pregnancy rate following ovarian stimulation only.15,16 as mentioned earlier, several authors described an increased risk of ovarian pregnancy among fertile patients undergoing ovulation induction treatment17 as well as ivf treatment.1821 apart from the aforementioned risk factors, possible explanations for the increased risk of ovarian pregnancy are injection of a high volume of transfer media and the patient in a tilted position.19 oliveira described a possible association between blastocyst transfer and intraovarian implantation, as they had no cases of ovarian pregnancies following transfer of embryos on days 2 and 3. surprisingly, several cases of ovarian pregnancies were described following in vitro fertilization - embryo transfer (ivf - et) in patients who had previously undergone bilateral salpingectomy. the exact mechanism for these is unclear but should encourage further evaluation of other potential pathophysiological mechanisms for the formation of ovarian pregnancies following fresh and frozen embryo transfer.22 an interesting proposed mechanism was described by shan.23 according to this suggestion, the cause for ovarian pregnancy is pelvic inflammation that involves the ovary. consequently, the inflammation of the ovary may affect the process of detachment of the cumulus - oocyte complex and prevent the release of the ovulated oocyte, leading to fertilization of the mature oocyte by the sperm arriving through the fallopian tube inside the ruptured follicle.22,23 despite its uncommon occurrence, perhaps ovarian stimulation and the resulting high estradiol concentration as well as the oocyte retrieval induce an inflammatory process in the ovary, which affects the oocyte release. combined with intercourse close to the time of oocyte retrieval may lead to spillage of sperm via the tubes through the puncture site of the ovarian follicle, leading to intrafollicular fertilization. several studies have indicated a possible relationship between supraphysiologic levels of estradiol in art cycles and ectopic pregnancy.15 in their study, wang found a higher prevalence of ectopic pregnancy following fresh embryo transfer compared to frozen embryo transfer, a subanalysis of the results showed more ectopic pregnancies among patients with peak serum estradiol concentration exceeding 4085 pg / ml (15,000 pmol / l).24 the peak serum estradiol for the patient presented in this case report did exceed this threshold (19,584 pmol / l). a possible mechanism for the effect of high estradiol concentration is provided by the following observations : it has been described that elevated estradiol leads to alteration in the normal physiologic function of the fallopian tube, increasing the likelihood for extrauterine implantation.25 these effects include impaired protein secretion,26 ciliary motion frequency,27 embryonic motility,28 and implantation.29 we can assume that the impact of high estradiol on the normal function of the tube can lead to nontubal ectopic pregnancy such as ovarian pregnancy in certain patients. another hypothesis that attempts to explain ovarian pregnancies post bilateral salpingectomy states that during embryo transfer an incidental puncture of the uterus occurs, and the embryo descents through the microscopic tract, resulting in ovarian pregnancy.30 a case report by hsu described a corneal fistula in patients who had previous salpingectomy. no abnormal findings during surgery and no history of any uterine manipulation were reported in our case. since imaging and serum hcg measurements are of limited contribution to the diagnosis of ovarian pregnancy, the american society for reproductive medicine recommends on a surgical intervention in any case of suspected ovarian pregnancy.32 surgical intervention may begin as explorative laparoscopy for suspected extrauterine pregnancy. the surgical treatment for ovarian pregnancy may include salpingo - oophorectomy, oophorectomy, wedge resection, and removal of gestational product. as long as the patient is stable, the aim should be to attempt to keep as much ovarian tissue as possible and avoid compromising ovarian reserve. medical intervention with i m methotrexate (mtx) injection is a reasonable option in hemodynamically stable patients with preoperative diagnosis of ovarian pregnancy. it should be noted that unlike treatment with mtx for tubal pregnancy, the use of mtx to treat ovarian pregnancy is not as well established. kudo and shamma and schwartz34 described a successful treatment for ovarian pregnancies with systemic mtx, while mittal showed promising results with an intragestational sac mtx injection. in stable patients with decreasing levels of hcg, expectant management with no intervention the risk for recurrence is very low, as only one case of recurrent ovarian pregnancy had been described thus far.9 we present a case of ovarian pregnancy following a fresh embryo blastocyst transfer that was managed surgically by removal of the gestational sac from the ovary while preserving the ovary. ovarian pregnancy is a challenging diagnosis ; however, clinicians should be aware of the possible risk of ovarian pregnancy in art treatment. it should be actively pursued in high - risk patients in order to prevent complications.	backgroundovarian pregnancy is a rare and challenging clinical phenomenon. recent studies have identified assisted reproductive treatments and infertility as risk factors. however, neither a definite mechanism nor clear risk factors were identified and therefore prevention strategies are yet unavailable.clinical casein this article, we present a case of ovarian pregnancy occurring following in vitro fertilization treatment and a fresh embryo transfer. the couple was diagnosed with unexplained infertility and no identifiable risk factors for extrauterine pregnancy. the diagnosis of ovarian pregnancy was made during explorative laparoscopy performed due to suspected extrauterine pregnancy. the patient had normal intra- and postoperative course.conclusionovarian pregnancy is an infrequent and a challenging diagnosis. yet, late diagnosis and lack of appropriate intervention may have long - term implications. several mechanisms and risk factors are proposed, and their acknowledgment may improve early diagnosis and prevention of complications.
careful monitoring of anchorage is one of the most important factors in successful orthodontic treatment. to obtain stable anchorage, osseointegrated implants have been used to obtain absolute anchorage without the need for patient cooperation. however, osseointegrated implants require a precise 2-stage protocol and considerable time for osseointegration. in addition, these implants are expensive and there is a limited area for their insertion due to their size. kanomi first introduced miniscrew implants (msis), which can be placed almost anywhere, in either the maxilla or the mandible, with a simple procedure. over time, the ease of placement and removal, effectiveness in anchorage without patient cooperation and benefit of their low cost has increased the popularity of these devices. many studies and successful clinical cases have been published describing the use of msis for orthodontic anchorage. however, msis can untimely be lost due to their mobility during the orthodontic treatment. therefore, the stability of msis must be further improved to prevent these failure rates. with msis, the reason for this stability is believed to be a result of mechanical interlocking. because msis are not osseointegrated, their anchorage potential is most likely influenced by the quantity of bone into which they are placed. in addition to bone quality and quantity, surgical technique and screw geometry are factors that affect primary stability. because clinicians have little control over the bone quantity available for screw placement, due to the presence of roots and anatomic landmarks, screw geometry and surgical technique remain the parameters to be improved for the success of the procedure. several in vitro studies have been conducted to enhance the bone - screw contact area and increase the force resistance and stability (anchorage) of miniscrews by changing the length and width of the screws. for the purpose of increasing the contact area at the bone - screw interface and obtaining maximum support from the cortical plate, instead of using wider screws or bicortical screws, we designed a new apparatus, a mini implant ring (mir), placed at the neck of the screw. the purpose of this in vitro study was to evaluate the effects of this mir on the anchorage force resistance (afr) and the stability of orthodontic msis. what is the effect of the mir apparatus on the afr and stability relative to cortical bone thicknesses (cbt) ? forty - eight self - drilling, titanium (ti-6al-4v) grade 5 cylindrical msis (tm, trimed, ankara, turkiye), 1.6 mm in diameter and 9 mm in length, were used for the study [figure 1 ]. the 24 mirs used in the study group had outer diameters of 5 mm and inner diameters of 2 mm [figure 2a c ]. they had four spines on the side, which were in contact with the cortical bone surface. the spines were 0.75 mm of length and made of titanium (ti-6al-4v) grade 5. a hole in the mir was designed to fit to the neck of the msi. a schematic and a clinical application of the mir the miniscrew implants used in the study (a, b) lateral and top views of the mini implant ring (mir). (c) dimensions of the mir diagram showing the application of the mini implant ring miniscrew, applied with the mini implant ring that was used in a case for en masse retraction bovine ilium was used as the bone model. the cortical thickness of the bone segments ranged from approximately 0.5 mm to 2.5 mm from the iliosacral joint toward the hip joint. these values are similar to the mean values of the cortical thicknesses of the human maxilla and mandible. the bone segments were sliced and eight bone slices, which had a minimum width of 5 mm, were obtained. care was taken to place the adjacent miniscrews, one from the control group and another from the study group, to ensure even distribution of the cortical thicknesses of the bone segments in both groups [figure 5 ]. miniscrew implants on bone slices and adjacent msis inserted, one from the control group and the other one from the test group the msis were inserted with the use of a handled screwdriver (trimed, ankara, turkiye) until the distance from the bone to the screw collar was 2 mm. in the mir group, the mir was placed with the help of a hand instrument, called an mir pusher (trimed, ankara, turkiye), for the spines to penetrate the cortical bone. in both groups, after insertion of the msis, final screwing, to 1 mm from final insertion depth, was performed using a torque screwdriver (n2 dpsk, nakamura, mfg co. ltd.) and the maximum insertion torque (mit) values were measured for each msi. the remaining 1 mm distance left after final insertion was the space left for the clinical soft - tissue. in the mir group, after insertion of all of the msis, the bone slices were sectioned into small blocks, which had a minimum of 4 mm of bone tissue around each screw [figure 6 ]. embedding the bone - screw block using a custom - made positioning device a device was designed for positioning the bone - screw block during the embedding in acrylic resin so that the msis were aligned perpendicular to the axis of mechanical testing. the msis were subjected to a tangential force load perpendicular to the screw using an instron test machine adjusted to a crosshead speed of 0.05 mm / s. during the loading, the displacement of the screws was measured up to a distance of 0.6 mm, which represented the adequate displacement without slippage that would result in clinical screw mobility and potential failure. after the torque measurement upon removal, the samples were sectioned through the center of the miniscrew hole to examine the cbt. the mean cortical thicknesses of the bone samples for the mir and control groups were 1.22 0.49 mm and 1.23 0.49 mm, respectively. each group was divided into two subgroups and these subgroups were designated as thick or thin according to whether their cbt was greater or less than 1.15 mm [table 1 ]. intergroup comparison of the cbt statistical tests were performed with the statistical package for the social sciences (spss) software, (spss inc., chicago, il, usa) version 15.0. a power analysis (gpower version 3.1.0, universitt kiel, germany) revealed that a sample size of 24 for each group would provide 95% power to detect significant differences at a significance level of p = 0.5. group differences for afr, removal torque and cbt were assessed by the independent samples t - test and mit was studied with the mann - whitney u - test. the chi - square test was performed to determine the differences in mobility. when p 0.05). intergroup comparison of the afr msis inserted with mirs showed significantly greater insertion torque values when compared with msis inserted without mirs (p 0.05). bone specimens with thick cortical bone had significantly greater removal torque values than specimens from the thin subgroups (p 0.05). cbt had an effect on the mobility of the miniscrews in the control group (p 0.05). the mean cortical thicknesses of the bone samples for the mir and control groups were 1.22 0.49 mm and 1.23 0.49 mm, respectively. each group was divided into two subgroups and these subgroups were designated as thick or thin. the mean cbt for the thick subgroups was significantly greater than that for the thin subgroups (p 0.05). msis inserted with mirs showed significantly greater insertion torque values when compared with msis inserted without mirs (p 0.05). bone specimens with thick cortical bone had significantly greater removal torque values than specimens from the thin subgroups (p 0.05). cbt had an effect on the mobility of the miniscrews in the control group (p 0.05). the stability of these small - sized appliances depends on parameters such as the properties of the hard and soft - tissues, screw design, insertion procedure and the amount of force applied. however, the key determinant for stationary anchorage is the quality and quantity of the bone into which the msis are placed. evaluated the effect of cbt on the success of msis and concluded that the insertion site should have a cbt of at least 1 mm. stated that when using msis in patients with a high mandibular plane angle, special care should be taken in the presence of thin cortical bone to avoid failures. it has been observed that the more screw - cortical bone contact there is, the greater stability and resistance to failure there will be. therefore, an appliance, the mir, was designed, which increased the cortical bone surface area in contact with the anchorage unit. in this study, the mir is a ring designed to increase the surface contact area of msis with cortical bone. nalbantgil., using finite element analysis, concluded that the spines on the miniplates were highly efficient in reducing the stress on the fixation screws. in general, with conventional screw implants, the load concentration has a tendency to occur on the first threads, leading to increased stress on the surrounding cortical bone and possibly resulting in resorption. if the load can be delivered to a larger bone surface, then the damage, which is relative to stress over an area, might be reduced. as this study confirms, the greater area of distributed load is, the greater the msi 's stability and resistance to loading will be. msis with larger diameters provide greater surface area, which increases torque and afr as a result of increased friction and greater support at the bone - to - screw interface. however, the insertion of an msi with a diameter greater than 1.6 mm needs careful consideration for placement and inserting these msis safely is generally very difficult because of anatomic limitations. these authors mentioned that if the range of cbt between groups is not too small, the effect of cbt on stability can be demonstrated. in our study, there was a significant difference in the thickness of the cortical bone between the groups. in addition, the anchorage potentials of the control group displayed significant differences between the subgroups with thin and thick cortical bones. however, the anchorage potentials of the subgroups with thin and thick cortical bones did not show any significant difference in the mir group. for the mir group, our hypothesis is that the mirs were so effective that the thickness of the cortical bone did not vary sufficiently to demonstrate an effect. as expected, a significant difference in insertion torque was found between the control and the mir group. an increase in the insertion torque was expected due to the penetration of the spines into the cortical bone. to prevent an undesirable increase in insertion torque, a hand instrument, which we called the mir pusher, was designed and used to push the mirs into cortical bone. the mean insertion torque for the mir group was 15.30 n / cm. this force was within the limits recommended by previous studies concerning success rates and was less than the limit necessary to avoid complications, such as the breakage of the msi. the main purpose of this study was to determine how much the mir would increase the force anchorage values of msis. we also wanted to determine the effects of mir appliances on mit and mrt because each of these measurements is a determinant of primary stability. insertion torque analysis was used to measure the mechanical retention achieved by screwing and removal torque analysis is a method used to assess the stability and the osseointegration capacity of msis. we did not want to experience any unwanted increases or decreases in terms of mit, mrt or mobility. we found a significant (p < 0.01) difference in terms of mit between the control and mir groups (this difference was within the desired limits) and we observed an improvement in terms of mrt and mobility, but the difference was not statistically significant. within the limits of this in vitro study, these findings suggest that the mir appliance increased the force anchorage resistance of msis without causing any biomechanical side - effects. in this in vitro study, however, in vivo reactions to immediate loading of msis can be replicated with in vitro studies because msi stability is believed to result from mechanical interlocking and it does not require a period for osseointegration. the bone samples used in this study, which were obtained from the ilium of bovines, enabled us to standardize the testing conditions. in the clinic further studies are needed to evaluate the biologic responses of soft- and hard - tissues as well as the success rate of screw anchorage, when mirs is used. within the limits of this in vitro study, the results of this investigation demonstrated that the newly designed appliance (mir) increased anchorage resistance and insertion torque, thereby increasing the primary stability and anchorage resistance of msis. the mir might have a favorable effect on msi stability in patients presenting with thin cortical bone.	objective : an appliance was designed to increase the cortical bone surface contact area of miniscrew implants (msis). the purpose of this in vitro study was to evaluate the effects of this appliance on the anchorage force resistance and the stability of orthodontic msis.materials and methods : a total of 48 msis were placed into bone specimens prepared from the ilium of bovines. half were placed with the newly designed apparatus and half were placed conventionally. all the specimens were subjected to tangential force loading perpendicular to the msi with lateral displacement of 0.6 mm, using an instron universal testing machine. the maximum removal torque of each tested specimen was also recorded. both study and control groups were divided into two subgroups based on whether they had thin and thick cortical bone.results:the test group had statistically higher force anchorage resistance and maximum insertion torque values than the control group (p < 0.001). the results were found to be more significant in cases in which the cortical bone was thin (p < 0.001).conclusions : within the limits of this in vitro study, the present findings suggest that the newly designed apparatus might have a favorable effect on msi stability in patients presenting with thin cortical bone. clinical studies are necessary to confirm the results that were observed in vitro.
radiofrequency catheter ablation (rfca) of idiopathic premature ventricular contractions (pvcs) is widely accepted as a safe and reliable therapy 1, 2. idiopathic pvcs are commonly originating from the right and left ventricular outflow tract (rvot and lvot, respectively), although a parahisian origin has been described 3, 4, 5, 6. in the 12lead electrocardiogram (ecg), parahisian pvcs can mimic an origin from the posterior rvot 3. in such cases, careful analysis of the intracardiac electrograms and mapping of both sides of the interventricular septum and of the aortic root is required to determine the optimal ablation site. a 71yearold man with ischemic cardiomyopathy who previously underwent secondary prevention defibrillator (icd) implantation was admitted with repeated episodes of sustained ventricular tachycardia (vt) resulting in antitachycardia pacing and shocks. on admission, the ecg showed sinus rhythm (sr) with normal qrs complexes (duration 87 msec) and repolarization, and bigeminal monomorphic pvcs (qrs duration 148 msec, inferior axis, left bundle branch block morphology with transition in v4, fig. echocardiography demonstrated reduced left ventricular (lv) ejection fraction (0.45) with akinetic basal and medium segments of the inferior wall and an additional septal dyskinesia. during telemetric ecg monitoring, frequent monomorphic pvcs, often bigeminal, were observed, and in the icd 's recordings, the pvc burden was > 20%. of note, the intracardiac electrograms stored during the vts and the pvcs had different morphology. after obtaining informed consent, an electrophysiology study was performed under fasting conditions and conscious sedation. after substrate modification ablation, confined to the lv inferior wall, we concentrated on mapping the bigeminal pvcs using conventional and 3d mapping (ensite navx, st jude medical, st. ablation was performed using an irrigated tip catheter (thermocool, biosense webster, diamond bar, ca, usa) in a temperaturecontrolled fashion 1035 watt (w), 42c. the pvcs were mapped starting initially in the rvot, but the earliest activation was observed in the parahisian region (preceding the qrs onset by 27 msec, fig. pacemapping here was attempted but without good results because of direct capture of the specific conduction system even using low output. application of radiofrequency (rf) energy here, increasing the power from 20 to 30 w, quickly eliminated the pvcs, without signs of av nodal conduction impairment. mapping of the right parahisian region did not show nearfield early activation or unipolar signals with steep downslope of the qs anymore, and additional rf applications here did not affect the pvcs. earliest activation (46 msec to the onset of the qrs) was seen under the right coronary aortic cusp (rcc) close to the membranous septum, where a hisbundle potential > 0.5 mv was recorded (fig. careful rf application at 10 w was attempted, but immediately stopped because of fast junctional beats. rf application was then tried in a slightly more apical position with up to 25 w without affecting the pvcs (fig. 3a). an intermediate position between the two application sites, with local activation (both in the bipolar and the unipolar recording) preceding the qrs onset by 44 msec, was attempted despite a hisbundle potential (0.15 mv) on the distal ablation bipolar recordings (fig. 3b). as shown in figure 3c, during initial rf energy application at 10 w however, after carefully increasing the power to 25 w, after 60 sec, the ah time increased from 60 to 97 msec, and as soon as rf application was stopped, the pvcs reappeared, and, gradually, the ah interval returned to the baseline value. another careful map of the right septum was performed, and a good combination of bipolar and unipolar signals was observed only on the midseptal tricuspid annulus (fig. 4a) close to the fast pathway area, where, despite lowenergy application, rf resulted in fast junctional beats. 4b). despite relatively late local activation (20 msec to the onset of the qrs), temporary elimination of the pvcs was achieved relatively late (40 sec) after the initiation of rf delivery at 35 w, with immediate reappearance of the pvcs after termination of energy application. although the patient already had dual chamber pacing capabilities, further rf applications were not given for the presumed high risk of av block. (a) morphology of the first pvc (qrs duration 148 msec, inferior axis with humps on the inferior leads). (b) earliest activation is seen at the rv septum above the hisbundle (preceding the qrs onset by 27 msec). in the position where a hisbundle potential is registered, the bipolar signal precedes the qrs onset by 25 msec with a slow downstroke slope in the unipolar signal ; close above, an earlier bipolar signal with rapid qs on the unipolar and no hisbundle potential is found. (c) two multipolar catheters are placed via the femoral vein at the right ventricular apex and at the hisbundle ; a third one is inserted via the internal jugular vein into the coronary sinus. right (30, left panel) and left (50, right panel) anterior oblique radiographic views show the ablation site. the activation time is the same on both sides of the interventricular septum, but the unipolar signal has a more rapid downstroke slope of the qs on the left compared to the right side. (c) radiographic position of the catheters shown in b. of note, abnormal lv substrate was confined to the inferior wall, and no low amplitude and/or fractionated potentials were seen on the septum. (a) unsuccessful attempt slightly more apical in the lv (far field hisbundle on the ablation proximal) despite early local activation time (42 msec) in the bipolar signal (but late beginning of the qs complex in the unipolar). (b) successful ablation site (44 msec, lowvoltage hisbundle on the ablation distal). (c) immediate disappearing of the pvcs at this position b with inadvertent ah prolongation despite lowenergy application after 60 sec. (a) last attempt on the rv septum (46 msec). in sr, on the distal ablation are recorded both atrial and ventricular signals with an hisbundle potential, while the proximal bipolar shows only an atrial signal without ventricle 11. despite low energy, fast junctional beats immediately occur. (b) local activation in the rcc (20 msec to the onset of the qrs). (c) computed tomography scan showing the relationship between the right, the left septum, and the aortic root. (d) 3d mapping of pvc 2 showing similar activation times on both sides of the interventricular septum. to the best of our knowledge, this is one of the very few cases of pvc rfca with a highamplitude hisbundle potential at the site of earliest activation. ecg findings suggesting a parahisian origin of pvcs are (1) a relatively narrow qrs with an inferior axis, (2) a monophasic rwave in lead i, (3) a qs pattern in v1, and (4) a relatively low rwave amplitude in the inferior leads, with lower rwave amplitude in lead iii than in lead ii 3, 4. in some cases, a superior axis with left or right bundle branch block pattern has also been described 5, 6. furthermore, there are no reported criteria to differentiate a right from a leftsided parahisian focus. despite these ecg criteria, as the present case shows, it can be challenging to identify a parahisian origin, especially if the pvc morphology is relatively broad and there is late transition. in these cases, the diagnosis of parahisian pvcs is mostly realized during the electrophysiologic procedure. careful and extensive mapping of both sides of the septum and of the aortic root is required, and initial ablation should ideally be attempted as far away as possible from the hisbundle in order to minimize inadvertent av node injury. the distance between the ectopic focus and the hisbundle and the av node, respectively, is of critical importance. in the largest published series on parahisian pvcs, a hisbundle potential was only registered on the proximal ablation electrode pair at the successful ablation site, whereas rfca was avoided when a hisbundle electrogram > 0.1 mv was recorded at the site of earliest ventricular activation. therefore, the reported rates of damage to av conduction 3, 4, 5, 6 may underestimate the true risk. recently, the bordeaux group described a very didactical case of successful elimination of parahisian pvcs 7. in that case, the initial rf application from the lvot and the aortic root did not affect the pvcs ; the ectopic focus was located on the right interventricular septum toward the distal hisbundle, where early local activation with qs unipolar electrogram morphology was seen, and the pvcs could be eliminated without damaging the av node. as discussed by the authors, the av block risk is high but the hisbundle is isolated from the adjacent myocardium by sheaths of fibrous tissue that presumably contribute to protection during rf energy application. in contrast, the av node is not surrounded by fibrous tissue 7, 8. furthermore, the left hisbundle emerges into the lvot immediately adjacent to the membranous septum, whereas the right bundle runs through deeper layers of the septum before emerging more apically on the surface 8. most likely, the ectopic focus in our case had a deeper location in the septum rather than being located endocardially, as it was possible to affect the pcvs (i.e., change of the morphology and temporary elimination) from both the right and left side of the septum as well as from the aorta but it was not possible to achieve permanent elimination from any of these locations. in contrast, in the presence of a right subendocardial focus as well as in cases of parahisian accessory pathways 9, an incremental rf energy strategy will be safe and effective. furthermore, close proximity of the focus to the hisbundle is suggested both by the intracardiac signals and by the ability to affect the pvcs only in combination with av node injury (ah prolongation and/or fast junctional beats) despite low rf energy. cryoenergy may be an option to reduce the rate of av block although the acute and longterm efficacy under these circumstances remains to be determined 10. on the other hand, a more detailed mapping approach using different electrodes sizes and interspacing might help in precisely defining the location of the focus, as well as intracardiac echo would allow direct visualization of the region of interest. radiofrequency catheter ablation of parahisian pvcs is challenging and may be associated with a high complication rate and usually requires a compromise between success and inadvertent av node injury. a careful approach with extensive mapping on both sides of the interventricular septum as well as in the aortic root and an incremental energy strategy starting away from the hisbundle may be prudent. finally, in some cases, complete abolition of the pvcs may only be achievable at the price of av block. jude medical, biosense webster, boston scientific, medtronic, astrazeneca, and pfizer, and has received speaker fees from st. jude medical, boston scientific, medtronic, biotronik, astrazeneca, bayer, berlin chemie, boehringer ingelheim, novartis, meda, pfizer, and sanofi aventis. all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, and/or revision of the manuscript.	key clinical messagecatheter ablation of parahisian premature ventricular contractions (pvcs) still represents a challenge and is a compromise between success and inadvertent av block. we describe a possible strategy to address pvcs from this location with highamplitude hisbundle potentials at the site of earliest activation.
the thermal springs harbor population of microorganisms with great commercial importance and interest to the researchers and industry working on enzymes, sugars, compatible solutes and antibiotics. diversity analysis of such extreme environments has got ample attention due to their diverse and unique ecology, chemistry and opportunity they provide to identify rare compounds and genes. despite the possibility of the presence of novel microbes with high economic and industrial use very few reports are available on the microbial diversity of hot springs from the indian subcontinent. cultivation - dependent studies are valuable for isolating novel organisms and exploring their properties but the cultivation independent methods offer a more comprehensive assessment of microbial diversity. the study was designed to investigate community composition by using metagenomic approach and a clone library based study of hot spring microbial mat samples. the findings revealed the presence of a large number of sequence reads from bacterial taxa arthronema which may represent novel species within this genus which was reported for the first time in association with hot spring. india is one of the tectonically active areas in the world inhabiting many hot springs. the jakrem hot spring (2524.450 n and 9132.414 e) is one of the less explored hot water springs in meghalaya, india. the temperature and ph of the hot spring are reported to be 46 c and 9 respectively. the only report available on this less explored hot water spring revealed the presence of species of mastigocladus and microcoleus of cyanophyceae. in the present investigation, the total community dna was isolated from the microbial mat samples of the spring using a fastdna spin kit (mp biomedicals, llc, usa). final dna concentration was quantified using a microplate reader (bmg labtech, jena, germany). the v3 region of the 16s rrna gene was amplified using 341f/518r primer combination (5cctacgggaggcagcag 3 ; 5attaccgcggctgctgg 3). amplicon was excised and purified by qia quick gel extraction kit (qiagen, valencia, ca, usa) according to the manufacturer 's manual. quality check on raw sequences was performed as per base quality score distributions, average base content per read and gc distribution in the reads. singletons, the unique otu that did not cluster with other sequences, were removed as it might be a consequence of sequencing errors and can result in spurious otus. chimeras were also removed using uchime, and pre - processed consensus v3 sequences were grouped into operational taxonomic units (otus) using the clustering program uclust at a similarity threshold of 0.97,. all the pre - processed reads were used to identify the otus using qiime program for constructing a representative sequence for each otu. the representative sequence was finally aligned against greengenes core set of sequences using pynast program. representative sequence for each otu was classified using rdp classifier and greengenes otu database and the sequences of those not classified were categorized as unknown. in the sample studied, the classified otus represent 13 distinct phyla dominated by firmicutes (61.60%), chloroflexi (21.37%) and cyanobacteria (12.96%) (fig. firmicutes grow chemo - organo - trophically on a number of organic substrates particularly in alkaline springs. firmicutes constituted 138 otus i.e. 19.88% of total otus and 5, 40,441 reads which are 61.59% of the total reads. chloroflexi constitutes 58 otus i.e. 8.35% of total otus and 1, 87,546 reads which is 21.37% of total reads whereas 237 otus i.e. 34.14% of total otus belong to the unknown phylum substantiating the earlier findings that non - acidic hot springs harbor more diverse and variable bacterial phyla than the acidic springs. prominent families in the metagenome were clostridiaceae (60.92%), chloroflexaceae (21.26%) and pseudanabaenaceae (12. a total of 44 genera including unknown bacterial genus (63.50%), chloroflexus (21.22%) and arthronema (12.69%) were obtained. the existence of significant percentage of arthronema sequences in the metagenome suggests that the microbial mat pigment richness in the microbial / algal mat of the hot spring may have massive commercial value as natural colorants in nutraceuticals, cosmetics and pharmaceutical industries. at the species level analysis, 12 bacterial species were recognized with the prominent presence of unknown species (99.38%) and rhodococcus fascians (0.44%). more than 99% of the unknown species strongly suggest that the jakrem hot spring harbors the wealth of uncultivable bacteria.	jakrem hot water spring is located in the west khasi hill district of the state of meghalaya, and is one of the most popular hot springs of the state. there is a populist belief among the inhabitants and people that the hot spring water has got curative properties against various skin ailments. this is the first report on v3 hyper - variable region of 16s rdna metagenome sequence employing illumina platform to profile the microbial community of this less known hot spring from meghalaya, india. metagenome comprised of 10, 74,120 raw sequences with a sequence length of 151 bp and 56.35% g + c content. metagenome sequence information is now available at ncbi, sra database accession no. srp056897. a total of 8, 77, 364 pre - processed reads were clustered into 694 otus (operational taxonomical units) comprising of 14 bacterial phyla including unknown phylum demonstrating 49 families. hot spring bacterial community is dominated by firmicutes (61.60%), chloroflexi (21.37%), cyanobacteria (12.96%) and unclassified bacteria (1.2%) respectively.
the recent national birth statistics reported a 15-year low in the rates of preterm birth (ptb) in the united states of america : 11.53%. this prevalence can be further stratified on the basis of ancestry where african - americans have the highest (16.53%), followed by native - americans (13.25%) and latin americans (11.58%). despite the overall nationwide drop in prevalence, the march of dimes (mod) 2013 ptb report card has deemed florida a grade d, having one of the highest prevalence (13.7%) of ptb in the united states. this could be due to the highly diverse population found in florida and a lack of clear public policies which could improve these rates. ptb is defined by the world health organization (who) as live births prior to the completion of 37 gestational weeks (gw), based on the mother s last menstrual cycle. currently, ptb accounts for up to 75% and 80% of perinatal morbidity and mortality, respectively,. of the worldwide 8 million infant deaths a year, approximately 1734% are attributable to ptb. there is a group of women at risk for sptb. from the total infants that survive after being delivered prematurely, about 1015% are significantly disabled. preterm infants are born with multiple short- and long - term health complications including bronchopulmonary dysplasia, necrotizing enterocolitis, retinopathy of prematurity, cognitive impairment, and an increased risk for adult onset diseases such as hypertension and diabetes [8, 9, 10 ]. another less well known complication is that when infants are born preterm, there is an interruption of normal renal organogenesis involving the vascular tree and kidney branching increasing the susceptibility of the surviving infants to develop hypertension and renal disease as adults. considering the vast amount of health conditions associated with ptb, a considerable amount of money is invested in the care of preterm infants in the usa which totals approximately $ 26 billion per year. the mod reports that the average cost of care for a preterm infant is 10 times greater than for a full - term (ft) infant. based on gw, ptb can be categorized into extreme (12th65.448.864.764.6prenatal care 0.1). socioeconomic differences is a known risk factor for ptb. in our study, this was only observed upon stratifying for iptb and sptb [or = 0.51 (95%ci = 0.260.99), p = 0.045 ] indicating lower education levels to be associated with sptb. on the contrary, access to prenatal care is important as it was shown to be a strong risk factor to ptb compared to tb (p < 0.001). however, no difference was observed in women with either sptb or iptb [or = 2.12 (95%ci = 0.509.57) ]. in table 2, we summarized the histological findings in women with iptb and sptb. women with sptb were twice as more likely to have chronic villitis during the histologic examination of theirs placentas. chronic villitis, defined by the presence of a lymphohistiocytic infiltrate in chorionic villi, is associated to preterm delivery, fetal growth restriction, and recurrent pregnancy loss. a recent study by rudzinski, gilroy, demonstrated that chronic villitis may represent host - versus - graft rejection by the mother. this finding points toward a possible role of the maternal immune system in the sptb. in our cohort, chronic villitis was present in 4.2% of iptb, 9.3% of sptb suggesting a tendency to happen in spontaneous preterm labor when the placentas had chronic villitis. the remainder of the studied variables was not significant, even upon stratifying for minority populations (data not shown). it is important to obtain quality phenotype data and characterize deliveries based on clinical and histological findings (clayton, sappenfield. our study confirms that race is an important risk factor in ptb. considering that hispanics have the highest birth rates and are the fastest growing population in the united states, the lower rates of morbidity and mortality indicate an interesting epidemiological paradox [1, 34 ]. it would therefore be of interest to further characterize this population by deep phenotyping or high throughput genotyping methods to elucidate this paradox in hispanics. another promising theory is the immunological reaction depicted in some preterm placentas with chronic villitis of unknown etiology since the fetus has a mixed genetic makeup from the father and the mother and the chronic inflammation in chorionic villi and sometimes in the decidua can represent a maternal immunological response to paternal antigenic stimulation. limitations in this study include the low number of samples being analyzed due to restrictions to collect prospective data maintained by the irb. we could increase statistical power by increasing the number of years for retrospective sampling, but could not collect prospective maternal information. it would be interesting to replicate the findings of this paper in either accumulating multiple years of birth outcomes of tertiary care centers in south florida. another limitation includes the vast socioeconomic and ancestry admixture in our studied cohort which were possibly confounding factors for determining significant associations. in conclusion, we found some demographic and physiological risk factors associated with ptb and histologically, the spontaneous preterm birth group had twice as many cases of chronic villitis as the induced preterm birth group suggesting an immunological maternal response to fetal paternal antigens.	we aim to identify the link between placental histological findings and obstetric reports to determine possible risk factors of spontaneous preterm birth (sptb). we prospectively ascertained birth records and outcomes from all deliveries in our hospital in 1 year. records were used to determine and stratify for either full - term or preterm [spontaneous or indicated (i) ] deliveries. we analyzed for risk factor association using 2 tests and common odds ratio estimates (spss v21.0). our cohort totaled 6088 deliveries : 236 iptb, 43 sptb, and 5809 term births. largely hispanic, we determined race, parity, prenatal care access, preeclampsia, gestational diabetes, and bmi to be highly associated with sptb (p < 0.01). histologically, placentas of women with sptb were twice as likely to have chronic villitis. we found that chronic villitis is associated with sptb. results of this study can be used in increasing the understanding of sptb.
sixty - three participants of the parenteral and oral insulin dpt-1 trials whose diabetes was diagnosed with two consecutive diabetic 2-h ogtts (initial and confirmatory) are included in the analyses. those in the intervention arm of the parenteral insulin trial (n = 41) were excluded because the parenteral insulin was received (as per protocol) between the two diabetic ogtts. also excluded were those (n = 8) whose interval between the two ogtts was greater than 3 months. the algorithm for determining risk in the dpt-1 has been described previously (9). participants were considered to have a 5-year risk above 50% and be eligible for the parenteral insulin trial if either the first - phase insulin response on intravenous glucose tolerance testing was below a defined threshold and/or there were ogtt abnormalities. if those metabolic criteria were not present but insulin autoantibodies were positive, the 5-year risk was considered to be 2650% and participants were eligible for the oral insulin trial. participants in the parenteral insulin trial intervention group received recombinant human ultralente insulin, whereas those in the oral insulin trial intervention group received recombinant human insulin crystals. the dose of oral glucose was 1.75 g / kg (maximum, 75 g carbohydrate). samples were obtained for plasma glucose and c - peptide measurements in the fasting state and at 30, 60, 90, and 120 min. insulin measurements were not obtained because there was concern over the formation of insulin autoantibodies. individuals with glucose values in the diabetic range at a routine visit were asked to return for confirmation by an ogtt within 60 days (some returned beyond 60 days) unless an ogtt was clinically contraindicated. participants were to continue the same study regimen they had been using before the initial diabetic ogtt. the age at the first of the diabetic ogtts was considered the age at diagnosis. the thresholds for diabetes were fasting glucose values 126 mg / dl and/or 2-h glucose values 200 mg / dl. the interassay coefficient of variation for the c - peptide assay was 6.9% in a reference pool with relatively high values and 7.8% in a reference pool with relatively low values. fasting c - peptide values in the undetectable range (< 0.2 ng / ml) were assigned a value of 0.1 ng / ml for the analyses. the statistical significance of percent change against a null hypothesis of no change was assessed with signed - rank tests. values for rates of change in peak c - peptide were obtained by dividing the difference in peak c - peptide values for an interval by the length of the interval. sas (version 9.1.3 ; sas institute, cary, nc) was used for the analyses. participants in the parenteral insulin trial intervention group received recombinant human ultralente insulin, whereas those in the oral insulin trial intervention group received recombinant human insulin crystals. the dose of oral glucose was 1.75 g / kg (maximum, 75 g carbohydrate). samples were obtained for plasma glucose and c - peptide measurements in the fasting state and at 30, 60, 90, and 120 min. insulin measurements were not obtained because there was concern over the formation of insulin autoantibodies. individuals with glucose values in the diabetic range at a routine visit were asked to return for confirmation by an ogtt within 60 days (some returned beyond 60 days) unless an ogtt was clinically contraindicated. participants were to continue the same study regimen they had been using before the initial diabetic ogtt. the age at the first of the diabetic ogtts was considered the age at diagnosis. the thresholds for diabetes were fasting glucose values 126 mg / dl and/or 2-h glucose values 200 mg / dl. the interassay coefficient of variation for the c - peptide assay was 6.9% in a reference pool with relatively high values and 7.8% in a reference pool with relatively low values. fasting c - peptide values in the undetectable range (< 0.2 ng / ml) the interassay coefficient of variation for the c - peptide assay was 6.9% in a reference pool with relatively high values and 7.8% in a reference pool with relatively low values. fasting c - peptide values in the undetectable range (< 0.2 ng / ml) were assigned a value of 0.1 ng / ml for the analyses. the statistical significance of percent change against a null hypothesis of no change was assessed with signed - rank tests. values for rates of change in peak c - peptide were obtained by dividing the difference in peak c - peptide values for an interval by the length of the interval. sas (version 9.1.3 ; sas institute, cary, nc) was used for the analyses. all had a complete ogtt in the diabetic range that was confirmed by a second complete ogtt within an interval of 3 months. of these, 31 were in the parenteral insulin trial and 32 were in the oral insulin trial (15 in the intervention group). the mean sd age at the first diabetic ogtt was 13.2 6.9 years. there was a tendency for glucose levels to increase between the first diabetic ogtt and the confirmatory diabetic ogtt with statistically significant increases at 90 (p = 0.006) and 120 min (p < 0.001) and for auc glucose (p = 0.016). table 2 shows the c - peptide levels for the initial and confirmatory ogtts. there were significant declines in c - peptide levels at each postchallenge time point and for auc and peak c - peptide values (p < 0.01 for all). the median percent change in peak c - peptide levels was 14.3% (p < 0.001). there was less of a decline in fasting c - peptide levels (6.7%, p = 0.416). when the fasting c - peptide to fasting glucose and the auc c - peptide to auc glucose ratios were examined, percent changes were appreciable for both the former (10.3%, p = 0.046) and the latter (16.7%, p < 0.001). the change in auc glucose values between the two diabetic ogtts was positively associated with the length of the interval between them (r = 0.32, p = 0.011), whereas there was an inverse correlation of change of peak c - peptide levels with that interval (r = 0.31, p = 0.014). thus, the fall in peak c - peptide levels increased with longer intervals. a scatter plot for the association of the change in peak c - peptide levels between the ogtts and the interval between the diabetic ogtts (with the removal of an outlier) is shown in fig. the correlation was almost identical (r = 0.31, p = 0.016) with the outlier excluded. with an allowance for the peak c - peptide levels from the first diabetic ogtt, the slope for the association of change in peak c - peptide levels with the interval between the diabetic ogtts was 0.56 ng ml month. of the 63 individuals included in the analysis, 55 had an ogtt 6 months before the initial diabetic ogtt. the median percent change for the peak c - peptide in that interval was 14.0% (p = 0.052). the percent change in the peak c - peptide from the last nondiabetic ogtt to the second diabetic ogtt (mean sd interval 7.5 1.3 months) was 23.8% (p < 0.001). the auc c - peptide to auc glucose percent change was even more marked (45.7%, p < 0.001) in that interval. figure 3 shows the median rates of change in peak c - peptide levels over intervals in the perionset period. the values were obtained by dividing the difference in peak c - peptide values for an interval by the length of the interval. there was minimal change (0.00 ng ml month, p = 0.468, n = 36) in peak c - peptide from 12 to 6 months before diagnosis. there was a decline in peak c - peptide levels from 6 months before diagnosis to diagnosis (0.10 ng ml month, p = 0.059, n = 55) and an even greater rate of decline from diagnosis to within 3 months after diagnosis (0.43 ng ml month, p = 0.002, n = 63). the data in this report show that, on average, c - peptide levels decreased substantially in the interval from diagnosis to 3 months after diagnosis. these changes occurred even with glucose levels still in a range associated with minimal or no symptoms. we previously examined metabolic progression before diagnosis in dpt-1 participants (4). in that report, peak c - peptide levels were consistent during a period of 30 to 6 months before diagnosis, after which levels declined. this report extends observations to the postdiagnosis period and suggests that there is an acceleration of postchallenge c - peptide loss once glucose levels are in the diabetic range. the median decline of 23.8% in peak c - peptide levels from the last nondiabetic ogtt to the confirmatory ogtt indicates that there is a marked loss of insulin secretion in the perionset period. the extent to which this loss is reversible can not be determined from the data. estimates for the rate of change of peak c - peptide levels in the postdiagnosis period were obtained in two ways. in one approach (fig. 2) a regression analysis was used, whereas in the other approach (fig. 3), the estimate was derived from an analysis based on rate of change calculated for each individual. glucose levels seem to have been maintained relative to the decline in c - peptide levels after diagnosis. this suggests the possibility that compensatory mechanisms for glucose homeostasis are at play, such as an increase in insulin sensitivity. because c - peptide levels are only indicative of insulin secretion, it is also possible that a slowing of insulin degradation could have contributed to the maintenance of glucose levels. for calculations of the rate of change in peak c - peptide levels this assumption is of particular importance in the interval from 6 months before diagnosis to diagnosis, as one can not discern from the data the pattern of c - peptide decline within that period. thus, the rate of decrease in c - peptide may be more rapid closer to diagnosis and similar to the rate of decline in c - peptide after diagnosis. also, it should be emphasized that the average change provides an overall picture ; individual patterns of change vary considerably. however, we excluded those receiving parenteral insulin from the analyses, and there was no overall effect from either insulin intervention. knowledge of the results of the first diabetic ogtt could have resulted in lifestyle changes (11) or perhaps even have caused some to attempt to lower glucose levels with medication. still, it is doubtful that such interventions would explain the large degree of c - peptide loss. no prior studies have examined metabolic changes from before diagnosis to after diagnosis with ogtt surveillance. also, no studies have assessed metabolic changes in individuals with newly diagnosed diabetes as close to the onset of type 1 diabetes. c - peptide levels appear to be much lower when type 1 diabetes is clinically diagnosed (1214) than when it is diagnosed through ogtt surveillance. it is important to emphasize that of all individuals in whom type 1 diabetes was diagnosed in the dpt-1, 75% were asymptomatic (9). how our observations relate to the rate of decline of insulin secretion in symptomatic patients with clinically diagnosed diabetes is unknown. studies of patients with clinically diagnosed diabetes suggest that there is a progressive loss of insulin secretion that can be decreased by effective glucose control (15,16). the marked rate of decline of c - peptide levels in the perionset period provides a strong rationale for developing early interventions to prevent or delay the progression to type 1 diabetes. moreover, the data suggest that postdiagnosis interventions should be developed for application as close to the diagnosis of type 1 diabetes as possible.	objective we examined metabolic changes in the period immediately after the diagnosis of type 1 diabetes and in the period leading up to its diagnosis in diabetes prevention trial type 1 (dpt-1) participants.research design and methods the study included oral insulin trial participants and parenteral insulin trial control subjects (n = 63) in whom diabetes was diagnosed by a 2-h diabetic oral glucose tolerance test (ogtt) that was confirmed by another diabetic ogtt within 3 months. differences in glucose and c - peptide levels between the ogtts were assessed.resultsglucose levels increased at 90 (p = 0.006) and 120 min (p < 0.001) from the initial diabetic ogtt to the confirmatory diabetic ogtt (mean sd interval 5.5 2.8 weeks). peak c - peptide levels fell substantially between the ogtts (median change 14.3%, p < 0.001). among the 55 individuals whose last nondiabetic ogtt was 6 months before the initial diabetic ogtt, peak c - peptide levels decreased between these two ogtts (median change 14.0%, p = 0.052). among those same individuals the median change in peak c - peptide levels from the last normal ogtt to the confirmatory ogtt (interval 7.5 1.3 months) was 23.8% (p < 0.001). median rates of change in peak c - peptide levels were 0.00 ng ml1 month1 (p = 0.468, n = 36) from 12 to 6 months before diagnosis, 0.10 ng ml1 month1 (p = 0.059, n = 55) from 6 months before diagnosis to diagnosis, and 0.43 ng ml1 month1 (p = 0.002, n = 63) from the initial diabetic ogtt to the confirmatory diabetic ogtt.conclusionsit seems that postchallenge c - peptide levels begin to decrease appreciably in the 6 months before diagnosis and decrease even more rapidly within 3 months after diagnosis.
mirnas, a subset of small non - coding single - strand rnas approximately 22 nucleotides (nt) long, have emerged as a group of important regulators of gene expression at the post - transcriptional level. the action of mature mirnas rely on the mirna - induced silencing complex (mirisc), in which mirna recognize target mrna by binding to the complementary sequence of 3 untranslated region of the target. mirisc promote target mrna encoded protein down - regulation by either degrade or repress the translation of mrna. recent studies have suggested mirnas are involved in the regulation of myocardial ischemia reperfusion (i / r) injury., cardiac stress including myocardial reperfusion injury could induce altered expression of mirnas, and studies using mirnas arrays or real - time quantitative pcr have consistently found that the mir-15 family are up - regulated in myocardial ischemia and heart failure. the mir-15 family members include mir-15a, mir-15b, mir-16, mir-195, mir-424 and mir-497., one study showed that mir-195 was up - regulated during cardiac hypertrophy, an overexpression of which could result in pathological cardiac growth and heart failure in transgenic mice. cardiomyocytes are conventionally thought to be terminally differentiated with limited regenerate capacity ; the amount of acute and chronic cell loss after myocardial infarction is the key factor that affects cardiac function and patient prognosis. reperfusion may induce more cell apoptosis, which is detrimental for cardiac function recovery. research involving tumor cells suggest mir-15a, mir-15b, and mir-16 regulate apoptosis by post - transcriptionally down- regulating bcl-2 protein. in the present study, we tested the hypotheses that mir-15a, mir-15b, mir-16 may play a role in myocardial reperfusion injury by regulating apoptosis. thirty male mice (kunming, eight to ten weeks) were randomly divided into control group and i / r group, then anesthetized intramuscularly with 2% sodium pentobarbital. ischemia was achieved by using a 7.0 prolene suture around the left anterior descending coronary artery (lad) and tied with a slipknot for 30 min, after which the knot was relaxed and the heart was allowed reperfusion for 24 hours. after reperfusion, mice were sacrificed and hearts were harvested for rna isolation and tunel assay (roche, germany). the determination of cell apoptosis was performed using tunel assay according to manufacturer 's instruction. briefly, the cardiac tissue from infarct border zones was sectioned at 50 m thickness and stained. the ratio of apoptotic (green) nuclei vs. total nuclei (blue) was calculated and compared with the control group. the hearts from sprague - dawley rats born within 24 hours were minced and digested with trypsin. the primary neonatal rat cardiomyocytes were cultured in dmem containing 10% neonatal bovine serum (nbs) and gentamicin. after synchronization, the medium was replaced with dmem free of nbs before the cells were exposed to hypoxia, which was induced by placing the culture dishes into a hypoxic incubator filled with 95% n2 and 5% co2 at 37c. after 6 hours hypoxia, the dishes were transferred to a normoxic incubator for 24 hours reoxygenation. trypan blue staining was used to determine cell death with the number of trypan blue - positive and trypan blue- negative cells counted by hemocytometer under microscope. after hypoxia / reoxygenation (h / r), (15) 10 primary neonatal cardiomyocytes were collected and washed with phosphate buffer solution (pbs), the cells were resuspended in binding buffer followed by annexin v - fitc and propiduim iodide (pi) double - stain according to the manufacturer 's instruction, and then the samples were examined by a flow cytometer (bd, usa). total rna was isolated from mice heart tissues or cultured cells using trizol (invitrogen, usa) according to the manufacturer 's instructions. the concentration of extracted rna was quantified by uv spectrophotometer, the a260/a280 needs to be about 1.82.0. then real - time pcr was used to quantify the expression of mir-15a, mir-15b, mir-16 with specific taqman assays (applied biosystems, usa) and taqman universal master mix (applied biosystems, usa). briefly, we constructed 15 l reverse transcript system with 10 ng total rna, and real - time pcr system of 10 l, containing eight l taqman universal pcr master mix, one microlitre taqman assay and one microlitre rt product. the 2 indicates the fold change in gene expression relative to the untreated control, the equation displayed as ct= (ct, target ct, u6)i / r or h / r (ct, target ct, u6)control. all animal experiments were approved by the animal research ethics committee of the chinese pla general hospital, beijing, china. thirty male mice (kunming, eight to ten weeks) were randomly divided into control group and i / r group, then anesthetized intramuscularly with 2% sodium pentobarbital. ischemia was achieved by using a 7.0 prolene suture around the left anterior descending coronary artery (lad) and tied with a slipknot for 30 min, after which the knot was relaxed and the heart was allowed reperfusion for 24 hours. after reperfusion, mice were sacrificed and hearts were harvested for rna isolation and tunel assay (roche, germany). the determination of cell apoptosis was performed using tunel assay according to manufacturer 's instruction. briefly, the cardiac tissue from infarct border zones was sectioned at 50 m thickness and stained. the ratio of apoptotic (green) nuclei vs. total nuclei (blue) was calculated and compared with the control group. the hearts from sprague - dawley rats born within 24 hours were minced and digested with trypsin. the primary neonatal rat cardiomyocytes were cultured in dmem containing 10% neonatal bovine serum (nbs) and gentamicin. after synchronization, the medium was replaced with dmem free of nbs before the cells were exposed to hypoxia, which was induced by placing the culture dishes into a hypoxic incubator filled with 95% n2 and 5% co2 at 37c. after 6 hours hypoxia, the dishes were transferred to a normoxic incubator for 24 hours reoxygenation. trypan blue staining was used to determine cell death with the number of trypan blue - positive and trypan blue- negative cells counted by hemocytometer under microscope. after hypoxia / reoxygenation (h / r), (15) 10 primary neonatal cardiomyocytes were collected and washed with phosphate buffer solution (pbs), the cells were resuspended in binding buffer followed by annexin v - fitc and propiduim iodide (pi) double - stain according to the manufacturer 's instruction, and then the samples were examined by a flow cytometer (bd, usa). total rna was isolated from mice heart tissues or cultured cells using trizol (invitrogen, usa) according to the manufacturer 's instructions. the concentration of extracted rna was quantified by uv spectrophotometer, the a260/a280 needs to be about 1.82.0. then real - time pcr was used to quantify the expression of mir-15a, mir-15b, mir-16 with specific taqman assays (applied biosystems, usa) and taqman universal master mix (applied biosystems, usa). briefly, we constructed 15 l reverse transcript system with 10 ng total rna, and real - time pcr system of 10 l, containing eight l taqman universal pcr master mix, one microlitre taqman assay and one microlitre rt product. the 2 relative quantification method was applied, and u6 served as internal reference. the 2 indicates the fold change in gene expression relative to the untreated control, the equation displayed as ct= (ct, target ct, u6)i / r or h / r (ct, target ct, u6)control. all animal experiments were approved by the animal research ethics committee of the chinese pla general hospital, beijing, china. the mice i / r model was successfully made by temporary occlusion of the lad coronary artery for 30 min while monitoring with ecg ; the st segment elevation could be observed in lead ii. after 24-h reperfusion, triphenyltetrazolium chloride (ttc) and evans blue double stain was performed to evaluate the extent of infarction area, the i / r group exhibited significant infarction area (i / r infarction size, 21.38 2.22%, representative photos of both control group and i / r group are shown in figure 1a. to further investigate the injury induced by i / r in vivo, as displayed in figure 1b, tunel assay showed the proportion of apoptotic cells were significantly increased in i / r group compared with control group (24.4% 9.4% vs. 2.2% 1.9%, p < 0.01, n = 5). figure 2 shows that the neonatal cardiomyocytes subjected to h / r presented significant cell death, and flow cytometry analysis confirmed that the cell death is mainly contributed by apoptosis (14.12% 0.92% vs. 2.22% 0.08%, p < 0.05 compared with control, n = 3), (figure 2b). to further explore the molecular mechanism of myocardial i / r injury, we performed real - time quantitative pcr analyses to investigate whether mir-15a, mir-15b and mir-16 were involved in i / r or h / r. as shown in figure 3a and b, we found the mir-15a and mir-15b expression levels were increased in the mice i / r model and cardiomyocytes exposed to h / r in vitro (p < 0.05). the mir-16 expression had no significant difference between i / r or h / r in the control group, both in vivo and in vitro. cardiomyocytes are cells with limited regenerative capacity, and their viability is very important for the maintenance of the heart function. in myocardial these apoptotic cell deaths account for the acute cell loss in the infarct area and the chronic cell loss in the infarct border zone. tunel data from this study revealed apoptosis was dramatically increased in hearts subjected to i / r. the data of annexin - v / pi labeling flow cytometry also revealed apoptosis was increased in the cultured cardiomyocytes subjected to h / r., over the past decade, there were many studies investigating the functions of mirnas, showing some of these rnas participate in regulating cardiovascular disease. some mirnas are up - regulated in response to i / r, as displayed by our results, while i / r could induce dynamic expression changes of mir-15a and mir-15b. also, our results indicated that cardiomyocytes exposed to hypoxia lead to similar change. these data suggest that mir-15a and mir-15b may play an important role during i / r injury. accumulating activated hepatic stellate cells, mir-15b and mir-16 were decreased and were found to be related to apoptosis by targeting bcl-2.. previous studies have found that the mitochondrial permeability transition (mptp) opening would lead to the collapse of the membrane potential and the efflux of pro- apoptosis factors is particularly important if myocardial i / r injury happened on the moment of blood flow restore.,. whereas mptp is regulated mainly by bcl-2 family, and especially bcl-2/bax is a crucial factor for mitochondrial apoptotic pathway by controlling mitochondrial membrane integrity.. since both mir-15a and mir-15b are the upstream regulators of bcl-2, mir-15a and mir-15b are up - regulated in myocardial i / r injury, mir-15a and mir-15b may regulate mitochondrial apoptotic pathway by targeting bcl-2. it is suggested that the knockdown of mir-15a and mir-15b could be a therapeutic strategy for myocardial i / r injury. the limitation of the current study is that we haven't directly investigated the effect of mir-15a and mir-15b on apoptosis of cardiomyocytes and the downstream signaling transduction through over expression or down expression, although the studies in tumor cells have provided evidence that mir-15a and mir-15b have an intimate relationship with apoptosis. in the future, we will attempt to verify the role of mir-15a and mir-15b in myocardial i / r induced apoptosis and the signaling pathway. both mir-15a and mir-15b are related to myocardial i / r injury, and these rnas may play an essential role in apoptosis by targeting bcl-2 and caspase signaling pathway. the down - regulation of mir-15a / b may be a promising strategy to reduce myocardial apoptosis induced by cardiac i / r injury. the limitation of the current study is that we haven't directly investigated the effect of mir-15a and mir-15b on apoptosis of cardiomyocytes and the downstream signaling transduction through over expression or down expression, although the studies in tumor cells have provided evidence that mir-15a and mir-15b have an intimate relationship with apoptosis. in the future, we will attempt to verify the role of mir-15a and mir-15b in myocardial i / r induced apoptosis and the signaling pathway. both mir-15a and mir-15b are related to myocardial i / r injury, and these rnas may play an essential role in apoptosis by targeting bcl-2 and caspase signaling pathway. the down - regulation of mir-15a / b may be a promising strategy to reduce myocardial apoptosis induced by cardiac i / r injury.	objectiveseveral studies have indicated that mir-15a, mir-15b and mir-16 may be the important regulators of apoptosis. since attenuate apoptosis could protect myocardium and reduce infarction size, the present study was aimed to find out whether these mirnas participate in regulating myocardial ischemia reperfusion (i / r) injury.methodsapoptosis in mice hearts subjected to i / r was detected by tunel assay in vivo, while flow cytometry analysis followed by annexin v / pi double stain in vitro was used to detect apoptosis in cultured cardiomyocytes which were subjected to hypoxia / reoxygenation (h / r). taqman real - time quantitative pcr was used to confirm whether mir-15a/15b/16 were involved in the regulation of cardiac i / r and h / r.resultscompared to those of the controls, i / r or h / r induced apoptosis of cardiomyocytes was significantly increased both in vivo (24.4% 9.4% vs. 2.2% 1.9%, p < 0.01, n = 5) and in vitro (14.12% 0.92% vs. 2.22% 0.08%). the expression of mir-15a and mir-15b, but not mir-16, was increased in the mice i / r model, and the results were consistent in the h / r model.conclusionsour data indicate mir-15 and mir-15b are up - regulated in response to cardiac i / r injury, therefore, down - regulation of mir-15a / b may be a promising strategy to reduce myocardial apoptosis induced by cardiac i / r injury.
historically, radical nephrectomy (rn) has been the mainstay treatment for renal cell carcinoma (rcc). increased incidental detection of small renal masses with the development of radiologic diagnostic tools has brought increased attention to treatment options and operation modality for small renal masses. with the evidence of comparable oncologic outcomes with partial nephrectomy (pn) and the risk of de novo renal failure with rn for a small renal tumor, attention is focused on expanding and setting the indications of pn in small renal tumors. in the last decade, pn has developed rapidly and supplanted rn as the standard treatment for t1a rcc, with comparable oncological outcomes, superior renal function preservation and better quality of life. although treatment for tumors larger than 4 cm remains controversial, emerging data indicates that pn for t1b rcc provides comparable cancer control compared with rn, as well as pn for t1a rcc. however, most of those studies are single - center, retrospective and observational studies with low - level evidence, and the results and interpretation are questionable because of uneven distribution of patients and patient selection bias. therefore, we conducted a pair - matched clinical outcome study in patients who underwent pn or rn for t1b renal tumors. factors that can affect oncologic and functional outcomes were matched, and data were distributed evenly with regard to age, sex, preoperative renal function, comorbidity, american society of anesthesiologists score, body mass index (bmi), tumor size, and histologic subtype. in this study, we evaluated the oncologic and functional outcomes of pn versus rn in t1b renal tumors within a pairmatched control cohort. after approval was obtained from the institutional review board at each center, we retrospectively reviewed our database. we identified 611 patients treated between 1999 and 2011 with rn or pn for a solitary, nx / no mo solid renal mass (4 - 7 cm) at five institutions in korea. after excluding patients with benign pathology and those with missing records, 577 (pn, 100 ; rn, 477) patients with pathologically confirmed pt1b remained for analysis. patients who underwent pn (n=100) were matched to patients who underwent rn (n=458). to eliminate the influence of the confounding factors and achieve equal distributions of patients between the two groups, we pair - matched the patients of the two groups using propensity scores. the propensity score included age, sex, comorbidities (hypertension, diabetes), bmi, tumor size and depth, histologic type, and preoperative estimated glomerular filtration rate (egfr). preoperative egfr was classified into five groups according to the current guidelines for chronic kidney disease (ckd). clinical data including age, sex, bmi, tumor size and location, and surgical approach were recorded. intraoperative and postoperative data including operative time, estimated blood loss (ebl), intraoperative transfusions and serum creatinine and hemoglobin levels on postoperative day 1 were recorded. complication rates, tumor recurrences, overall survival (os) rate, cancer - specific survival (css) rate, progression - free survival (pfs) rate, and renal function outcomes were documented. the os, css, and pfs rates were measured until the last date of follow - up. patients were evaluated for postoperative recurrence by chest x - ray and computed tomography scan every 6 months for the first 3 years. renal function evaluation included serum creatinine and egfr on preoperative, one day and 3 months postoperative and annually thereafter. the egfr was calculated for each creatinine value based on the cockcroft - gault equation. postoperative new - onset ckd was defined as egfr lower than 60 ml / min/1.73 m, according to the national kidney foundation dialysis outcomes quality initiative clinical practice guidelines. procedures performed included pure laparoscopic, handassisted laparoscopic, robot - assisted laparoscopic and open rn and pn. surgical modality and approach were decided by surgeon preference and tumor characteristics. clinical and pathological variables were compared between patients who underwent pn and rn using the chi - square test and student s t test as appropriate. the os, css, pfs, and new - onset ckd rates were analyzed using the kaplan - meier method. post operative renal functions between groups were compared as the mean percent of decline in egfr from baselines at postoperative 3, 12, 24, 36, and 48 months by student s t test. to compare the survival rate between groups, was then used to compare the os, css and pfs rates of patients who underwent pn as opposed to rn. the associations between variables and the outcome parameters are presented with the hazard ratios and 95% confidence intervals. null hypotheses of no difference were rejected if p - values were less than 0.05, or, equivalently, if the 95% confidence intervals (cis) of hazard ratio estimates excluded 1. after approval was obtained from the institutional review board at each center, we retrospectively reviewed our database. we identified 611 patients treated between 1999 and 2011 with rn or pn for a solitary, nx / no mo solid renal mass (4 - 7 cm) at five institutions in korea. after excluding patients with benign pathology and those with missing records, 577 (pn, 100 ; rn, 477) patients with pathologically confirmed pt1b remained for analysis. patients who underwent pn (n=100) were matched to patients who underwent rn (n=458). to eliminate the influence of the confounding factors and achieve equal distributions of patients between the two groups, we pair - matched the patients of the two groups using propensity scores. the propensity score included age, sex, comorbidities (hypertension, diabetes), bmi, tumor size and depth, histologic type, and preoperative estimated glomerular filtration rate (egfr). preoperative egfr was classified into five groups according to the current guidelines for chronic kidney disease (ckd). clinical data including age, sex, bmi, tumor size and location, and surgical approach were recorded. intraoperative and postoperative data including operative time, estimated blood loss (ebl), intraoperative transfusions and serum creatinine and hemoglobin levels on postoperative day 1 were recorded. complication rates, tumor recurrences, overall survival (os) rate, cancer - specific survival (css) rate, progression - free survival (pfs) rate, and renal function outcomes were documented. the os, css, and pfs rates were measured until the last date of follow - up. patients were evaluated for postoperative recurrence by chest x - ray and computed tomography scan every 6 months for the first 3 years. after that period renal function evaluation included serum creatinine and egfr on preoperative, one day and 3 months postoperative and annually thereafter. the egfr was calculated for each creatinine value based on the cockcroft - gault equation. postoperative new - onset ckd was defined as egfr lower than 60 ml / min/1.73 m, according to the national kidney foundation dialysis outcomes quality initiative clinical practice guidelines. procedures performed included pure laparoscopic, handassisted laparoscopic, robot - assisted laparoscopic and open rn and pn. surgical modality and clinical and pathological variables were compared between patients who underwent pn and rn using the chi - square test and student s t test as appropriate. the os, css, pfs, and new - onset ckd rates were analyzed using the kaplan - meier method. post operative renal functions between groups were compared as the mean percent of decline in egfr from baselines at postoperative 3, 12, 24, 36, and 48 months by student s t test. to compare the survival rate between groups, was then used to compare the os, css and pfs rates of patients who underwent pn as opposed to rn. the associations between variables and the outcome parameters are presented with the hazard ratios and 95% confidence intervals. null hypotheses of no difference were rejected if p - values were less than 0.05, or, equivalently, if the 95% confidence intervals (cis) of hazard ratio estimates excluded 1. patient demographics and surgical and perioperative data from both groups are summarized in table 1. the two groups were similar in age, sex, bmi, and number and type of comorbidity. preoperative mean creatinine and egfr were similar between the groups : 0.98 pn (range, 0.4 to 2.0) and 1.04 rn (range, 0.62 to 1.75) (p=0.28), 80.46 pn (range, 24.5 to 177.7), and 88.79 rn (range, 24.9 to 192.0) (p=0.41), respectively. the mean pathologic tumor size was similar between the two groups : 4.90 cm pn and 4.91 cm rn (p=0.98). the ratio of the pathological variables was also similar between the two groups (p=0.3). laparoscopic surgery was performed in 130 patients. among them, 64 patients underwent pure laparoscopic surgery (pn, n=37 ; rn, n=27), 14 patients underwent handassisted laparoscopic surgery (pn, n=1 ; rn, n=13), and 52 patients underwent robot - assisted laparoscopic surgery (pn, n=18 ; rn, n=34). open surgery was performed in 70 patients (pn, n=44 ; rn, n=26). perioperative parameters including mean operative time, ebl and transfusion rate between the two groups did not differ significantly (p > 0.05). the mean change in preoperative hemoglobin (hb) to postoperative hb was similar between groups (p=0.16). renal pedicle clamping was conducted only in the pn group, in 96 patients (warm ischemia, n=82 ; cold ischemia, n=14), and the mean ischemic time was 29.97 minutes (range, 9 to 68 minutes). intraoperative complications in the pn group consisted of three organ injuries (one spleen injury, one major vessel injury, and one ureter injury) and four pleural injuries. in the rn group, there were two organ injuries (one major vessel injury and one duodenal injury) and three pleural injuries. the postoperative complication rate was comparable in patients treated with pn and rn (p=0.09). postoperative complications for pn included five patients with grade i, two patients with grade ii, and one patient with grade iiia complications. in the rn group, there were four cases of grade i and two cases of grade ii postoperative complications. in the pn group, three patients had a positive surgical margin (psm), and of those, two were treated laparoscopically and one treated with open pn. the median follow - up duration in the rn group was 48.1 and 42.6 months for the pn group. tumor progression was found in six of 100 patients after pn. of those, three patients developed local recurrence and three patients developed distant metastases after a median duration of 97.0 months (95% ci, 85.05 to 109.04 months). in the pn group, three patients had a psm, and none of these developed local or distant recurrence after a median followup of 37 months (range, 19 to 45 months). in the rn group, 12 of 100 patients developed distant metastases after a median of 110.7 months (95% ci, 98.54 to 123.00 months). the pfs rate was comparable between the two groups (log - rank, p=0.66). the 5- and 10-year pfs rates were 86.4% and 79.2% for the pn group and 86.0% and 66.0% in the rn group, respectively (table 3, fig. the css rates for patients with t1b rcc treated with pn or rn were comparable (log - rank, p=0.52) (table 3). in the pn group, the estimated 10-year css rate for pn was 85.7%, similar to the rn rate of 84.4%. the cox proportional hazards model adjusting for propensity scores also did not differ in css rate between groups (hazard ratio [hr ], 2.01 ; 95% ci, 0.22 to 18.33). the estimated 10-year os rate was significantly higher in the pn group (85.7%) than the rn group (73.3%) in the kaplan - meier analysis (log - rank, p=0.003). using the cox proportional hazards model, tumor size (p=0.947), type of surgery (p=0.131), and postoperative renal function (p=0.146) forty - six patients (pn, 22 ; rn, 24) had pre - existing ckd before surgery. there was no difference in baseline egfr between pn and rn (p=0.41). renal function declined in both groups from preoperative to 3 months postoperative (80.46 ; range, 24.5 to 177.7) to 71.99 ml / min/1.73 m (range, 9.6 to 172.3 ml / min/1.73 m) and 88.79 ml / min/1.73 m (range, 24.9 to 192.0 ml / min/1.73 m) to 59.27 (range, 29.8 to 137.4) in the pn and rn groups, respectively). the mean percent decline of egfr from baseline to postoperative 3, 12, 24, 36, and 48 months were all greater in the rn group compared to the pn group (p 0.99). patient demographics and surgical and perioperative data from both groups are summarized in table 1. the two groups were similar in age, sex, bmi, and number and type of comorbidity. preoperative mean creatinine and egfr were similar between the groups : 0.98 pn (range, 0.4 to 2.0) and 1.04 rn (range, 0.62 to 1.75) (p=0.28), 80.46 pn (range, 24.5 to 177.7), and 88.79 rn (range, 24.9 to 192.0) (p=0.41), respectively. the mean pathologic tumor size was similar between the two groups : 4.90 cm pn and 4.91 cm rn (p=0.98). the ratio of the pathological variables was also similar between the two groups (p=0.3). laparoscopic surgery was performed in 130 patients. among them, 64 patients underwent pure laparoscopic surgery (pn, n=37 ; rn, n=27), 14 patients underwent handassisted laparoscopic surgery (pn, n=1 ; rn, n=13), and 52 patients underwent robot - assisted laparoscopic surgery (pn, n=18 ; rn, n=34). open surgery was performed in 70 patients (pn, n=44 ; rn, n=26). perioperative parameters including mean operative time, ebl and transfusion rate between the two groups did not differ significantly (p > 0.05). the mean change in preoperative hemoglobin (hb) to postoperative hb renal pedicle clamping was conducted only in the pn group, in 96 patients (warm ischemia, n=82 ; cold ischemia, n=14), and the mean ischemic time was 29.97 minutes (range, 9 to 68 minutes). intraoperative complications in the pn group consisted of three organ injuries (one spleen injury, one major vessel injury, and one ureter injury) and four pleural injuries. in the rn group, there were two organ injuries (one major vessel injury and one duodenal injury) and three pleural injuries. the postoperative complication rate was comparable in patients treated with pn and rn (p=0.09). postoperative complications for pn included five patients with grade i, two patients with grade ii, and one patient with grade iiia complications. in the rn group, there were four cases of grade i and two cases of grade ii postoperative complications. in the pn group, three patients had a positive surgical margin (psm), and of those, two were treated laparoscopically and one treated with open pn. the median follow - up duration in the rn group was 48.1 and 42.6 months for the pn group. tumor progression was found in six of 100 patients after pn. of those, three patients developed local recurrence and three patients developed distant metastases after a median duration of 97.0 months (95% ci, 85.05 to 109.04 months). in the pn group, three patients had a psm, and none of these developed local or distant recurrence after a median followup of 37 months (range, 19 to 45 months). in the rn group, 12 of 100 patients developed distant metastases after a median of 110.7 months (95% ci, 98.54 to 123.00 months). the pfs rate was comparable between the two groups (log - rank, p=0.66). the 5- and 10-year pfs rates were 86.4% and 79.2% for the pn group and 86.0% and 66.0% in the rn group, respectively (table 3, fig. the css rates for patients with t1b rcc treated with pn or rn were comparable (log - rank, p=0.52) (table 3). in the pn group, the estimated 10-year css rate for pn was 85.7%, similar to the rn rate of 84.4%. the cox proportional hazards model adjusting for propensity scores also did not differ in css rate between groups (hazard ratio [hr ], 2.01 ; 95% ci, 0.22 to 18.33). the estimated 10-year os rate was significantly higher in the pn group (85.7%) than the rn group (73.3%) in the kaplan - meier analysis (log - rank, p=0.003). using the cox proportional hazards model, tumor size (p=0.947), type of surgery (p=0.131), and postoperative renal function (p=0.146) forty - six patients (pn, 22 ; rn, 24) had pre - existing ckd before surgery. there was no difference in baseline egfr between pn and rn (p=0.41). renal function declined in both groups from preoperative to 3 months postoperative (80.46 ; range, 24.5 to 177.7) to 71.99 ml / min/1.73 m (range, 9.6 to 172.3 ml / min/1.73 m) and 88.79 ml / min/1.73 m (range, 24.9 to 192.0 ml / min/1.73 m) to 59.27 (range, 29.8 to 137.4) in the pn and rn groups, respectively). the mean percent decline of egfr from baseline to postoperative 3, 12, 24, 36, and 48 months were all greater in the rn group compared to the pn group (p 0.99). our results with t1b rcc demonstrated comparable 5- and estimated 10-year pfs and estimated 10-year css rates and superior estimated 10-year os for patients treated with pn versus rn. five- and estimated 10-year pfs rates were 86.4%/79.2% for pn and 86.0%/66.0% for rn. estimated 10-year css rate was 85.7% for pn and 84.4% for rn. as the tendency changes toward performing more pn procedures for t1b rcc, studies demonstrate that the oncologic outcome and survival rate are comparable in patients treated with pn compared with rn. reported that pn led to higher 5-year css and 5-year tumor recurrence rates compared to rn in 4 to 7 cm rcc using univariate analysis. similarly, thompson. found no significant difference in 5- and 10-year css rates in patients with t1b who underwent pn or rn, although there was a tendency for a high rate of solitary kidney or ckd in the pn group, implying the possibility of patient selection bias. reported that there was no significant difference in the 5-year css rate in patients with t1b rcc treated with pn versus rn (82.5% and 85%, p=0.161, respectively). based on these reports and the results of the present study, we suggest that pn is oncologically safe and has comparable, at least not inferior outcomes, compared with rn for the treatment of t1b rcc. a major benefit of pn is renal function preservation and the derived potential positive effects on os and quality of life. reported that a postoperative decline in gfr of 16.6% and 23.5% was observed in patients treated with pn and rn for t1b rcc, respectively. reported that pn for ct1b resulted in a similar postoperative egfr level to that of ct1a. in addition, the probability of freedom from the new onset of ckd after pn did not differ between ct1b or ct1a tumors. in our study, renal function, estimated by egfr, decreased after both pn and rn, but pn led to better postoperative renal function preservation compared to rn. the change in egfr from baseline to 3 months and 4 years was 10.52%/8.04% for pn and 33.24%/35.12% for rn. pn was less likely to induce newonset ckd and end - stage ckd compared with rn. the present study demonstrates the feasibility and validity of pn for t1b rcc with respect to oncologic outcome and superior renal function preservation. in the present study, renal function decreased sharply during the first 3 months after pn and rn and was maintained for 4 years. the percent decline of egfr from baseline to postoperative follow - up time was significantly smaller in the patients treated with pn compared with rn. although technical concerns relative to the effect of renal artery occlusion or reperfusion injury and warm ischemia, and the association of resection volume and remnant renal function in t1b rcc require additional consideration and investigation, it seems clear that the functional outcome after pn is superior to that after rn in patients with t1b rcc. several studies show an advantage in os for patients who underwent pn, mainly attributed to the renal function preservation after surgery. however, in a randomized trial by van poppel., 10-year os was greater in patients treated with rn (81.1%) than in patients treated with pn (75.7%) (p=0.03). they criticized the superior outcome of pn in other studies, saying those results depend heavily on patient selection. in the present study, postoperative renal function manifested as egfr was better preserved in the pn group, and the rn group had more frequent new - onset postoperative ckd. the os rate was significantly higher in the pn group compared with the rn group (p=0.003). while our study, in design, may also fundamentally present results of careful patient selection when choosing candidates for pn, neither has our study shown that renal function following rn or pn significantly affected os. while we hypothesized that postoperative renal function may prognosticate os, in multivariate cox regression analysis, postoperative egfr was not associated with os, despite a significant higher ratio of new - onset of ckd in the rn group than the pn group. also, there was no significant prognostic factor for os using the multivariate cox regression analysis. the critical opinions and concerns of pn for rcc include multifocality and microscopic satellite of rcc and incomplete tumor resection. multifocality is a characteristic of rcc and occurs in approximately 16% and 47% of rcc patients in the form of a satellite lesion. however, the local recurrence rate after pn for t1b rcc was 1% to 5.4%, lower than the high rate of multifocality or microscopic satellite tumor lesions. since the local recurrence rate of pn for t1a rcc is 0%-10%, we suggest no significant increase in t1b rcc. reported that, after pn for t1a and t1b rcc, local recurrence rates were comparable, 5% and 1%, respectively (p=0.57). in the present study, the local recurrence rate was 3%, similar to other studies. pn for larger renal tumors can be technically challenging and requires extensive experience, causing concerns of higher perioperative complications and incomplete tumor resection. the risk of perioperative complications with pn is slightly higher than with rn. in a prospective, randomized study, van poppel. reported that pn is associated with increased hemorrhage (3.1% and 1.2%), urinary fistula (4.4% and 0%, p < 0.001) and re - operation due to side effects (4.4% and 2.4%). in the present study, there was no significant difference in mean operative time, ebl or transfusion rate, and the overall intraoperative complication rate was similar between the groups. however, patard. reported that the psm rate was comparable in small tumors and tumors larger than 4 cm. psm after pn for t1b rcc occurs in 0%-13.3% of cases. whether or not psm is a risk factor for tumor recurrence after pn, which is still controversial reported that patients with psm after pn were not at increased risk for local recurrence or metastasis after a mean follow - up time of 3.4 years. psm rates after pn are reported differ by surgical approach and tend to increase in minimally - invasive approaches (0.7%-4% in laparoscopic approach, 3.9%-5.7% in robot - assisted approach) compared to an open approach (0%-7%). in the present study, psm was 3%, and none of the patients developed local or distant recurrence after a median followup of 37 months (19 - 45 months). since rn was established as a surgical treatment for rcc, it has been the standard of care in treating rcc. during the last decade although the cutoff of 4 cm in the treatment of rcc generated controversy after t1 substratification was introduced in 2002, it has been validated by a number of studies showing differences between t1a and t1b rcc in a 5-year css. however, emerging data about pn for t1b rcc indicate that pn provides cancer control and survival comparable to rn. the present study is valuable in that it adds the evidence of feasibility of pn for t1b rcc based on data that is evenly pair - matched and includes both basic patient characteristics and tumor characteristics. also, in the present study, both oncologic and functional outcomes were simultaneously compared between pn and rn. it is important to evaluate the benefit of os in the patients treated with pn. although we conducted a pair - matched study and the data were distributed evenly with regard to patient and tumor characteristics, it was nevertheless a retrospective design. to overcome the disadvantages of a retrospective study, this method is suggested as a way to overcome the effects of observed covariates and allowing focus on the treatment method. through propensity score matching, studies may overcome the difficulties of conducting large randomized prospective studies where covariates other than the purposed outcome can not be manipulated, and thus may introduce new bias if the sample size is not sufficiently large. however, propensity score matching has its failings, as it can not compensate for unobserved covariates such as, in our study, the lack of cause of death (except death from rcc) and occurrence of cardiovascular events. unidentified potential confounders may have resulted in some early cases of rn showing significant change on survival. our study suggests that pn shows similar css and pfs compared with rn in t1b rcc in a pair - matched clinical outcomes study. though there are several limitations of the present study, our results suggest additional support for the feasibility of pn in t1b renal tumors. a large - scale prospective study is needed to clarify the association of os and postoperative renal function.	purposethe study was to compare the oncologic and functional outcomes of partial nephrectomy (pn) and radical nephrectomy (rn) for pathologically proven t1b renal cell carcinoma using pair - matched groups.materials and methodswe reviewed our prospectively maintained database for rn and pn in t1b renal tumors surgically treated between 1999 and 2011 at five institutions in korea. of 611 patients treated with pn or rn for a solitary and nx / n0 m0 renal mass (4 - 7 cm), 577 (pn, 100 ; rn, 477) patients with pathologically confirmed pt1b remained for analysis. study subjects were grouped by pn or rn, then matched by age, sex, comorbidities, body mass index, tumor size and depth, histologic type, and preoperative estimated glomerular filtration rate (egfr) using propensities score. to evaluate oncologic outcomes, overall survival (os), cancer - specific survival (css), and progression - free survival (pfs) rates were analyzed. the functional outcomes were evaluated by postoperative egfr.resultsthe median follow - up in the rn group was 48.1 and 42.6 months in the pn group. the estimated 10-year css rate (pn 85.7% vs. rn 84.4%, p=0.52) and 5- and estimated 10-year pfs rates (pn : 86.4% and 79.2% vs. rn : 86.0% and 66.1%, p=0.66) did not differ significantly between groups. the estimated 10-year os rate was significantly higher in the pn group (85.7%) compared to the rn group (73.3%) (p=0.003). pn was less likely to induce new - onset chronic kidney disease (ckd) and end - stage ckd compared with rn.conclusionour study suggests that patients treated with pn demonstrate a superior os rate and postoperative renal function with analogous css and pfs rates compared with pair - matched patients treated with rn.
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.	spiroketals organize small molecule structures into well - defined, three - dimensional configurations that make them good ligands of proteins. we recently discovered a tandem cycloisomerization dimerization reaction of alkynyl hemiketals that delivered polycyclic, enol - ether - containing spiroketals. here we describe rearrangements of those compounds, triggered by epoxidation of their enol ethers that completely remodel their structures, essentially turning them inside out. due to the high level of substitution on the carbon skeletons of the substrates and products, characterization resorted to x - ray crystallography and advanced computation and nmr techniques to solve the structures of representative compounds. in particular, a new proton - detected adequate nmr experiment (1,1-hd - adequate) enabled the unequivocal assignment of the carbon skeleton of one of the new compounds. solution of the structures of the representative compounds allowed for the assignment of product structures for the other compounds in two separate series. both the rearrangement and the methods used for structural determination of the products are valuable tools for the preparation of characterization of new small molecule compounds.
	near - infrared (nir) persistent luminescence nanoparticles (plnps), possessing unique nir pl properties, have recently emerged as important materials for a wide variety of applications in chemistry and biology, for which they must endure high - temperature solid - state annealing reactions and subsequent complicated physical post - treatments. herein, we report on a first direct aqueous - phase chemical synthesis route to nir plnps and present their enhanced in vivo renewable nir pl. our method leads to monodisperse plnps as small as ca. 8 nm. such sub-10 nm nanocrystals are readily dispersed and functionalized, and can form stable colloidal solutions in aqueous solution and cell culture medium for biological applications. under biotissue - penetrable red - light excitation, we found that such nanocrystals possess superior renewable pl photoluminescence in vitro and in vivo compared to their larger counterparts currently made by existing methods. we believe that this solid - state - reaction - free chemical approach overcomes the current key roadblock in regard to plnp development, and thus will pave the way to broad use of these advanced miniature luminous pearls in photonics and biophotonics.
twin pregnancies occur in approximately 1% of all pregnancies, but approximately 10% of perinatal complications occur in twin pregnancies 1,2. because the perinatal mortality rates are 3 - 6 times higher in twin pregnancies than singleton pregnancies, prenatal management and safe delivery of twin pregnancies are important issues in obstetrics. in particular, the second twin may have a greater risk of adverse perinatal outcome than the first twin, because of difficulties in delivery due to uterine inertia and abnormal lie or high presentation after the vaginal delivery of the first twin, and increased risk of premature placental separation and cord prolapse 3,4. although some large - scale epidemiologic studies have reported that there was no difference in the risk of perinatal complications according to birth order 5,6 ; recent studies have shown that the risk of perinatal mortality and neonatal morbidity was higher in the second twin than the first twin 7,8, and the inter - twin delivery interval have been suggested as an important risk factor associated with the incidence of delivery - related complications in second twins 9. recently, in an effort to reduce complications in twin deliveries, the rate of planned cesarean delivery has been increased in multiple deliveries. however, the safe time limit of the inter - twin delivery interval has not been defined, and there are controversies regarding the optimal mode of delivery of twin gestations. meta - analysis did not evidence significant increased adverse neonatal outcome of both twins after vaginal delivery in comparison with cesarean 8,9. conversely, some suggest systematic planned cesarean delivery for all twin gestations could protect second twins from increased neonatal mortality and morbidity 10,11. indeed, cesarean delivery may increase the prevalence of fetal and maternal complications 12. accordingly, additional research on the risk of hypoxia during labor in second twins, and determining whether or not cesarean section may reduce the risk of hypoxia is needed. the current study was performed to investigate the perinatal outcomes of the second twin according to mode of delivery. in particular, to provide objective evidence of peripartum asphyxial insult and neonatal outcome, we focused on acid base status of the fetus as measured in umbilical arterial blood gas analysis just after delivery 13,14. mary hospital of the catholic university between 1 january 2006 and 31 december 2008 were reviewed retrospectively. the cases of emergency cesarean delivery due to failure to progress or fetal distress during labor, intrauterine death of either one of the twins before labor, discordant twins defined by the discrepancy of birth weight by more than 20%, and pregnancies complicated by preeclampsia, intrauterine growth restriction, twin - to - twin transfusion syndrome, and fetal anomalies were excluded. a total of 79 twin gestations in which the umbilical arterial blood gas values were available for both twins were selected. if the first twin was in the cephalic presentation, vaginal delivery was attempted for all women regardless of parity and the presentation of the second twin. multiparas whose first twin was in the breech presentation, were allowed a trial of labor. a cesarean delivery was performed if the pregnant woman had a history of cesarean delivery or uterine surgery or on maternal request. as a result, among the 79 cases in the current study, 40 pregnant women had cesarean deliveries and 39 women had vaginal deliveries. to compare the perinatal complications between two groups, we investigated maternal age, parity, gestational age, birth weight, mode of delivery, and fetal sex from the delivery records of each case. if the 5-minute apgar score was 15 minutes (10.3% ; table 1). the mean umbilical arterial blood ph, carbon dioxide tension, bicarbonate and base - deficit were 7.28 0.09, 47.84 10.45 mmhg, 21.80 3.40 the neonates with an umbilical arterial ph of 15 minutes (10.3% ; table 1). the mean umbilical arterial blood ph, carbon dioxide tension, bicarbonate and base - deficit were 7.28 0.09, 47.84 10.45 mmhg, 21.80 3.40 the neonates with an umbilical arterial ph of < 7.2 were 6 in first twins (7.6%) and 14 in second twins (17.7%). severe fetal acidemia with a ph < 7.0 occurred in 3 cases, all of which were from the vaginal delivery group. three neonates had a 5-minute apgar score of less than 7, but did n't show any neurologic morbidity. in a comparison of the first twins between the vaginal delivery and cesarean delivery groups, none of the umbilical arterial gas parameters showed statistically significant differences, and the prognosis of the second twin in each pregnancy was assessed using inter - twin difference of the umbilical arterial gas parameters and apgar scores between the first and the second twins (table 3). the mean inter - twin differences of umbilical arterial ph are 0.02 0.07 in the vaginal delivery group and 0.020.05 in the cesarean delivery group, showing no significant difference according to the mode of delivery. with respect to the other umbilical arterial blood gas parameters, the differences between the first and second twins were not significantly different according to mode of delivery. in the analysis of apgar scores, the difference in 1-minute apgar scores of more than 2 occurred in 8 (20.5%) twin gestations in the vaginal delivery group, which is significantly more than difference in the cesarean delivery group (5.0 %) (p = 0.048). in addition, the difference in 5-minute apgar scores of more than 1 showed a significantly higher incidence in the vaginal delivery group than in the cesarean delivery group (38.5 % vs. 17.5 % ; p = 0.038). excluding 11 twin gestations with an inter - twin delivery interval of more than 10 minutes we compared 28 cases of vaginal deliveries with the 40 cases of cesarean sections. no significant differences were noted in the umbilical arterial blood gas factors and apgar scores between the two groups (table 4). the delivery of the second twin is complicated by uterine inertia following the delivery of the first twin, and the abnormal fetal lie or high presentation of the second twin 3,4. moreover for these reasons, many studies have been conducted on safe modes of deliveries for second twins, but the conclusions were not consistent. several retrospective analyses and meta - analyses have reported that the prognosis of twins was not different according to delivery mode 5,6,15, but population - based studies reported that the mortality rate of second twins is higher in vaginal deliveries 7,8. this study shows that the risk of intrauterine fetal hypoxia in second twins was not different between vaginal deliveries and cesarean deliveries. although the 1- and 5-minute apgar scores were significantly lower in second twins delivered vaginally than second twins delivered by cesarean section, inter - twin difference of umbilical arterial ph value was not different according to mode of delivery. in particular, compared to previous studies in which the mean values of the second twin group were compared with the mean values of the first twin groups, our analysis may be more accurate as the difference between the first and second twin was analyzed in each delivery. for the mode of delivery of twins, the american college of obstetricians and gynecologists (acog) recommended vaginal delivery in the case of twins in cephalic - cephalic presentation, 16. however, some have argued for planned cesarean delivery for all twin gestations 10,11. because controversies still exist over the optimal mode of delivery for twin gestation moreover, cesarean deliveries can cause other pregnancy complications, including early delivery, low birth weight, and increased risk of placenta previa, placenta accreta, emergency cesarean section, and premature delivery in the next pregnancy 17. an important difference between second twins according to the mode of delivery is the inter - twin delivery interval time. as shown in the current study, the inter - twin delivery interval time was significantly longer in vaginal delivery group (8.2 8.2 minutes) than in cesarean delivery group (< 2 minutes) ; 28.2% of twin gestations delivered vaginally had inter - twin delivery interval time 10 minutes. some previous studies which concluded that the umbilical arterial blood gas of the second twin is worse in the vaginal delivery group 18,19, reported the mean inter - twin delivery interval times were 16 - 18 minutes, which were rather longer compared to the mean time in the current study (8.2 minutes). according to schmiz. 20 who argued that perinatal outcomes were not different according to the mode of delivery, the mean inter - twin delivery interval time was 4.9 minutes, which was much shorter than previous reports 18,19. therefore the difference in the inter - twin delivery interval may be the most important factor to causing the inconsistent prognosis of the second twins. in particular, when we compared the differences of umbilical arterial blood gas in twin sibling between vaginal delivery group in which inter - twin delivery interval time was less than 10 minutes and cesarean delivery group, the umbilical arterial blood gas were not significantly different. for twin deliveries, it is necessary to make an effort to reduce the inter - twin interval time and to establish stricter indications for trial of labor not to take inter - twin delivery interval time more than about 10 minutes rather than apply elective cesarean delivery uniformly to all twin gestations. a limitation of our study was the small number of women investigated, preventing any formal conclusion from being drawing. however, our study provides data to support the planed vaginal delivery in selected women. in conclusion, the results of this study showed that there was no difference in umbilical arterial blood gas parameters and the neonatal morbidity according to the mode of delivery. accordingly, for twin deliveries, it is relatively safe to plan for a vaginal delivery as long as the other obstetric conditions are available for vaginal delivery, but an effort should be made to reduce the inter - twin delivery interval time.	purpose : to compare umbilical arterial gas parameters in the second twin of twin pregnancies according to the mode of deliverymethods : we retrospectively analyzed the medical records of twin deliveries after 34 weeks of gestation for 3 years. excluding the cases which underwent emergency cesarean delivery during trial of labor, a total of 79 twin gestations had umbilical arterial blood gas values available and were and divided into cesarean delivery group (n=40) and vaginal delivery group (n=39). the mean differences of umbilical arterial blood parameters and the apgar score between the first and second twin in each pregnancy were compared according the mode of delivery.results : the differences of umbilical arterial gas parameters between twin siblings showed no significant difference according to the mode of delivery. with regard to the 1 minute and 5 minute apgar scores, the differences between twin siblings are significantly increased in vaginal delivery group compared to cesarean delivery group (p=0.048, and p=0.038, respectively). in comparing the 28 cases delivered vaginally with an inter - twin delivery interval < 10 minutes and 40 cases delivered by cesarean section, no significant differences were observed in the umbilical arterial gas parameters and apgar scores.conclusion : the inter - twin umbilical arterial blood gas parameters according to the mode of delivery showed no difference. for twin deliveries, it is relatively safe to plan for a vaginal delivery, but an effort should be made to reduce the inter - twin delivery interval time.
pancreatitis - panniculitis - polyarthritis (ppp) syndrome is characterized by the development of erythematous cutaneous nodules typically affecting the lower extremities as well as poly- or oligoarthritis in the setting of acute or chronic pancreatitis. the pathologic hallmark is fat necrosis of the affected tissues, but its pathophysiology has not yet been elucidated. it has been hypothesized that high circulating levels of pancreatic enzymes extravasate into the peripheral tissue causing local lipolysis, tissue necrosis and secondary inflammation. the bony or articular lesions are often mistaken for osteomyelitis or de novo arthritis delaying definitive treatment of the underlying pancreatitis. we report a case of ppp syndrome following prolonged pancreatitis secondary to blunt abdominal trauma. further, we have looked into its prevalence in the largest tertiary - care pediatric hospital in canada. a previously healthy 6-year old boy suffered a bicycle handle bar injury to the abdomen, resulting in transection of the pancreas between the head and body. he subsequently developed pancreatitis including abdominal pain and elevation of amylase (252 u / l, normal range 20110 u / l), which increased to 1444 u / l by hospital day 1, and lipase of 983 u / l (normal range 060 he was re - admitted with abdominal pain, fever, hyperamylasemia and hyperlipasemia (peak amylase and lipase : 2362 u / l and 3807 u / l respectively). the patient failed repeated attempts of advancing enteral feeding leading to the decision to perform an endoscopic retrograde cholangiopancreatography (ercp) and an endoscopic ultrasound (eus) to establish cyst drainage. challenged by the needed to organize an outside adult gastroenterologist and rental of the equipment, cyst - drainage via cyst - gastrostomy was not established until 10 weeks after injury. five weeks following the pancreatic injury the patient developed swelling and erythema of his right index and ring fingers (fig. subsequently his left middle finger, right great toe and his left 3rd and 5th toes showed similar lesions. plain radiographic examination demonstrated multiple permeative lucencies in the affected phalanges, metacarpals and metatarsals with preservation of the adjacent joints (fig. 2). magnetic resonance imaging (mri) of the right hand revealed multiple medullary bone infarctions with phlegmonous appearing collections in the affected areas (fig. rheumatologic evaluation for differential diagnosis of dactylitis was negative for anti - nuclear antibodies, rheumatoid factor and hla b-27. surgical exploration of the affected fingers of the right hand, indicated due to non - response to antibiotics, showed multiple cortical erosions with breach of the periosteum allowing the medullary canal to communicate with the subcutaneous tissues. free cream - colored fluid was sampled from the right index (metacarpal and middle phalanx) and ring (middle phalanx) fingers for diagnostic analysis. histopathology demonstrated necrotic tissue debris, which had undergone saponification, with absence of any inflammatory cellular infiltrate (fig. 4). microbiological cultures of the cream - colored fluid were negative for any pathogens. extensive pathological and biochemical investigations to further look into the diagnosis and possible mechanisms of ppp revealed low levels of triglycerides (2.1 mmol) and high levels of fatty acids (10,443.5 m, randox enzymatic method), reflecting a lipolytic process. no amylase activity was detected (0 u / l) ; however the lipase activity exceeded the serum lipase by 4 times (15062.5 u / l) and was further specified, using rabbit antisera against recombinant human pancreatic triglyceride lipase (ptl) and recombinant human carboxyl ester lipase (cel), as pancreas triglyceride lipase (fig. his abdominal symptoms resolved, his pancreatic enzymes decreased to an amylase of 138 u / l and lipase of 330 u / l and his digital lesions improved. four months later the pseudocyst and the digital lesions had completely recovered as confirmed by serial hand x - rays, and the pancreas enzymes had normalized. we performed a retrospective chart review of patients with pancreatitis seen at the hospital for sick children (hsc), toronto, between 1992 and 2012 (approved by the research ethics board). we only identified one other patient of 265 children presenting with pancreatitis who had developed ppp and who was reported previously, yielding an incidence of 7 per 1000 pancreatitis cases. in brief, this is a 4-year old boy with progressive renal failure due to steroid refractory nephrotic syndrome and sclerosing glomerulonephritis of unknown cause, who developed acute pancreatitis (peak serum amylase 1800 u / l and lipase 1327 u / l). he was kept on bowel rest and parenteral nutrition ; eight days later he developed erythematous tender nodules on his back, right flank and knees. a biopsy of these lesions confirmed lobular panniculitis with fat necrosis and signs of saponification. we performed a retrospective chart review of patients with pancreatitis seen at the hospital for sick children (hsc), toronto, between 1992 and 2012 (approved by the research ethics board). we only identified one other patient of 265 children presenting with pancreatitis who had developed ppp and who was reported previously, yielding an incidence of 7 per 1000 pancreatitis cases. in brief, this is a 4-year old boy with progressive renal failure due to steroid refractory nephrotic syndrome and sclerosing glomerulonephritis of unknown cause, who developed acute pancreatitis (peak serum amylase 1800 u / l and lipase 1327 u / l). he was kept on bowel rest and parenteral nutrition ; eight days later he developed erythematous tender nodules on his back, right flank and knees. a biopsy of these lesions confirmed lobular panniculitis with fat necrosis and signs of saponification. we report the case of a 6-year old boy presenting with panniculitis and intramedullary fat necrosis caused by traumatic pancreatitis. prolonged pancreatitis due to late resolution of the pseudocyst may have caused the development of panniculitis. nevertheless, previous reports argue against a simple linear relation between duration and magnitude of elevated pancreatic enzymes and the onset of peripheral tissue damage. osteoarticular lesions have appeared weeks or months after the initial pancreatic event and can even predate the identification of the pancreatic disease. the incidence of ppp syndrome is extremely low ; a handful of pediatric cases have been published : five cases of pancreatitis with panniculitis and osteoarticular lesions and two with panniculitis alone [611 ]. only two patients with ppp have been identified in our hospital over the last 20 years. the most widely accepted theory states that serum lipase extravasates into the peripheral tissues and penetrates the periarticular space, thus causing lipolysis and secondary inflammation. in our patient, we detected grossly elevated concentrations of pancreatic triglyceride lipase at the site of the peripheral inflammation. described higher levels of lipase in the synovial fluid of a patient with pancreatitis and polyarthritis and forstrm. in a skin tissue specimen from a patient with panniculitis. high lipase concentrations in the aspirated fluid of the peripheral lesions may not only be due to extravasation, but can either be explained by slow tissue clearance of this enzyme, or by a process that involves local production of lipase. peripheral fat tissue expresses adipose triglyceride lipase and as recently shown, also pancreatic lipases, which when activated induce fat necrosis. the process observed in ppp syndrome could thus involve locally residing adipocytes, and the isolated pancreatic triglyceride lipase in the patient s fluid may stem from the patient s pancreas or his tissue adipocytes. interestingly, as seen in other cases, panniculitis occurred in the patient s peripheries targeting the osteoarticular space, which may indicate the higher vulnerability of this tissue. recently demonstrated that fatty acids increase the production of pro - inflammatory cytokines in synovial fibroblasts and human chondrocytes in vitro. the high concentration of fatty acids in the digital fluid of our patient may therefore have been sufficient to induce inflammation in this tissue. we suggest that management of ppp - associated panniculitis and polyarthritis should mainly be directed toward evaluating and treating of the underlying pancreatitis. our two cases indicated that resolution of the panniculitis was dependent on the resolution of the pancreatitis, which is in agreement with previous reports. thus, clinical knowledge about the association of panniculitis and/or polyarthritis with pancreatitis is essential to avoid unnecessary investigations of the peripheral lesions, and rather direct treatment strategies toward resolution of the pancreatitis. the pathophysiological findings in our case support earlier reports suggesting that pancreatitis induced panniculitis derive from a lipolytic process in susceptible peripheral tissues. the resulting accumulation of fatty acids in turn causes tissue inflammation and destruction. while this process is most likely initiated by extravasated lipase, exacerbation of the tissue destruction by local activated tissue adipocytes may explain the missing correlation between severity of the pancreatitis and onset of panniculitis.	pancreatitis - panniculitis - polyarthritis (ppp) syndrome is rare and its physiopathology unclear. a 6-year old boy suffered of traumatic pancreatitis complicated by ppp syndrome. extensive investigations demonstrated high levels of pancreatic lipase and fatty acids in the affected peripheral tissues. these findings support the sequence of peripheral lipolysis and fatty acid accumulation inducing tissue inflammation.
the field of movement disorders, which are also known as extrapyramidal disorders, is growing rapidly. it is now recognized that disorders of the basal ganglia have a genetic basis and it is believed that molecular genetic data will provide clues to the pathophysiology of normal and abnormal motor control. an understanding of pathogenic processes and the significant role of genetics has led to many experiments that, perhaps, in the future will result in more effective treatment of diseases such as parkinson 's (pd) or huntington s disease (hd). however, the unclear pathogenesis of many neurodegenerative diseases and the lack of simple and readily available indicators of the early period of these diseases remain crucial issues in the causal treatment of these diseases. progress in molecular genetic studies, leading to the detection of genetic mutations and loci, has contributed to the understanding of mechanisms of neurodegeneration and to clarify the pathogenesis of some neurodegenerative diseases. molecular studies have also found application in the diagnosis of neurodegenerative diseases, increasing the range of genetic counseling and enabling more accurate diagnosis. currently, diagnostic tests, including the exploration of known inherited disease - causing mutations in an autosomaldominant or autosomal recessive manner, are carried out in patients with a clinical diagnosis of a particular disease. tests prior to the onset of a disease are performed at the request of adult individuals with families in which molecular studies have previously identified a genetic mutation linked to the disease in question. moreover, prenatal diagnosis is carried out if one of the parents is known to have the disease. currently used molecular diagnostics based on dna analysis can identify 9 neurodegenerative diseases, namely spinal cerebellar ataxia inherited in an autosomal dominant manner, dentate - rubro - pallido - luysian atrophy (drpla), friedreich s disease, ataxia with oculomotorapraxia induced mutation of aprataxin gene (aptx), hd, dystonia type 1 (dyt1), wilson s disease (wd), and some cases of pd caused by gene mutations [1 - 3 ]. the online mendelian inheritance in man (omim) database lists over 500 entries for disorders of which ataxia or a movement disorder is a component, but this paper presents only selected issues on the molecular basis and diagnostic capabilities of certain diseases of the extrapyramidal system. parkinson s disease is a progressive disorder of the central nervous system (cns), manifesting a clinical slowdown in mobility, muscle rigidity and tremor at rest, resulting from loss of midbrain substantia nigra (sn) cells, which results in a dramatic reduction of the neurotransmitter dopamine in the striatum. parkinson s disease is one of the most common degenerative diseases of the cns. according to estimates of the world health organization (who), approximately 5 million people suffer from pd. in europe aprroximately 1 million americans and about 100 000 british suffer from pd [6, 7 ]. as is clear from epidemiological studies in the united states, on average, every nine minutes a new case of pd however, there are cases of much earlier onset, called early - onset parkinsonism (before 40 years of age) and juvenile parkinsonism, onset before 30 years. it is also known that the incidence of pd is about 20 in 100 000 each year [8 - 11 ]. the four main symptoms of pd are resting tremor, slowness of movement, muscle rigidity, and altered postural stability. most often the first clinical symptom of pd is a tremor of one hand in addition to the tremors, disability in one upper limb or dystonic setting for walking may occur. characteristic of pd is also inclined silhouette of the body due to increased paraspinal muscle tension, and patients walk using small steps. a reduction of the amplitude of movement has also been observed, manifested by micrographia and hypomimia. over time, there are disturbances of balance that can lead to frequent falls [10, 12 ]. as is well known in pd, in addition to the movement disorders patients also present with neuropsychiatric disorders and autonomic dysfunction. the role of genetic factors in the etiology of pd has been controversial for many years. it is currently believed that the etiology of pd is multifactorial, indicating the contribution of both environmental and genetic factors. indicate that the risk of developing pd in the general population over 80 years of age is about 2%, and increases to about 5 - 6% if the disease is present in a parent or sibling. if the symptoms of the disease occur in two family members, the risk is 20 - 40%.. showed that the risk of developing of pd in first degree relatives of patients within a geographical area for age 75 was 2% while in the control group was 1%. epidemiological studies in a group of 203 sibling pairs have showed pd manifestation in all subjects. furthermore, a pet study on 17 twins estimated a concordance of 45% in monozygotes and 29% in dizygotes, supporting the genetic hypothesis of pd pathogenesis. a greater risk of pd manifestation has been shown among biological relatives than among spouses. for many familial forms of pd (fpd), the genes or loci responsible for the manifestation of the disease have been identified and described. it is known that fpd, depending on the causal mutations, can be inherited in either an autosomal dominant or in an autosomal recessive manner. however, it is believed that certain genetic variants may also play a role in the modulation of the risk for the sporadic form of pd (spd). the first genetic mutation, located on the long arm of chromosome 4, was detected in a large italian - american family, coming from the contrusi village in the province of salerno in southern italy, with typical for pd clinical and neuropathological symptoms. since then, numerous further genes and mutations involved in the pathogenesis of pd have been described. the currently identified genes associated with the occurrence of fpd are shown in (table 1). it is now believed that genetic diagnostic testing can be performed in some patients with fpd and those with early onset of disease (before age 50). however, according to recent studies, mutations affecting lrrk protein-2 (dardarin) and parkin protein can also cause spd. genetic studies indicate that nearly 20% of patients with initial symptoms of pd before an age of 40 years may have a mutation of the prkn gene encoding the parkin protein. an increasing body of evidence points to the influence of mutations of this gene as a risk factor of pd at a later age (> 40 years). moreover, it has been shown that, in some populations, as many as 40% of all pd patients have a mutation of the lrrk2 gene. furthermore, there are newer reports in the literature of additional genes involved in the pathogenesis of pd and their possible interactions. genome - wide association studies (gwas) indicate a strong association to the snca and mapt gene [20 - 26 ]. the first meta - analysis of datasets from pd gwas identified five novel risk loci for pd (acmsd, stk39, mccc1/lamp3, syt11 and ccdc62/ hip1r) and six that had previously been identified (mapt, snca, hla - drb5, bst1, gak and lrrk2). a recent meta - analysis revealed another five pd risk loci (park16, stx1b, fgf20, stbd1 and gpnmb). other genetic disorders may also be occasionally present with parkinsonism, including tau mutations, which may sometimes cause a progressive supranuclear palsy (psp)-like presentation, sca3 mutations which may be present with parkinsonism as well as ataxia, the juvenile presentation of hd, and wd. some genes related with pd may also be included in the pathogenesis of parkinsonism plus syndromes. although parkinsonism plus syndromes have been classified based on phenomenology, the studies of the underlying pathology have changed our understanding of the relationship between these disorders. it is currently known that, in pick disease and tau - positive frontotemporal dementia there is a predominance of 3-repeat isoform of tau protein, whereas corticobasal degeneration and progressive supranuclear palsy exhibit the 4-repeat tau isoform. it is also known that the presence of different protein isoforms is conditional, based on the presence of a particular genetic variant. on the other hand, in some cases of ubiquitin positive and tau negative frontotemporal dementias, mutations in the progranulin gene and in tdp43 (a nuclear factor that functions in regulating transcription and splicing) tdp43 has also been found to underlie familial cases of amyotrophic lateral sclerosis (als) and guam - parkinson dementia complex, suggesting a pathologic role ; while in lewybodies dementia and multiple system atrophy (msa), disturbances in synuclein (asn) protein structure and the presence of lewy bodies and fibrillary aggregates formed by this protein have been shown. although msa was previously widely considered to be a nongenetic disorder, recently several genes have been associated with an increased risk of msa, including the snca gene encoding asn first and foremost, but also other genes involved in oxidative stress, mitochondrial dysfunction, inflammatory processes, as well as parkinsonism- and ataxia - related genes have been implicated as susceptibility factors. it has been demonstrated that clinical symptoms of snca multiplication in patients sometimes resemble symptoms usually seen in msa, suggesting that the clinical phenotype can be more variable and does not necessarily resemble that of pd. moreover, sequencing studies, gene dosage measurements, and microsatellite testing have demonstrated a significant association of snca variants with msa [35 - 38 ]. furthermore, it is currently suggested that additional candidate genes for msa may be among others : prkn, pten - induced putative kinase 1 (pink1) [39, 40 ], mapt [41, 42 ], slc1a4, sqstm1, eif4ebpi. in addition, polymorphisms in il-1, il-1, il-8, and icam-1 have been reported to be associated with an increased risk of msa [44 - 46 ]. the adhc1 g78x mutation has also been shown to be associated with msa in the british, but not in the german population, whereas no significant associations were detected on adh7 gene in these populations [47, 48 ]. the severity of chorea movement can vary greatly : from discrete to such that it prevents normal function. chorea may occur as a result of various factors, which include the history of both the autosomal dominant (including hd, neuroacanthocytosis, benign familial chorea) and the autosomal recessive (such as wd) hereditary diseases. it is caused by a mutation in the gene encoding the huntingtin protein, and is located on the short arm of chromosome 4 (4p16.3.) ; it consists of a pathological increase in the quantity of cag triplets. the wild - type contains a cag repeat, coding for a polyglutamine stretch in the protein at that site in the range between 6 and 26 copies. the number of trinucleotide repeats varies between generations due to the existence of meiotic instability, which in normal alleles is rare (60), where the juvenile form of hd was observed [52 - 54 ]. the age at which the disease becomes apparent, may also be determined by other genetic factors, such as genetic polymorphisms of the glur6 gene, located on chromosome 6, or the apolipoprotein e4 gene. moreover, since a huntingtin - associated protein (hap 1) is enriched in the brain, this may represent a possible basis for the selective brain pathology in hd. the diagnosis of hd is based on clinical criteria, family history and genetic testing. when chorea is the first and most prominent sign of the disease, analyzing the history would be the initial and most valuable step. in many cases the underlying cause is another general internal disorder or an iatrogenic disorder. only very few genetically determined disorders are responsible for choreatic syndromes. in about 1% of the cases clinically diagnosed as hd by the clinician, the genetic test does not confirm the diagnosis. these cases are called phenocopies and are defined by a clinical diagnosis of hd with chorea, psychiatric and or cognitive signs, and an autosomal dominant pattern of inheritance or family history (table 2). wilson s disease or hepato - lenticular degeneration, is a rare disorder of the metabolism of copper [58 - 60 ]. the incidence of wd in most of the world s population is estimated to be approximately 1:30.000. this disease is more common in china, japan (1:10.000), sardinia (1:7000), iceland and the canary islands (1:2600) [58 - 60 ]. wilson s disease is caused by mutations in the gene encoding the p - type adenosine triphosphatase (atp7b) located at chromosomal position 13q14-q21. nearly half of the patients have impaired liver function : hepatitis (chronic or acute), cirrhosis and hyperacutehepatitis. neuropsychiatric disorders are seen in the majority of patients, which may lead to an incorrect diagnosis, often schizophrenia or alcohol dependence. testing for mutations of the atp7b gene is performed if the results of biochemical tests (including the concentration of copper in daily urine) or liver biopsy are inconclusive and do not allow for the diagnosis. indications for genetic testing are wd in first - degree relatives of patients, if patients are not found to have a keiser - fleischer ring, and the concentration of ceruloplasmin is slightly reduced and patients have impaired liver function. so far, the presence of missense and nonsense point mutations has been demonstrated, as have deletions, duplications and insertions. the individual mutations occur with different frequencies in different regions of the world. in poland, the most common (72% of alleles) is believed to be mutation c.3207c > a, located in exon 14 of the atp7b gene. this mutation was also observed with high frequency in other european countries including germany, russia, austria, hungary, greece, sweden, denmark, and england [65 - 72 ]. because of the wide variety of mutations in the atp7b gene, as well as a large variety of phenotypes of wd, a comparative analysis was performed to determine whether the genotype can be inferred by the phenotype of the disease. the comparative analysis of the phenotype of patients with the wd p.h1069q mutation and patients with other mutations in atp7b found that mutation p.h1069q (as either homo - or heterozygous) is associated with significantly less severe disturbances in the metabolism of copper, and with later onset of the first clinical signs of wd in comparison with other mutations. it is believed that the occurrence of wd in patients homozygous for the p.h1069q mutation is caused by a defect in the treatment of atp - ase7b and its accelerated degradation. despite the observed genotype - phenotype relation, significant variations in the phenotype of the disease among people with the same type of mutationhave been found in all analyses. it is believed that the phenotypic variability in wd is largely due to the influence of other modifying factors, among which include the dietary intake of copper, the activity of antioxidant mechanisms, and the activity of other proteins involved in the metabolism of copper. dystonia is a group of symptoms involving persistent muscle spasms, often of a repetitive twisting movement, sometimes with the formation of fixed positions. involuntary movements often increase with purposeful movement, which is sometimes called action dystonia. sometimes muscle spasms are associated only with a specific function, such as writer 's cramp, when symptoms occur only with attempting writing, and are not present in other activities. as the disease progresses, dystonic movements can be triggered by motor activity in the uninvolved areas of the body. with further progression of the disease, dystonia may be a separate disease, but it can also be a symptom of the course of other diseases. according to the symptomatic classification of dystonia, it may be generalized, focal, multifocal, segmental, or unilateral (hemidystonia). according to classification based on age of onset, dystonia is divided into dystonia onset in childhood (under 12 years), beginning at a young age (between 13 and 20 years old) and dystonia onset in adulthood (after age 20). according to recently published epidemiological studies, dystonia is particularly frequent in the population of ashkenazi jews, in which the rate is about 1 per 6000. genetic studies have allowed the identification of 15 types of familial dystonia, which are listed chronologically according to their description, from dyt1 to dyt15, although there is currently even a dyt25 form (table 3). the genetically determined generalized dystonias include : the earliest described generalized dystonia dyt1, idiopathic torsion dystonia dyt2, x - linked dystonia, which can be both generalized and segmental (dyt3), and 2 forms of dystonia responsive to levodopa (dyt5 and dyt14). the most well - known, idiopathic torsion dystonia dyt 1, described at the beginning of the last century, is dystonia associated with an autosomal dominant mutation located on chromosome 9 (9q34) [80 - 83 ]. this gene encodes the torsin protein, whose role under normal conditions is connected totransmembrane transport in the dopaminergic system, involving a cell s response to stress and the process of intracellular protein metabolism. the mutation (a 3-bp deletion in the coding sequence of the tor1a gene) leads to the loss of the ability of torsin to bind to a target protein. this mutation is found in diverse ethnicities, either inherited as a founder mutation or de novo, and only two additional mutations of unclear pathogenicity (p.r288q and p.f205i) have been described in isolated a typical cases [85, 86 ]. what is interesting is that the penetrance of the mutated gene is only about 40%, which means that not all carriers of these mutations have symptoms of the disease [80 - 83 ]. dyt 4 is described in several families and is called whispering dysphonia. for dyt 4, the p.r2 g mutation in the tubb4a gene (dyt4, encoding isoform it is known that this form of dystonia is inherited in anautosomal dominant manner, while dyt 6 is related with an autosomal dominant mutation in gene thap1 (thanatos associated protein-1) for which the genetic location is not yet fully known (chromosome 8 p21-q22), with onset of symptoms in adulthood. dyt 7 is associated with an autosomal dominant mutation on chromosome 18p11.3, where an unknown gene product leads to focal dystonia with onset in adulthood. furthermore, it is known that dystonia dyt 13 is related with an autosomal dominant mutation on chromosome 1p36 with an unknown gene product, leading to focal or segmental, usually mild, dystonia. paroxysmal dystonias are the three successively described dyt 8, 9 and 10, while among dyt 11 and dyt 12 belong to the genetically determined form of dystonia with specific symptomatology. dyt 11 is an autosomal dominant myoclonic dystonia associated with the mutation located on chromosome 7q21-q31 and chromosome 11q23. the gene product, which is subject to mutation, in the first case is the gene for the protein -sarcoglycan, and in the second case is the gene for the dopamine d2 receptor protein. clinically, dyt 11 manifest smyoclonic muscle break up mainly of the shoulder and neck, accompanied by dystonia. inheritance is autosomal dominant and the mutation is located on chromosome 19q13 in the gene for atpase associated with the sodium - potassium pump. dyt 15 is a recently identified myoclonic dystonia genetically different from dyt 11 [79, 90 ]. it is believed that genetic testing in dyt 1 is justified in patients with primary dystonia with onset of symptoms before 26 years of age and in patients with primary dystonia with alater start but with a family in which early - onset dystonia has occurred. some centers worldwide are also able to provide genetic tests for dyt 11 and dyt 5. spinocerebellar ataxias (sca) are an autosomal dominant clinically and genetically heterogeneous group of disorders. to date, 27 loci and genetic mutations have been identified in different types of spinal cerebellar ataxia (sca1-sca27, table 4). the disease usually begins in adulthood, most often between 30 and 45 years old, and has a progressive course usually leading to severe disability. it is assumed that the most common type of the disease is sca3, which appears to be very rare in poland (not a single case found to date), while the most common types seen in poland are sca1 (the most) and sca2. a characteristic feature of most forms of sca is the phenomenon of genetic anticipation, caused by trinucleotide repeat instability. this phenomenon depends on the earlier onset of symptoms in subsequent generations, as well as increasingly more severe course. in all types of sca the cerebellar syndrome is similar, dominating increasingly problematic balance and gait abnormalities accompanied by dysarthria and ataxia of the upper limbs [92 - 94 ]. extrapyramidal symptoms are sometimes observed (chorea, pd), as well as abnormal eye movements, symptoms of upper motor neuron damage, dementia, and peripheral neuropathy. the genetic basis of the majority of sca cases is associated with instability of cag trinucleotide repeats found in the coding regions of the various genes sca1, sca2, sca3, sca6, sca7 and sca17 [95, 96 ]. however, two genes have been identified in which the unstable sequences are in the untranslated ends of genes, and their expansion underlies two types of the disease - sca8 and sca12. in sca10 the repeat instability has been shown in fivenucleotide sequences located in an intron [93, 94 ]. except for sca6, wherein said defect in the gene encoding the subunit calcium channel cacna1a, and for sca17 with a tbp - defective gene, other genes associated with particular types of sca encode proteins of unknown function and lack homology to other known proteins present. it was also found that the multiplication of the number of cag repeats in the cacna1a gene leads to the onset of symptoms characteristic of sca6 and point mutations in the same gene are associated with the presence of other diseases : episodic ataxia type 2 (ea2) and familial hemiplegic migraine (fhm). drpla is often included in the group of sca, as there is also a reported effect in the number of cag trinucleotide repeats and ataxia is the predominant clinical symptom. the most common form of cerebellar ataxia is inherited in therecessive friedreich disease (fd). an increased number of gaa trinucleotide repeats and point mutations have been detected in the locus encoding the protein frataxin on chromosome 9q13 - 21.1. in the gene encoding frataxin, the correct number of repetitions is 6 - 28, while number ranging from 66 to 1700 has been reported in patients. it is believed that frataxin is involved in iron transport, and in the course of fd there is a significant accumulation of iron in the mitochondria. i, missense mutations were detected in the aptx gene located on chromosome 9q13, encoding the aprataxin protein, which belong to the histidine triad family. early symptoms (typically at 20 years of age) and a rapid development of the disease have been observed. early in the onset, there is a cerebellar syndrome and oculomotor apraxia present, and later peripheral neuropathy, chorea and dementia are seen. ataxia with oculomotor apraxia type ii is a recently described ataxia in which mutations were located on chromosome 9q34. clinical symptoms occur later in life, the disease progression is slower, and increased levels of serum -fetoprotein (afp) have been detected. in the genes associated with the occurrence of sca, the normal range of microsatellite repeats is characterized by a high degree of polymorphism. a specific cag range that is stable and however, the presence of intermediate alleles (ia) has been shown to not to cause symptoms of the disease, but is characterized by reduced stability and an increased risk of the emergence of new pathogenic mutations in the number of cag repeats. molecular diagnosis of sca is based on dna analysis to determine the number of cag repeats in the genes associated with the occurrence of various types of sca. it is possible to confirm the clinical diagnosis and to determine the type of ataxia, if it is shown that the number of cag repeats in one of the studied genes exceeded the normal range. the diagnosis of six autosomal dominant forms of spinal cerebellar ataxia is possible using dna testing : sca1, sca2, sca3, sca6, sca7 and drpla.	knowledge on the genetics of movement disorders has advanced significantly in recent years. it is now recognized that disorders of the basal ganglia have genetic basis and it is suggested that molecular genetic data will provide clues to the pathophysiology of normal and abnormal motor control. progress in molecular genetic studies, leading to the detection of genetic mutations and loci, has contributed to the understanding of mechanisms of neurodegeneration and has helped clarify the pathogenesis of some neurodegenerative diseases. molecular studies have also found application in the diagnosis of neurodegenerative diseases, increasing the range of genetic counseling and enabling a more accurate diagno - sis. it seems that understanding pathogenic processes and the significant role of genetics has led to many experiments that may in the future will result in more effective treatment of such diseases as parkinson s or huntington s. currently used molecular diagnostics based on dna analysis can identify 9 neurodegenerative diseases, including spinal cerebellar ataxia inherited in an autosomal dominant manner, dentate - rubro - pallido - luysian atrophy, friedreich s disease, ataxia with ocu - lomotorapraxia, huntington 's disease, dystonia type 1, wilson s disease, and some cases of parkinson 's disease.
i m was developed as a program planning framework for the development of theory- and evidence - based health promotion interventions (bartholomew, parcel, kok, gottlieb, & fernndez, 2011). i m provides guidelines and tools for the empirical and theoretical foundation of health promotion programs, for the application of theory, for the translation of theory into actual intervention activities and materials, for the management of program adoption and implementation, and for the collaboration between health educators, researchers, priority groups and stake - holders. i m enables health promoters to develop interventions that include theory - based intervention strategies and materials that may accomplish program objectives, and that match priority populations and intervention contexts. i m guides program planners to map the path of intervention development from recognizing a need or problem to testing solutions. it describes the intervention development process in six steps (see table 1). in everyday language (schaalma & kok, 2009) : (1) what is the problem ? (2) what does the program planner need to change, and why ? (3) how can these changes be accomplished with theory- and evidence - based methods that can be expected to work ? (4) how can the methods be delivered in a way that makes sense and will be implemented ? (5) how can we facilitate sustained implementation ? (6) did the program work out the way we planned ? each of the six steps comprises several tasks, and completing of the tasks in a step creates a foundation for the subsequent step. the completion of all of the steps serves as a blueprint for designing, implementing and evaluating an intervention based on a foundation of theoretical, empirical and practical information. an i m approach is characterized by four perspectives that are applied during the entire program planning process and in all steps (bartholomew., 2011). from a participation perspective, the framework suggests that the priority population and program implementers should be involved in all aspects of decision making to make sure that the program is based on the needs of the community. from a multi - theory perspective, i m stimulates an eclectic use of theories. because theories are by definition abstractions of reality, and therefore explain only part of it, i m suggests that real - life problems should be approached from multiple theories (buunk & van vugt, 2008). from a systems perspective, interventions are seen as events occurring in systems, with other factors within a system possibly reinforcing or dampening the influence of an intervention on the target behavior or environmental change. finally, from an ecological perspective, planning with i m recognizes the relevance of social and physical environmental conditions that influence behaviors. these social and physical environmental conditions may have a much stronger impact on the target behavior than individual - related factors (kok, gottlieb, commers, & smerecnik, 2008). in this paper, we focus on i m steps 2, 3, 4, and 5 because they are most relevant for the main question of this paper : what are alternatives to fear appeals ? we assume that in conducting step 1 the program planner will focus on the analysis of the problem behaviors and environmental conditions and will have no reason to be focusing on intervention. likewise, evaluation (step 6) focuses on whatever has been developed in 15 and decisions regarding fear appeals are not made in this last step. in step 2 in step 4, fear appeals may be introduced by other professionals involved in text writing and materials development. and in step 5, implementers and decision makers in health promotion organizations may overrule program planners in a misguided preference for fear appeals. a full example of an i m application on adolescent hiv / sti prevention can be found at the web address given in the schaalma and kok (2011) reference. in applying i m, determinants have a function in understanding behavior and in changing behavior. in step 2, the determinants of the risky as well as the health - promoting behaviors are analyzed, while in step 3, theoretical methods for change are identified that may effectively change these determinants. the first impression of the causes of a health behavior problem does not always represent the correct definition of the problem. an example of poorly explored causation is an intended campaign to increase child restraint devices (crds) to protect young children against the consequences of an automobile accident. at first program planners began to plan a campaign based on the assumption that parents did not use the devices based on underestimation of risk. as it turned out, the reasons for parents not using crds was not an underestimation of risk (more than 90% intended to use crds) but a lack of self - efficacy and skills for coping when a child becomes restless and disruptive (pieterse, kok, & verbeek, 1992). a fear appeal intervention would have made the situation for the parents even worse ; what they needed instead was skills training and self - efficacy improvement. a second example is about hiv prevention ; specifically, why do adolescents have sexual intercourse without using condoms ? one explanation, of course, is that they do not recognize the severity of an hiv infection and their own susceptibility to become infected with hiv. however, as it turns out, the major determinants of using or not using condoms are outcome expectations, subjective norms, self - efficacy, responsibility, and embarrassment (bartholomew., 2011, fear appeals would not promote condom use ; what is needed is interventions increasing skills and self - efficacy. how to find the right determinants to target in an intervention ? intervention mapping suggests four core processes : (1) brainstorming in the planning group involving representatives from the target population, stakeholders, and implementers ; (2) searching the empirical literature for determinants of behavior ; (3) identifying and applying appropriate theories on determinants ; and (4) conducting additional qualitative and quantitative research for unanswered questions (bartholomew., 2011, pp. how to identify appropriate theories ? by searching the literature on the topic for theories, by matching constructs from the brainstorm to theories, and by applying frequently used theories (buunk & van vugt, 2008). this construct might lead to the theoretical construct of self - efficacy in social cognitive theory (mcallister., regarding frequently used theories, some agreement is emerging in the health promotion and behavioral health fields regarding important determinants of behavior, based on theoretical constructs across theories. five major behavioral science theorists agreed on a set of eight variables as key determinants of behavior, later called the integrated behavioral model (montao & kasprzyk, 2008). the determinants from the integrated behavioral model are presented in table 2 with the first three variables thought to be necessary and sufficient for behavior change and the remaining five as influencing the direction and strength of behavioral intention. lack of fear not perceiving the negative health consequences of life - endangering behaviors is very seldom an important determinant of risky behavior. determinants of behavior from the integrated behavioral model (montao & kasprzyk, 2008) a number of systematic reviews and meta - analyses in the health promotion field show that adequate use of theory - based methods increases the effectiveness of interventions to change behavior (albarracn., 2005 ; de bruin., 2010b ; fisher, fisher, bryan, & misovich, 2002 ; mullen, green, & persinger, 1985 ; peters. in the third step of i m, all program objectives are organized by determinant. then theoretical methods are identified that may help reach the program objectives, which in turn are translated into practical applications. a theory - based change method is a technique for changing a behavioral determinant of individuals or environmental agents, while a practical application delivers the method to fit with the priority group and the program setting. there are no magic bullets ; some methods can be used for a range of determinants, some only for a specific determinant. there is increasing interest in systematic descriptions of health promotion interventions, the theoretical methods they contain, and the determinants that are targeted for change. for instance, abraham and michie (2008) provide a theory - linked taxonomy of generally applicable individual health behavior change techniques. those behavior change techniques derived from reviews were then linked with theory - based determinants (michie, johnston, francis, hardeman, & eccles, 2008), comparable to the i m approach. other authors combined the taxonomy with the i m approach, to develop a checklist for coding methods in patient education interventions (de bruin. (2009) described processes (and their measures) for use by communities and local governments in planning and monitoring environmental and policy - level interventions for obesity prevention. however, these taxonomies define behavior change methods ; they do not describe whether that method is used appropriately or inappropriately : the parameters for use or the conditions under which methods are effective. 2011) provide tables with theoretical methods and their limiting conditions for every major determinant and for all higher environmental levels. see table 3 for examples of basic theoretical methods at the individual level, table 4 for methods to change risk perception, and table 5 for methods to improve self - efficacy and skills. examples of basic methods at the individual level (adapted from bartholomew., 2011, pp. 327329) examples of methods to change awareness and risk perception (adapted from bartholomew., 2011, pp. 333334) examples of methods to change skills, capability, and self - efficacy and to overcome barriers (adapted from bartholomew., 2011, pp. 342344) it is important to bear in mind that theoretical methods are only effective under certain conditions (schaalma and kok, 2009). modeling, for instance, is only effective when the model is reinforced, and observers pay attention, have sufficient self - efficacy and skills, identify with the model, and observe a coping model instead of a mastery model (mcalister., 2008). other theoretical methods have other conditions that need to be met ; for example, goal setting is effective when the chosen goal is challenging but feasible (latham and locke, 2007). as mentioned earlier, fear arousal as a method requires high self - efficacy expectations about the behavior ; a condition that is very rare because of the complex nature of most behavior change settings. however, it may be clear from these examples that many potential alternative methods are available. step 4 of i m concerns the actual development of the program, integrating the various applications that were chosen in the previous step. program planners decide about the overall structure of the program, themes, channels, and vehicles of the program. they work with other professionals and they pilot - test the relevant elements of the program. even when program planners are aware of the limitations of fear appeals, the other professionals that contribute to this step in program production may not be. fear appeals may appear in the text writing, the choice of illustrations, or in the filming. also in the actual delivery of the program by program peer educators or implementers, the message may be. there is always a moment in the program planning process where someone will suggest using a harder or the program planner is the one responsible for the correct translation of theoretical methods into practical applications based on the parameters of the methods and is often the only team member who is sufficiently trained to do so. program planners should respect other professionals ' expertise but at the same time be able to effectively communicate their own expertise. essential in step 4 is pilot - testing, which is meant as a guarantee against implementing inadequate program materials. individual components of the intervention program should be pilot - tested on their effectiveness before final production and implementation, which can be done relatively easily using experimental research designs (whittingham, ruiter, castermans, huiberts, & kok, 2008). program implementers are the people who participate in program activities to deliver the program to participants or beneficiaries. for example, teachers present health education programs to students, and nurses often deliver programs to patients. gate - keepers, even though they are not program implementers, may be program adopters because they are necessary to get the program to the program participants. for example, school principals may not be the user of a health education curriculum, but their support is critical for program adoption. (2014, this issue) experienced difficulties in convincing their managers and collaborating organizations to implement well - planned alternatives to fear appeals. in the implementation step, fear appeals may be promoted by program implementers and gatekeepers or decision makers, who have insufficient knowledge of the available evidence. effective health education and promotion programs will have little impact if they are never used or if they are discontinued while still needed to create the desired health impact (durlak & dupre, 2008 ; oldenburg & glanz, 2008 ; rogers, 2003). in step 5 of i m, the focus is on planning an implementation protocol to ensure that the program developed in the previous steps will be used and maintained over time, for as long as it is needed. program planners may have to convince implementers and decision makers in their own organization and in collaborating organizations that fear appeals are ineffective and that alternatives are available. the methods discussed above and illustrated in tables 5 can be applied to interventions to accomplish program adoption, implementation, and sustainability. program planners can address personal determinants of implementers such as knowledge of program compatibility and relative advantage, outcome expectations for the program, and self - efficacy for doing the program activities with methods at the individual level, such as persuasive communication, tailoring, and modeling. however, implementation almost always involves organizational change, and program planners have to apply methods at environmental levels as well. organizational theory can be used to understand the determinants of implementation and to select methods for organizational change that will support program implementation (rutten, roger, abu - omar, & frahsa, 2009 ; weiner, lewis, & linnan, 2009). examples of basic methods at the environmental level are presented in table 6 and examples of methods at the organizational level in table 7 (bartholomew., examples of basic methods at the environmental level (adapted from bartholomew., 2011, pp. 347348) examples of methods to change organizations (adapted from bartholomew., 2011, pp. 352353) we elaborated on aspects of i m that would foster our objectives that intervention developers should be better trained in, and more insistent on (1) performing adequate determinant analyses, (2) applying theory- and evidence - based behavior change methods, and (3) convincing their program implementers, managers and collaborating organizations to implement well - planned alternatives to fear appeals. non - behavioral scientists think that fear appeals will trigger people 's risk awareness and motivate them to change based on the assumption that risky or unhealthy behavior is determined by a lack of risk awareness. even if that is the case, underestimation of the severity of consequences is often less important that underestimation of personal susceptibility (ruiter., 2001). moreover, most risk behavior is determined by other determinants intentions, skills, and environmental factors while intention is determined by attitude, social norms, personal norms, affective reactions, and self - efficacy. exposing at - risk people to fear appeals while the actual determinants of the risky behavior are, for example, environmental factors, self - efficacy, and skills, will lead to defensive reactions, including less attention to the fear - arousing message (kessels, ruiter, & jansma, 2010 ; ruiter & kok, 2012). before considering fear appeals, program planners should carefully identify important and changeable determinants of the behavior they want to change, using theory and qualitative and quantitative research (bartholomew. for example, schaalma and colleagues developed an std / hiv prevention program for 13 - 15 year old school students (schaalma & kok, 2011). of course they mentioned the severity of std / hiv infections, but not in an emotional way. the focus of the program was on personal susceptibility, condom use as response efficacy, and the promotion of self - efficacy regarding carrying condoms, negotiating with the partner, correct condom use and maintenance of condom use (schaalma & kok, 2011). they applied theoretical methods such as modeling, scenario - based risk information, and guided practice. to get the schools to implement the program, they applied technical assistance and increasing stakeholder influence, in this case through the municipal public health services. their program was shown to be effective for all targeted determinants, and led to an increase in condom use. as another example, de bruin and colleagues developed a program for hiv nurses to increase therapy adherence with hiv + patients (de bruin, hospers, van den borne, kok, & prins, 2005). again, they explained the health consequences of low adherence, but not in an emotional way. the focus of the program was on self - management and coping with difficult situations. de bruin and colleagues applied theoretical methods such as participation and tailoring, self - monitoring, goal setting, and planning coping responses. to get the hospital and the nurses to implement the program, they applied participatory problem solving, technical assistance, and organizational diagnosis and feedback. the program was shown to be effective in improving adherence (de bruin., 2010a). the i m protocol helps program planners to identify methods and applications for raising risk awareness and for changing any other determinant : not like a cook book, but by suggesting theory- and evidence - based behavior change methods linked with determinants, and by providing the conditions under which these method will be more or less effective. i m has been described as tiresome, complex, elaborate, expensive and time - consuming. to a degree, that may be true. at the same time, however, all authors who assessed i m critically also indicated that i m helped in bringing the development of interventions to a higher level. in the end, advantages outweighed disadvantages (kok & mesters, 2011). implementation almost always involves organizational change, schools, worksites, hospitals, local governments, etc. the same i m process that guides program planners in developing interventions for the at - risk populations and environmental decision makers also guides program planners to develop interventions for dissemination, adoption, implementation, and sustainability. in applying i m, determinants have a function in understanding behavior and in changing behavior. in step 2, the determinants of the risky as well as the health - promoting behaviors are analyzed, while in step 3, theoretical methods for change are identified that may effectively change these determinants. the first impression of the causes of a health behavior problem does not always represent the correct definition of the problem. an example of poorly explored causation is an intended campaign to increase child restraint devices (crds) to protect young children against the consequences of an automobile accident. at first program planners began to plan a campaign based on the assumption that parents did not use the devices based on underestimation of risk. as it turned out, the reasons for parents not using crds was not an underestimation of risk (more than 90% intended to use crds) but a lack of self - efficacy and skills for coping when a child becomes restless and disruptive (pieterse, kok, & verbeek, 1992). a fear appeal intervention would have made the situation for the parents even worse ; what they needed instead was skills training and self - efficacy improvement. a second example is about hiv prevention ; specifically, why do adolescents have sexual intercourse without using condoms ? one explanation, of course, is that they do not recognize the severity of an hiv infection and their own susceptibility to become infected with hiv. however, as it turns out, the major determinants of using or not using condoms are outcome expectations, subjective norms, self - efficacy, responsibility, and embarrassment (bartholomew., 2011, fear appeals would not promote condom use ; what is needed is interventions increasing skills and self - efficacy. how to find the right determinants to target in an intervention ? intervention mapping suggests four core processes : (1) brainstorming in the planning group involving representatives from the target population, stakeholders, and implementers ; (2) searching the empirical literature for determinants of behavior ; (3) identifying and applying appropriate theories on determinants ; and (4) conducting additional qualitative and quantitative research for unanswered questions (bartholomew., 2011, pp. how to identify appropriate theories ? by searching the literature on the topic for theories, by matching constructs from the brainstorm to theories, and by applying frequently used theories (buunk & van vugt, 2008). this construct might lead to the theoretical construct of self - efficacy in social cognitive theory (mcallister. regarding frequently used theories, some agreement is emerging in the health promotion and behavioral health fields regarding important determinants of behavior, based on theoretical constructs across theories. five major behavioral science theorists agreed on a set of eight variables as key determinants of behavior, later called the integrated behavioral model (montao & kasprzyk, 2008). the determinants from the integrated behavioral model are presented in table 2 with the first three variables thought to be necessary and sufficient for behavior change and the remaining five as influencing the direction and strength of behavioral intention. lack of fear not perceiving the negative health consequences of life - endangering behaviors is very seldom an important determinant of risky behavior. a number of systematic reviews and meta - analyses in the health promotion field show that adequate use of theory - based methods increases the effectiveness of interventions to change behavior (albarracn., 2005 ; de bruin., 2010b ; fisher, fisher, bryan, & misovich, 2002 ; mullen, green, & persinger, 1985 ; peters., 2013 ; van achterberg., then theoretical methods are identified that may help reach the program objectives, which in turn are translated into practical applications. a theory - based change method is a technique for changing a behavioral determinant of individuals or environmental agents, while a practical application delivers the method to fit with the priority group and the program setting. some methods can be used for a range of determinants, some only for a specific determinant. there is increasing interest in systematic descriptions of health promotion interventions, the theoretical methods they contain, and the determinants that are targeted for change. for instance, abraham and michie (2008) provide a theory - linked taxonomy of generally applicable individual health behavior change techniques. those behavior change techniques derived from reviews were then linked with theory - based determinants (michie, johnston, francis, hardeman, & eccles, 2008), comparable to the i m approach. other authors combined the taxonomy with the i m approach, to develop a checklist for coding methods in patient education interventions (de bruin. (2009) described processes (and their measures) for use by communities and local governments in planning and monitoring environmental and policy - level interventions for obesity prevention. however, these taxonomies define behavior change methods ; they do not describe whether that method is used appropriately or inappropriately : the parameters for use or the conditions under which methods are effective. bartholomew. (2011) provide tables with theoretical methods and their limiting conditions for every major determinant and for all higher environmental levels. see table 3 for examples of basic theoretical methods at the individual level, table 4 for methods to change risk perception, and table 5 for methods to improve self - efficacy and skills. examples of basic methods at the individual level (adapted from bartholomew., 2011, pp. 327329) examples of methods to change awareness and risk perception (adapted from bartholomew., 2011, pp. 333334) examples of methods to change skills, capability, and self - efficacy and to overcome barriers (adapted from bartholomew., 2011, pp. 342344) it is important to bear in mind that theoretical methods are only effective under certain conditions (schaalma and kok, 2009). modeling, for instance, is only effective when the model is reinforced, and observers pay attention, have sufficient self - efficacy and skills, identify with the model, and observe a coping model instead of a mastery model (mcalister., 2008). other theoretical methods have other conditions that need to be met ; for example, goal setting is effective when the chosen goal is challenging but feasible (latham and locke, 2007). as mentioned earlier, fear arousal as a method requires high self - efficacy expectations about the behavior ; a condition that is very rare because of the complex nature of most behavior change settings. however, it may be clear from these examples that many potential alternative methods are available. step 4 of i m concerns the actual development of the program, integrating the various applications that were chosen in the previous step. program planners decide about the overall structure of the program, themes, channels, and vehicles of the program. they work with other professionals and they pilot - test the relevant elements of the program. even when program planners are aware of the limitations of fear appeals, the other professionals that contribute to this step in program production may not be. fear appeals may appear in the text writing, the choice of illustrations, or in the filming. also in the actual delivery of the program by program peer educators or implementers, the message may be. there is always a moment in the program planning process where someone will suggest using a harder or, the program planner is the one responsible for the correct translation of theoretical methods into practical applications based on the parameters of the methods and is often the only team member who is sufficiently trained to do so. program planners should respect other professionals ' expertise but at the same time be able to effectively communicate their own expertise. essential in step 4 is pilot - testing, which is meant as a guarantee against implementing inadequate program materials. individual components of the intervention program should be pilot - tested on their effectiveness before final production and implementation, which can be done relatively easily using experimental research designs (whittingham, ruiter, castermans, huiberts, & kok, 2008). program implementers are the people who participate in program activities to deliver the program to participants or beneficiaries. for example, teachers present health education programs to students, and nurses often deliver programs to patients. gate - keepers, even though they are not program implementers, may be program adopters because they are necessary to get the program to the program participants. for example, school principals may not be the user of a health education curriculum, but their support is critical for program adoption. (2014, this issue) experienced difficulties in convincing their managers and collaborating organizations to implement well - planned alternatives to fear appeals. in the implementation step, fear appeals may be promoted by program implementers and gatekeepers or decision makers, who have insufficient knowledge of the available evidence. effective health education and promotion programs will have little impact if they are never used or if they are discontinued while still needed to create the desired health impact (durlak & dupre, 2008 ; oldenburg & glanz, 2008 ; rogers, 2003). in step 5 of i m, the focus is on planning an implementation protocol to ensure that the program developed in the previous steps will be used and maintained over time, for as long as it is needed. program planners may have to convince implementers and decision makers in their own organization and in collaborating organizations that fear appeals are ineffective and that alternatives are available. the methods discussed above and illustrated in tables 5 can be applied to interventions to accomplish program adoption, implementation, and sustainability. program planners can address personal determinants of implementers such as knowledge of program compatibility and relative advantage, outcome expectations for the program, and self - efficacy for doing the program activities with methods at the individual level, such as persuasive communication, tailoring, and modeling. however, implementation almost always involves organizational change, and program planners have to apply methods at environmental levels as well. organizational theory can be used to understand the determinants of implementation and to select methods for organizational change that will support program implementation (rutten, roger, abu - omar, & frahsa, 2009 ; weiner, lewis, & linnan, 2009). examples of basic methods at the environmental level are presented in table 6 and examples of methods at the organizational level in table 7 (bartholomew., 2011). examples of basic methods at the environmental level (adapted from bartholomew., 2011, pp. 347348) examples of methods to change organizations (adapted from bartholomew., 2011, pp. we elaborated on aspects of i m that would foster our objectives that intervention developers should be better trained in, and more insistent on (1) performing adequate determinant analyses, (2) applying theory- and evidence - based behavior change methods, and (3) convincing their program implementers, managers and collaborating organizations to implement well - planned alternatives to fear appeals. non - behavioral scientists think that fear appeals will trigger people 's risk awareness and motivate them to change based on the assumption that risky or unhealthy behavior is determined by a lack of risk awareness. even if that is the case, underestimation of the severity of consequences is often less important that underestimation of personal susceptibility (ruiter., 2001). moreover, most risk behavior is determined by other determinants intentions, skills, and environmental factors while intention is determined by attitude, social norms, personal norms, affective reactions, and self - efficacy. exposing at - risk people to fear appeals while the actual determinants of the risky behavior are, for example, environmental factors, self - efficacy, and skills, will lead to defensive reactions, including less attention to the fear - arousing message (kessels, ruiter, & jansma, 2010 ; ruiter & kok, 2012). before considering fear appeals, program planners should carefully identify important and changeable determinants of the behavior they want to change, using theory and qualitative and quantitative research (bartholomew., for example, schaalma and colleagues developed an std / hiv prevention program for 13 - 15 year old school students (schaalma & kok, 2011). of course they mentioned the severity of std / hiv infections, but not in an emotional way. the focus of the program was on personal susceptibility, condom use as response efficacy, and the promotion of self - efficacy regarding carrying condoms, negotiating with the partner, correct condom use and maintenance of condom use (schaalma & kok, 2011). they applied theoretical methods such as modeling, scenario - based risk information, and guided practice. to get the schools to implement the program, they applied technical assistance and increasing stakeholder influence, in this case through the municipal public health services. their program was shown to be effective for all targeted determinants, and led to an increase in condom use. as another example, de bruin and colleagues developed a program for hiv nurses to increase therapy adherence with hiv + patients (de bruin, hospers, van den borne, kok, & prins, 2005). again, they explained the health consequences of low adherence, but not in an emotional way. the focus of the program was on self - management and coping with difficult situations. de bruin and colleagues applied theoretical methods such as participation and tailoring, self - monitoring, goal setting, and planning coping responses. to get the hospital and the nurses to implement the program, they applied participatory problem solving, technical assistance, and organizational diagnosis and feedback. the program was shown to be effective in improving adherence (de bruin., 2010a). the i m protocol helps program planners to identify methods and applications for raising risk awareness and for changing any other determinant : not like a cook book, but by suggesting theory- and evidence - based behavior change methods linked with determinants, and by providing the conditions under which these method will be more or less effective. i m has been described as tiresome, complex, elaborate, expensive and time - consuming. to a degree, that may be true. at the same time, however, all authors who assessed i m critically also indicated that i m helped in bringing the development of interventions to a higher level. in the end, advantages outweighed disadvantages (kok & mesters, 2011). implementation almost always involves organizational change, schools, worksites, hospitals, local governments, etc. the same i m process that guides program planners in developing interventions for the at - risk populations and environmental decision makers also guides program planners to develop interventions for dissemination, adoption, implementation, and sustainability. fear appeals in health education are effective rarely, and only under exceptional circumstances of high efficacy. instead of applying fear appeals, health promoters should identify effective alternatives for fear arousal and carefully develop theory- and evidence - based programs, specifically : identify important and changeable determinants of the risk behavior, choose appropriate intervention methods and applications, and develop interventions for dissemination, adoption, implementation and sustainability of those theory- and evidence - based programs.	fear arousal vividly showing people the negative health consequences of life - endangering behaviors is popular as a method to raise awareness of risk behaviors and to change them into health - promoting behaviors. however, most data suggest that, under conditions of low efficacy, the resulting reaction will be defensive. instead of applying fear appeals, health promoters should identify effective alternatives to fear arousal by carefully developing theory- and evidence - based programs. the intervention mapping (i m) protocol helps program planners to optimize chances for effectiveness. i m describes the intervention development process in six steps : (1) assessing the problem and community capacities, (2) specifying program objectives, (3) selecting theory - based intervention methods and practical applications, (4) designing and organizing the program, (5) planning, adoption, and implementation, and (6) developing an evaluation plan. authors who used i m indicated that it helped in bringing the development of interventions to a higher level.
abnormal uterine bleeding (aub), defined as a change in any or a combination of frequency, duration, or amount of bleeding, is a common gynecological complaint that affects 10 - 30% of reproductive - aged women and constitute about one - third of all outpatient gynecological visits. the abnormal bleeding patterns can be annoying and adversely affect the quality of woman 's life since unpredictable or heavy bleeding can lead to psychological, social, medical, and sexual problems and thus necessitate appropriate and adequate management. apart from bleeding related to pregnancy, menorrhagia in reproductive - aged women can result from different causes including dysfunctional uterine bleeding (dub), organic lesions as fibroids, adenomyosis as well as systemic causes such as coagulopathies (e.g., von willebrand 's disease or use of anticoagulants). different treatment modalities for such problem are available, yet the levonorgestrel intrauterine system (lng - ius) has recently provided a good treatment option effective in treating such complaint and at the same time, having a reliable contraceptive effect which is desired by such age group. we aim to highlight the role of lng - ius in the treatment of aub in reproductive - aged women. the lng - ius, which is marketed under the name of mirena (bayer health care pharmaceuticals inc, wayne, nj), is similar in shape to the cu - t intrauterine device with the vertical stem containing a mixture of 52 mg of levonorgestrel and polydimethylsiloxane (pmds) surrounded by rate - controlling pdms capsule. figure 1 shows hysteroscopic view of lng - ius properly placed inside the uterus. the lng - ius allows a steady local release of 20 ug of levonorgestrel per day. the intrauterine concentrations of levonorgestrel released by intrauterine system have been estimated to be 1000 times higher than that with a levonorgestrel subdermal implant. this high level of levonorgestrel in the endometrium induces dramatic effects leading to the unique mode of contraceptive and therapeutic action of the lng - ius. initially developed to decrease the risk of expulsion of the intrauterine contraceptive device by reducing myometrial contractility, lng - ius has been found to result in dramatic reduction in the menstrual blood loss which led researchers to investigate its role as an alternative to surgery for the treatment of aub especially in reproductive - aged women. the use of lng - ius was found to reduce menstrual blood flow which subsequently led to rise in the serum ferritin levels and hemoglobin levels in women with normal menstrual blood loss as well as in those with heavy menstruation. it was found to reduce blood loss by 86% after 3 months of use and up to 97% by 12 months of use. more recent evidence shows that in women with heavy menstrual blood loss, the lng - ius can normalize blood flow, with up to 35% of women being amenorrheic at 24 months. several studies have compared the efficacy of lng - ius in treating menorrhagia with other treatment modalities. women with menorrhagia treated with lng - ius were found to have more reduction in the menstrual blood flow than those who used non - steroidal anti - inflammatory drugs or anti - fibrinolytic agents (tranexamic acid). moreover, when compared to norethisterone 5 mg (three times a day from day 5 to day 26 of the cycle) in a three cycle randomized study, lng - ius was found to be more effective in reducing menstrual blood flow (94% in the lng - ius group and 87% in the oral norethisterone group). in the same study, only 22% of women wished to continue treatment in the norethisterone arm compared to 76% of women using the lng - ius. hysterectomy has long been the definitive treatment for refractory cases with dub who failed to respond to medical treatment. however, since the late 1990s, evidence started accumulating to suggest that the lng - ius can be a safe and effective medical alternative to hysterectomy. this was proved in two studies which documented that 64 - 82% of patients who had the lng - ius inserted for temporary control of their aub, pending hysterectomy declined proceeding with the hysterectomy. lng - ius was found to be almost equally as effective as endometrial ablation or resection which emerged as a new minimally invasive procedure for the treatment of menorrhagia. who randomized women to either an endometrial resection or lng - ius to control their heavy menstrual bleeding. this study demonstrated a reduction in the pictorial blood loss assessment chart (pbac) at 12 months by 79% in the lng - ius users and 89% in those women who had undergone a resection. amenorrhea or infrequent bleeding was reported by 65% of women using the lng - ius compared with 71% who underwent a resection. satisfaction was high in both groups, with no significant difference in health - related quality - of - life scores. a cochrane systematic review appraised the literature and concluded that surgery, especially hysterectomy, reduces menstrual bleeding at 1 year more than medical treatments but lng - ius appears equally effective in improving quality of life. it is estimated that about 13% of women with menorrhagia suffer from von willebrand 's disease. when the effect of the lng - ius on hemostasis in women with menorrhagia was studied, it showed high expression of fibrinolytic inhibitors (plasminogen activator inhibitor 1 and 2) and up - regulated urokinase - type plasminogen activator receptor expression in the endometrium. this may explain why the lng - ius successfully treated heavy menstrual blood loss in cases with inherited bleeding disorders. lng - ius is effective in reducing the duration and amount of menstrual bleeding in women with menorrhagia associated with the use of oral anticoagulation. women with menorrhagia treated with lng - ius were found to have more reduction in the menstrual blood flow than those who used non - steroidal anti - inflammatory drugs or anti - fibrinolytic agents (tranexamic acid). moreover, when compared to norethisterone 5 mg (three times a day from day 5 to day 26 of the cycle) in a three cycle randomized study, lng - ius was found to be more effective in reducing menstrual blood flow (94% in the lng - ius group and 87% in the oral norethisterone group). in the same study, only 22% of women wished to continue treatment in the norethisterone arm compared to 76% of women using the lng - ius. hysterectomy has long been the definitive treatment for refractory cases with dub who failed to respond to medical treatment. however, since the late 1990s, evidence started accumulating to suggest that the lng - ius can be a safe and effective medical alternative to hysterectomy. this was proved in two studies which documented that 64 - 82% of patients who had the lng - ius inserted for temporary control of their aub, pending hysterectomy declined proceeding with the hysterectomy. lng - ius was found to be almost equally as effective as endometrial ablation or resection which emerged as a new minimally invasive procedure for the treatment of menorrhagia. who randomized women to either an endometrial resection or lng - ius to control their heavy menstrual bleeding. this study demonstrated a reduction in the pictorial blood loss assessment chart (pbac) at 12 months by 79% in the lng - ius users and 89% in those women who had undergone a resection. amenorrhea or infrequent bleeding was reported by 65% of women using the lng - ius compared with 71% who underwent a resection. satisfaction was high in both groups, with no significant difference in health - related quality - of - life scores. a cochrane systematic review appraised the literature and concluded that surgery, especially hysterectomy, reduces menstrual bleeding at 1 year more than medical treatments but lng - ius appears equally effective in improving quality of life. it is estimated that about 13% of women with menorrhagia suffer from von willebrand 's disease. when the effect of the lng - ius on hemostasis in women with menorrhagia was studied, it showed high expression of fibrinolytic inhibitors (plasminogen activator inhibitor 1 and 2) and up - regulated urokinase - type plasminogen activator receptor expression in the endometrium. this may explain why the lng - ius successfully treated heavy menstrual blood loss in cases with inherited bleeding disorders. lng - ius is effective in reducing the duration and amount of menstrual bleeding in women with menorrhagia associated with the use of oral anticoagulation. results from trials of the lng - ius to treat leiomyoma - related menorrhagia are conflicting. although grigorieva., reported reduced blood loss and improved hematocrits in these women, mercorio. there is some evidence that expulsion rates may be increased in the presence of submucous fibroids. recently, kriplani. reported that use of the lng - ius appears to lead to a significant reduction in the uterine volume of women with menorrhagia, as well as reducing the mbl in women with uterine leiomyomas. women with adenomyosis - related menorrhagia may benefit as well from the use of lng - ius with normalization of their menstrual flow. it may also be a useful adjuvant therapy following endometrial resection to help adenomyosis - related menorrhagia. gonadotropin releasing hormone agonists (gnrh - a) also are one of the effective methods to decrease the menstrual blood loss in patients with dub or aub due to pathological uterine lesions, but their side effects prevent their long term use. however, lng - ius has been shown to have comparable results, and also comparable clinical efficacy to gnrh analogues or progestins for the symptomatic treatment of endometriosis. for women in their reproductive years, the lng - ius has become one of the most acceptable medical treatments for menorrhagia, reducing referrals to specialists, and decreasing the recourse to operative treatments. it is easy to insert, has a sustained effect, cost - effective, and well tolerated besides providing reliable contraception. in a recent risk - benefit review, it has been brought out that lng - ius has a positive effect on most quality - of - life domains, comparable to those achieved with hysterectomy or endometrial ablation, and is consistently a cost - effective option across a variety of countries and settings. it concluded that lng - ius is an effective treatment option for women with heavy menstrual bleeding, including those with underlying organic pathology or bleeding disorders. as has been brought out that lng - ius appears equally effective as hysterectomy in improving quality of life in patients of dub, it can serve to bring down the incidence of hysterectomies.	abnormal uterine bleeding is a common gynecological complaint affecting 10 - 30% of women in midlife and constitute about one - third of all outpatient gynecological visits. it adversely affects the quality of woman 's life and can lead to psychological, social, medical, and sexual problems and thus necessitating appropriate and adequate management. different treatment modalities for such problems are available, yet the levonorgestrel intrauterine system (lng - ius) has recently provided a good treatment option effective in treating such complaints and at the same time, having a reliable contraceptive effect which is desired by such age group. for women in their reproductive years, the lng - ius has become one of the most acceptable medical treatments for menorrhagia, reducing referrals to specialists, and decreasing the recourse to operative treatments. it is easy to insert, has a sustained effect, cost - effective, and well tolerated besides providing reliable contraception.
granular cell tumors (gct) are relatively infrequent lesions that were initially described by abrikossoff as part of a series of 5 tumors of the tongue, which he termed myoblastoma. in 1931, he described the first case of this kind of neoplasm located in the esophagus and since then approximately 200 cases of gct have been documented. in the esophagus, the lesion is generally restricted to the submucosal layer, although occasionally it can also affect the mucosal layers and muscularis propria of the organ. histologically, different cell types, for example histiocytes, fibroblasts, or intestinal mesenchymal cells, have been reported to be the source of this tumor. however, the most widely accepted theory was that it was of myogenic origin, hence its former name of granular cell myoblastoma. since 1950 and with the introduction of histochemical techniques, it has clearly been shown that it is of neurogenic origin, arising from the schwann cells which, in the esophagus, form part of the submucosal neuronal plexus. there are many cases of endoscopic mucosal resection (emr) as treatment for gcts. however, to our knowledge, there are few reports of endoscopic submucosal dissection (esd). this article reports a case of a gct in the esophagus which was resected by esd. a 51-year - old man received an endoscopic examination after an esophageal lesion was found in an upper gastrointestinal (gi) x - ray series health examination. at the time of diagnosis he was completely asymptomatic and endoscopy identified an elevated yellow lesion, located 37 cm distal from the incisor teeth with a submucosal aspect and a depression in the center, measuring approximately 12 mm, which was rounded and coated with normal mucosa. iodine staining showed a minimally unstained top of the tumor (fig. 1). endoscopic ultrasonography (eus) using a 20 mhz catheter probe and the water filling method identified a submucosal tumor with a diameter of 10 mm. it was solid and the border was unclear but the bottom of the tumor was demarcated from the adjacent normal muscle layer, and was therefore confirmed to be located in the submucosal layer. the tumor was hypoechoic, but was hyperechoic compared with the adjacent muscle layer (fig. 2). histological analysis of tissue obtained with standard endoscopic forceps revealed stroma infiltrated by cells with eosinophilic cytoplasm of granular aspect whose central nuclei showed no evidence of atypia or mitotic figures (fig. 3). an upper gi x - ray series showed an elevated lesion with slight depression at the lower esophagus (fig. a computed tomography scan of the chest and abdomen revealed the solid mass in the distal esophagus but no additional pathology.fig. a an elevated yellow lesion located 37 cm distal from the incisor teeth, with a submucosal aspect and a depression in the center, was identified. b lugol staining showed minimal unstained top of the tumorfig. 2endoscopic ultrasonography showing a tumor which was solid, hypoechoic, with a diameter of 10 mm (t). this tumor was located in the submucosal layer (white arrow) and demarcated from the muscle layer (black arrow)fig. 3the histological analysis revealed stroma infiltrated by cells with eosinophilic cytoplasm of granular appearance whose central nuclei showed no evidence of atypia or mitotic figuresfig. 4an upper gastrointestinal x - ray series showed an elevated lesion with slight depression at the lower esophagus (arrow) upper gastrointestinal endoscopy. a an elevated yellow lesion located 37 cm distal from the incisor teeth, with a submucosal aspect and a depression in the center, was identified. b lugol staining showed minimal unstained top of the tumor endoscopic ultrasonography showing a tumor which was solid, hypoechoic, with a diameter of 10 mm (t). this tumor was located in the submucosal layer (white arrow) and demarcated from the muscle layer (black arrow) the histological analysis revealed stroma infiltrated by cells with eosinophilic cytoplasm of granular appearance whose central nuclei showed no evidence of atypia or mitotic figures an upper gastrointestinal x - ray series showed an elevated lesion with slight depression at the lower esophagus (arrow) the patient subsequently underwent an esd using sodium hyaluronate, a small - caliber tip transparent hood (st hood ; fujifilm, japan) and a water jet short needle knife (flush knife ; fujifilm) according to the method developed by toyonaga. the esd procedure was carried out using a single - channel upper gi endoscope (olympus, japan) and a high - frequency generator with an automatically controlled system (endocut mode ; icc200 ; erbe elektromedizin, germany). an st hood was fitted on the tip of the endoscope to obtain a constant view and to create tension on the connective tissue for dissection. lugol staining was performed to mark the margins of the lesion. with use of the flush knife, dots were placed about 3 mm outside the margins at 2 mm intervals. following an initial injection of 2 ml saline, a submucosal fluid cushion was created using a solution prepared with a mixture of hyaluronic acid, saline, indigo carmine, and epinephrine. approximately 2 ml of the solution at a time was injected into the submucosal layer, and repeated until the mucosa was elevated. after lifting the lesion, the mucosa was gently cut with the flush knife, using the endocut mode. en - bloc resection with tumor - free lateral / basal margins was accomplished (fig. the resected specimen contained a massive tumor of 11 7 mm, with eosinophilic cytoplasm with a granular appearance located from the lamina propria mucosae to the submucosal layer (fig. this method enabled submucosal incision under direct visualization, and thus enabled precise determination of both the lateral and vertical margins to be resected. follow - up endoscopy 6 weeks after removal of tumor showed scar tissue from mucosal dissection ; no masses or nodules were detected.fig. 5endoscopic submucosal dissection. a lower esophagus where the tumor and mucosa were removed. b macroscopically, the surgical margin of resected lesion was free from tumor cellsfig. 6the resected specimen contained an 11 7 mm tumor with eosinophilic cytoplasm with a granular appearance located from just below the squamous epithelium to the lamina propria mucosae endoscopic submucosal dissection. b macroscopically, the surgical margin of resected lesion was free from tumor cells the resected specimen contained an 11 7 mm tumor with eosinophilic cytoplasm with a granular appearance located from just below the squamous epithelium to the lamina propria mucosae although gcts are uncommon tumors in the gi tract, nearly one - third occur in the esophagus [6, 7 ], the most common site of origin of gi gcts. most esophageal gcts are found incidentally during endoscopy of the upper gi performed for other reasons. although a gct is usually asymptomatic, when the tumor is larger than 1 cm, it may cause dysphagia. the tumor usually appears as a yellowish - white, firm, sessile submucosal mass. differential diagnosis should include an esophageal cyst, epithelial lesions, for example glycogenic acanthosis, an inflammatory polyp, squamous papilloma, and other submucosal tumors, for example leiomyoma, lipoma, and hamartoma. gcts were originally considered to be of myogenic origin, but electron microscopic and immunohistochemical studies confirmed the schwann - cell origin of the tumors. histologically, these tumors consist of polygonal and fusiform cells in compact nests. cells have small dark nuclei and abundant, fine, granular eosinophilic acid - schiff - positive, diastase - resistant cytoplasm. gcts of the skin, larynx, and esophagus are known to induce pseudoepitheliomatous hyperplasia in the malpighian epithelium. although the natural history of the tumor is unclear, most esophageal gcts have a benign clinical course. however, approximately 1.52.7% of all cases reported in the literature were regarded as malignant variants. the infiltrative growth pattern and the presence of metastases are important features in differentiating between malignant and benign tumors because they may be of very similar appearance histologically. malignant lesions are usually larger than 4 cm, with evidence of rapid recent growth, tend to recur locally after resection, and may have subtle histologic features, for example nuclear pleomorphism, increased nuclear size, tumor cell necrosis, large nucleoli, mitotic figures (2 or more/10 high power fields), and tumor cell spindling. the optimum treatment for gcts remains controversial, but the current treatment options are a conservative approach with regular endoscopic follow - up for tumors 20 mm in diameter, benign gcts causing symptoms, or when malignancy is suspected. if no malignant changes are detected in the removed specimen, additional treatment or follow - up is not considered necessary. emr was recently reported to be an effective treatment, and eus is thought to be most useful for deciding if a tumor meets the criteria for endoscopic removal, including small size (< 20 mm) and non - attachment to the muscle layer [14, 15 ]. in a series of 650 esophageal mucosal cancers removed with emr, makuuchi reported an incidence of complications of 4.8% (perforation 0.7%, bleeding 3.1%, stricture 1.6%). esd for esophageal pathology has recently been established, and the affected mucosa is incised and removed using a variety of endoscopic electrosurgical knives. using esd, a wider range of the mucosa takahashi. reported that the incidence of complications was not significantly different between esd and emr perforation 2.6%, mediastinal emphysema 4.3%, pneumonia 2.6%, and stenosis 17.2%. in our case, after submucosal injection of sodium hyaluronate to maintain sufficient thickening of the submucosal tissue, dissection of the mucosa and submucosa with a flush knife was performed by the method developed by toyonaga. we preferred esd for its more accurate resection than conventional emr, and sodium hyaluronate solution for its ability to maintain submucosal elevation for a longer time. additional esophagectomy with lymph node dissection was not required, because no submucosal invasion or vessel permeation was seen in the endoscopically resected specimen. endoscopic resection is less invasive and, for smaller tumors, results in fewer complications than esophagectomy ; on the other hand there is a case report of emr of a malignant gct with a diameter of 10 mm. with regard to gcts from 10 to 20 mm in diameter, we believe endoscopic resection (emr or esd) may be preferable, as described elsewhere [19, 20 ]. concerning esophageal gcts exceeding 20 mm, esophagectomy had been performed until quite recently. however, esophagectomy is the therapeutic option with the highest mortality. because esd has been established as safer and less invasive therapy, it is expected to be the best therapeutic option for mucosal or submucosal esophageal gcts exceeding 20 mm. in summary, endoscopic and endosonographic evaluation of the lesion defines the location and extent of the tumor and its suitability for endoscopic treatment.	granular cell tumors of the esophagus are rare neoplasms and their diagnosis is mainly based on histopathologic examination of endoscopic biopsies. with the development of endoscopic techniques, there has been a marked increase in local treatment modalities for early esophageal neoplasms. in this case report, we describe the removal of a granular cell tumor by the endoscopic submucosal dissection technique, and briefly discuss the literature on clinicopathologic aspects and management of granular cell tumors.
the control of cellular adhesion and motility is one of the crucial mechanisms responsible for tumor initiation and progression. the genes involved are also contributors to malignancy along with genes responsible for cell proliferation and survival. i propose to summarize the current knowledge on one very important tumor suppressor gene, viz. e - cadherin is one of the most important molecules in cell - cell adhesion in epithelial tissues. it is localized on the surfaces of epithelial cells in regions of cell - cell contact known as adherens junctions. as a member of a large family of genes coding for calcium - dependent cell adhesion molecules (cams), the cadherin glycoproteins are expressed by a variety of tissues, mediating adhesion through homotypic binding. n - cadherins being the best characterized play important roles in the formation of tissues during gastrulation, neurulation and organogenesis. it is essential for the formation and maintenance of epithelia, was first identified in chicken, and was originally called l - cam. the mouse counterpart of this protein, uvomorulin, has 80% identity in both nucleotide and amino acid sequences to the human counterpart. besides its role in normal cells, this highly conserved gene can play a major role in malignant cell transformation, and especially in tumor development and progression. the suppression of e - cadherin expression is regarded as one of the main molecular events responsible for dysfunction in cell - cell adhesion. most tumors have abnormal cellular architecture, and loss of tissue integrity can lead to local invasion. thus, loss of function of e - cadherin tumor suppressor protein correlates with increased invasiveness and metastasis of tumors, resulting in it being referred to as the " suppressor of invasion " gene. isolated the full - length gene by using recombinant lambda phage, cosmid and p1 phage clones. further analysis of the gene showed 15 introns ranging from 120 bp (intron 4) to 65 kb (intron 2). the intron - exon boundaries are highly conserved in comparison with other " classical cadherins ", and in intron 1 a 5 ' high - density cpg island was identified that may have a role in transcription regulation. this island covers the region from exon 1 to exon 2 of the human e - cadherin gene, while other exons lacked such features (fig. positions of exons are shown in color boxes with the base pair number of each exon. the region from exon 1 to exon 2, a sequence of about 1500 bp, is a high - density cpg island. the chromosomal location of cdh1 on 16q22.1 was later confirmed by fluorescent in situ hybridization (fish) analysis. it is interesting that the human p - cadherin gene was recently located only 32 kb upstream from e - cadherin, and also the m - cadherin gene was positioned on chromosome 16q24.1-qter, which further suggests clustering of cadherin genes originating probably from gene duplication, while possible co - evolution might be explained by gene conversion. cdh1 encodes a 120 kda glycoprotein with a large extracellular domain, a single transmembrane segment and a short cytoplasmic domain, which interacts with the actin cytoskeleton through linker molecules, alpha- beta- and gamma - catenins. on the cytoplasmic side of the membrane, a bundle of actin filaments is linked to the e - cadherin molecules via a protein complex. alpha - catenin and either beta- or gamma - catenins are included in this complex. beta- and gamma - catenins share significant homology and bind to a specific domain at the e - cadherin c - terminus. alpha - catenin links the bound beta- or gamma - catenin to the actin cytoskeleton. cytoplasmic membrane ; aj adherens junction ; ed extracellular domain ; i d intracellular domain ; ac actin cytoskeleton ; 1-beta - catenin ; 2-alpha - catenin ; 3-p120. alpha - catenin has structural similarities with vinculin, one of the key components of fibroblast membrane attachment sites of microfilaments, beta - catenin shows homology to armadillo of drosophila melanogaster and gamma - catenin is identical to plakoglobin, a protein found in desmosomes. the c - terminal cytoplasmic domain of ~150 residues is highly conserved in sequence, and has been shown experimentally to regulate the cell - cell binding function of the extracellular domain of e - cadherin, possibly through interaction with the cytoskeleton. the juxtamembrane region of the cadherin cytoplasmic tail has been identified as a functionally active region supporting cadherin clustering and adhesive strength ; one of the interacting proteins involved in clustering and cell adhesion is p120ctn. the structure of the extracellular domain of classical e - cadherin contains five tandem repeats of a 100-residue - amino - acid - motif, and the biggest part of n - terminal of these repeats contains the sites with adhesive activity. this part of the molecule also has binding sites for calcium ions situated in the pockets between the repeats. the amino acid sequences that form the cabinding pockets are highly conserved between different members of the cadherin family and between different species. cell - cell adhesion is mediated through homotypic interactions of e - cadherin extracellular domains in a process of lateral dimerization. parallel dimers are able to interdigitate with dimers from neighbouring cells forming the points of adhesion. those findings introduce a cadherin - cadherin interface at the cellular surface. until recently cadherins were thought to be involved only in homophilic interactions ; however, e - cadherin has now been shown to be a ligand for two integrins, alphaebeta7 and alpha2beta1. the first interaction might serve to retain intraepithelial lymphocytes in mucosal tissue, while the second may contribute to the organization of epithelial multilayers. cadherins have been identified in a large variety of species, including mammals, xenopus, drosophila, caenorhabditis elegans. many new cadherin family members have been isolated and the cadherin group of genes has grown very fast in the last decade. proteins of several isolated molecules show a great deal of homology with the " classical " cadherins. other cadherin like molecules share with the classical cadherins putative cabinding motifs in repeated extracellular domains but diverge considerably in various regions, particularly in the cytoplasmic domains. even more deviations were observed for new cadherins, such as k - cadherin and li - cadherin, while the ret protooncogene product also shows some similarity to cadherin. although the extracellular domain has several similar repeats with putative cabinding motifs, this transmembrane tyrosine kinase is considered to be unrelated to the cadherins. since the ret gene lacks matching of the splice sites to the cdh1 gene, this might be the explanation. it is possible that this kinase has acquired its cadherin - like motifs by convergent evolution. expression of e - cadherin in embryonic development is very early, at the two - cell stage. epithelial differentiation and polarization (processes fundamental to cell differentiation) occur early in ontogeny in the morula stage, when the embryo compacts and each cell polarizes along its apicobasal axis to generate an epithelial - like phenotype. e - cadherin plays an important role in the adhesion of the blastomeres, and early embryo 's ability to compact. e - cadherin is expressed in the membrane even before compaction of the morula occurs, is distributed in a non - polar manner, and does not exhibit adhesive function. the mechanism that renders e - cadherin functional is unknown, but it does include phosphorylation of the protein. controlled epithelial - mesenchymal conversion is the most important exhibit of e - cadherin 's function in development. rietmacher and co - workers introduced a targeted mutation in mouse embryonal stem cells and generated a mouse without e - cadherin sequences essential for cabinding and for adhesive function. on the other hand, the homozygous e - cadherin -/- embryos showed severe abnormalities before implantation. this included failure to maintain a polarized and compacted state and also failure to form a trophectoderm epithelium ; they distort at the blastocyst stage, making the mutation lethal. embrios is probably due to the presence of e - cadherin proteins from maternal sources. investigation of zebrafish e - cadherin expression during early embryogenesis confirmed observed expression in blastomeres, but also led to the detection of a protein expressed in the anterior mesoderm during gastrulation and developing epithelial structures. in the developing nervous system, cdh1 was detected at the pharyngula stage in the midbrain - hindbrain boundary and was preceded by wnt 1 expression. as far as normal adult epithelial tissue structure and integrity is concerned, e - cadherin is also involved in its maintenance and homeostasis. cadherin mediated adhesion is a dynamic process that is regulated by several signal transduction pathways. there is also evidence that cadherins are not only targets for signaling pathways that regulate adhesion, but may themselves send signals that regulate basic cellular processes, such as migration, proliferation, apoptosis and cell differentiation. the image of individual adhesion molecule performing its function, or linear downstream signalling cascade is somewhat abandoned scheme. instead of separating and dividing into distinct fields, the cellular mechanisms of signalling and adhesion are nowadays thought to be closely connected mechanisms where components have double (or more) functions and interconnect in a signalling - structural network. the clearest example is recently discovered interaction of e - cadherin with epithelial growth factor receptor (egfr). the recently discovered wnt / wingless pathway, of mouse and drosophila respectively is one of the most interesting kind of signal transduction, in which key components have multiple functions in adhesion and signalling. information on wnt signalling can be found on the wnt gene site and at the connection map at science stke web site. in vertebrate cells, it is named after wnt proteins, a family of highly conserved secreted signaling molecules that regulate cell - to - cell interactions during embryogenesis. insights into the mechanisms of wnt action have emerged from several systems : genetics in drosophila and caenorhabditis elegans ; biochemistry in cell culture ; and ectopic gene expression in xenopus embryos. many wnt genes in the mouse have been mutated, leading to very specific developmental defects. as currently understood, wnt proteins bind to receptors of the frizzled family on the cell surface. through several cytoplasmic relay components, the signal is transduced to beta - catenin, which then enters the nucleus and forms a complex with tcf to activate transcription of wnt target genes. it has been well documented that wnt genes, together with other components of wnt signalling pathway, are implicated in cancer. another tantalizing compartment of the wnt signalling pathway lies in downstream transcriptional activation. in response to wnt signalling, cytoplasmic beta - catenin is stabilized, accumulates in the cytoplasm and enters the nucleus, where it finds a partner, a member of the dna binding protein family lef / tcf (t cell factor - lymphoid enhancer factor). one of the target genes for -catenin / tcf encodes c - myc protein, explaining why constitutive activation of the wnt pathway can lead to cancer. progressive accumulation of somatic mutations in a number of different genes characterizes the process of tumorigenesis. many genes involved in the process of tumorigenesis are components of one of a great many signal transduction pathways through which signals traffic via molecular networks. it is now apparent that epithelial malignancy can in certain aspects be explained by alterations in the adhesive properties of neoplastic cells. this process is similar to developmental events but with the important difference that it is uncontrolled. malignant carcinoma cells are characterized in general by poor intercellular adhesion, loss of the differentiated epithelial morphology and increased cellular motility. downregulation or a complete shutdown of e - cadherin expression, mutation of the e - cadherin gene, or other mechanisms that interfere with the integrity of the adherens junctions, are observed in carcinoma cells. in human tumors, the loss of e - cadherin - mediated cell adhesion correlates with the loss of the epithelial morphology and with the acquisition of metastatic potential by the carcinoma cells. loss of heterozygosity on 16q is detected frequently in metastasizing malignancies derived from the liver, prostate, and breast. mutations in cdh1 have been described in a number of human cancers including breast, stomach, endometrium, ovary and thyroid. transgenic mouse model with loss of e - cadherin expression developed invasive carcinoma from well - differentiated adenomas and finally germ - line mutations have recently been reported in early onset, diffuse - type stomach cancers. many immunohistochemical studies have examined changes in expression of the e - cadherin gene in human malignancies. in almost all non - colonic tumors examined, the patterns of changes in the expression of this gene have been similar to that seen in colorectal cancer, i. e. loss of protein expression is positively correlated to loss of tumor differentiation. in oesophageal, pulmonary, squamous head and neck tumors, pancreatic and cervical tumors, loss of expression has been correlated with a high grade and an advanced stage of the disorder, with poor prognosis. it has been reported that inactivating mutations of e - cadherin gene are highly frequent in infiltrating lobular breast carcinomas and diffuse gastric carcinomas. these mutations mostly occur in combination with loss of heterozygosity of the wild - type allele. another interesting paper on e - cadherin expression demonstrates its reappearance in metastatic cells. significant increase in re - expression of e - cadherin, together with alpha and beta - catenin, was observed in metastatic deposit of primary lobular breast cancers. lobular breast cancers harbour e - cadherin mutations as well as losses of the gene locus, even in situ disease. that suggests that the mutated protein has a role even before invasion process started. the findings reveal another dimension of adhesion molecules in tumor metastasis : reestablishment of cellular contact may prevent apoptosis. i would like to cite m. iiyas " adhesion molecule expression is the phoenix in tumor metastasis ". and finally, let us not forget yet another level of cdh1 expression regulation, i. e. e - cadherin promotor hypermethylation. this process has been known to be an inactivating event in some tumors and is currently extensively researched. becker. suggested that e - cadherin mutations contribute to the histopathologic appearance of stomach cancer because 13 of 26 diffuse gastric carcinomas, which had reduced homophilic cell - to - cell interactions, had abnormal gene transcripts that were not seen in non - cancerous tissue from same patients. in addition to a few missense mutations, those were mainly splice site and truncation mutations caused by insertions, deletions, and nonsense mutations. there was a major difference in mutation type between diffuse gastric and infiltrative lobular breast cancers. in diffuse gastric tumors, the predominant defects were exon skippings, which caused in - frame deletions. in contrast, most mutations found in infiltrating lobular breast cancers were out - of - frame mutations, which yield secreted truncated e - cadherin products. in most cases these mutations occurred in combination with loss of heterozygosity. guilford. reported germline mutations in the cdh1 gene in 3 familial gastric cancer kindreds of maori origin from new zealand.. analyzed 8 uk gastric cancer kindreds and identified novel germline cdh1 mutations (a nonsense and a splice site mutation) in 2 families. both mutations were predicted to truncate the e - cadherin protein in the signal peptide domain. gayther. also described germline cdh1 mutations in familial gastric cancer. in a family with a strong history of diffuse gastric carcinoma, chun. found the 1558insc germline mutation in the cdh1 gene the gastric cancer was of the early onset, histologically diffuse type. in another family with early onset diffuse gastric cancer, guilford. found that the 30-year - old proband was heterozygous for a - to - t transition at nucleotide 2095, which resulted in a nonsense mutation. the mutation was predicted to result in an e - cadherin peptide lacking both the transmembrane and cytoplasmic domains. same authors described a family in which multiple members with gastric cancer were heterozygous for the insertion of an additional c residue in a run of 5 cytosines at positions 2382 to 2386. the resulting frameshift led to an e - cadherin molecule lacking about half of its cytoplasmic domain. in another diffuse gastric cancer family, a heterozygous g - to - t transversion at nucleotide 70 in exon 2 of the cdh1 gene, was also identified. the mutation converted a glutamic acid (glu24) to a tag stop codon in the signal peptide of the e - cadherin precursor protein. identified a splice acceptor site mutation, an a - to - g transition at position -2 from nucleotide 49 at the start of exon 2 of the cdh1 gene and also a germline g - to - a transition at nucleotide 59 in exon 2. the mutation, a trp20-to - ter substitution was predicted to truncate the e - cadherin gene product in the signal peptide domain, which is cleaved from the n terminus of the mature protein. in a family segregating diffuse gastric cancer, gayther. found a 2095c - t transition in the cdh1 gene, resulting in a truncating mutation, arg598 to ter. same authors found a 1-bp insertion in the cdh1 gene in the proband of a family with familial diffuse gastric cancer. insertion of a g after nucleotide 1711 created a frameshift that would truncate the protein at codon 587. another 1-bp insertion (c) at nucleotide 1588 in exon 11 was also identified in a family segregating diffuse gastric cancer. mutations were also found in other types of carcinoma. in an endometrial carcinoma, risinger. identified a gca - to - aca transition in codon 617 predicting a substitution of thr for ala in the e - cadherin molecule. they also identified a ctg - to - gtg transversion resulting in a leu711-to - val amino acid substitution in e - cadherin., same authors identified an agc - to - ggc transition in codon 838 resulting in substitution of glycine for serine. the tumor tissue also showed somatic loss of heterozygosity. in an infiltrative lobular breast carcinoma, berx. found a gaa (glu)-to - taa (stop) nonsense mutation in the cdh1 gene. tumor - specific loss of heterozygosity of chromosomal region 16q22.1 was demonstrated in this case. here the number of mutations is growing each day and can be reached at the following web site. i should also mention the type of genetic changes that my group has encountered so far.. indicated yet another type of genomic instability of cdh1 gene encountered in tumor tissue. when searching for allelic loss i. e. loss of heterozygosity (loh) of the cdh1 gene in clear cell renal cell carcinoma, we came across replication error (rer) positive samples, an instability problem that characterizes tumor development and progression. replication / repair machinery seems to be targeted in 10% of our clear cell renal cell carcinoma sample. reduced expression of e - cadherin is regarded as one of the main molecular events involved in dysfunction of the cell - cell adhesion system, triggering cancer invasion and metastasis. one of the most crucial exhibit of e - cadherin 's function in development is the controlled epithelial - mesenchymal conversion. the involvement of e - cadherin in wnt signaling, indicates that same molecule may have different functions and that e - cadherin can regulate cellular response generated by external signals the cell receives. in this way it can regulate migration, proliferation, apoptosis and cell differentiation. the method of blocking e - cadherin downregulation in tumors is one of the important future approaches in gene therapy. to target this molecule is the logical path to prevent metastazing potential of almost any epithelial tumor. nevertheless, it will not be an easy enterprise since its downregulation is caused by many different mechanisms, ranging from mutations and gross deletions to repression of gene transcription, as well as signal transduction stimulation of e - cadherin adhesion complex formation. this work was supported by grant 0108215 from ministry of science and technology, republic of croatia.	e - cadherin tumor suppressor genes are particularly active area of research in development and tumorigenesis. the calcium - dependent interactions among e - cadherin molecules are critical for the formation and maintenance of adherent junctions in areas of epithelial cell - cell contact. loss of e - cadherin - mediated - adhesion characterises the transition from benign lesions to invasive, metastatic cancer. nevertheless, there is evidence that e - cadherins may also play a role in the wnt signal transduction pathway, together with other key molecules involved in it, such as beta - catenins and adenomatous poliposis coli gene products.the structure and function of e - cadherin, gene and protein, in normal as well as in tumor cells are reviewed in this paper.
midline splitting cervical laminoplasty (mscl) has been considered an effective and safe method for decompressing multisegmental cervical lesions caused by cervical spondylotic myelopathy (csm) or ossification of the posterior longitudinal ligament (opll) as well as for achieving immediate stability of these lesions.4,5) in the beginning of this procedure, autologous grafts from the iliac crest were used to keep the opened lamina splitted.2) however, pain and discomfort at the donor site of the iliac crest were not uncommon, probably due to injury to the superior gluteal nerve,11) and sunken - down of grafted bone spacer occurred. and then hydroxyapatite (ha) was introduced as a spacer material, but the fusion rate between ha and cervical lamina is not good,9) which may cause sunken - down or pull out. in addition, ha has the higher potency of infection than autologous grafts.7) in contrast, allogeneic bone grafts can avoid the complications associated with harvesting autologous bone. allograft materials are obtained from deceased human donors, undergoes rigorous safety screening, and can be transplanted into the patient at the time of surgery. the shape of allografts is well developed and capability to keep splitted lamina also increased.6) however, the fusion rate of mscl using allogeneic bone spacers has not been studied well. the purpose of this study was to examine the clinical and radiological outcomes in patients undergoing mscl using allogeneic bone spacers, especially compared the results between csm and opll. a total of 27 consecutive patients with compressive cervical myelopathy including csm and opll underwent mscl using allogeneic bone spacers, allo - spine lamina spacer (cg bio, seoul, korea) between april 2012 and september 2013. patients with cervical kyphosis, evidence of cervical instability and serious medical problems were excluded for this surgery. therefore, we retrospectively analyzed the outcomes of 17 patients (4 men, 13 women). mean follow - up duration was 11.3 months (range, 6 - 19 months). three patients of csm had myelopathy due to dynamic factor such as fall - down and traffic accident, and the others due to static factor such as soft disc herniation and congenital canal stenosis. the numbers and locations of spacers were : 11 at c3 level, 17 at c4, 16 at c5, 12 at c6, and 3 at c7. he made gutters at the bilateral laminofacet junctions with a 3 mm diameter diamond type burr, and split the midline spinous process with a 2 mm diameter diamond type burr. the allogeneic bone spacers were inserted between the splitted spinous processes and fixed with 1 - 0 black silk. cervical x - rays were taken preoperatively, immediate post - operatively, and after 3, 6 months. computed tomography (ct) change in overall cervical lordosis was assessed on x - ray images (figure 1). spinal canal dimension and anteroposterior (ap) distance between the posterior surface of the vertebral body and the anterior surface of allogeneic bone spacer were assessed on axial ct images, which were measured at the level of the vertebral pedicles. the primary clinical outcome was the rate of change in the japanese orthopedic association (joa) scores (0 - 17) between preoperative and postoperative 6 month period. recovery from myelopathy at 6 months was calculated using the formula : (6 month joa score - pre operative joa score)/(17-pre operative joa score). contact status between lamina and allogeneic bone spacer immediately after surgery, and fusion between lamina and allogeneic bone spacer at 6 months were assayed as hirabayashi method and ichikawa classification (figures 2 and 3).2,3) d or e statuses were classified as fused status. the factors that affected the rate of fusion between lamina and allogeneic bone spacer were analyzed by uni- and multivariate logistic regression. age, sex, type of disease (csm or opll), level of operation, contact status, smoking, diabetes mellitus (dm) and hypertension were included in these analyses. groups were compared using the paired t - test, chi square test, fisher 's exact test, mann - whitney test, linear mixed model, and uni- and multivariate logistic regression. mean cervical lordosis, which was defined as the angle between c2 and c7 inferior endplates, changed from 10.38.7 to 15.012.6 degrees in the csm group and from 11.711.1 to 13.611.0 degrees in the opll group (p=0.602)(figure 4). these data reveal the mean angle of cervical lordosis was not changed significantly and the occurrence of post - operative kyphosis was negligible. the spinal canal dimension was significantly increased after surgery from 183.141.1 mm to 295.646.2 mm in the csm group (p<0.001) and from 145.538.7 mm to 255.856.6 mm in the opll group (p<0.001). in addition, the dimensions were evaluated as 299.034.2 mm (p=0.785) and 248.252.2 mm (p=0.216), respectively in post - operative 6 months. the spinal canal dimensions were increased with statistical significance after mscl surgery in both csm and opll, but there was no statistical difference between two groups (p=0.554) (figure 5). the ap distance of the spinal canal on axial ct images was increased immediately after surgery from 9.6 1.6 mm to 17.92.0 mm in the csm group (p<0.001) and from 6.51.6 mm to 15.02.7 mm in the opll group (p<0.001), but slightly decreased to 17.72.1 mm (p=0.400) and 14.82.8 mm (p=0.312), respectively at 6 months (figure 6). there was also no statistical difference in increase of ap distance between two groups (p=0.924). mean joa scores changed from 14.32.0 to 16.30.5 in the csm group and from 12.04.9 to 14.62.8 in the opll group, with mean calculated recovery rates of 76.422.6% and 62.527.2%, respectively (p=0.257)(table 2). there was no post - operative infection. excellent contact was achieved with 9.1% of spacers at c3, 35.3% at c4, 81.3% at c5, 100% at c6, and 66.7% at c7 (p<0.001). contacts between the spacer and the lamina become better in lower cervical level except c7 (table 3). in the respect of immediate post - operative contact status for csm and opll, excellent in 14 levels (66.7%) and 20 levels (52.6%), good in 7 (33.3%) and 16 (42.1%), and fair at 0 (0%) and 2 (5.3%), the post - operative 6 month fusion status between lamina and allogeneic bone spacer, classified as d or e status were 30 (51%) in total, 10 (47.6%) in csm, and 20 (52.6%) in opll (p=0.789). excellent immediate post - operative contact status between lamina and allogeneic bone spacer had a significantly higher probability of fusion (table 4). in the respect of immediate post - operative bonding state affecting fusion rate, excellent group showed fusion in 24 (71.0%), good in 5 (22.0%), and fair in 1 (50.0%). in univariate logistic regression analysis, surgical level of allogeneic bone spacer (p=0.043), immediate post - operative contact status of spacer (p=0.001), and absence of dm (p=0.048) were significant factors. however, multivariate logistic regression analysis showed only contact status of lamina and bone spacer as significant factor of fusion (p=0.024)(table 6). there was no significant difference in clinical recovery rate between csm and opll in previous studies.1,12) our results in this present study also support the data, in which post - operative clinical outcomes were significantly improved in csm as well as opll, but no significant difference between two groups. the reason why there was no difference in clinical outcome between two groups could be primarily because this clinical outcome was affected by decompression of the spinal cord and post - operative expansion of spinal canal dimensions were similarly increased in two groups. in the previous report on mscl using ha spacer,9) the overall fusion rate between lamina and ha was 27.46% and wingless type ha, which is similar shape as the allogeneic bone spacer used in this study showed 48.8%.9) in our present study, the fusion rate was 51%, which is slightly higher than wingless type ha. therefore, allogeneic bone spacer seems to have higher potential for bone fusion even though it was sterilized with antibiotics, chemicals, and gamma ray. but it had the potential of disease transmission such as viral hepatitis, tuberculosis, syphilis, septicemia and malignancy.10) in multivariate analysis, initial contact status between lamina and allogeneic bone spacer significantly affected fusion status. nagashima.8) demonstrated in animal experiments that a high degree of new bone formation occurred at the interface between the spacer and the spinous process. we think that the splitted spinous process becomes higher in lower cervical levels and contacts between the spacer and the lamina become better in lower cervical level which explains the significance in univariate analysis. the limitations of our present study are that this study is not prospective analysis and there were small cases in some classified groups such as c7 level and good / fair contact status. to study further results and outcome about mscl using allogeneic bone spacer, additive analysis may be needed with prospective, larger cases and longer follow - up duration. this present study suggests that csm and opll did not show difference of surgical outcome in mscl using allogeneic bone spacer. in addition, we should consider the contact status between lamina and bone spacer for the better fusion rates for this surgery.	objectiveto analyze factors associated with fusion using allogeneic bone spacers for midline splitting cervical laminoplasty (mscl).methodsduring april 2012 and september 2013, seventeen patients with cervical spondylotic myelopathy (csm) or ossification of posterior longitudinal ligament (opll) underwent mscl with allogeneic bone spacers by a single surgeon. mean follow up periods was 11.3 months (range, 6 - 19 months). clinical outcomes were evaluated by the japanese orthopedic association (joa) scores at preoperative and postoperative 6 months. simple cervical x - rays were taken preoperatively, immediate postoperatively, 3, and 6 months after operation. computed tomography (ct) scans were performed preoperatively, immediate postoperatively and 6 months postoperatively. the differences between two diseases were analyzed on cervical lordosis, canal dimension, anteroposterior (ap) distance, fusion between lamina and allogeneic bone spacer and affecting factors of fusion.resultsall surgeries were performed on 59 levels. there were no significant differences on the changes of lordosis (p=0.602), canal dimension (p=0.554), and ap distance (p=0.924) as well as joa scores (p=0.257) between csm and opll groups. overall fusion rate was 51%. multivariate analysis on the factor for the fusion rates between lamina and spacers showed that the immediate postoperative contact status between lamina and spacers in ct as significant factor of fusion (p=0.024).conclusionthe present study suggests that csm and opll did not show difference of surgical outcome in mscl using allogeneic bone spacer. in addition, we should consider the contact status between lamina and bone spacer for the better fusion rates for this surgery.
	primary optic nerve sheath meningiomas (onsm) are rare, benign and slow growing tumor involving the intra - orbital / intra - canalicular segment of the optic nerve. untreated, they can potentially lead to visual deterioration. magnetic resonance (mr) is the gold standard imaging modality for diagnosing the entity. often, a clinical dilemma exists to narrow the differential diagnosis of an enhancing intra - orbital mass on mr. molecular imaging provides a high degree of precision in diagnosing meningioma in view of relatively high levels of somatostatin receptor expression by these tumors. the following case demonstrates the potential clinical utility of somatostatin receptor spect using 99mtc- labeled hynic - toc in clinical diagnosis of onsm.
the type species pseudomonas aeruginosa has become a major opportunistic pathogen for humans and this is probably the underlying incentive for most research that is currently performed on p. aeruginosa. hence if we want to talk about advances in understanding pseudomonas we can confine ourselves to p. aeruginosa. thanks to the revolution of next generation sequencing, most new knowledge at least in quantitative terms has been generated during the last few months and years in pseudomonas genomics. we are now starting to understand the evolution and population biology of p. aeruginosa and its interaction with the environment at the whole genome scale, which can provide us with some general insight into the diversity, ecology and evolution of the microbial world. p. aeruginosa is a fascinating ubiquitous microorganism that can thrive at low densities within the range of 4c to 42c in inanimate aquatic habitats and can colonize the surface of animate hosts ranging from worms and flies to plants and mammals. the p. aeruginosa population has an epidemic structure. with the exception of a genomic region around the origin of replication, dna segments can freely recombine between clonal complexes, although the individual clonal frames remain remarkably stable over time (our own unpublished genome sequencing data). by genotyping a large collection of strains from environmental and disease habitats with a custom - made multi - marker array we have identified more than a thousand different clonal complexes, members of the two major clones c and pa14 were sampled from salt and fresh water, secluded national reserves, anthropogenically polluted sites, plants, wild and domestic animals and acute and chronic human infections.. however, the 20 next frequent clones predominate in particular geographic areas and/or habitats. numerous clones still have no representative among the subset of human infections and, conversely, clones that had previously caused outbreaks of nosocomial infection still lack an environmental isolate in our strain collection. these data suggest that the p. aeruginosa population consists of global and local generalists on the one hand and niche specialists on the other. to verify this statement most information about the genetic repertoire of p. aeruginosa has been gathered from the genomes of the reference strains pao1 and pa14. matching phenotypes were seen for orthologs of metabolic enzymes, whereas complex phenotypes (such as virulence traits) were combinatorial in nature, that is, the encoded function of a gene was modified by epistatic interactions. even a rather simple trait such as colony morphology was induced by mutagenesis of a peculiar gene in one strain, but not in another. these pilot studies provide evidence that phenotypic diversity in the p. aeruginosa population can not solely be ascribed to sequence variation and/or the presence or absence of genes, but rather results from an interplay between trait - shaping key genes, genetic modifiers and clonal genomic background. however, prior to addressing this complex issue, we need an inventory of genes and sequence variants to describe the genomic diversity of p. aeruginosa. ongoing genome sequencing projects in our own and other laboratories indicate that the core genome common to all p. aeruginosa consists of somewhat more than 4,000 genes. the so - called regions of genome plasticity make up the variable accessory genome of about a further 10,000 genes. variable subsets of this accessory gene pool are present in individual strains and clonal complexes. on top of that, between dozens and hundreds of unique genes previously unknown in p. aeruginosa have regularly been observed whenever a strain of a yet uncharacterized clonal complex was subjected to genome sequencing [12 - 16 ]. since more than 1,000 clonal complexes have already been identified in p. aeruginosa, we can estimate a pool of 100,000 further private genes that are rare or very rare in the p. aeruginosa population. the publication of the p. aeruginosa pao1 genome sequence in the year 2000 classified 2,500 pao1 open reading frames as hypotheticals of unknown function. since then, 300 of this number have been functionally characterized by the pseudomonas community, so one can imagine the challenge remaining to achieve a comprehensive insight into the encoded genetic repertoire of the p. aeruginosa pan - genome. another subset of genes that had been underappreciated prior to the advent of rna - sequencing are small regulatory rnas (srnas) that, for example, shape the adaptive response to environmental cues by titrating regulatory proteins or base - pairing with target mrnas. rna - sequencing of pao1 or pa14 bacteria grown in planktonic culture in luria - bertani (lb) broth has already identified over 500 novel small rnas or cis - antisense rnas in addition to the previously known 44 srnas. further srnas will probably show up under other experimental conditions and in other clonal complexes, because non - coding rnas are preferentially localized in the accessory genome. the closest homologs of genes of the accessory genome are typically found in - and -proteobacteria, mostly in other pseudomonads, enterobacteriaceae, and species of the genera burkholderia, ralstonia and cupriavidus that had been classified as pseudomonads prior to the introduction of 16s rdna sequence - based molecular taxonomy. burkholderia, ralstonia and cupriavidus bacteria are still called honorary pseudomonads because, like the true pseudomonads, they are metabolically versatile and can utilize a broad range of compounds as carbon and nitrogen sources and even degrade halogenated xenobiotics. the operons that encode these extraordinary metabolic capabilities are typically located in genomic islands, which are shuffled across genus and species barriers by horizontal gene transfer. an intriguing example is a pagi-2 island that was detected with 100% sequence identity in a cupriavidus metallidurans isolate from heavy - metal ion polluted soil and some 1,000 miles apart in a p. aeruginosa isolate from the chronically infected airways of a patient with cystic fibrosis. in other words, the gc - rich p. aeruginosa apparently exchanges its gene pool of the accessory genome with other gc - rich proteobacteria that enjoy a similar lifestyle and metabolic versatility. the intraspecies transfer of genomic islands among p. aeruginosa strains has been determined to occur at a frequency of 10 to 10 [28 - 30 ], meaning that only a minority of cells within the p. aeruginosa community is capable of horizontal transfer of the island. the activation occurs at random and is associated with a loss of fitness to grow, which apparently restricts the number of transfer - competent donors to a few cells. genome mobility in p. aeruginosa is typically accomplished by horizontal gene transfer of plasmids, transposons, genomic islands and bacteriophages. recent work has made p. aeruginosa into a model organism for the study of bacterial genome integrity in the context of the co - evolution of phage and bacteria. these systems consist of clustered regularly interspaced short palindromic repeats (crispr) coupled with cas (crispr - associated) genes. similar to rna interference in eukaryotes, these crispr / cas systems use small rnas for sequence - specific detection and neutralization of invading genomes. in p. aeruginosa pa14, the type i - f crispr / cas system was shown to prevent the replication of phages and, conversely, the genomes of phages infecting strain pa14 harbour the first known anti - crispr genes that mediate the inhibition of a crispr / cas system. p. aeruginosa causes a wide range of infections in humans that can vary from local to systemic, subacute to chronic, and superficial and self - limiting to life - threatening. chronic airway infections with p. aeruginosa are regularly seen in patients with advanced stages of chronic obstructive pulmonary disease and individuals suffering from the autosomal recessive trait cystic fibrosis. the basic defect of a perturbed ion, water and ph homeostasis predisposes the cystic fibrosis airways to colonization with opportunistic pathogens, predominantly staphylococcus aureus and p. aeruginosa. once p. aeruginosa has taken up residence in the cystic fibrosis lungs for more than a year, the organism is notoriously resistant to eradication by chemotherapy. unless the patients become co - infected by a transmissible strain, more than 50% of patients will still be harbouring the initially acquired clone after 20 years of colonization. the chronic infections of the stomach with helicobacter pylori and of the cystic fibrosis airways with p. aeruginosa are the only cases of infections with extracellular bacteria in humans where the causative agent persists in its niche for decades and does not disseminate into other organs. these two scenarios provide a unique opportunity to monitor the genome evolution of a human pathogen in a disease habitat over many years. maynard olson 's group has investigated the genetic adaptation of p. aeruginosa to cystic fibrosis airways in the single child of a cystic fibrosis newborn cohort who became chronically colonized with p. aeruginosa during infancy. numerous loci were hit by mutation in the child 's serial airway isolates collected over an 8-year observation period. of these, the gene loci encoding multidrug efflux pumps and regulators of quorum sensing and alginate biosynthesis were subsequently confirmed by genotyping of a large strain collection to be hot - spots of mutation of p. aeruginosa isolates in cystic fibrosis lungs. the cystic fibrosis clinics in copenhagen and hanover have regularly collected p. aeruginosa from the airways of all their patients who became chronically colonized in the 1970s and 1980s. the analysis of these unique strain collections taken from numerous patients should provide a more comprehensive insight into the microevolution of p. aeruginosa during the chronic infection of cystic fibrosis airways than the single case of the infant from the us and hence whole genome sequencing of serial isolates has been performed at both sites. patients from the hanover clinic became colonized with clones that are found at a similar frequency in the environment and other cystic fibrosis clinics. in contrast, the copenhagen patient cohort became chronically colonized with two highly related clones (dk1 and dk2) by patient - to - patient spread. at the hanover clinic, hence the microevolution of the initially acquired clone in patients ' airways could be followed over 30 years (currently ongoing). we started with the investigation of microevolution of the globally most abundant clones c and pa14 in two cystic fibrosis patients ' airways. both clones underwent phenotypic conversion that is typical for the cystic fibrosis lung habitat, that is, they became less motile, secreted less siderophores and virulence effectors and became deficient in the lipopolysaccharide (lps) o - antigen, but they remained non - mucoid. late isolates were only slightly compromised in competitive growth in the presence of first isolates which, did not confirm the common belief in the literature that adaptation in the cystic fibrosis lungs is accompanied by a loss of global fitness of p. aeruginosa. for clone c, a loss - of - function mutation in the dna repair gene mutl generated a bushy structure of multiple hypermutable clades [44 - 46 ] that accumulated close to 1,000 de novo mutations, but interestingly the mutation rate returned to normal in the mutl - deficient lineages after 10 years of colonization. the initial pa14 clade diverged into three clades during the first years of colonization, but after 15 years only one clade persisted in the cystic fibrosis lungs indicating that only this clade was endowed with the features to cope with the on - going remodelling and dedifferentiation of the chronically inflamed airways. we are currently investigating the genome evolution of p. aeruginosa in contrasting patient cohorts with a very mild or very severe course of their infection (our own unpublished data). exopolysaccharide biosynthesis, antimicrobial resistance and global regulators of lifestyle and metabolism were found to be common functional categories whose genes were hit by mutations in all p. for example, the p. aeruginosa clone inhabiting the most severely affected lungs generated progeny with stop mutations or drastic amino acid changes in key genes of lifestyle, whereas the p. aeruginosa clone that has persisted in a patient with normal lung function had diverged into co - existing clades which, accumulated benign, probably modifying amino acid substitutions, but no stop mutations. these two cases demonstrate that the acquisition of a few mutations can make a major impact on bacterial phenotype. the fact that a single loss - of - function mutation can have dramatic consequences is also the take home lesson of intraclonal genome comparisons. clones cha and tb were chosen for analysis because the clone members cha and tbcf10839 confer unusual and severe traits of virulence. both strains undermine man 's major host defence against p. aeruginosa, that is, killing by macrophages or neutrophils. by virtue of its type iii secretion system, strain cha destroys host defence cells by a mechanism called pack swarming whereby dozens of bacteria simultaneously attack and destroy the mammalian cell. for genome comparison, a clone cha isolate from the river ruhr and the first p. aeruginosa isolate from a cystic fibrosis patient from another clinic were chosen. each strain was carrying a few specific elements in its accessory genome and some dozen strain - specific single nucleotide polymorphisms (snps) reflecting the diverse spatiotemporal origin of the strains. however, the exceptional pathogenicity of strain cha was attributed to a combined deletion of the lactate dehydrogenase and gacs genes, the latter being the major switch between the sessile biofilm - forming and the planktonic exoprotein - secreting lifestyles. disruption of gacs created a strain that is both mucoid and proficient in the secretion of virulence effectors. the two cystic fibrosis strains tbcf121838 and tbcf10839 just differed in one gene deletion, less than 10 snps and one insertion of a genomic island from each other, but their phenotype was dramatically different in transcriptome, proteome, metabolome and adherence. tbcf121838 was efficiently killed by neutrophils, whereas tbcf10839 could grow and multiply in polymorphonuclear cells (pmns). the atypical virulence of strain tbcf10839 was caused by the pleiotropic consequences of a deletion within the pilq gene that is essential for pilus biogenesis. the other pilins were still synthesized, secreted by alternative routes and facilitated the intracellular survival of tbcf10839 in pmns. in conclusion, microevolution in p. aeruginosa can generate novel complex traits by a few or even single mutations, provided that predisposing mutational events had occurred before in the clonal lineage. complementary to our study on the genome evolution of distinct clones in a cohort of patients seen at the hanover clinic, a retrospective study has been performed on the genome evolution of the transmissible dk2 clone in a cohort of patients seen at the copenhagen cystic fibrosis clinic [51 - 54 ]. genome sequencing of 55 bacterial isolates collected from 21 cystic fibrosis patients over 38 years uncovered 8,530 mutations. overall, no evidence was found for either intragenic bias of mutations or for positive selection within coding regions. the 65 most frequently mutated genes, however, showed clear signs of positive selection. a large part of these genes should be involved in niche adaptation because they are associated with the composition of the cell envelope, antibiotic resistance and evasion of the host response. besides these investigations on the dk1 and dk2 clones from denmark, genomic and phenotypic analyses are being pursued on further transmissible p. aeruginosa strains that have caused patient - to - patient spread in cystic fibrosis clinics, namely in australia, canada and the united kingdom [55 - 64 ]. a consortium from these three countries headed by fiona brinkman (vancouver), roger levesque (quebec city) and craig winstanley (liverpool) will sequence a further 1000 p. it is anticipated that we will be able to link the data to the pseudomonas.com website, integrate the information with patient data registries in cystic fibrosis and develop a user - friendly pipeline for researchers and clinicians. this report focuses on our progress in understanding pseudomonas in the context of genomics and its application to evolution, epidemiology and clinical microbiology. besides these global genome - wide approaches, several major topics of interest were discussed at the pseudomonas 2013 conference : (a) the world of non - coding rnas ; (b) the dissection of sigma factor regulons ; (c) structure and function of the type ii, type iii and type vi secretion systems (t6sss) [68 - 80 ] ; (d) the physiological functions of secondary metabolites such as phenazines [81 - 85 ] ; (e) quorum sensing inhibitors ; (f) the sessile biofilm - forming lifestyle [88 - 94 ] ; (g) and the interaction of p. aeruginosa with its competitors in the microbial community [73,77,78, 82 - 85,95 ]. of these topics, t6sss are molecular nanomachines allowing gram - negative bacteria to transport and inject proteins into a wide variety of target cells. the t6ss is composed of 13 core components and displays structural similarities with the tail - tube of bacteriophages. the phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject dna. t6ss is considered to be an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. the p. aeruginosa pao1 genome encodes three t6sss, namely h1-, h2-, and h3-t6ss. p. aeruginosa h1-t6ss targets at least three effector proteins (tse1 - 3) to recipient gram - negative cells. the tse2 protein is a cytoplasmic effector that induces the quiescence of target cell proliferation, thus providing a pronounced fitness advantage for p. aeruginosa donor cells. the amidase tse1 and the muramidase tse3 are injected into the periplasm of target bacteria in a cell contact - dependent manner and degrade the peptidoglycan leading to the death of the recipients. to protect itself from the action of the tse toxins, p. aeruginosa uses specific periplasmically localized immunity proteins. the role of h3-t6ss is still unknown, but the function and regulation of h2-t6ss are starting to be elucidated. h2-t6ss secretes a phospholipase d that releases choline from phosphatidylcholine and thereby leads to a severely perturbed membrane phospholipid composition in the recipient cell. thus the h2-t6ss delivers an enzyme that is active against both prokaryotic and eukaryotic membranes. this strategy of membrane targeting may contribute to the fitness of p. aeruginosa in interspecies competition under t6s - conducive conditions.	pseudomonas aeruginosa, the type species of pseudomonads, is an opportunistic pathogen that colonizes a wide range of niches. current genome sequencing projects are producing previously inconceivable detail about the population biology and evolution of p. aeruginosa. its pan - genome has a larger genetic repertoire than the human genome, which explains the broad metabolic capabilities of p. aeruginosa and its ubiquitous distribution in aquatic habitats. p. aeruginosa may persist in the airways of individuals with cystic fibrosis for decades. the ongoing whole - genome analyses of serial isolates from cystic fibrosis patients provide the so far singular opportunity to monitor the microevolution of a bacterial pathogen during chronic infection over thousands of generations. although the evolution in cystic fibrosis lungs is neutral overall, some pathoadaptive mutations are selected during the within - host evolutionary process. even a single mutation may be sufficient to generate novel complex traits provided that predisposing mutational events have previously occurred in the clonal lineage.
for those readers who are not familiar with the molecular composition of gap junctions, we will briefly recollect the substructure of this cell contact and refer to recent detailed reviews (duffy. 2002 ; gaietta. 2002 ; goodenough and paul 2003 ; meier and dermietzel 2006 ; willecke. gap junctions are formed by hemichannels (connexons), which consist of an oligomer of six proteins (connexins). at present, at least 20 genes seem to be present in the human and rodent genome (willecke. 2002), which may oligomerize in monomeric or heteromeric patterns to form a hemichannel. a complete gap junction channel is formed by two hemichannels in mirror symmetry (fig. 1). heterotypic configurations of different isoforms are allowed for some connexins while others occur exclusively in homotypic configuration. in the history of gap junctions, the junctional plaque has always been considered to occur in a naked form without cytoplasmic adjuncts like scaffolding proteins or cytoskeletal elements (hirokawa and heuser 1982). however, accruing evidence indicates that gap junctions are associated with a complex system of scaffolding and cytoskeletal proteins, which seem to assemble in cell specific patterns (duffy. only apposed connexons allow intercellular transfer of ions (ionic coupling) and small metabolites (metabolic coupling). unapposed connexons seem to perform per se functions general structure of a gap junction plaque. only apposed connexons allow intercellular transfer of ions (ionic coupling) and small metabolites (metabolic coupling). unapposed connexons seem to perform per se functions in the following, we will put main emphasis on heart and brain tissues for which most of the data on gap junctions and cell adhesion have been reviewed. in the terminal intercalated discs between cardiomyocytes, cx43 celebrates an example for integrating its hemichannel and cell coupling functions (gros and jongsma 1996 ; shaw and rudy 1997 ; van veen. asides the terminal intercalated discs, gap junctions are also localised in the lateral sarcolemma of the heart (fig. 2), and thus described to form site - to - site and end - to - end connections (yao. cardiomyocytes also represent unopposed connexons with hemichannel function in the lateral sarcolemma (saez. bar indicates 25 m a shows cultured cardiomyocytes immunolabelled with an anti - cx43 antibody (red). bar indicates 25 m to ensure its trafficking and functional integration into a gap junction plaque or in form of unpaired connexons (hemichannels) into the plasma membrane, cx43 has to interact with other proteins. in the intercalated discs of coupled cardiomyocytes for instance, gap junction plaques are embedded into adherens junctions, which are primarily formed by cadherins (matsuda. 2003 ; niessen 2007), the gap junction ensures the propagation of action potentials along the cardiomyocytes (gros and jongsma 1996 ; shaw and rudy 1997). multiple models describe the pathway from connexon assembly to the initial gap junction formation and interaction with cadherins in the adherens junction. the most common examples are based on half - life time determined trafficking and junctional protein (cadherins) mediated activation of cx43. the half - life time of cx43 is restricted between 1 and 3 h, and implicates a dynamic process of assembly, insertion and replacement of connexons, and pairing of connexons to form gap junctions (beardslee. cx43 synthesis was shown to be located on membrane bound ribosomes, where connexin proteins are rapidly oligomerized into homo or heteromeric connexons (evans. final packing into hemichannel loaded vesicles occurs in the trans - golgi network as shown by musil and goudenough (1993) followed by directed transport along microtubules to multiple insertion sites in the membrane (akhmanova and hoogenraad 2005 ; jordan. 1999 ; lauf. 2002 ; mimori - kiyosue. 2005 ; shaw. 2007). once arrived at the gap junction borders at the membrane, connexons are assumed to be inserted via flipping events into the membrane and to diffuse into the centre of the plaque, whilst elderly connexin proteins are shifted to the plaque periphery for subsequent disposal (gaietta. 2002 ; laird 2005 ; segretain and falk 2004). the finding that mislocated cx43 gap junction plaques in the ischemic myocardium are associated with similarly misplaced adherens junctions (matsushita. 1999), underlines an interdependence between cx43 and the adherens junction related cadherins (angst. 1997 ; li. 2005 ; luo and radice 2003 ; matsushita. e - cadherin transfections into gap junction incompetent cells, allowed the transfectants to build out functional gap junctions (matsushita. furthermore, n - cadherin knockout mice (luo and radice 2003) and conditional knockdown of n - cadherin in the heart caused mislocalisation and compromized expression of gap junctions. conditional knockdown of n - cadherin in the heart was additionally shown to lead to arrhythmogenic death (li. 2005), which may involve aberrant regulation of gap junction function (for reviews see : duffy. 2007) recently, shaw. (2007) described microtubule - mediated target - delivered transport of cx43 via microtubule plus - end - tracking proteins (+ tips) and interaction partners such as p150(glued) (berrueta. 1999), a component of the dynein / dynactin complex, which in turn is potent to tether microtubules at the adherens junctions (chausovsky. studies implicating fluorescence recovery after photobleaching (frap) on cx43-yfp transfected hela cells that do not endogenously express cx43, revealed a rapid cx43 delivery to gap junction plaques. deconvolution clarified that microtubules extend directly to the gap junction plaques at the cell s border and total internal reflection fluorescence (tirf) microscopy and time lapse imaging revealed the appearance of a preferential and prolonged association of microtubule plus ends with the plaques. (2007) were able to show that gap junction plaque formation was disrupted by sirna knockdown of the dimeric + tip eb1. eb1 associates directly with the plus ends of microtubules and provides, in turn, dual binding sites for adherens junction related proteins like p150(glued) and -catenin. furthermore, in this setting gap junction plaques could also be disrupted via nocodazol and taxol treatment, peptides, which compromise the homophilic cadherin - cadherin interaction in adherens junctions. while nocodazol interrupts formation of microtubules by depolimerization, taxol lets microtubules remain stable, but interferes with their eb1 interaction partner (nakata and hirokawa 2003).fig. 3model for microtubulus mediated delivery of vesicle - bound connexons to adherens junctions (adapted from shaw. eb1 in turn binds to p150 (glued), a component of the dynein / dynactin complex, which interacts with -catenin through p120-catenin with the adherens junction. this interaction is understood to tether the microtubule to the junction and to serve as a gateway for connexon delivery model for microtubulus mediated delivery of vesicle - bound connexons to adherens junctions (adapted from shaw. eb1 in turn binds to p150 (glued), a component of the dynein / dynactin complex, which interacts with -catenin through p120-catenin with the adherens junction. this interaction is understood to tether the microtubule to the junction and to serve as a gateway for connexon delivery in this context, actin is discussed to act as an initial sensor of cell - cell interaction, driving the localisation of adherens junctions with assistance from rho - gtpases (noren. how close cytoskeletal (re-)organization and intracellular connexin distribution are related is demonstrated for polarized intestinal cells during shigella flexneri invasion (clair. 1964), the gram negative enteric bacillus requires rhogtpases, src and abl / arg tyrosine kinases for actin polymerization and formation of cytoplasmic extensions of surrounding cells (burton. the invasion and dissemination of the bacteria causes intense inflammatory responses, and especially atp - dependent paracrine signalling induced by cx hemichannel opening (tran van nhieu. e - cadherins were indispensable for the intercellular spreading of s. flexneri (sansonetti. 1994). it is assumed that a cytoskeletal reorganization toward the formation zone of gap junctions is induced in this process to allow the spread of the bacteria (clair. 2004), which might be of importance for incoming phagocytic cells during bacterial infection (ferrari. 1997 ; griffiths. 1995 ; john. 2001 ; korcok. 2004). the necessity of gap junction adhesion via regulation by its cytoskeleton interaction partners p120 catenin, integrin and actin has become well identified in the developing brain (xu. 2001) and provide guidance of the developing neurons to the target zones of the cortical plate, where they are meant to become pyramidal cells of the adult cortex (rakic 1971, 1972, 1988). electron microscopy showed, that during the process of migration of neuronal precursors, gap junctions occur between radial fibres and migrating neurons and nestin and nestin+ cells (huang. 1998a). the most important gap junction protein isoforms are cx43 and cx26 in developing brain tissue (dermietzel. until now there was evidence that gap junctions between radial glia and migrating neurons served for chemical and electrical communication. elias. (2007) recently found that cx26 and cx43 are expressed in -iii tubulin positive migrating neurons in the contacting regions close to vimentin positive radial glial fibres. using a rna knockdown of cx43 and cx26 by short hairpin rna (shrna) constructs in rat, the authors were able to demonstrate a reduced fractioning of neurons in the cortical plate. in addition, transplantation of cx26 and cx43 shrna knocked down donor cells into e17 wildtype mice revealed an intact engrafting of the donor cells into the host brain, but no migration. immunocytochemistry of the shrna knocked down transplanted neurons in the recipient brains showed no compromised cell cycle exit and no alterations of differentiation. furthermore, the expression of the adherens related proteins zo-1, n - cadherin and -integrin was not altered, indicating that gap junctions mediate glial - guided radial migration of developing neurons in the cortex. this migration was additionally demonstrated to rely on the adhesive and not on the channel properties of cx26 or cx43 (elias. dominant negative connexin mutants lacking channel properties were still able to form adhesive contacts. in reverse experiments, the authors demonstrated that channel, but no adhesion forming mutants, were unable to rescue the cx43 shrna induced migration defect. finally, time lapse imaging of cx43/cx26 shrna expressing neurons affirmed their inability to stabilize their processes and to continue to extend along the radial glia. gap junction regulated polarized cell movements and directional migration are not restricted to developmental processes within the central nervous system (elias. studies focusing on cx43 expression of cardiac neural crest cells indicated a clear relationship between their migratory properties and cx43 expression (huang. 2002 ; lo. 1999 ; reaume. 1995 ; sullivan and lo 1995 ; xu. neural crest cells are ectomesenchymal cells emerging from epithelial mesenchymal cell transformation in the dorsal neural tube from where they disperse throughout the embryo to generate a variety of tissues (kirby and waldo 1995 ; xu. neural crest cells from different axial levels of the neural tube use multiple migratory pathways to reach their terminal destinations. cardiac neural crest cells (cncs) have been shown to migrate along a circumpharyngeal pathway to reach the aortic arches and the heart (kirby. this deployment has been shown to be modulated by cx43 and cytoskeletal interaction partners with the extracellular matrix (xu. the finding, that dynamic di- and reassembly of focal contacts is essential for polarized cell movements and directional cell migration moved the heterodimeric receptor group of integrins into the centre of related studies. integrins cluster to form focal domains within the cell membrane, linking the extracellular matrix to the actin cytoskeleton. since, it could be shown that neural crest cells express multiple integrins (delannet. 1994 ; monier - gavelle and duband 1997) and perturbation studies provide evidence that integrins modulate the migratory behaviour of neural crest cells, (strachan and condic 2003, 2004, 2008) the question arose whether integrin signalling might be affected in cx43 expressing versus cx43 knock out cells (xu. 2006). in neural tube explants of the post - otic hindbrain folds from e8.5 mice, underlying either an cx43 knockout or cx43 overexpression, neural crest cells were generated that emerge from the same axial level as cncs, which migrate to the heart. in contrast to overexpressing cncs, the cx43 deficient cnc type was characterized by a severe loss of directionality and reduced adhesion whilst being cultured on a fibronectin matrix. furthermore, an increase in the fibronectin matrix density leads to reductions in the migratory speed of cx43 deficient cncs, as shown via time lapse videomicroscopy (xu. in fact double - immunostaining against 1-integrin and vinculin as markers for focal adhesion, was significantly reduced in cx43 deficient cncs, indicating a reduction in the actin - cytoskeletal linkage for matrix adhesion. a modulatory influence of cx43 on the actin cytoskeleton became evident in rhodamine - phalloidine stainings, where cx43 knockout cncs represented shorter stress fibre bundles (xu. additionally, these bundles exhibit no anchoring via vinculin to focal adhesions, as being observed for cx43 overexpressing cncs. furthermore, adhesion and migration of cx43 deficient cncs on a fibronectin matrix could be inhibited by semaphorin application, which is described to act as a potent blocker of integrin activation (brown. immunoprecipitation, western blot and immunocytochemistry pointed out that cx43 in cncs does not co - localise with 1-integrin, but with vinculin and actin - filaments (osborne. this finding is supported by the co - localisation of cx43 with several actin binding proteins, such as ezrin, iqgap, -actinin and drebrin (butkevich. 2004). no correlation between gap junctional coupling properties and the density of fibronectin matrix was found for cx43 knockouts and cx43 overexpressing cncs in dye coupling experiments, although an upregulation of gap junction communication with altered integrin - matrix interactions has previously been described for other cell types (czyz. asides their function as gap junction forming elements, unpaired connexons have been shown to modulate the cells migratory and adhesive functions whilst being in permanent crosstalk with an elaborate complex of cytoskeletal interaction partners. how important the connexin - cytoskeleton interaction is, becomes elucidated in case of pathology. the interaction normally becomes active in cell sorting mechanisms during development (wheelock and johnson 2003). since, shaw. (2007) have shown that cx43 can reach adherens junctions via microtubule directed delivery, it may be suggested that gap junctions are formed preferentially with cells, expressing the same type of cadherin (wheelock and johnson 2003). additional studies provide evidence, that loss of e - cadherin or upregulation of n - cadherin can increase tumour invasiveness and cx43 downregulation in malignant cells (mesnil 2002). it is thought that gap junction channels and their interactions with molecules such as p120 catenin, integrin and the actin cytoskeleton are important for neural crest cell migration (xu. 2001, 2006), and that glioblastoma invasion of the brain parenchyma requires functional gap junctions between tumour cells and astrocytes (lin. furthermore, the migration of lung and skin cancer cells has also been associated with gap junction expression, although no clear mechanism has been proposed so far (ito. as already indicated, cx43 knockout mice reveal comprised conotruncal heart development, which is associated with a reduction in the number of cardiac neural crest cells targeted to the heart (xu. mutations in cx43 which seem to influence cytoskeletal organization in a strong manner range from disease patterns like deafness, cataracts, germ cell developmental defects, ocludentodigital dysplasia to cardial outflow abnormalities (polontchouk. gap junction channels, in particular in form of hemichannels, constitute a new player in the complex interaction of cell adhesion and cytoskeletal activation, which underlies directed migration during development and in mature tissue. it is a romantic phase where anything may go, but time has to approve what will remain forever.	recent evidence indicates, that gap junction forming proteins do not only contribute to intercellular communication (kanno and saffitz in cardiovasc pathol 10:169177, 2001 ; saez. in physiol rev 83:13591400, 2003), ion homeostasis and volume control (goldberg. in j biol chem 277:3672536730, 2002 ; saez. in physiol rev 83:13591400, 2003). they also serve biological functions in a mechanical sense, supporting adherent connections between neighbouring cells of epithelial and non - epithelial tissues (clair. in exp cell res 314:12501265, 2008 ; shaw. in cell 128:547560, 2007), where they stabilize migratory pathways in the developing central nervous system (elias. in nature 448:901907, 2007 ; malatesta. in development 127:52535263, 2000 ; noctor. in nature 409:714720, 2001 ; rakic in brain res 33:471476, 1971 ; j comp neurol 145:6183 1972 ; science 241:170176, 1988), or mediate polarized movements and directionality of neural crest cells during organogenesis (kirby and waldo in circ res 77:211215, 1995 ; xu. in development 133:36293639, 2006). since, most data describing adhesive properties of gap junctions delt with connexin 43 (cx43) (beardslee. in circ res 83:629635, 1998), we will focus our brief review on this isoform.
age - related macular degeneration (amd) is a chronic degenerative process and is the leading cause of severe vision loss in people over the age of 60 in developed countries. the neovascular (wet or exudative) form of amd, which accounts for approximately 10% of cases, results in rapid deterioration in visual acuity often with permanent severe loss of vision. the identification of the pathophysiologic mechanisms at the basis of neovascular amd, particularly the role of vascular endothelial growth factor (vegf), has led to the development and use of intravitreally delivered anti - vegf agents, which target and cause regression of choroidal neovascularization [3, 4 ] and have become the standard of care. currently, there are three clinically available agents, ranibizumab (lucentis, genentech, south san francisco, ca), bevacizumab (avastin, genentech), and a more recent addition aflibercept (eylea, regeneron, tarrytown, ny, usa). each of these drugs has been tested in large multicenter, randomized, controlled clinical trials and found to have comparable effects on treatment of nave patients [58 ]. to date, no clear evidence has been presented demonstrating a significant difference between aflibercept and any other agent for the treatment of either nave or resistant neovascular amd. indeed, patients that are deemed unresponsive to treatment with any one agent are regularly offered an alternative drug. treatment protocols with aflibercept also differ among studies, with a loading dose of three monthly injections followed either by a bimonthly or by a pro re nata regimen. recently, a study examining the response to anti - vegf treatment, using all three drugs, of patients with diabetic macular edema demonstrated that eyes with severe vision loss were more likely to benefit from the use of aflibercept. this suggests that there may be a difference in drug response among some patient subgroups. in this paper we aimed to perform a meta - analysis of the published literature on the efficacy of aflibercept in patients with neovascular amd resistant to previous treatment with ranibizumab and/or bevacizumab. we evaluated the changes in their visual acuity and central retinal thickness (crt) following the switch to aflibercept. major databases including pubmed (medline), embase, the science citation index expanded, and the cochrane central register of controlled trials (central) in the cochrane library were searched for studies comparing visual acuity and/or change in crt on optical coherence tomography (oct) of eyes with resistant neovascular amd on prior anti - vegf treatment switching to aflibercept, published in english from january 2012 to may 2015. the medical search headings ranibizumab, bevacizumab, avastin, anti - vegf, lucentis, aflibercept, eylea, and combinations of these were used, so were the keywords persistent, resistant, and recurrent and the keywords switching, the reference lists of articles identified were examined to find additional relevant studies that had not been identified by the database searches. we only included comparative clinical studies with the same group of patients treated with anti - vegf prior to switching to aflibercept and that had full text available in english. the final inclusion of articles was determined by consensus between the authors ssg and otn. we followed the preferred reporting items for systematic reviews and meta - analysis (prisma) guidelines in designing, performing, and reporting the systematic review. included studies were required to (1) assess visual acuity and/or crt of patients with persistent and resistant neovascular amd treated with one anti - vegf drug and then switched to aflibercept, (2) have a minimum follow - up of six months after switching to aflibercept, (3) have the treatment regimen used clearly stated, and (4) be published in english. data recorded included patient and study characteristics, bcva, crt, and statistics used for the study. in order to evaluate the reliability of the comparative evidence, two authors (ssg and otn) independently assessed the risk of bias of the included studies using a modified version of the newcastle - ottawa scale for assessing the quality of prospective and retrospective studies in meta - analysis [11, 12 ]. each paper was awarded a score in four categories, patient selection (03), treatment comparability (06), statistical methods (03), and outcome (06). studies achieving ten or more points were considered to be of high quality. only these studies were included in the final analysis. prospective studies scored higher on patient selection than retrospective studies as were multicenter studies, as they are more likely to be representative of the entire patient population. use of fluorescein fundus angiography (ffa), oct, or combination of both received an increasingly higher score on treatment comparability. outcome and timing assessments of best corrected visual acuity (bcva) and crt as well as reporting of follow - up were all evaluated. for the purpose of the analysis, all bcva values were converted to the log minimum angle of resolution (logmar). original data were extracted from the studies and analyzed and the standardized mean change was used to calculate intervention effects. the standardized mean change was used to compute the estimates of treatment for correlated designs. the standardized mean change has been shown to be a more appropriate measure of effect size for the direct comparison of data from studies using a pretest and posttest design without control groups [1315 ]. for the purposes of this meta - analysis, we used the mean, standard deviation (sd), and the correlation for pre / postswitching data. in all studies, the correlation between pre- and postswitching data was not reported and values were obtained either directly from the authors or by calculating the correlation using the p value / t - test values, means, standard deviation, and the number of patients included from the published data. using these values we were then able to calculate the standardized mean change for each group. studies for which such information could not be obtained were excluded from the meta - analysis (n = 5). the standardized mean change was calculated as the difference between the means of the posttreatment switching values and the baseline divided by the pooled standard deviation. five studies lacked enough statistical information to be included in this meta - analysis and their outcomes were reviewed for information only [1620 ]. studies specific standardized mean change was pooled using fixed effect models with the mantel - haenszel method if heterogeneity was negligible or using random effect models with the dersimonian - laird method when heterogeneity was significant [21, 22 ]. interstudies heterogeneity was assessed using the cochran q test and i tests, with a p 50% suggesting a high interstudy heterogeneity. in an attempt to identify the source of heterogeneity in the data, we performed a metaregression analysis on confounders such as the type of study, the previous total length of treatment, previous total number of anti - vegf injections prior to switching, and mean bcva at baseline. a p value < 0.05 was regarded as significant results and all tests were 2 sided. all statistical analyses were performed using commercially available software comprehensive meta - analysis (cma, biostat, englewood, nj, usa, v 3.0). the estimates of treatment effect are presented as weighted mean difference (wmd) and graphically as forest plots. sensitivity analysis was carried out by including only studies deemed with a quality score of 90% or above (13 points and above) and excluding each study from the analysis of each outcome measure to confirm the stability of our findings. the search strategy initially generated 28 relevant clinical studies, of which 13 scored higher than 10 on the quality assessment. of these, seven studies had sufficient outcome data and statistical information to be included in the meta - analysis (four retrospective studies [2427 ] and 3 prospective studies [2830 ], figure 1). all these studies were included in the meta - analysis of visual and/or crt following switching to aflibercept, six studies in the analysis of bcva (232 eyes of 225 patients) [2426, 2830 ] and five in the analysis of crt (266 eyes of 259 patients) [2428 ]. table 1 details study characteristics, quality, and comparability assessments, and table 2 shows the analyzed outcome measures. median patient age at time of treatment switching was 78 years (range 70.180.3 years), with a median female percentage of 55.96% (range 30.970.5%). at the time of treatment switching patients had been diagnosed with neovascular amd for a median of 40.65 months (range 20.544.1 months) and eyes had already been given a median of 28.6 anti - vegf injections (range 9.642.0 injections). the median time between the last anti - vegf injection and beginning of aflibercept treatment was 35.0 days (range 33.346.5 days) and eyes were treated with a median of 5.1 aflibercept injections (range 4.505.60 injections). the median bcva at baseline was 0.53 logmar (range 0.320.64 logmar) and crt on oct was 413 m (range 336.3448.3 m). the overall results of the meta - analysis showed a small but statistically significant improvement in bcva at six months after switching to aflibercept (wmd 0.142, 95% ci 0.006 to 0.28 ; p = 0.04). the random model was used as heterogeneity of the data (i) was 81.02%. exploring possible confounders on bcva we performed a weighted regression on the six studies included. the type of study design was the only moderator that showed a significant effect on the bcva at six months (q = 8.71, p = 0.003), with prospective studies demonstrating a greater estimate of treatment effect on change in bcva (wmd 0.407, 95% ci 0.02 to 0.79, p < 0.038, figure 2) than retrospective studies (wmd 0.104, 95% ci 0.04 to 0.25 ; p = 0.16). other covariates such as mean number of anti - vegf injections prior to switching to aflibercept, length of time of treatment with anti - vegf prior to switching, mean bcva at switching, and the type of treatment had no effect on the overall results. overall, patients with their current treatment resistant amd had a significant improvement in crt at six months following switching to aflibercept (wmd 0.36, 95% ci 0.49 to 0.24 ; p < 0.0001, figure 3). the random model was used as heterogeneity of the data (i) was 66.79%. the weighted regression on the 5 studies included for covariates (mean number of anti - vegf prior to switching to aflibercept, total length of treatment with anti - vegf prior to switching, mean bcva at switching, and mean crt at switching) showed no significant effect on crt at 6 months. we performed a sensitivity analysis, to determine the effect of each study on the overall result, by removing each of the studies one at a time and recalculating the summary wmd. the overall pooled wmd remained stable indicating that our results were not influenced by any single study. we performed a further sensitivity analysis of all studies scoring above 90% of the maximal score (13 points and above, wmd 0.204, ci from 0.012 to 0.395, p = 0.037) and excluding each study from the analysis of each outcome measure [26, 2830 ]. although the funnel plot showed evidence of publication bias, the small number of studies limits this analysis, as confirmed by egger 's test (p = 0.03). the same tests did not suggest publication bias for the anatomical outcome (p = 0.43). this study systematically reviewed and analyzed the evidence in the literature of the effect of switching to treatment with aflibercept in eyes with neovascular amd resistant to previous anti - vegf treatment. overall, we included four retrospective and three prospective studies that had a follow - up period of at least six months (three retrospective studies had a follow - up period of up to twelve months). while we were able to identify other studies that examined the effect of switching to aflibercept, insufficient outcome information prevented these from being included in this analysis. our meta - analysis demonstrated a small but significant improvement in bcva following switching to aflibercept. while this improvement in bcva was indeed small and of limited clinical significance, it was driven by a significant improvement noted in the analyzed prospective studies (ranging between 0.1 and 0.14 logmar), suggesting that the retrospective design of the studies where no effect was found may have influenced their outcome. the metaregression analysis did not demonstrate any effects on bcva from confounders such as mean number of anti - vegf injections, mean length of treatment or the drug used prior to switching, which is in keeping with results from other studies [19, 27 ]. while most studies used a standard treatment protocol following switching, of a loading dose of three monthly injections followed by a bimonthly regime, other studies used a pro re nata regimen, neither of which resulted in a difference in treatment effect. the results of this meta - analysis demonstrated a significant improvement in crt following the treatment switch to aflibercept. this was a consistent finding in all the studies and was maintained during the longer follow - up up to twelve months [27, 31, 32 ]. while there was a definite improvement in retinal thickness, this did not correlate with significant restoration of visual function. repeated use of any drug can result over time in tachyphylaxis with resultant loss of clinical effect [3335 ], which can often be overcome by switching to another drug with an alternative mode of action. thus, restoration of effect may be achieved following switching, resulting in clearing of retinal fluid and reduction in crt. however, the lack of a concomitant gain in visual function suggests it may be related either to an accumulating effect of long term neovascularization, such as development of retinal gliosis, or to progression of retinal atrophic changes that have been documented to occur in neovascular amd eyes treated with anti - vegf injections [3638 ]. while this meta - analysis included all current studies examining treatment switching to aflibercept in eyes with neovascular amd, it nevertheless suffers from several limitations, mainly related to the quality of the studies analyzed. most studies performed to date were nonrandomized retrospective treatment comparison studies, which introduce potential confounding biases due to an inability to adjust for patient baseline demographic characteristics or disease severity. though we found a significant improvement in crt on oct and bcva, study design and population varied across studies and this variation was reflected in significant heterogeneity in the estimated comparative effect for the analysis of both bcva and crt. we found that the type of study had a significant impact on our results when analyzing bcva, supporting the view that information gathered from prospective studies may offer clearer conclusions and that overinterpretation of retrospective studies should be avoided. sensitivity and subgroup analysis of only high quality studies did not reveal any difference in the results and suggests that these results indeed represent the culmination of all current studies. the main strength of our meta - analysis is in incorporating all previous studies, resulting in a large sample size and allowing us to focus the results of all previous studies. to date, this is the first systematic meta - analysis evaluating the visual and anatomical outcomes of patients with resistant amd converted to aflibercept. our analysis provides substantial evidence that following switching there is a significant anatomical effect, resulting in crt thinning. however, the visual function change was far more modest and while there is evidence to support that aflibercept has a comparable effect to other anti - vegf agents in maintaining vision, any potential significant benefit should be regarded with caution. while this study has clarified the known effect of aflibercept in treatment failure neovascular amd eyes, future results, especially from prospective studies, may offer new insights into the different effects of these agents.	with the introduction of aflibercept, eyes with neovascular age - related macular degeneration (amd) not responding well to injections of ranibizumab or bevacizumab can be switched to treatment with aflibercept. we carried out a meta - analysis to analyze all available evidence of visual and anatomical outcomes of eyes with resistant neovascular amd switched to aflibercept at six months. data from seven retrospective and prospective studies looking at change in best corrected visual acuity (bcva) and central retinal thickness (crt) were included. weighted mean difference (wmd) and 95% ci were estimated using the standardized mean change method. the overall results of the meta - analysis showed a small but statistically significant improvement in bcva six months following treatment switch to aflibercept (wmd 0.142, 95% ci 0.006 to 0.28 ; p = 0.04), and the effect was more significant in data gathered from prospective studies (wmd 0.407, 95% ci 0.023 to 0.791, p = 0.038). there was a significant improvement in crt following treatment switch to aflibercept (wmd 0.36, 95% ci 0.485 to 0.235 ; p < 0.0001). our meta - analysis indicates that following treatment switch to aflibercept patients may have a significant improvement in crt with stabilization or even some improvement in their visual acuity.
hyperthyroidism occurs in approximately 2/1000 pregnancies with the majority of those cases being graves ' disease, an autoimmune disorder caused by tsh - receptor stimulating autoantibodies. propylthiouracil (ptu) is the primary treatment of hyperthyroidism in the us followed by methimazole (mmi) while carbimazole (cmi) is not distributed in the us. untreated hyperthyroidism during pregnancy leads to developmental toxicity which includes spontaneous abortion, prematurity, growth restriction, and fetal death [1, 2 ]. it is not clear if untreated hyperthyroidism during pregnancy leads to structural malformations [3, 4 ] ; however one dataset suggested that the risk of malformations was greater than expected among infants from untreated than treated pregnancies. additionally, there is concern that one of the medical therapeutic strategies, mmi, may be a weak developmental toxicant, producing structural malformations, as well as fetal goiter. part of this concern stems from the observation that cmi is a weak developmental toxicant and one of its metabolites is mmi. on the one hand, as just mentioned, treatment of hyperthyroidism during pregnancy may result in developmental toxicity, as the available drugs cross the placenta and can cause fetal goiter. on the other hand, while the data remain unclear, there is evidence suggesting that maternal hypothyroidism is associated with impaired fetal neurodevelopment. consequently the clinician must balance the use of the antithyroid medications against the potential developmental consequences of inadequate or aggressive therapy, with a limited set of therapeutic options. several assumptions about ptu and mmi have guided the medical treatment strategies for graves ' disease during pregnancy in the us [2, 68 ]. it has been assumed that placental transport of ptu is less than mmi, it has been assumed that fetal and neonatal thyroid effects of ptu is less than with mmi (based on lower placental transport), it has been assumed that mmi exposure during pregnancy leads to greater impairment of neurocognitive development than with ptu exposure, and it has been assumed that mmi produces structural defects and that ptu does not. a thoughtful review and analysis of literature by mandel has demonstrated that these assumptions are not correct [2, 6 ]. given the uncommon nature of graves ' disease in pregnancy and the weak link between the antithyroid medications and malformations, we have taken a different approach to assess the potential developmental toxicity of the drugs which are being used to treat the disease in pregnancy. several years ago we created datasets to evaluate the structural determinants of developmental toxicity in experimental animals and humans [913 ]. statistical analyses demonstrated that animal models are reasonable predictors of human developmental toxicity, and that rules could be agreed upon among experts in developmental toxicity for evaluating animal and human data. subsequently we evaluated the utility of structure - activity relationship (sar) models generated by multicase for studying and predicting developmental toxicity in diverse species including humans. this later dataset of chemicals assessed for human developmental toxicity has subsequently been utilized to create a more transparent and robust model of developmental toxicity using the categorical - sar (cat - sar) expert system. briefly, the cat - sar expert system diverges from other sar expert systems wherein there is a high degree of user flexibility in both learning set development and model parameterization. cat - sar analysis allows the user to specify adjustable modeling attributes including the selection of size of the 2-dimensional fragments, whether or not to include hydrogen atoms in the analysis, and rules for identifying important fragments for the final model. hence, the selection of compounds included in the learning set and control over various model attributes provides the user with the ability to rigorously explore the relationships between chemical structure and biological activity. application of the cat - sar expert system to a toxicological or pharmacological endpoint is thus not constrained wherein a given set of data must fit the attributes of a predefined and often proprietary modeling process. data on human developmental toxicity were derived from the teratogen information system and a database that utilized the us fda guidelines as described previously. the chemicals in this database were specifically characterized with respect to risk for human developmental toxicity including death, growth retardation and functional and structural abnormalities. the cat - sar approach is a computational sar or in silico toxicity analysis and prediction expert system. in previous analyses, the cat - sar program was able to achieve an overall concordance between observed and predicted values of 92% for a set of chemicals assessed for their ability to induce respiratory hypersensitivity, 80%90% for a set of environmental estrogen mimics, and 78%84% for a set of rat mammary carcinogens. cat - sar models are built through a comparison of structural features found amongst categorized compounds in the model 's learning set. essentially, the cat - sar approach is transparent in the development of the learning set, the identification of fragments, and the determination of significant or important ones. moreover, the approach allows user intervention and model optimization throughout the modeling process. this method includes the ability to examine the entire fragment base and to explore and optimize the fragments that have perceived biological relevance. moreover, since cat - sar analyzes categorical data and 2-dimensional fragments rather than intact chemicals, the program can examine noncongeneric datasets that are divided into categories of activity rather than degrees of potency as in the case of quantitative sar (qsar). thus, unlike hansch and conformational molecular field analysis (comfa) approaches that require continuous - type data, cat - sar works by identifying molecular attributes associated with biological activity by comparing attributes of active (e.g., teratogenic) to inactive (e.g., nonteratogenic) compounds. the models and subsequent predictions based on this dichotomy can then be used to examine structural features associated with teratogenicity and predict the likelihood of teratogenic activity of unknown compounds, respectively. overall, the cat - sar models discussed herein for developmental toxicity demonstrate a high degree of predictivity and mechanistically interpretability and can be useful for screening new drug candidates for developmental toxicity as well as for investigating their therapeutic and toxic mechanisms of action. the cat - sar models are built through a comparison of structural features found amongst two designated categories of compounds in the model 's learning set. as mentioned, for these analyses the categories were developmental - toxicity and nondevelopmental - toxicity. the cat - sar learning set consists of the chemical name, its structure as a mol2 file, and its categorical designation (e.g., one or zero for active and inactive). typically, organic salts are included as the freebase and simple mixtures and technical grade preparations may be included as the major or active component, metals, metalo - organic compounds and polymers, and mixtures of unknown composition are not included. using the tripos sybyl hqsar module, each chemical was fragmented in silico into all possible fragments meeting user - specified criteria. hqsar allows the user to select attributes for fragment determination including atom counts (i.e., the size of the fragments), bond types, atomic connections (i.e., the arrangement of atoms in the fragment), explicit hydrogen atoms, chirality, and hydrogen bond donor and acceptor groups. fragments can be linear, branched, or cyclic moieties. for analysis of the developmental toxicity dataset the models developed contained fragments between three and seven atoms in size and considered atoms, bond types, and atomic connections. upon completion of the fragmentation routine a sybyl hqsar add - on is used to produce a compound - fragment data matrix. in the matrix, thus for each chemical, a tabulation of all its fragments is recorded across the table rows and for each fragment all chemicals that contain it are tabulated down the columns. rather, the compound - fragment matrix is subsequently analyzed with the cat - sar expert system in order to identify structural features associated with the categorized active and inactive compounds. the cat - sar program, human developmental toxicity database, and the compound - fragments matrix are available through the corresponding author. a measure of each fragment 's association with biological activity is next determined. to ascertain an association between chemical descriptors (i.e., fragments) and a chemical 's activity (or inactivity), a set of rules is used to choose important from unimportant descriptors. the first selection rule (the number rule) is the number of chemicals identified in the learning set that possesses each particular fragment. the second selection rule (the proportion rule) is the proportion of active or inactive chemicals that then possesses the particular fragment. although previously published cat - sar models required the user to select specific values for the number and proportion rules, a new routine was implemented here to determine optimal values for the number and proportion rules. for this exercise the values for the number rule were allowed to range between one and eight and the initial values for the proportion rule were allowed to range between 0.50 and 0.95. the resulting list of important fragments can then be used for mechanistic analysis, or to predict the activity of an unknown compound. in the latter circumstance, the model determines which, if any, fragments from the model 's learning set the test compound contains. if none are present, no prediction of activity is made for the compound (i.e., no default prediction). if one or more fragments are present, the number of active and inactive compounds containing each fragment is determined. here the fragment sum (fragsum) method calculates the average probability of the active and inactive fragments contained in a compound and is weighted to the number of active and inactive compounds that go into deriving each fragment. for example, if a compound contains two fragments, one being found in 9/10 active compounds in the learning set (90% active) and the other being found in 3/3 inactive compounds (0% active), the unknown compound will be predicted to be have a probability of activity of 69% (9/10 actives + 0/3 actives = 9/13 actives or 69% chance of activity). as described, a cat - sar prediction of activity or inactivity is based on two separate fragment sets (i.e., the active fragments and the inactive ones) and the predicted activity of a chemical is based on the average probability of all the active and inactive compounds contributing to its structure. therefore, to classify compounds back to an active or inactive category (i.e., rather than a probability of activity), the program identifies an optimal cutoff point that best separates the prediction of active and inactive compounds based on the probabilistic values of activity derived from a model validation analysis. depending on the application of the model, the cutoff point that separates active from inactive categorization, for example can be adjusted wherein a model with the best overall concordance can be selected (i.e., a most predictive model), one with equal sensitivity and specificity (i.e., a balanced model that does not overly predictive active compounds at the cost of wrongly predicting inactive one and viceversa), or one with high sensitivity (i.e., a risk averse model). a self - fit, leave - one - out (loo), and multiple leave - many - out (lmo) cross - validations were conducted for each model (see table 1). for the self - fit, a model was developed from the complete learning set of 323 compounds and that model was used to predict the activity of each compound in the learning set to determine the general robustness of the model. for the loo validation, each chemical, one at a time, was removed from the model 's total fragment set, and an n-1 model was derived.. predicted vs. experimental values for each chemical were then compared and the model 's overall concordance, sensitivity, and specificity were determined, where, (1)concordance = correct predictionstotal predictions, sensitivity = correct positive predictionstotal positive predictions, specificity = correct negative predictionstotal negative predictions. for the lmo validation, randomly selected sets of 5% of the chemicals were removed from the model, and the n-5% model was derived for each set.. predicted versus experimental values for the chemicals in the left out sets were then compared and the n-5% model 's concordance, sensitivity, and specificity were determined. this was repeated 10 000 times to compute the model 's average concordance, sensitivity, and specificity. model 1 was designed to have near - equal sensitivity and specificity while model 2 was designed to demonstrate the best overall concordance between observed and predicted values (table 1). overall, 26 401 unique chemical fragments between 37 nonhydrogen atoms were derived for the developmental toxicity database (table 1). of these, for model 1, 1815 were associated with developmental toxicants and 2707 with nontoxicants (4522 total significant fragments). and for model 2, 1027 were associated with developmental toxicants and 2413 with nontoxicants (3440 total significant fragments). the self - fit analysis yielded concordance between observed and predicted results of 94% (274/291) for the balanced sensitivity and specificity model (model 1) and 99% (250/252) for the best concordance model (model 2). these high concordance rates indicate a robust model wherein there is sufficient structural information contained in the learning set to distinguish between active and inactive compounds. the loo cross - validation yielded a concordance of 79% for the balanced sensitivity and specificity model (model 1) and 81% for the best concordance one (model 2). the lmo cross - validation yielded an average concordance of 78% for the balanced sensitivity and specificity model (model 1) and 80% for the best concordance one (model 2). overall, since the loo and lmo validation results are in near agreement, it is estimated that the cat - sar developmental toxicity model is ~80%, accurate for predicting the developmental toxicity potential of chemicals not included in the model 's learning set. in order to better judge how well these two models performed in general, one can consider the accuracy or reproducibility of a standard in vitro toxicological test. the us national toxicology program 's (ntp) salmonella mutagenicity database, which is derived from a standardized protocol, has been estimated to be about 85% reproducible (49). based on these findings wherein the concordance between observed and predicted values for human developmental toxicity is ~80% the cat - sar human developmental toxicity models thus appear to be as predictive as data used to develop sar models from standardized in vitro assays. for these analyses, the balanced sensitivity and specificity model (model 1) was used for analysis of ptu, mmi, and cmi. this model identified 13 fragments within the three antithyroid medications relating to developmental toxicity (figure 1). nine of the 13 fragments were considered to be associated with developmental toxicants and 4 of the 13 fragments were considered to be associated with non - developmental toxicants. fragments 2184, 2182, and 6241 are found in fluorouracil, for which there is a small amount of human data on developmental toxicity. however the compound is known to interfere with dna synthesis and has been shown to be a developmental toxicant in rat, mouse, rabbit, hamster, guinea pig, and in nonhuman primates (, teris micromedex accessed 06 july 2009). fragments 2184, 2182, and 6229 are found in a series of barbiturates used as sedative - hypnotics and anticonvulsants. amobarbital has been associated in several human studies with cardiovascular malformations although no experimental animal studies appear to have been conducted. phenobarbital use throughout pregnancy, as an anticonvulsant, has been associated with facial and cardiovascular malformations in humans and experimental animals (teris micromedex accessed 06 july 2009). mephobarbital is metabolized to phenobarbital and has been associated with increased risk of facial malformations (teris micromedex accessed 06 july 2009). there is little animal and human data on metharbital, used as an anticonvulsant, or on butalbital used as a sedative hypnotic. fragments 6172 and 6206 are found in thioguanine used as an antineoplastic and in treatment of crohn 's disease. thioguanine has been demonstrated to produce malformations in experimental animals and in humans (, teris micromedex accessed 06 july 2009). the additional fragments associated with developmental toxicity, 9479, 9564, and 9565, are found in a series of tetracycline like antibiotics. tetracycline is identified as a developmental toxicant because of incorporation into teeth and evidence of alteration in bone growth (, teris micromedex accessed 06 july 2009) ; because of structural similarity and mechanism of action the other tetracyclines are also considered to be developmental toxicants. there were 4 fragments which were found in molecules thought not to be developmental toxicants : 6279, 6298, 6308, and 6332. the molecules these fragments were found in are cephalosporin, antibiotics and across this class of compounds there is evidence of the absence of risk for developmental toxicity in both experimental animals and humans (, teris micromedex accessed 06 july 2009). the three antithyroid drugs analyzed represent the limited choices available to the health care provider who is caring for a woman with graves 's disease during pregnancy. these are old drugs, all available as generics ; however despite their long history of use there is little data on the developmental risks resulting from use and exposure during pregnancy. all three drugs are capable of crossing the placenta and can cause fetal goiter. thus while ptu has been suggested to be without risk of developmental toxicity in humans, that assumption appears to reflect a shared attitude rather than presence of data demonstrating developmental safety. an additional concern with the use of ptu is liver toxicity leading to liver failure and need for transplant. analysis of data submitted to the us food and drug administration (fda) suggests that children may be at increased risk for ptu - induced liver failure, and cases have been observed of fetal hepatic toxicity in women treated with ptu (fda adverse events reporting system data obtained through the freedom of information act). additionally there have been cases of infants with malformations observed in women treated with ptu, and structural malformations have been observed in women with graves 's disease who were not treated during pregnancy. surgery and radioiodine are typically avoided, leaving propylthiouracil and methimazole in the us. cmi, a metabolic precursor of mmi, is available in some countries outside of the us. in the us ptu is recommended because of concern about developmental toxicity from mmi and cmi. in summary the three drugs available to treat graves 's disease in pregnancy all appear, based on structural analysis, to be capable of producing developmental toxicity. given the need to treat women with graves 's disease during pregnancy it is essential to develop new molecules with structural attributes which provide suppression of thyroid function while minimizing the risk for developmental toxicity. based on the results described herein, the cat - sar method would be a useful approach in screening compounds for developmental toxicity as well as for investigating their therapeutic and potential toxic mechanism of action.	the choice of therapeutic strategies for hyperthyroidism during pregnancy is limited. surgery and radioiodine are typically avoided, leaving propylthiouracil and methimazole in the us. carbimazole, a metabolic precursor of methimazole, is available in some countries outside of the us. in the us propylthiouracil is recommended because of concern about developmental toxicity from methimazole and carbimazole. despite this recommendation, the data on developmental toxicity of all three agents are extremely limited and insufficient to support a policy given the broad use of methimazole and carbimazole around the world. in the absence of new human or animal data we describe the development of a new structure - activity relationship (sar) model for developmental toxicity using the cat - sar expert system. the sar model was developed from data for 323 compounds evaluated for human developmental toxicity with 130 categorized as developmental toxicants and 193 as nontoxicants. model cross - validation yielded a concordance between observed and predicted results between 79% to 81%. based on this model, propylthiouracil, methimazole, and carbimazole were observed to share some structural features relating to human developmental toxicity. thus given the need to treat women with graves 's disease during pregnancy, new molecules with minimized risk for developmental toxicity are needed. to help meet this challenge, the cat - sar method would be a useful in screening new drug candidates for developmental toxicity as well as for investigating their mechanism of action.
warren in the late 1970s, and we and others more recently (reviewed in [1214 ]), have analyzed the types of blood vessels that are found in tumors. these studies have shown that the tumor vasculature is highly heterogeneous, and, for the most part, very different from that found in normal tissues. examining several human cancers, we were able to identify six distinct tumor blood vessel types (table 1). further, using an adenovirus engineered to express vegf - a, we were able to replicate each of these in the form of tumor surrogate vessels in immunodeficient mice [14, 15 ]. this model system allowed us to elucidate the steps and mechanisms by which each vessel type formed initially and evolved over time (fig. 1). unlike tumors, which express high levels of vegf - a over extended periods of time, ad - vegf - a gives rise to a single pulse of vegf - a. this vegf - a pulse is initially of similar magnitude to that found in many tumors. however, unlike in tumors, local vegf - a levels fall off dramatically over a matter of weeks because the adenoviral insert carrying the transgene is not integrated into the cellular genome and so is discarded from the cell. in contrast, tumors continue to express vegf - a at high levels over long periods of time ; thus, they continually induce the formation of new blood vessels, while, concurrently, causing previously formed vessels to differentiate into more stable forms. given this mixture of early and late vessels, it becomes difficult to follow progression of newly formed vessels as they evolve over time in tumors. in contrast, at ad - vegf - a injection sites, the formation of new blood vessels is restricted temporally and their evolution into late vessels can therefore be followed sequentially. studies with the ad - vegf - a model demonstrated that four of the six tumor vessel surrogates develop by angiogenesis, i.e., they derive initially from preexisting small blood vessels, namely, venules and capillaries. in addition, both vegf - a - secreting tumors and ad - vegf - a induce abnormal arteriogenesis and venogenesis, i.e., they cause pre - existing arteries and veins to enlarge and remodel and these feed and drain the angiogenic vascular bed. there is every reason to believe that similar processes are going on in tumors.table 1classification of tumor / tumor surrogate blood vesselsprocess involvedvessel typevessel propertiesangiogenesismother vessels (mv)large, thin - walled, hyperpermeable, lightly fenestrated pericyte - poor sinusoids that are engorged with red blood cells.capillariesformed from mv by a process that involves intra - luminal bridging and intussusception.glomeruloid microvascular proliferations (gmp)poorly organized vascular structures that resemble renal glomeruli macroscopically. gmp are comprised of endothelial cells and pericytes with minimal vascular lumens and reduplicated basement membranes.vascular malformations (vm)mother vessels that have acquired an often asymmetric coat of smooth muscle cells and/or fibrous connective tissue.arterio-venogenesisfeeder arteries (fa)enlarged, often tortuous arteries and veins that are derived from preexisting arteries and veins. they extend radially from the tumor mass, supplying and draining the angiogenic vessels within.draining veins (dv)fig. 1schematic diagram of the steps by which vegf - a - induces angiogenesis and arterio - venogenesis. the blood vessels responsive to anti - vegf - a therapy are enclosed within the box outlined with a dashed line.. 1 in) classification of tumor / tumor surrogate blood vessels schematic diagram of the steps by which vegf - a - induces angiogenesis and arterio - venogenesis. the blood vessels responsive to anti - vegf - a therapy are enclosed within the box outlined with a dashed line. mother vessels (mv) are the first new type of angiogenic blood vessel to appear, both in tumors and also in response to ad - vegf - a [15, 16 ] (fig. 1). mv are greatly enlarged sinusoids that are highly permeable to plasma proteins and to other circulating macromolecules [13, 14 ]. they begin to develop from preexisting venules and capillaries within hours of injection of tumor cells or of ad - vegf - a into mouse tissues. we predicted that vascular basement membrane (bm) degradation would be an important step in mv development because bm are non - compliant (non - elastic) structures that normally restrict microvessel expansion. swayne had demonstrated the importance of bm in maintaining microvessel size in studies by demonstrating that progressive increases in intravascular pressure were only able to increase vascular cross - sectional area by ~30 % before vessels burst, i.e., far less than the three to five - fold increase in area typical of mv. testing this hypothesis, we found that over the course of a few days after injecting ad - vegf - a or tumor cells into mouse tissues, bm staining for laminin and type iv collagen, the most abundant components of vascular bm, was progressively lost in developing mv. gene chip studies revealed that cathepsin transcripts were increased locally, and this finding was confirmed and extended by rt - pcr and at the protein level by immunohistochemistry. further, western blots revealed that activated forms of three cathepsins, b, s, and l, increased substantially as mv developed, and immunohistochemistry selectively localized increased cathepsin activity to the pericytes associated with developing mv. in normal tissues the action of cathepsins is opposed by a family of endogenous inhibitors called cysteine protease inhibitors (cpi). as mv formed, expression of these inhibitors progressively decreased in both endothelial cells and pericytes. thus, bm degradation was induced in mv by increased expression of cathepsins and decreased expression of cpi, i.e., by an upsetting of the cathepsin / cpi balance that normally maintains bm integrity and so microvascular size. as a consequence of bm degradation, pericytes lost their attachments to endothelial cells, and endothelial cells, no longer restrained by bm or attached pericytes, underwent cellular thinning as their lumens expanded in response to intravascular pressure. increased lumen size requires an increase in endothelial cell surface area and so an increase in plasma membrane. this was provided, at least in part, by vesiculo - vacuolar organelles (vvos), clusters of hundreds of interconnected vesicles and vacuoles contained within the cytoplasm of normal venular endothelial cells. vvos have an important role in the transport of macromolecules across venules in the acute vascular hyperpermeability induced by vegf - a, histamine, etc. the membrane stored in vvos amounts to more than twice that found in the plasma membranes of normal venular endothelial cells. as the formerly cuboidal endothelial cells of normal venules flattened, vvos fused with the plasma membrane, contributing to the plasma membrane expansion necessary for mv formation. mv are typically unstable blood vessels as their lack of pericytes, basement membrane support, and sluggish blood flow make them susceptible to thrombosis or collapse. mv are therefore transitional structures that evolve into one or another type of daughter vessel : capillaries, glomeruloid microvascular proliferations (gmp) and vascular malformations (vm) [13, 14 ] (fig. 1). capillaries form from mv by a process of internal bridging as endothelial cells extend thin, tip - cell - like processes into the mv lumen rather than externally as in vascular sprouting [13, 14 ]. these endothelial cell processes grow to form transluminal bridges that divide mvs into smaller, capillary - sized structures that eventually separate from each other by a process of intussusception. gmp result from a proliferation of endothelial cells and pericytes that fill mv lumens and divide them into much smaller channels that are enveloped by redundant layers of bm [15, 22 ]. like mv, gmp are hyperpermeable to macromolecules, but, because they are poorly perfused, account for relatively little plasma extravasation. finally, vascular malformations (vm) derive from mv that have acquired a supporting smooth muscle cell coating. vm are readily distinguished from normal arteries and veins by their inappropriately large size (for their location) and by their thinner and often asymmetric muscular coat. they, and the capillaries that form in parallel, are not hyperpermeable to plasma proteins. in addition to inducing angiogenesis, tumors and ad - vegf - a also stimulate abnormal arteriogenesis and venogenesis, leading to the formation of the large, often tortuous feeder arteries (fa) and draining veins (dv) that supply and drain the tumor microvasculature [13, 14 ]. the mechanisms by which these vessels develop have been little investigated. however, once induced to form in response to ad - vegf - a, fa and dv, like vm, persisted indefinitely. a number of reasons have been offered for the disappointing ineffectiveness of anti - vegf / vegfr drugs in treating human cancers [2325 ]. these include the need for better dosing strategies ; the frailty of cancer patients who are much sicker than tumor - bearing mice and therefore can not withstand the toxicities associated with high dose therapy ; and vascular normalization, a period of time following anti - vegf / vegfr therapy in which tumor vessels normalize structurally and lose their hyperpermeability. an additional important reason is that an impaired vascular supply may not result in the killing of all tumor cells, leaving survivors hypoxic. hypoxic tumor cells reprogram their transcriptional profile to increase the synthesis of vegf - a as well as panoply of other growth factors that together can overwhelm anti - angiogenesis therapy. however, they seem insufficient to explain fully the lack of anti - vegf / vegfr drug effectiveness in cancer patients relative to those in mice, and other possibilities must be considered. the major successes of anti - vegf - a / vegfr therapies in mice have been in the prevention of the growth of freshly transplanted mouse tumors and tumor xenografts [23, 26 ] (for additional references, see [27, 28 ]). in contrast, when anti - vegf / vegfr therapy is delayed and administered to mice that have established autochthonous or transplanted tumors, the benefits are of lesser magnitude [23, 26 ]. obviously the difference in treatment regimen timing is important because freshly transplanted tumors do not mimic human cancer ; in patients, cancers have been growing for months or years before they are discovered and treated. the better question, then, becomes, why does anti - vegf - a / vegfr therapy work better on newly transplanted tumors than on established tumors ? we suggest that the different types of blood vessels that tumors induce are likely to provide important insights. as noted, tumor blood vessels are heterogeneous and form linearly over time by two distinct but interrelated processes, angiogenesis and arterio - venogenesis (fig. 1). the initial or early vessels induced by vegf - a are mv and actively remodeling arteries and veins ; these subsequently evolve over a period of weeks to months into stable late vessels, i.e., capillaries, vm, fa and dv. based on this understanding of tumor vessel development early and late vessels might differ in their susceptibility to anti - vegf - a / vegfr therapy. early vessels are susceptible to anti - vegf - a / vegfr therapy whereas late vessels, though formed from early vessels that are induced by vegf - a, have lost their vegf - a dependence and have therefore become resistant to anti - vegf - a / vegfr therapy. established tumors, whether mouse or human, are expected to contain a mixture of early and late vessels. early vessels would predominate initially ; however, as tumors grow over time, the population of late vessels would be expected to become proportionately greater. late vessels are less dependent on exogenous vegf - a than early vessels and 2. late vessels are relatively resistant to anti - vegf - a / vegfr therapy compared with early vessels. vessels should be less dependent on the vegf / vegfr axis than early vessels. initial experiments early vessels such as mv and gmp stained strongly for vegfr-2, the vegf - a receptor responsible for both vegf - a - induced angiogenesis and increased permeability ; in contrast, staining for vegfr-2 was greatly reduced or largely negative in vm, fa and dv. this finding, consistent with our hypothesis, suggests that vegfr-2 signaling is less important in late than in more directly, we evaluated the effects of two drugs that act at different stages in vegf - a signaling [28, 29 ]. one such drug, rapamycin, inhibits mtor, a downstream target of the vegf - a - akt pathway. when administered before and coincident with ad - vegf - a injection into tissues, rapamycin effectively inhibited local vascular akt and s-6 phosphorylation and prevented the formation of mv and the vascular hyperpermeability and edema that accompany their formation. when administered slightly later, at a time when mv had already formed, differentiation of mv into gmp and vm was greatly reduced. however, when administered still later, when vm, fa and dv predominated, rapamycin had little effect. very recently we obtained similar results with a second drug, aflibercept (vegf - trap). aflibercept is a human soluble decoy receptor protein with high affinity for all of the vegf - a isoforms, as well as for vegf - b and placental growth factor. when administered prior to or shortly after ad - vegf - a, mv and gmp formation were prevented and both angiogenesis and arterio - venogenesis were strikingly inhibited, as judged both by histology and by quantitative analysis using a double tracer method that measured total intravascular plasma volume as well as leaked plasma however, when given 2 months after ad - vegf - a injection, vegf trap had no significant effect on the vm, fa and dv that had by this time become well established. together these experiments indicate that early and late vessels differ substantially, not only in structure but also in their dependence on exogenous vegf - a, and therefore their responsiveness to anti - vegf therapy (fig. 1). this does not imply, however, that the endothelial cells of late vessels are entirely vegf - a - independent. pericytes and smooth muscle cells that are in intimate contact with endothelial cells are known to be a source of vegf - a and may well be supplying them with adequate vegf - a for survival. such a paracellular source of vegf - a is not likely to be easily inhibited by anti - vegf - a drugs, especially by large molecules such as aflibercept that can not effectively enter the closely apposed smooth muscle cell- or pericyte - endothelial cell interface. the data presented above indicate that anti - vegf / vegfr therapy has serious limitations. bevacizumab, accompanied by chemotherapy, is able to prolong life marginally in colon cancer and to delay progression modestly in several other cancers. it seems unlikely that these results can be much improved upon with other drugs targeting the vegf - a / vegfr axis. however, a distinction needs to be made between anti - vegf - a / vegfr and anti - vascular therapy. the results targeting the former should not be interpreted as a negation of the latter. rather, the point to be made is that there is a need for new targets beyond, or in addition to, the vegf - a / vegfr axis. in particular, there is a need to find molecules in or on late blood vessels that can serve as new targets. we expect our ad - vegf - a approach to be useful in finding such targets. with it we can develop large numbers of tumor surrogate blood vessels of each type at local tissue sites in relatively pure form and at different stages of their progression. this ad - vegf - a approach differs from another useful approach, that of purifying blood vessels and their endothelial cells directly from cancers using sage technology. this latter approach requires many hours during which changes in gene expression may take place. also, the endothelial cells harvested come from blood vessels of all different types, not just from those late vessels that may be most fruitful for targeting. work in progress is making use of the ad - vegf - a approach to identify genes that are highly expressed in tissues harvested at varying times after ad - vegf - a injection are being harvested and subjected to gene chip and rt - pcr analysis. the hope is to find new potential targets that will allow us to target late blood vessels. we appreciate the difficulties that may accompany this approach. at least by histology, late vessels such as fa, dv and vm differ less from their normal counterparts than do the early vessels such as mv and gmp that have no clearly established normal counterparts. nonetheless, we have already identified several potential molecular targets that we will be investigating further in the months ahead and hope that one or more of these may be useful therapeutically. finally, it should be noted that our ad - vegf - a model might also have a second use. it can provide a valuable tool for screening anti - vascular drugs, as it allows the assessment of drug effectiveness on each type of tumor surrogate blood vessel. we suggest that this model can serve as a rapid and relatively inexpensive means for screening the effectiveness of new anti - vascular drugs as they are developed.	therapies directed against vegf - a and its receptors are effective in treating many mouse tumors but have been less so in treating human cancer patients. to elucidate the reasons that might be responsible for this difference in response, we investigated the nature of the blood vessels that appear in human and mouse cancers and the tumor surrogate blood vessels that develop in immunodeficient mice in response to an adenovirus expressing vegf - a164. both tumor and tumor surrogate blood vessels are heterogeneous and form by two distinct processes, angiogenesis and arterio - venogenesis. the first new angiogenic blood vessels to form are mother vessels (mv) ; mv arise from preexisting venules and capillaries and evolve over time into glomeruloid microvascular proliferations (gmp) and subsequently into capillaries and vascular malformations (vm). arterio - venogenesis results from the remodeling and enlargement of preexisting arteries and veins, leading to the formation of feeder arteries (fa) and draining veins (dv) that supply and drain angiogenic vessels. of these different blood vessel types, only the two that form first, mv and gmp, were highly responsive to anti - vegf therapy, whereas late-formed capillaries, vm, fa and dv were relatively unresponsive. this finding may explain, at least in part, the relatively poor response of human cancers to anti - vegf / vegfr therapies, because human cancers, present for months or years prior to discovery, are expected to contain a large proportion of late - formed blood vessels. the future of anti - vascular cancer therapy may depend on finding new targets on late vessels, apart from those associated with the vegf / vegfr axis.
infectious bursal disease (ibd) is an acute, highly contagious viral disease of young chickens that primarily affects lymphoid tissues [13 ]. it is caused by a member of the genus avibirnavirus in the family birnaviridae [4, 5 ]. the disease was first described by cosgrove in around gumboro, delaware, usa. first described the disease in south western nigeria and was confirmed by onunkwo in. since then several studies have shown that the disease is of major concern to the poultry industry in the country [912 ]. infectious bursal disease virus (ibdv) has tropism to actively dividing precursor b lymphocytes, primarily in the bursa of fabricius, but other immune organs are also involved. the disease is mainly controlled by rigorous sanitary measures and vaccination through the use of either live or killed vaccines. infectious bursal disease intermedite or intermediate plus vaccines are commonly used to protect broilers and commercial pullets replacements from field ibdv exposure [1418 ]. despite vaccinations outbreaks of ibd are occasionally reported in vaccinated flocks of broiler and pullets with varying degree of mortality rates. reversion of these attenuated vaccinal strains to more virulent phenotypes under field and experimental conditions has been frequently reported [19, 20 ] possibly due to a lack of ibdv polymerase fidelity during vaccine viral genome replication in the host cells. infectious bursal disease virus is very stable and has been reported to resist many disinfectants at certain concentrations and or conditions [3, 21 ]. the virus remained viable by exposure for 1 hour at 30c to 0.5% phenol and 0.125% thimerosal.. found that the virus survived 60c but not 70c for 30 minutes, and exposure to 0.5% chloramine after 10 minutes and 0.5% formalin for 6 hours destroyed the virus. in addition, iodine complex had deleterious effects on the virus at 23c for 2 minutes. certainly, the hardy nature of this virus is one reason for its persistent survival in poultry houses even when thorough cleaning and disinfection procedures are followed [3, 23 ]. there are several claims by some practicing veterinarians and animal scientists that disinfectants like hypo, v - ox, and iodine are effective for the control of virulent ibd. currently, there is no report available on the efficacy of these agents as treatment regimen for ibd in field or clinical trials in nigeria. this paper describes the clinicopathological correlation of virulent ibd outbreaks in two successive flocks of commercial chickens raised under battery cage system in attempted treatments. outbreaks of suspected ibd case were reported to the avian clinic of veterinary teaching hospital, ahmadu bello university, zaria, from a commercial poultry farm in kaduna metropolis located at km 3.7, kaduna international airport road, kaduna. two subsequent outbreaks of acute ibd in pullets raised under battery cage system in the same farm were investigated. repeated farm visits were made and analysis was made on the clinical presentations of the disease, farm records including source of the chicks, breed, vaccination, age, major signs observed, intervention, and the mortality rate. moribund and dead birds were collected and thorough postmortem examination was conducted. the first outbreak was encountered in a flock of 8,413, 4.3-week - old brown pullets (isa brown). the flock had received newcastle disease vaccines at days 10 and 20 of age and ibd vaccines at days 8, 18, and 31 of age using bioveta ornibur (intermediate strain, bioveta, s.r.o. ltd., czech republic) and b2k indovax (invasive intermediate strain, b.p. vet., india) vaccines at 10 ml reconstitution per bird per os, respectively. clinical signs such as dullness and ruffled feathers were observed at about 5 hours after bursa b2k indovax ibd vaccine administration. twenty birds were lost on the first day and immediately floricol (florfenicol, vic animal health, russia) at 1 ml / l of water was given for a day and v - ox (mixture of inorganic peroxygen compounds ; polchem hygiene laboratories pvt. g / l of water, vitavet (multivitamins, vitamins a, bs, e, d, and k, pharma - swede, egypt) at 1 g/2 l of water, and glucose at 1 g / l of water were administered for 3 days. on days 2 and 3 of the outbreak, floricol was changed to doxy - gen (doxycycline and gentamycin, kepro, holland) at 1 g/2 l of water and on day 4 doxy - gen was switched to oxyfuravit (oxytetracycline hcl, furaltadone hcl, vitamins (a, b2, b12, c, d3, e, and k3), nicotinic acid, dl - methionine, and lysine, maridav, ghana) at 1 g / l of water and substituted v - ox for polidine (iodine, alkylphenoxypolyglycol ether and phosphoric acid ; animal care services konsult, nigeria) at 1 ml per 2 liters of water for 5 days. despite medications, the morbidity and mortality worsened and lasted for 9 days. the birds that survived (347) were disposed off and the house was thoroughly cleaned, washed, and disinfected. the second outbreak occurred in a flock of 9,000, 4-week - old brown pullets. the chicks were vaccinated against nd at days 10 and 20 of age and ibd at days 8 and 18 of age using abic ibd vaccines through oral and intranasal routes, respectively. during the outbreak, oxyfuravit was administered in feed and water at the rate of 10 g/5 kg and 1 g / l, respectively, together with polidine at 1 ml / l. v - ox, neoceryl plus (neomycin, erythromycin, oxytetracycline, colistin sulphate, streptomycin, and multivitamins, alfasan international bv, holland) at 1 g / l, and tylodox (tylosin tartrate and doxycycline ; kepro, holland) at 1 g / l together with multivitamins were administered for 3 days each. however, despite the interventions, the morbidity and mortality also lasted for 9 days. after the first outbreak, the house was rigorously cleaned, washed, and disinfected using cid20 (alkyldimethoylammonium chloride, glutaraldehyde, formaldehyde, glyoxal, and isopropanol ; cid lines, leper - belgium), at dilution of 1 : 200, glutasan (glutaraldehyde and alkyldimethoylammonium chloride ; pine oil, usa), at 1 : 400 of water, terminator iii (ortho - phenylphenol, ortho - benzyl - para - chlorophenol, and para - tertiary - amylphenol ; neospark, india) at 2 ml / l of water, and hypo (sodium hypochlorite ; multipro enterprises ltd., nigeria) at 2 bursae of fabricius (bfs) were aseptically harvested into universal bottles and stored at 20c for viral detection. tissues including skeletal muscles, spleen, bf, and kidneys were fixed in 10% neutral buffered formalin for histological examination. the fixed tissues were processed routinely for histopathology stained with haematoxylin and eosin and examined with the light microscope. frozen samples of the bfs were homogenised into 50% w / v suspension in phosphate buffered saline (pbs). the homogenate was centrifuged at 2000 rpm for 30 minutes and the supernatant was harvested and tested for ibd virus using agid test as described by oie. clinical signs observed in chicks in the two outbreaks were ruffled feathers, depression, hurdling together, anorexia, prostration, and whitish diarrhoea. mortality recorded spiked within 5 days of onset and then declined but lasted for 9 days in both cases. morbidity and mortality recorded were 100% and 95.8%, respectively, for case 1 and 80% and 43.3%, respectively, for case 2. there were petechial and ecchymotic haemorrhages on the pectoral, thigh and leg muscles and caecal tonsils and at the junction between proventriculus and ventriculus. the liver was severely congested and the spleen was enlarged and mottled but in some cases atrophied. in most cases the bursa of fabricius was edematous and haemorrhagic with yellowish gelatinous exudate on the mucosal surface. were swollen and pale (figure 2). microscopic examination of tissues showed moderate haemorrhages in the muscles and kidneys (figures 3(a) and 3(b)) and the spleen showed moderate lymphoid depletion in the lymphoid nodules (figure 4(a)). there was marked interfollicular oedema and depletion of lymphocytes from the lymphoid nodules in the bfs (figure 4(b)). other lymphoid nodules of the bf showed degeneration and necrosis of lymphocytes and cystic cavitations with heterophil infiltrates (figure 4(b)). escherichia coli were isolated from the liver and the bursal homogenate gave positive reactions to the ibdv known antiserum. the purpose of this investigation was to determine whether polidine or v - ox at 1 ml / l could significantly reduce typical ibd lesions in chicks infected with ibdv. however, the clinical manifestations and gross lesions observed in this study are similar to those reported previously [12, 2432 ] that chickens infected with ibdv exhibit anorexia, prostration, and white diarrhoea while grossly the bfs appear yellowish, hemorrhagic, and turgid with prominent striations, oedema, and caseous material found and varying degrees of hemorrhages in the thigh and breast muscles and at the junction between gizzard and proventriculus. the microscopic lesions seen in this study are similar to those reported [3, 12, 25, 27, 28, 3238 ] that found that bursae from ibdv exposed birds showed lymphoid depletion in the bursal follicles, interfollicular oedema, cellular debris in the medullary areas with necrosis, and/or eosinophilic cystic cavitations. these typical lesions confirmed that the attempted treatment claims can not stand for control of ibd. the observed morbidity and mortality are suggestive of vibd and agree with the report by asif. [12, 28 ], el - mahdy., and ezeibe. that chickens infected with virulent ibdv could experience high morbidity rate of 80100% and mortality rate of 4090% depending on the presence of secondary bacterial complication. the sudden onset, high morbidity, spiking mortality pattern, and sharp recovery from clinical signs are suggestive of the disease. however, the course of the disease lasted longer than what was reported by cosgrove, cho and edgar, okoye and uzoukwu, mbuko. mortality lasted for 9 days and peaked at day 5 in both cases. this could be attributed to the management system (battery cages) of the birds. the pullets in these cases were brooded under battery cages which provide minimum contacts of the chicks with one another and their droppings. although aerosol route of the disease transmission exists, faeco - oral route is the major route by which susceptible chick can be infected. the disease might spread very fast in deep litter management system due to free contact of the infected and noninfected birds. also in the deep litter, the birds have direct access to their droppings, and as such the feed and water can be contaminated by the droppings of infected birds as suggested by saif and eterradossi and saif. the high mortality recorded in the first outbreak may be due to the fact that it was the first outbreak of the disease in the farm and also the type of vaccines and vaccination programme employed may not have protected the birds against the field ibd virus. there is the likelihood that the intermediate vaccines administered at days 8 and 18 were interfered by maternally derived antibodies (mdas) and therefore the chicks were unprotected and the intermediate plus vaccine given at day 31 may have exacerbated the condition. several studies have indicated that high mdas at the time of ibdv vaccination might interfere with the vaccine response, neutralise the vaccine virus, and delay or even prevent the induction of humoral immunity [15, 33, 4345 ]. however, virulent strains of ibdv of same serotype have been reported to overcome high mdas in commercial flocks vaccinated with vaccines developed from different variants, causing up to 60% to 70% mortality. although vaccination of chickens has remained the principal method to control this disease [27, 47 ], the effectiveness of vaccinations relys on variants of the virus circulating in the area. studied the relationship between field and foreign vaccine strains in nigeria and reported that when ibdv strains spread from their region of origin to a different region they mutate alongside indigenous field strains. the antigenic differences between field and vaccine viral strain could be responsible for vaccine failures. therefore, vaccines being used in the country should be those made from strains of the virus circulating in nigeria. the source of the second outbreak for the subsequent batch of pullets may be due to the persistence of the virus in the environment between outbreaks, since ibd virus is very stable and resistant to many disinfectants [21, 42 ]. it is also pertinent to note that the brooding house was thoroughly cleaned and disinfected using the following disinfectants (polidine, v - ox, cid20, terminator, and glutasan) following the first outbreak before restocking. this further confirms previous report that once an outbreak of ibd occurs in a farm it will continue to occur with reduced mortality [3, 49 ] as seen in this investigation. the escherichia coli isolated from the bacteriological investigation was not surprising due to the immunosuppressive effects of the ibdv. consequences of immunosuppression due to ibd include poor response to vaccinations, gangrenous dermatitis, inclusion body hepatitis - anaemia syndrome, and escherichia coli infection [51, 52 ]. escherichia coli is known to be the abundant normal flora of gastrointestinal tracts of poultry. there are several claims by field veterinarians and animal health workers that certain disinfectants (e.g., iodine, sodium hypoclorite, and v - ox) when given orally are effective for the treatment of ibd. in these cases, the chemicals administered seemed not to have been effective as the disease ran its normal course with a very high mortality rate in case 1 and mortality rate of about 40% in case 2. in conclusion, the ibd vaccines currently being used to vaccinate birds against ibd in nigeria may be antigenically different from the ibd virus circulating in our environment. there is therefore the need for effective sanitary measures and adequate decontamination with sufficient fallow period before restocking of birds after an ibd outbreak in a poultry farm. the findings of this study have shown that polidine and v - ox at 1 ml per litre of water given orally in chickens will not prevent typical ibd lesions in case of field exposure. also, effort should be made to produce vaccine locally with the strains of the circulating ibd virus.	clinical and pathological investigations were conducted on outbreaks of infectious bursal disease (ibd) in pullets under brooding using the battery cage system in a commercial poultry farm in kaduna, nigeria. two consecutive outbreaks of ibd on the same farm were studied. the onset of the disease and morbidity and mortality rates were recorded. postmortem examinations were conducted and gross lesions recorded. tissues were collected and fixed in 10% buffered formalin and processed for histopathological examinations. in the first outbreak, 80 to 100% of the chicks were affected at the age of 4 to 5 weeks and mortality rate was 95.8% and lasted for 9 days. in the second outbreak, the mortality rate was 43.3% and it also lasted for 9 days. at the onset of the disease, the birds were also 4-week - old like in case 1. the disease was diagnosed based on clinical signs, pathology, and agar gel immunodiffusion test (agid). clinical signs, gross lesions, and histopathological findings were characteristic of virulent infectious bursal disease. after the first outbreak (case 1) the house was disinfected using polidine (iodophor compound), v - ox (inorganic peroxygen compounds), cid20 (quaternary ammonium chloride, aldehydes, and alcohol), terminator iii (phenols), and glutasan (aldehyde and quaternary ammonium chloride). but they failed to eliminate the ibd virus from the poultry pen.
dentin is a mineralized tissue constituting the body of a tooth, serving as a protective covering for the pulp and as a support for overlying enamel and cementum. dentin dysplasia is an autosomal - dominant trait, affecting either the primary or both the primary and secondary dentitions for approximately one patient in every 100,000. dentin dysplasia was first reported in 1922 by ballschmiedel who described six children in one family whose teeth had short, blunted roots with pulpal occlusion that he called rootless teeth. rushton later described a similar condition in an individual without evidence of genetic inheritance and he termed as dental dysplasia. this condition is rarely encountered in dental practice. generally, two main classes of dentin dysplasia are recognized based on clinical and radiographic appearance witkop later described type i as radicular dentin dysplasia and type ii as coronal dentin dysplasia to indicate the parts of the teeth that are primarily involved. eastman., in 1977 and ciola., in 1978 described two conditions for which they suggested the name dentin dysplasia type iii. ciola., case described radiographic findings characteristic of both dentin dysplasia type i and ii and proposed type iii classification in such cases. the case of eastman., appears different from type i and type ii, being focal rather than generalized. in type i (radicular) dentin dysplasia, both primary and permanent dentitions are affected, although the teeth are in normal morphologic appearance and color. the teeth characteristically exhibit extreme mobility and are commonly exfoliated prematurely. in type ii (coronal), both the dentitions are affected ; however, the deciduous teeth have a bluish or amber discoloration, while the permanent dentition appears normal. primary teeth show total pulp obliteration and permanent teeth show thistle - tube pulp configuration and pulp stones in pulp chambers. radiographically, in dentin dysplasia type i, the roots are sharp with conical, apical constrictions. pre - eruptive pulpal obliteration occurs leading to a crescent - shaped pulpal remnant and total pulpal obliteration in the deciduous teeth. in type ii, the pulp chambers and root canals are shaped like a thistle tube with an accumulation of pulp stones with no periapical radiolucencies. this article describes a rare case of dentin dysplasia type i in an 18-year - old female patient, highlighting the clinical, radiographic features of the defect and was confirmed by ground sectioning of the specimen and histopathologic examination. an 18-year - old female patient reported to our department with the complaint of poor esthetic appearance due to missing upper and lower front teeth since 4 years and seeking prosthetic treatment. [figure 1 ] the morphologic appearance and color of the permanent teeth were normal. in maxillary arch, the missing teeth were from right canine region to left canine region and in mandibular arch, all the incisors were missing. oral hygiene was poor and there were plaque and calculus deposits in all the quadrants. intra oral photograph of a patient showing missing anterior teeth in both jaws with rest of the dentition exhibiting normal coronal color and morphology panoramic radiograph [figure 2 ] revealed adequate enamel thickness on the cuspal tips of all the teeth. there was no root formation in most of the teeth while some of the teeth exhibited short, blunted and malformed roots of only a few millimeters with obliterated pulp chambers. well - defined periapical radiolucencies were present in association with the apex of the mandibular left first molar and right first and second molar. panoramic radiograph shows no root formation in most of the teeth with total obliteration of the pulp chambers and periapical radiolucency in lower right mandibular first molar, left mandibular first and second molar although the patient had attained her physical maturity and was ready for definitive dental treatment but her physical, financial and psychological situations were important to be considered in selecting the final treatment plan. dietary and oral hygiene instructions were given to the patient and extraction of the teeth that were mobile was advised along with the rehabilitation of the missing teeth with prosthesis. the right maxillary first molar tooth was extracted and sent to department of oral pathology and microbiology. the tooth crown was of normal dimensions measuring around 7.5 mm but the roots were short and measured about 6 mm. one half of the cut tooth specimen was decalcified in 10% nitric acid and processed for hematoxylin - eosin (h and e) staining. gross specimen of permanent right maxillary first molar tooth the ground section [figure 4a and b ] showed normal crown outline with normal enamel covering. a thin layer of superficial mantle dentin appeared but the pulp chamber was totally obliterated by an unusual calcified material consisting of atypical tubular dentin and osteodentin covered by thin discontinuous layer of cementum. normal dentinal tubule formation appeared to have been blocked and the new dentin so formed around obstacle, gave rise to the characteristic appearance of lava flow around the boulder [figure 5 ]. stereomicroscopic view of ground section of permanent right maxillary first molar showing obliteration of pulp chamber with abnormal dentin formation (ground section, x20) stereomicroscopic view of hematoxylin - eosin stained permanent right maxillary first molar showing obliteration of pulp chamber with abnormal dentin formation (h&e stain, x20) histopathologic examination of the decalcified hematoxylin - eosin section [figure 6 ] showed total obliteration of pulp that was filled with tubular dentin, osteodentin and irregular, globular masses of dentin [figure 7 and 8 ]. based on the clinical, radiographic, gross features and histological examination of the ground section and decalcified section, diagnosis of dentin dysplasia type i was made. (polarizing microscopy, 100) photomicrograph showing decalcified section showing abnormal dentin formed around the obstacles (h&e stain, x100) photomicrograph of decalcified section showing osteodentin with empty lacunae devoid of osteocytes (h&e stain, x100) there are some systemic disorders [table 1 ] that are associated with dentin - dysplasia - like alterations but no such findings were found in this patient on general and radiological examination. it is usually an autosomal dominant condition, but in our patient there was no familial history of the disease, so she can be considered as a first generation sufferer. logan., proposed that the dental papilla is responsible for the abnormalities in root development. they suggested that multiple degenerative foci within the papilla become calcified, leading to reduced growth and final obliteration of the pulp space. sauk., in a scanning electron microscope study, postulated that dentin dysplasia is a defect in the epithelial component of the developing tooth germ in which the invagination of the root sheath occurs too soon and, in a sequence of futile attempts to correct itself, results in a stunted root form with an unusual whorl - like pattern of dentin obliterating the pulp chambers. wesley., disagreed with this suggestion and proposed that the condition is caused by an abnormal interaction of odontoblasts with ameloblasts leading to abnormal differentiation and/or function of these odontoblasts. witkop suggested that the dysplasia results from epithelial cells from the sheath of hertwig breaking off and migrating into dental papilla, where they induce odontoblast differentiation and dentin formation. melnick., suggested that the abnormal root morphology is caused by abnormal differentiation and/or function of the odontoblasts. systemic diseases correlated with dentin - dysplasia - like alterations dentin dysplasia type i should be differentiated from other conditions like dentin dysplasia type ii, dentinogenesis imperfecta and odontodysplasia [table 2 ]. in our patient, normal morphology of crown, short, blunted and malformed roots with pulpal obliteration and periapical radiolucency and ground section revealing stream flowing around boulders are characteristic for diagnosis of dentin dysplasia type i. there were no morphological variation in the dentition of our patient but there are reports that have suggested possible variations in the morphology of teeth affected by this type of dysplasia. difference between dentin dysplasia type i, ii, dentinogenesis imperfect and regional odontodysplasia management of patients with dentin dysplasia has posed several problems to the dentists. extraction has been suggested as a treatment alternative for teeth with pulp necrosis and periapical abscess. endodontic treatment is contraindicated in teeth with total obliteration of root canals and pulp chambers. another approach for the treatment of teeth with dentin dysplasia has included peri - apical surgery and retrograde filling, which is recommended in teeth with long roots. follow - up and routine conservative treatment is another choice of treatment plan in dentin dysplasia. orthodontic treatment is suggested ; however, further resorption of the roots, loosening of teeth and premature exfoliation may occur due to the resistance of the short roots to the orthodontic forces. successful oral rehabilitation with complete denture after extraction of all teeth and curettage of cysts has also been proposed. since these patients usually have early exfoliation of the teeth and consequently, maxillomandibular bony atrophy, treatment with a combination of onlay bone grafting and a sinus lift technique to accomplish implant placement can be used successfully. dentin dysplasia type i is an unusual abnormality of dentin which leads to premature exfoliation of the teeth. the treatment of children with dentin dysplasia aims in effective preventive care as because of the early loss of teeth due to shortened roots and periodontitis. so, meticulous oral hygiene measures and dietary instructions must be established and maintained for the retention of teeth to help children have natural teeth as long as possible. in this regard, dentist has an important role in early diagnosis of this disorder and in guiding patients in the selection of measures to prolong the retention of affected teeth.	dentin dysplasia is a rare disturbance of dentin formation characterized by normal enamel but atypical dentin formation with abnormal pupal morphology. the teeth appear clinically normal in morphologic appearance and color. the teeth characteristically exhibit extreme mobility and are commonly exfoliated prematurely. radiograph shows obliteration of all pulp chambers, short, blunted and malformed or absent roots with periapical radiolucencies involving apparently intact tooth. this case is reported here because of its rarity along with the description of various clinical, radiological and histological features.
low back pain (lbp) is one of the most common complaints in the western society.1,2 ninety percent of the population in the united states suffer from low back pain at one or multiple points in time in their lifetime.2 although lbp is a common chronic pain syndrome, in most cases a specific diagnosis can not be established. it can arise due to spinal injury spinal disc problems, osteoarthritis, spinal stenosis, compression fractures, spinal tumors, etc.3 nonsteroidal anti - inflammatory drugs (nsaids) are generally used for acute lbp. opioids are the alkaloid analgesics used for the treatment of moderate to severe pain syndromes. these opioids work on the,, and receptors in the central nervous system;4 however, due to the wide presence of these receptors in the body, opioids not only suppress the noxious stimuli effects, but also have undesirable side effects.5,6 the concerns arising from the use of analgesic medications have increased the interest in nonpharmacological therapies for lbp. nonpharmacological treatment modalities for pain relief include heat, cold acupuncture, electrotherapy, and massage.7 the use of cold and hot methods has been shown to cause tissue and nerve injury.810 among the electrotherapy modalities are transcutaneous electrical nerve stimulation (tens) with various pulse modulation, electroacupuncture, and percutaneous electrical nerve stimulation (pens).1113 unfortunately, these modalities go amiss with respect to their provided duration and magnitude of analgesia. study of tens has produced results with limited statistical significance,14 and the american academy of neurology has advised against the use of tens in chronic lbp, stating that the strongest evidence indicates that it is ineffective for this syndrome.15 one accepted explanation for lbp symptoms is that patients have myofascial pain syndrome, a condition characterized by painful active myofascial trigger points (atps) in muscles.16,17 in the last 1015 years, much clinical and basic science research into atps has been published, including epidemiological, diagnostic, therapeutic, and pathophysiological studies.1721 the pathogenesis of atps is probably related to sensitized sensory peripheral free nerve endings (nociceptors) associated with dysfunctional endplates.16 in a histological study, small nerve fibers were commonly found near the sensitive atps.22 therefore, the sensitive loci in the region of muscle atps are probably related to sensitized nerve fibers (nociceptors). local pain could be explained by the tissue ischemia resulting from prolonged muscle contraction, with accumulation of acids and chemicals such as serotonin, histamine, kinins, and prostaglandins.23 studies have suggested that the development of atps is dependent on an integrative mechanism in the spinal cord. when the input from the nociceptors in an original receptive field (pain from atps) persists, central sensitization in the spinal cord may develop, and the receptive field corresponding to the original dorsal horn neuron may be expanded (referred pain). through this mechanism, new common treatments of atps typically include minimally invasive intervention, such as injections with local anesthetics, corticosteroids, botulinum toxin, or dry needling.24 serious complications, although of rare occurrence, have been reported (eg, pneumothorax, hematoma, intravascular injection of local anesthetics, and intrathecal injections).25 hyperstimulation analgesia is an alternative modality, in which localized, intense, low - rate electrical pulses are applied to small surface areas at atp locations to stimulate peripheral nerve endings (a fibers), thus causing the release of endogenous endorphins.26,27 hyperstimulation anesthesia has been investigated in several controlled studies, showing a positive response in 87% of patients.26,2830 considerable evidence suggests that this type of neurostimulation analgesia is achieved through the activation of extra - segmental antinociceptive mechanisms, which accelerate the release of endogenous endorphins, serotonin, and cortisol.27,3134 while the most common physical finding of atps has been considered the palpation of a hypersensitive nodule of muscle fiber of harder - than - normal consistency the identification of such nodule appears to be very dependent on the subjective experience of the physician. there is no accepted reference standard for the clinical diagnosis of atps, and data on the reliability of physical examination are conflicting, and a 2009 review of nine studies examining the reliability of atp diagnosis found that physical examination could not be recommended as reliable for the diagnosis of atps.24 attempts to confirm the presence of myofascial trigger points using magnetic resonance elastography have been described.35 recently, sikdar have tried to use ultrasound to visualize and characterize atps.36 they found that atps appeared as focal, hypoechoic regions of elliptical shape, with a size of 0.16 cm.36 the presence of atps causes a localized decrease in skin resistance compared with the surrounding area.37,38 the hypoxic state in the pain area increases nociceptors and other sensitizing substances in the area, and this biochemical change induces greater blood flow and secretion from sweat glands, via stimulation of the autonomic nervous system.39 these physiologic differences may account for acute variations in electrodermal measurements at the pathologic site. atps are defined as small - diameter (34 mm), circumscribed low - skin - resistance areas.38 localized decrease in skin resistance is frequently associated with clinical atps that are richly innervated by myelinated a fibers40,41 the smallest in diameter (0.21.5 m) and most commonly present myelinated axons in peripheral nerves. their extremely small size prevents their identification by any imaging modality38 electrical skin impedance measurements are considered to be vulnerable to certain sources of imprecision, including instrument error resulting from the size, pressure, and the duration of probe application as well as from local skin conditions, such as variable thickness, hydration, and integrity of the stratum corneum.38,42 the hyperstimulation analgesia procedure is not extensively utilized in the clinical setting due to the necessity of locating appropriate atps. this necessity requires previous knowledge of the potential locations and the identification of atps associated with lbp and makes such treatments time consuming and cumbersome.2628 some devices offer the capability of measuring skin impedance for the location of atps, with the aim of applying hyperstimulation to them;43 however these are manually held devices that prolong the procedure, by firstly allowing application of hyperstimulation to a single point and secondly, by offering limited accuracy (due to their measurement of a single point at a time) that does not take advantage of the electrodermal information of the entire region of interest. a novel automated robotic system, the soleve (nervomatrix ltd, netanya, israel) (figure 1), utilizes an array of miniature probes (figure 2), allowing the measurement of skin impedance over the back at 1000 points in less than a couple of minutes.44 the system visualizes and analyzes the data to locate areas of low impedance compared with surrounding areas, thus indicating atps appropriate for hyperstimulation (figure 3). therapeutic neurostimulation, using modulated, intense electrical pulses, is then applied locally to specific painful atps, providing highly effective pain relief by stimulating the release of endorphins.3134 the effectiveness of the soleve system was investigated in 19 patients diagnosed with nonspecific chronic lbp.44 fifteen of the patients were female (79%), and four were men (21%), with a mean age of 52.1 10.8 years. each session included a < 1-minute, automatic impedance screening, followed by a 20-minute treatment. the primary outcome measurement consisted of changes in pain intensity, as measured on a 100 mm - long pain visual analog scale (vas) obtained at enrollment, pre-, and 2 hours posttreatment. the mean standard deviation (sd) baseline vas score for all patients was 61 14 mm (figure 4). following treatment, vas scores decreased by 39 17 mm (p < 0.001) compared with the baseline scores. notably, the vas scores of all but one patient decreased by more than 20 mm after the fourth treatment, representing a marked improvement in 95% of enrolled patients. a novel, noninvasive, image - guided, targeted neurostimulation modality that combines impedance imaging to locate atps and treatment based on the image analysis was found very effective clinically in 95% of patients after a series of four treatments. this promising result warrants future investigation and randomized, controlled, longitudinal studies in the treatment of lbp.	low back pain in patients with myofascial pain syndrome is characterized by painful active myofascial trigger points (atps) in muscles. this article reviews a novel, noninvasive modality that combines simultaneous imaging and treatment, thus taking advantage of the electrodermal information available from imaged atps to deliver localized neurostimulation, to stimulate peripheral nerve endings (a fibers) and in turn, to release endogenous endorphins. hyperstimulation analgesia with localized, intense, low - rate electrical pulses applied to painful atps was found to be effective in 95% patients with chronic nonspecific low back pain, in a clinical validation study.
ludwig 's angina is a rare consequence of periodontal infection that can result in severe upper airway obstruction and potentially death. prior to our report, there have only been two cases reported in pregnant women. we report the first case that required immediate delivery secondary to worsening fetal and maternal health status. this patient is a 24-year - old with a history of hepatitis c infection, intravenous drug use, and ten - pack year smoking history who presented as transfer of care during her thirty - third week of gestation. initially, she presented with a tooth abscess that was treated with outpatient medication therapy that included oral antibiotic (amoxicillin - clavulanate) and analgesic medications. despite reported compliance, she represented two days later at a community hospital with a low - grade fever and left - sided neck swelling consistent with angioedema. she was counseled and consented for elective endotracheal intubation given the concern for significant airway compromise. her diagnosis was ludwig 's angina in pregnancy and she was transferred to our tertiary hospital 's obstetrical intensive care unit. upon admission, the patient was arousable, responsive to commands, and able to communicate despite the in situ endotracheal tube. she was normotensive, mildly tachycardic, and saturating well on room air without the need for assisted ventilation. her palate and dentition were not fully visible, but her trachea appeared midline. antenatal testing revealed a reactive fetal heart rate tracing, sonographic fetal biometry was consistent with her dates, and the fetus had a normal anatomic survey. the otolaryngology service at our center confirmed the diagnosis of ludwig 's angina, initiated parenteral piperacillin - tazobactam therapy, and recommended against steroids to prevent further maternal immunosupression. an incision and drainage of her neck abscess was performed in the operating room followed by removal of her endotracheal tube and placement of a tracheotomy tube. a gram stain of the abscess revealed gram - negative rods and her antibiotic therapy was continued. throughout this therapy, antenatal testing on the first postoperative day, secondary to presumed septic shock, she became hypotensive with increasing oxygen demands that required vasopressive and ventilatory support. she was additionally diagnosed with acute respiratory distress syndrome (ards) based on her presentation and chest radiographic findings of bilateral opacities. despite several efforts, the patient 's status did not improve and the fetal heart rate tracing became persistently nonreassuring with repetitive late decelerations. at this time, the maternal fetal medicine specialist proceeded with cesarean delivery for nonreassuring fetal indications. she had an urgent repeat lower transverse cesarean section, delivering a female infant weighing 2120 grams with apgars of 2, 6, and 7. the newborn was intubated, admitted to the neonatal intensive care unit, and started on empiric ampicillin and gentamicin therapy. immediately following delivery of the neonate, the patient returned to the medical intensive care unit. on postpartum day number one, she was weaned off of vasopressor therapy and transitioned to a trach collar. the tracheotomy was first used continuously for five days, then intermittently, and successfully removed on postpartum day number eight. the newborn did well in the neonatal intensive care unit : her blood cultures were negative, antibiotics were discontinued, she was extubated on day of life 2, and she was progressively weaned off of oxygen therapy. the patient was then discharged home on postoperative day nine with oral antibiotic therapy and plans for follow - up. karl ludwig who observed deadly forms of tooth infection, ludwig 's angina is a rare case found in medicine. typically, as an odontogenic infection of the 2nd and 3rd molars, it has direct spread, rather than hematogenous or lymphatic, to the submandibular, sublingual, and submental spaces. this can lead to rapid and profound neck swelling and in some cases death by airway compression. the infection and edema are limited by the deep cervical fascia, mandible, and hyoid. as a result, the tongue and floor of the mouth are elevated and posteriorly displaced with compromise of the airway, which can result in abrupt asphyxiation. infection often results from streptococci, staphylococci, or bacteroides, although 50% of infections are polymicrobial. management of a patient with ludwig 's angina requires early aggressive therapy with antibiotics, incision and drainage of any abscess, and protection of the airway [1, 2 ]. fifty percent of pregnant women annually in the united states are affected by periodontal disease involving chronic anaerobic oral infection. the pregnant patient with a maxillofacial infection requires special attention given the physiologic changes of pregnancy, which puts the patients at increased risk of complications. such changes include increased sodium retention, dilutional anemia, decreased colloid pressure, and an upper airway that is affected by capillary engorgement and subsequent edema of the nasal passages, larynx, oropharynx, and trachea. chronic gingivitis, friable oral mucosa, and mild immunosuppresion of pregnancy play an important role in outcome. further, changes in gastrointestinal motility and other physiologic systems put the pregnant patient at increased risk of acute respiratory distress syndrome, pneumonia, sepsis, amniotic fluid embolism, disseminated intravascular coagulation, and pulmonary edema secondary to aspiration. adverse pregnancy outcomes such as preterm birth, low birth weight, fetal growth restriction, preeclampsia, and perinatal mortality are associated with periodontal disease [36 ]. there are only 2 prior case reports of pregnant women with ludwig 's angina in the present literature, neither of which required delivery as part of the treatment plan [7, 8 ]. in our case, delivery was indicated given the nonreassuring fetal tracing that was likely associated with fetal hypoxia or acidosis. the physiological changes associated with this preterm delivery may have resulted in improved ventilation status. functional residual capacity (frc) decreases on average by 18% in the third trimester and is further compromised in the case of ards. given the increase in frc that occurs postpartum, this improvement in gas exchange may have improved her hypoxemic state. respiratory compliance may also improve post delivery following a downward displacement of the diaphragm. a case series of 10 intubated pregnant patients demonstrated a 28% reduction in fraction of inspired oxygen requirement by 24 hours after delivery. delivery is also associated with improved hemodynamic changes, specifically related to cardiac output, when the delivery of the neonate relieves compression placed on the vena cava. broad - spectrum antibiotic coverage, including the addition of vancomycin therapy, likely contributed to resolution of this patient 's septic condition. it is unclear as to whether these physiological changes imply a direct cause and effect relationship for the improvement of our patient 's health status. it is controversial whether pregnant patients with acute respiratory failure such as ards will improve with delivery in situations when immediate delivery is not indicated [10, 11 ]. delivery should continue to be reserved for obstetrical indications [10, 11 ]. for this rare antepartum medical presentation of ludwig 's angina, the decision to perform a cesarean section avoided a potentially fatal outcome for the neonate.	at 33 weeks of gestation, a 24-year - old developed ludwig 's angina that worsened despite aggressive therapy. this is the first reported case of ludwig 's angina in pregnancy that required an emergent cesarean section for fetal indications. delivery may have contributed to improvement in the mother 's health status.
acute rhinosinusitis is a common disease, which still has challenges in its diagnostics and treatment. it usually responds well to conservative treatment and about 80% of patients will recover within 2 weeks without antibiotics.1 however, in some cases, the symptoms can be severe and/or first - line treatment may have been ineffective. in these patients, it helps to confirm the diagnosis and to differentiate from other types of facial pain, provides reliable sample for bacterial analysis, and may also relieve facial pain and pressure. antral irrigation has for long been the gold standard procedure for the diagnosis of rhinosinusitis in clinical research studies and it was also recommended for treatment of maxillary sinusitis in selected cases.2,3 however, the usefulness of antral irrigation in the treatment of rhinosinusitis has been surprisingly scantily studied and the studies that have been done are old.4 for the time being, there is no published evidence that antral irrigation would reduce symptoms or recovery time in acute rhinosinusitis and its use has gradually decreased. recently, optional methods for the collection of microbial samples have been introduced.5,6 in 2011, desrosiers and his coworkers7 stated that when culture is required for unusual evolution or complication of acute rhinosinusitis, sampling should be performed either by maxillary tap or endoscopically guided culture. in european position paper on rhinosinusitis and nasal polyposis (epos) 2012, antral irrigation was found to be useful for confirming bacteriological diagnosis in intensive care unit patients,8 but it was not included in diagnostics or treatment of acute maxillary rhino - sinusitis in nonhospitalized, nonsedated patients. apprehensions of painfulness during antral irrigation may also have an effect on its use in diagnostics and treatment.5,9 a recommendation for treatment by antral irrigation in severe or prolonged cases after appropriate medical therapy has failed is still included in finnish guidelines for rhinosinusitis based on good clinical experience.10,11 clinical trials are needed to validate or discard this practice. as the first step in evaluation of antral irrigation, we wanted to investigate patients experience of this procedure and whether it could be recommended to be suitable for outpatient practice. we also asked the patients to evaluate facial pain before and right after the procedure. the study was conducted during the year 2011 at an outpatient clinic of the ear, nose, and throat (ent) hospital of the helsinki university hospital (huh), in an ent specialist s private practice in helsinki, and at a communal health center in htri, a small town in central finland. it recommends antral irrigation for severe or prolonged symptoms of acute rhinosinusitis after appropriate medical therapy has failed. the procedure removes secretions from the maxillary sinus, opens the maxillary ostium for a moment, and gives an opportunity to take a bacterial sample. it usually relieves facial pain in acute rhinosinusitis.10,11 the patients in our study were treated according to this guideline. in the year 2011, a sinus x - ray was still commonly taken before sending the patient to huh for consultation. local anesthesia is introduced to the inferior meatus with a cotton - tipped applicator soaked in local anesthetic or with a mixture of lidocaine hydrochloride and prilocaine hydrochloride (emla cream, astra - zeneca) instilled with a suction needle and a syringe. maxillary sinus puncture is performed with a straight needle (circumference : 1.32 mm, length : 12 cm) under the inferior turbinate. the stylet is removed and a 100 ml syringe is connected to the needle with a prefilled silicon tube and a connector. irrigation is performed with warm (38c) physiological saline solution with the patient sitting in a forward leaning position. the patients were informed about the study at the end of their visit. if the patient was willing to participate, he / she completed an informed consent form and filled in a questionnaire before leaving the clinic or at home on the same day. the questionnaires were handed to the nurses or returned by prepaid mail in a closed envelope. the physicians completed their part of the questionnaire at the end of the patients visit. the researchers opened and analyzed the data after all the patients had returned the questionnaires. the patients questionnaire included questions about previous experiences of antral irrigation, as well as current symptoms both before and immediately after the procedure. the patients evaluated the severity of facial pain and nasal obstruction on a 10 cm visual analog scale (vas). vas values > 0 to 3 were graded as mild, > 3 to 7 as moderate, and > 7 as severe.12 the same scale was also used in determining the severity of pain caused by antral irrigation. additionally, they evaluated using vas their sensation of other common medical procedures, such as having a blood sample taken or dental restoration. the physicians questionnaire inquired about their degree and level of experience, way of application, and choice of local anesthetics, as well as the doctors impression about the discomfort caused by the irrigation to the patients. the study was approved by the operative ethics committee of the helsinki and uusimaa hospital district. the wilcoxon signed - rank test was conducted for paired values, eg, when pain was compared before and after antral irrigation. spearman s rank correlation test was used to assess the connection between the severities of different symptoms. all analyses were conducted with the statistical software ncss 8 (number cruncher statistical systems, llc, kaysville, ut, usa). local anesthesia is introduced to the inferior meatus with a cotton - tipped applicator soaked in local anesthetic or with a mixture of lidocaine hydrochloride and prilocaine hydrochloride (emla cream, astra - zeneca) instilled with a suction needle and a syringe. maxillary sinus puncture is performed with a straight needle (circumference : 1.32 mm, length : 12 cm) under the inferior turbinate. the stylet is removed and a 100 ml syringe is connected to the needle with a prefilled silicon tube and a connector. irrigation is performed with warm (38c) physiological saline solution with the patient sitting in a forward leaning position. the patients were informed about the study at the end of their visit. if the patient was willing to participate, he / she completed an informed consent form and filled in a questionnaire before leaving the clinic or at home on the same day. the questionnaires were handed to the nurses or returned by prepaid mail in a closed envelope. the physicians completed their part of the questionnaire at the end of the patients visit. the researchers opened and analyzed the data after all the patients had returned the questionnaires. the patients questionnaire included questions about previous experiences of antral irrigation, as well as current symptoms both before and immediately after the procedure. the patients evaluated the severity of facial pain and nasal obstruction on a 10 cm visual analog scale (vas). vas values > 0 to 3 were graded as mild, > 3 to 7 as moderate, and > 7 as severe.12 the same scale was also used in determining the severity of pain caused by antral irrigation. additionally, they evaluated using vas their sensation of other common medical procedures, such as having a blood sample taken or dental restoration. the physicians questionnaire inquired about their degree and level of experience, way of application, and choice of local anesthetics, as well as the doctors impression about the discomfort caused by the irrigation to the patients. the study was approved by the operative ethics committee of the helsinki and uusimaa hospital district. the wilcoxon signed - rank test was conducted for paired values, eg, when pain was compared before and after antral irrigation. spearman s rank correlation test was used to assess the connection between the severities of different symptoms. all analyses were conducted with the statistical software ncss 8 (number cruncher statistical systems, llc, kaysville, ut, usa). the study population consisted of 121 patients (age range : 1882 years ; mean : 39.2 years ; 62% female) undergoing antral irrigation because of symptoms of acute rhinosinusitis. most patients (100 patients, 82.6%) were from huh, 11 (9.1%) patients from a private outpatient practice in helsinki, and 10 (8.2%) from a communal health center in central finland. in most cases (n = 69, 58%), antral irrigation was performed by ent residents from huh, followed by ent specialists (n = 26, 22%), medical students under supervision of an ent specialist (n = 17, 14%), or general practitioners (n = 6, 7%). more than half of the patients (71 patients, 59%) had undergone antral irrigation earlier. the mean duration of symptoms of rhinosinusitis was 31 days (median : 19 days ; range : 0330 days). antral irrigation was divided into phases : application of the anesthetic, insertion of the needle (maxillary sinus puncture), and the act of rinsing. patients experienced the pain caused by antral irrigation as mild (table 1, fig. application of the local anesthetic (mean : 2.8 ; median : 2.5 ; range : 010) was the most unpleasant part, followed by insertion of the needle, ie, puncture (mean : 2.4 ; median : 1.5 ; range : 010). the duration of the patients symptoms prior to antral irrigation, the amount or type of effusion obtained from the sinus, or patients former experiences of irrigation were not related to the experience of pain during antral irrigation. local anesthesia was achieved with emlacream in 44 patients (36%), lidocaine adrenaline solution mixture (lidocaine hydrochloride 40 mg / ml and adrenaline 1:1000) in 54 patients (45%), or cocaine adrenaline solution in 23 patients (19%). there was no difference in pain assessments between the anesthetic solutions either during application of the anesthetic (p = 0.77) or while inserting the needle in the maxillary sinus (p = 0.9). patients were also asked to evaluate the pain caused by other minor medical procedures (table 1). having a blood sample taken and dental restoration with anesthetic were found to be less painful than antral irrigation, whereas extraction of wisdom tooth and dental restoration without anesthetic were found more painful than irrigation. the physicians evaluation of pain in antral irrigation did not differ from the patients assessment (application of anesthetics : p = 0.34 ; maxillary sinus puncture : p = 0.80). they assessed pain to be less severe during insertion of the needle than during application of anesthetic (patients : p = 0.10 ; physicians : p = 0.001). patients experience of pain and discomfort during antral irrigation was not related to physicians professional experience (application of anesthetics : p = 0.70 ; maxillary sinus puncture : p = 0.56). physicians anxiety also did not correlate with the patients experience of pain (application of anesthetics : p = 0.49 ; maxillary puncture : p = 0.77). in the free text section of the questionnaire, patients most common comments on antral irrigation were as follows : not painful / went well / not bad (n = 24), not as bad as expected (n = 17), relieves pressure / pain (n = 11), recommendable (n = 7) ; or unpleasant (n = 19), unpleasant but not painful (n = 10), exciting / frightening (n = 8), and painful/ quite painful (n = 8). more than half of the patients (72 patients, 59.5%) replied that they would undergo antral irrigation again by all means and almost all the rest (43 patients, 35.5%) would agree to a repuncture in case their physicians found it medically indicated. none of the patients chose the alternative i would definitely refuse a repuncture, while five patients (4.1%) did not answer the question. twenty - six patients (21%) had severe pain and 44 patients (36%) had moderate pain before antral irrigation. number of patients with severe or moderate pain decreased to half after antral irrigation (table 2). patients mean facial pain caused by rhinosinusitis was relieved almost immediately after antral irrigation (table 3, fig. the mean assessment of facial pain on a vas scale was 4.4 (range : 010). after some minutes in the waiting room after antral irrigation or the same day at home, the mean vas was 1.7 (range : 010) (p < 0.001). pus obtained from the maxillary sinus or former experiences of antral irrigation were not related to the decrease of pain. the study population consisted of 121 patients (age range : 1882 years ; mean : 39.2 years ; 62% female) undergoing antral irrigation because of symptoms of acute rhinosinusitis. most patients (100 patients, 82.6%) were from huh, 11 (9.1%) patients from a private outpatient practice in helsinki, and 10 (8.2%) from a communal health center in central finland. in most cases (n = 69, 58%), antral irrigation was performed by ent residents from huh, followed by ent specialists (n = 26, 22%), medical students under supervision of an ent specialist (n = 17, 14%), or general practitioners (n = 6, 7%). more than half of the patients (71 patients, 59%) had undergone antral irrigation earlier. the mean duration of symptoms of rhinosinusitis was 31 days (median : 19 days ; range : 0330 days). antral irrigation was divided into phases : application of the anesthetic, insertion of the needle (maxillary sinus puncture), and the act of rinsing. patients experienced the pain caused by antral irrigation as mild (table 1, fig. application of the local anesthetic (mean : 2.8 ; median : 2.5 ; range : 010) was the most unpleasant part, followed by insertion of the needle, ie, puncture (mean : 2.4 ; median : 1.5 ; range : 010). the duration of the patients symptoms prior to antral irrigation, the amount or type of effusion obtained from the sinus, or patients former experiences of irrigation were not related to the experience of pain during antral irrigation. local anesthesia was achieved with emlacream in 44 patients (36%), lidocaine adrenaline solution mixture (lidocaine hydrochloride 40 mg / ml and adrenaline 1:1000) in 54 patients (45%), or cocaine adrenaline solution in 23 patients (19%). there was no difference in pain assessments between the anesthetic solutions either during application of the anesthetic (p = 0.77) or while inserting the needle in the maxillary sinus (p = 0.9). patients were also asked to evaluate the pain caused by other minor medical procedures (table 1). having a blood sample taken and dental restoration with anesthetic were found to be less painful than antral irrigation, whereas extraction of wisdom tooth and dental restoration without anesthetic were found more painful than irrigation. the physicians evaluation of pain in antral irrigation did not differ from the patients assessment (application of anesthetics : p = 0.34 ; maxillary sinus puncture : p = 0.80). they assessed pain to be less severe during insertion of the needle than during application of anesthetic (patients : p = 0.10 ; physicians : p = 0.001). patients experience of pain and discomfort during antral irrigation was not related to physicians professional experience (application of anesthetics : p = 0.70 ; maxillary sinus puncture : p = 0.56). physicians anxiety also did not correlate with the patients experience of pain (application of anesthetics : p = 0.49 ; maxillary puncture : p = 0.77). in the free text section of the questionnaire, patients most common comments on antral irrigation were as follows : not painful / went well / not bad (n = 24), not as bad as expected (n = 17), relieves pressure / pain (n = 11), recommendable (n = 7) ; or unpleasant (n = 19), unpleasant but not painful (n = 10), exciting / frightening (n = 8), and painful/ quite painful (n = 8). more than half of the patients (72 patients, 59.5%) replied that they would undergo antral irrigation again by all means and almost all the rest (43 patients, 35.5%) would agree to a repuncture in case their physicians found it medically indicated. none of the patients chose the alternative i would definitely refuse a repuncture, while five patients (4.1%) did not answer the question. twenty - six patients (21%) had severe pain and 44 patients (36%) had moderate pain before antral irrigation. number of patients with severe or moderate pain decreased to half after antral irrigation (table 2). patients mean facial pain caused by rhinosinusitis was relieved almost immediately after antral irrigation (table 3, fig. the mean assessment of facial pain on a vas scale was 4.4 (range : 010). after some minutes in the waiting room after antral irrigation or the same day at home, the mean vas was 1.7 (range : 010) (p < 0.001). pus obtained from the maxillary sinus or former experiences of antral irrigation were not related to the decrease of pain. patients assessed pain and discomfort caused by antral irrigation to be mild, and the vast majority would not hesitate to undergo the procedure again. when patients evaluated pain in other minor medical procedures, dental calculus scaling was found to be the most closely comparable to antral irrigation. additionally, we noted a relief in facial pain after the procedure. pain and discomfort are always subjective and therefore patients point of view should be carefully evaluated. the only study13 we found on antral irrigation and patient experience was performed in united kingdom and published in 1987. it stated that 91% of 200 patients were willing to undergo the puncture again.13 in the present study, this number was 95%. the mean vas of pain, unpleasantness, and side effects during antral irrigation in the english study was only 2130 (maximum : 100). this finding is well in accordance with our findings of mild pain, score <3 in mean vas, during antral irrigation. when comparing antral irrigation to other minor medical procedures, pain during dental calculus scaling was reported to be closest (table 1). informing the patients of minor procedures that are comparable to antral irrigation may help to relieve their anxiety before the procedure. in the present study, neither physicians professional experience nor their anxiety furthermore, physician s evaluation of patient s pain was close to the patient s own assessment, so the patients reported experiences during antral irrigation were likely to be well considered. in the present study, pain was not increased due to the procedure ; instead, a quick relief of facial pain after antral irrigation was noticed. some patients had sought help due to prolonged symptoms of acute rhinosinusitis with only mild pain or no pain, but most of the patients had moderate or severe pain. decrease of pain right after the procedure is promising, but this finding should be interpreted with caution and studied with a controlled study design with longer follow - up. questions like whether antral irrigation relieves symptoms in the long run, prevents chronic disease, or decreases treatment costs were not in the scope of this study and need to be determined in a different setting. patients experiences of pain from previous procedures may be a potential cause of bias due to the time passed. however, when patients evaluated their former antral irrigation and the present one, the latest procedure was experienced to be less unpleasant. this suggests that patients assessment of pain in former minor procedures had probably not decreased with time. pain during antral irrigation is usually mild and should not hinder this procedure when indicated. further studies are needed to better elucidate the role of antral irrigation in diagnosis and treatment of acute rhinosinusitis.	backgroundantral irrigation earlier had an important role in the diagnosis and treatment of rhinosinusitis. nowadays, it is often considered too unpleasant. however, the experience of patients of this procedure has been very seldom evaluated. nor has the effect on pain in rhinosinusitis been evaluated. the aim of this study was to evaluate patients experience of discomfort and pain during antral irrigation. we also assessed facial pain caused by rhinosinusitis before the procedure and pain soon after the procedure.methodsdoctors and 121 patients completed their questionnaires independently after antral irrigation in a university clinic, in a private hospital, and at a communal health center.resultspatients experienced mild pain during antral irrigation (mean and median visual analog scale score : < 3). their experience of pain during antral irrigation was closely comparable to pain during dental calculus scaling. facial pain assessed before antral irrigation decreased quickly after the procedure.conclusionsantral irrigation was well tolerated as an outpatient procedure. the procedure seems to relieve facial pain caused by the disease quickly. the role of antral irrigation in the treatment of acute rhinosinusitis will need further investigation.
many social interactions are self - beneficial if we behave positively and pro - cooperatively towards others. opportunities to benefit from cooperation are widespread, and reflect the extrinsic fact that the natural environment is often best harvested, insofar as rewards can be accrued and threats avoided, by working together. but the decision to cooperate is not always straightforward, as in some situations it leaves us vulnerable to exploitation by others. game theory specifies a set of potential social interactions in which outcomes of cooperation and defection systematically differ, allowing both experimentalists and theoreticians to probe an individual 's propensity for cooperation in different situations (camerer, 2003). these outcomes typically vary in the extent to which competitive actions may seem preferable and where a short - sighted temptation to exploit the cooperativeness of others has a capacity to subvert cooperation later. fortunately, the ability to look beyond the immediate returns of defection towards longer - term cooperation allows humans to escape from otherwise competitive equilibria, and this can be viewed as a hallmark of rational, sophisticated behaviour. however, humans appear to behave positively towards each other in situations in which there is no capacity to benefit from long - term cooperation : for instance, when they play single games in which they never meet the same opponent again, and when their identities are kept anonymous (fehr., 1993 ; berg., 1995 ; fehr and fischbacher, 2003). this removes the capacity for both direct reciprocity (tit - for - tat) (trivers, 1971 ; axelrod, 1984), and the ability to earn a cooperative and trustworthy reputation that can be communicated by a third - party (harbaugh, 1998 ; bateson. furthermore, they will do this even if it is costly to themselves (xiao and houser, 2005 ; henrich., this appears to be genuinely altruistic, being strictly irrational since it incurs a direct personal cost with no conceivable long - term benefit. arguments against altruistic interpretations of experimentally observed behaviour include suggestions that individuals do not understand the rules of the game, are prone to misbelieve they (or their kin) will interact with opponents again in the future, or falsely infer they are being secretly observed and accordingly act to preserve their reputation in the eyes of experimenters (smith, 1976). however, the widespread observation of altruism (both rewarding and punishing) across cultures (henrich., 2001), and within meticulously designed experiments conducted by behavioural economists provide compelling support for its presence as a clear behavioural disposition. furthermore, in fmri experiments, altruistic actions correlate with brain activity, suggesting that they derive from some sort of intended or motivated behaviour and are not an expression of mere effector noise the very existence of altruism raises the difficult question as to why evolution has allowed otherwise highly sophisticated brains to behave so selflessly. this directs attention towards the decision - making systems that subserve economic and social behaviour (lee, 2006, 2008 ; behrens., 2009), and questions whether they are structured in such a way that yields altruism either inadvertently, or necessarily. the broader consequence is that if they do, then this reframes the question regarding the ultimate (evolutionary) causes of altruism towards the evolution of these very decision systems, and away from the phenomenological reality of altruism per se. in this paper, we first review the structure of distinct human decision - making systems by considering a goal - directed (cognitive) system, a habitual system, and an innate (pavlovian) action system and their interactions. we consider how these systems might operate in social contexts where the key problem is how to make optimal decisions when outcomes depend on the uncertainty associated with other agents and their motives. in the face of such computational complexity, we then consider how optimal actions can be approximated by habit - based decision - making when outcomes are reliably predicted. in this context through habits we also consider the problem of evaluating the best policy when the payoff matrix is unknown but where individuals have an opportunity to learn from others. we frame observation as an inverse reinforcement learning problem, and consider value functions (including goals and subgoals) that are inferred from others actions, as well as by simpler strategies such as imitation. notably, with incomplete information a consequence of not being around to observe the long - term benefits of pro - cooperative behaviours, altruistic outcomes may be inferred as surrogate goals. in this context, altruism arises through optimal inference with incomplete information. studies of decision - making in behavioural neuroscience and psychology have tended to concentrate on elemental decision - making problems, such as reward accrual in simple, stochastic, non - social environments. this enterprise has been very successful and has combined ingenious experimental designs with more classical focal brain lesion paradigms to yield insights into the underlying structure of decision - making systems. one key emerging insight is the likelihood that there is no singly monolithic decision - making system in the brain. indeed, the best evidence suggest there are at least three distinct decision - making systems comprising a goal - directed, habitual, and innate (pavlovian) system with behavioural control being an admixture of cooperation or independence (dickinson and balleine, 2002 ; dayan, 2008). goal - directed decision - making systems function by building an internal model of the environment. in the simplest case in more complicated instances, it involves detailed knowledge of the structure of the environment and one 's position within it. although a goal - directed system may subsume several distinct sub - mechanisms, a wide variety of evidence suggest it localises to prefrontal cortex (daw., 2006 ; kim., 2006 ; valentin hippocampus (corbit and balleine, 2000 ; kumaran and maguire, 2006 ; lengyel and dayan, 2007) and dorsomedial striatum (balleine and dickinson, 1998 ; corbit., 2003 ; yin., habits, on the other hand, lack specific knowledge of the outcome of their decisions. in the parlance of computer science their values are cached, and represent only a scalar quantity which describes how good or bad an action is (daw., 2005). in animal learning, such values are characterised by their insensitivity to devaluation : changes in state (e.g. moving from hunger to satiety) do not alter the value of the action, since there is no access to the new value of the goal (dickinson and balleine, 1994 ; daw., 2005). this efficiency derives entirely from the way in which they learn : rewards reinforce actions that are statistically predictive of their occurrence, with reinforced actions acquiring value through simple associative learning rules (rescorla and wagner, 1972 ; holman, 1975 ; adams and dickinson, 1981). these are well described by reinforcement learning algorithms (such as q learning and sarsa ; sutton and barto, 1998), and localise to dorsolateral striatum (o'doherty., 2003 ; tricomi., control over decisions is often dynamic and frequently transfers from goal - directed mechanisms (early in a task) to a habit - based system (late in a task). indeed, this transfer can be manipulated by selective lesions to the neural substrates that underlie each of these systems (balleine., 2009). in formalising accounts of how these systems interact current views centre on the idea of control being mediated by the respective uncertainties with which each system predicts outcomes, a view that provides a reasonable normative account of experimental findings (daw., 2005). at a broader level, the evolutionary rationale for such a dual system is based on computational cost, since habits are vastly less resource demanding than goal - directed mechanisms. lastly, animals including humans have an innate, hard - wired, decision system. this is often referred to as a pavlovian system, characterised by the expression of values and responses acquired through simple state - based associative learning. unconditioned and conditioned pavlovian responses represent an evolutionarily acquired behavioural repertoire that reflect basic, reliable knowledge gleaned from an organisms evolutionary history : embodying such knowledge structures that approaching sweet tasting fruit and withdrawing from bitter tasting fruit are inherently useful responses to enact. but whereas, on average, this inbuilt knowledge structure is enormously valuable to a nave individual, it may also be a curse in the (usually) uncommon situations in which it is incorrect. the competitive (inhibitory) interaction between decisions based on experience (instrumental habit and goal - directed mechanisms) and those based on pavlovian impulse localises to brain regions such as the amygdala and ventral striatum (cardinal., 2002 ; seymour and dolan, 2008). this interaction reflects the classic tension between apparently emotional irrational and rational cognitive systems whereby the emotional expresses an apparent irrationality by way of some peculiarity of the environment. a challenge for decision neuroscience is to understand how basic decision - making systems operate within socially interactive environments. consider the game in table 1 : the repeated prisoner 's dilemma. subjects must choose between one of two actions : cooperate or defect, and their payoff depends on this and the choice of the opponent. now consider a goal - directed, cognitive decision - making policy in the game, which has the ability to consider multiple future hypothetical scenarios (figure 1a). if you neither know, nor care, what the other player does then the best strategy is to defect on the first round, since the outcome is always better regardless of what the other player does. for the same reason, even if you know what he / she will do, it is still better to defect. an example payoff matrix of two - player prisoner 's dilemma game in which each player can choose either to cooperate or defect. the left - side numbers represent the payoffs for the first player and the right - side numbers represent the payoffs for the second. payoffs are symmetric, and chosen so that the sum of the payoffs is greatest when both choose cooperate and least when both players choose defect. however, each player earns the most if he chooses to defect when the other cooperates. thus, the unique subgame perfect nash equilibrium of this game is for both players to defect. (a) in goal - directed learning, players learn the probability of other player 's action : cooperation (c) or defection (d) based on the history of their actions (h) as p(c\|h) and p(d\|h). they estimate the value of their own actions : v(c) and v(d) using the prediction from the learned model and the expected reward from the pair of actions. (b) in social games, the model of others leads a recursive process : my model of your action includes a model of your estimate of my actions, and so on. cooperation in the prisoner 's dilemma depends on these recursive representations ; since when i decide to cooperate this time, i must estimate that you are going to cooperate next time as you believe that i am going to cooperate with you. however, it is also clear that in the long run, both players are better off if they cooperate : this mutually prescribes the best exploitation of environmental resources. clearly, you need some way of both knowing that your opponent is committed to cooperation as well as a means of signalling to him / her your intention to cooperate. that is, you need to know that she is sophisticated enough to realise that cooperation is worthwhile, and you yourself need to be sophisticated enough to realise this. there is nothing truly altruistic about this, since you are both just trying to maximise your own payoff in an environment that contains another intelligent agent. thus, the existence of another intelligent agent in the environment makes the problem more complex than simpler decision - making problems that exploit inanimate environments. in the latter, the payoff probability usually depends fully on the observable states (they are fully observable markov decision problems ; bellman, 1957). that is, although the payoff may be probabilistic (either involving risk or ambiguity or both), your predictions depend in no way on how you came to arrive at that state in the first place. in social interactions, this assumption does not apply because outcomes depend on what the state thinks about you. if you have recently behaved uncooperatively that is, the outcome depends on unobservable states in the environment (making the problem partially observable). if you find yourself in a seemingly identical state to a previous occasion, for instance playing opponent x in the game y, then the expected payoffs are not independent of how you got there, since opponent x may have a memory of you. consequently, social decision - making benefits greatly from constructing some sort of internal model of the key aspects of the environment. in social games this model needs to capture the intentions of the other player (a component of theory of mind). indeed, your model should also include your opponent 's estimate of your intentions : with this model, you can strategically plan to signal to your opponent your intention to cooperate, knowing that it will change their model of you (figure 1b). accordingly, they should then be more willing to cooperate with you, and you will both be better off in the long run. it can be seen that this sort of model of others intentions, and their model of your intentions, captures features of reciprocity, trust, and reputation formation. indeed maintaining cooperation is in everyone 's selfish interest in repeated games when the end of play is not in sight. it does, however, require players to be able to resist the short - term temptation to exploit this mutual reciprocity by the treachery of defection. of course, there is no reason why an internal representation of an other - agent 's belief model need stop at a knowing the representation of your intentions in their mind. at the next level, it could include your understanding that they know that you know that they know your intentions, and so on. that there are infinite levels of embedded beliefs that make any perfect decision - policy intractable, has inspired models of strategic behaviour that either bound the upper limit of reciprocal beliefs (an example of bounded rationality) (camerer. 2008), or estimate the level of reciprocal belief in their opponent directly (yoshida., 2008). experimental evidence indicates that in repeated games with the same opponent, people reliably cooperate, as theory predicts. critically, however, the theory predicts that people should n't cooperate towards the end of repeated exchanges, when they play people that they will never meet again and who ca n't communicate with others that can. the observation that people do cooperate in these situations suggests something is either incorrect about the goal - directed model, or as we suggest, other decision - making systems compete to bias behaviour. in simple environments, habits allow you to navigate towards goals and avoid harm with speed and computational efficiency. habits operate by allowing recently experienced rewards to reinforce actions that are statistically predictive of them. if an outcome is reliably predicted by an action, then the value of that action becomes high. the action set available to an individual at any one time is elicited by the configuration of cues and contexts in the environment, which represents the current state. importantly, habits do n't themselves have access to any specific representation of their outcome, they merely know their value on an ordinal value scale imagine you are playing a selfish but sophisticated opponent in endless rounds of the prisoner 's dilemma. early in the game, your model - based system has the ability to consider multiple future rounds of the game, in which mutual cooperation is evaluated as valuable, since you know your opponent also knows this. cooperate begin to reliably predict rewarding outcomes, and so the habit learning system, operating concurrently with goal - orientated systems, acquires greater predictive certainty. as this accrues, control is transferred to the habit system, and the computational cost of considering multiple future rounds is relieved. automatic. the critical feature of this type of habit learning is what defines the state by which the habit can be elicited. in animal learning theory discriminative stimulus, and is typically experimentally determined by the presence of a cue (mackintosh, 1983). however, the discriminative stimulus in social games is more complex, and in principle could be determined by the nature of the game being played (prisoner 's dilemma, stag - hunt and so on) or by the identity of the opponent. below, we consider both possibilities : imagine that you ignore the identity of your opponent, and by good fortune play the prisoners dilemma with multiple cooperative opponents : i.e. you exist within a population of sophisticated cooperators (figure 2a). different types of social interaction will have distinct payoff matrices : some will benefit cooperation, others will not. if you know which game you are playing when you engage in an action, then if your action (e.g. to cooperate) is reliably rewarded it will be accessible to acquisition by a habit learning system that simply encodes that in a given game, cooperation or competition is reliably beneficial. an agent plays an action a when in a particular state that is defined by the game type, e.g. game y1. if the outcome is rewarded, then the action is reinforced, and is more likely to be emitted when the same state is encountered again. an agent (in green) plays action a (cooperate) when interacting with a particular agent (who defines the state, or discriminative stimulus sd as x1, for example). if the outcome is rewarding, then the reward reinforces action a, such that it 's value v(x1,a) increases, and is more likely to be chosen again in the same state in future. indeed even if the payoff matrix is not known, for instance in a novel game in an uncertain environment, a reasonable strategy may be to play by trial and error. this entails exploring different actions and seeing what the outcome is, in which case actions can be reinforced directly by habit systems. simulation studies demonstrate how readily cooperative equilibria can be reached by simple associative algorithms (such as q learning) without any model - based control at all (littman, 1994 ; claus and boutilier, 1998 ; hu and wellman, 2004). alternatively, you may choose to ignore the payoff matrix of the game, but concentrate instead on the identity of your opponent (figure 2b). for instance, if you play a specific opponent in a variety of games, and she reliably cooperates with you to your benefit, then you may learn the habitual action to cooperate whenever you play her. in this way, she becomes a positive discriminative stimulus that evokes actions that engage pro - cooperatively with her. the above mechanisms may acquire control of behaviour if several criteria are satisfied : the state and/or opponent are clearly discernable ; the game (i.e. its payoff matrix) is relatively static (or changes slowly) allowing equilibria to be reached ; and your internal preferences are stable. however, habit mechanisms are less reliable in the face of perceptual uncertainty, in which case an internal belief model of possible states may be required ; if there are sudden changes in the environment that require rapid new learning, or a search for causal antecedents ; or if your motivational state changes substantially (cooperation for food becomes less valuable when you are sated). switch off in situations in which they behave poorly, rather their influence on control diminishes when their predictions become unreliable (daw., although providing a plausible mechanism for social decision - making it turns out that, to date, evidence for habitised control of social behaviour is largely indirect. first, simple reinforcement learning algorithms do a remarkably good job at predicting behaviour in experiments across a variety of games (erev and roth, 1998, 2007). second, neuroimaging studies show opponent - specific value - related responses accruing according to opponents cooperativity / competitiveness in games (singer., 2004). third, neuroimaging studies have also identified dynamic reinforcement learning - like (prediction error) signals during games (king - casas., 2005). fourth, in single neuron recordings from non - human primates, lateral inter - parietal sulcus neurons in monkeys appear to encode value signals predicted by reinforcement learning in mixed - strategy games (seo., 2009), which adds to previous observations that neurons in dorsolateral prefrontal and anterior cingulate cortex encode quantities related to choice and reinforcement history, respectively (barraclough. in reality, humans might be expected to habitise their actions in the context of state information that incorporates both opponent and game type. although a diversity of subtly different payoff matrices may be common in experiments, it is likely that social interactions in different scenarios represent a relatively discrete set of payoff matrices. when there are small differences between different games, habit systems may generalise across salient features that have characteristic predictive power for beneficial outcomes. one especially important social scenario arises when a person interacts with others who are significantly more expert at social interaction. this can occur for a number of reasons : if the payoff matrix that defines the interaction is unknown to us but known to others either through their experience or private information ; because information about other players is known to them but not to us again through either experience or their own vicariously acquired knowledge ; of if they are more sophisticated for instance they are more mature or intellectually able. in these situations, you have the choice to engage in interactions and acquire the information directly through your own experience or, better, to observe apparently successful social agents and vicariously acquire knowledge. as long as success is discernable, as a hallmark of social expertise, then observational learning is likely to yield useful information. the computational problem becomes how to interpret the actions of others, and use observed actions to optimise your own. computationally, inverse reinforcement learning describes this problem of how to reverse engineer observed actions to evaluate their values and goals, and is particularly difficult in situations in which actions do not immediately lead to their benefits. unfortunately social interactions often display exactly this property : the benefits of cooperation are often long - term, through reputation formation and establishment of trust, and unless an observer has observational access to extended sequences of actions and their ultimate outcomes, the problem becomes even harder. in general, there are two broad classes of solution. imitation is the observational twin of habit learning, insofar as the resulting action has no specific representation of the outcome : it simply learns that a particular action is reliably performed in state s. the actions it bears are habit - like, elicited by a discriminative state that represents the environment in which they were learned. accordingly, the ease of imitation depends on the discernability of the state of the observer. in figure 3a, we illustrate this for a situation in which the state is defined by the game type : as long as it is clear to the subject that they are playing, say game y = prisoners dilemma, then the imitated action will be cooperate when playing game y. the imitated state - action pair could equally well be defined by the identity of the opponent. in this case, the resulting action will be cooperate when playing opponent x. note that the values of the actions can also be inferred by the frequency with which they are elicited by observation, allowing imitation to encode action values, and not just stimulus - responses. an observer estimates the value of action a from other players actions and simply imitates an action which maximises the payoff in a particular environment, which can be defined by game or opponent (or both). game = y and it does not take into account the opponent 's type (x) who they are playing with. this means the observer assumes that the players using a model - based learning ; i.e. they have a forward model of their opponents. for example, in a repeated prisoner 's dilemma, cooperative actions (a1) will predict a state of the other players happiness (s1) which leads mutual cooperation in the future. the value of action a is calculated as the value of state (e.g. other player 's emotional state), v(s), multiplied by the probability of occurrence of the state followed by the action, p(s\|a). the second strategy is more complex, and involves trying to reverse engineer actions so as to evaluate their value or actual outcome (ng and russell, 2000). this requires constructing some sort of internal model of the action. for sequential actions, a computationally useful strategy is to represent subgoals intermediate outcome states that appear to be reliable pre - requisites to eventual success (abbeel and ng, 2004). in the case of cooperative games, these subgoals ought to include the welfare of the other cooperators, since this is a powerful determinant of future cooperation. for example, in a repeated prisoner 's dilemma, sophisticated cooperators will themselves predict reward when their opponents cooperate with them, since they have a forward model of future beneficial interactions. assuming their reward - predicting state is discernable by observations of their emotional state s (their happiness), that is, it follows that the inference that eliciting the state of happiness in another player is a valid predictor of an agent 's success (figure 3b). although in the case of the agent being observed this is merely an intermediary state in ultimately selfish reciprocal interactions, this information (and its selfishness) is not available to the observer. even so, it is still valuable knowledge as long as the observer is fortunate enough to use the information in situations in which it actually is beneficial : i.e. in repeated social exchanges. as long as repeated social exchanges outnumber un - repeated exchanges, then observational inference is likely to be a better strategy than ignoring others. observational learning in games, and especially putative inverse reinforcement learning, remains relatively under - explored. it is well known that humans use both model - free (imitative) and model - based (inverse - inference) strategies when learning non - social actions through observation (heyes and dawson, 1990). recent imaging evidence shows that people learn values through instruction using similar neural mechanisms involved in personal experience based learning (behrens., 2008), and make inferences about values by pure third - party observation (klucharev., 2009). furthermore, pro - social feelings towards others (empathic reward), and it 's neural representation, have been shown to be modulated by perceived similarity with that person (mobbs., 2009), as one might predict from perspective - taking theories of social observation (wolpert., 2003) we have argued that consideration of the neurobiological mechanisms of learning and decision - making in humans can yield an explanatory account of true altruism. at the heart of this account are the learning systems that allow the brain to optimise reward and efficiency in complex environments. critically, since evolution is likely to operate primarily over learning and decision mechanisms, and not the content of those systems how they learn, not what they learn, the ensuing altruistic behaviours are perfectly permissible, despite the fact that they may in some instances become strictly irrational. this is strengthened by the fact that habit - based and observational learning systems have uses way beyond social decision - making per se. the latter, for instance, is elegantly utilised in complex behaviours such as food preparation, tool use, and even language. hence evolutionary selection for such mechanisms may be driven by a much broader range of decision - making problems than purely social interaction. accordingly the value of the inherent flexibility of learning systems is that it allows them to adapt to a wide range of potentially new and unexpected situations, appropriate for the diversity of the natural environment. but this flexibility carries the cost of inadvertently allowing individually economically disadvantageous actions to emerge, albeit rarely. part of this supposition incorporates the fact that an innate representation of the caveats of flexible learning in social decision - making (for instance : do n't cooperate in one - shot, anonymous exchanges in large groups) is itself cripplingly complex and maladaptive to novelty (it itself becomes a form of impulsivity). in other words, any social decision - making system that attempted to capture the enormous range of possible encounters and interactions, and individually specify optimal policies, would impair rather than augment decision - making under uncertainty. as such, efficient learning based systems are likely to be selected in the course of evolution. learning based accounts differ from the conventional approach of studying cooperation in behavioural economics, which often considers static, heuristic decision - policies, such as tit - for - tat, cooperate and punish, and free - ride. such models typically succumb to free - riders, including sophisticated (higher - order) free - riders that cooperate but do n't enforce or encourage cooperation in others. however, a valuable insight of these models has been the recognition that resistance to free - riders can be provided by acquisition (and defence) of cultural norms of behaviour (boyd and richerson, 1988 ; boyd., 2003 ; bowles and gintis, 2004). key underlying components of norm - abidance are likely to be observational learning and inference based mechanisms, since these form simple elements of cultural learning. the current paucity of biologically implemented algorithmic models and mechanisms of observational and cultural learning is therefore likely to be an important area of future research. in particular, the relative privacy of culturally acquired information within specific groups is likely to be an important factor in the development of parochialism, which may further allow group - based selection of altruistic behaviour (bernhard., 2006 ; choi and bowles, 2007). such mechanisms are thought to underlie many aspects of human impulsivity and irrationality, through their occasionally inflexible competition with instrumental actions (dayan., 2006). if cooperation was so consistently advantageous through human social evolution, that it is quite possible there might be some innate coding. indeed, the environment in which the social brain evolved is likely to have had a much higher proportion of repeated interactions with the same individuals than our modern environment in which cooperation can occasionally be economically disadvantageous. innate actions can be thought of as action priors over and above which more sophisticated goal - directed instrumental actions can assume control as experience accrues. their achilles heel, however, is the fact that they appear often difficult to overcome (inhibit) completely : they have a residual and significant weight that consistently biases actions in their favour. if such innate coding of cooperation exists in the human brain, then it follows that altruism would be akin to more basic forms of impulsivity. we note that control by innate systems is characterised by the intrinsic (typically emotional) value of a stimulus, as well as by the action it elicits. accordingly, the states associated with putatively pro - social innate actions could include that following the act of sharing, generosity or generation of equity (tomasello., 2005). in this way, they become intrinsic internal rewards that, phenomenologically, are elicited because they are personally satisfying (and akin to non - social innate behaviours such as novelty - seeking (wittmann., 2008)). the complexity of different putative accounts of human altruism appeals to neuroscience as an arbitrator (camerer., 2004b). distinguishing different decision systems purely on anatomical grounds may be difficult, however : brain regions such as the striatum, orbitofrontal cortex, amygdala and hippocampus for instance, appear to be convergence areas for all decision systems. for example the observation of activation of striatum in a study on altruistic punishment (de quervain., 2004), whilst providing a convincing illustration of the fact that such behaviour has a clear proximate basis, says little about the nature of that behaviour in terms of whether it is innate or learned. this underlines the importance for brain imaging techniques that have the ability to distinguish between competing models based on identifying coding of their underlying central parameters (o'doherty., 2007), in situations in which behaviour alone is necessarily ambiguous (yoshida., 2008). both habit - based and observation - based accounts of pro - social behaviour make specific experimental predictions. first, if the identities of others can act as discriminative stimuli, then cooperation should carry over between different games with the same individual. second, if game types can act as discriminative stimuli, then cooperation should carry over between the same game with different individuals. third, the duration of play should predict the degree of unfolding of cooperation towards the end of repeated games, since extended durations permit stronger habit formation and less susceptibility to anticipatory defection. fourth, the operation of associative learning mechanisms should be determinable by the use of co - incident cues associated with previous cooperative or uncooperative players, which ought to bias individuals behaviour in future games : in fact evidence already exists for this (vlaev and chater, 2006 ; chater., 2008). fifth, observational learning can be studied directly by allowing individuals to passively watch interactions between others before engaging in similar games, or different games with the observed opponents. indeed evidence does exist that previous observation has an influence on future social behaviour, in that people do seem to be biased towards the behaviour of others. what is more difficult to establish is exactly how this information is represented : either as a cached imitated value, or as a model - based representation. finally, we note that learning based accounts of altruism are by no means immune to exploitation by selfish and intelligent learning agents. any sophisticated model of other agents behaviour can incorporate the fact that they are habit and observational learners. consequently, highly sophisticated models of other agents could in theory incorporate representations of their different decision systems : thus knowing that people are habit learners gives predictive insight into what is likely to guide their behaviour in various situations. whereas determining this might not always be simple to an agent from passive observation, it might be in part revealed by probing : intentionally behaving in a certain way (such as maliciously cultivating pro - social cultures) to manipulate how values are acquired by others, so that they can be exploited later. 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 origin of altruism remains one of the most enduring puzzles of human behaviour. indeed, true altruism is often thought either not to exist, or to arise merely as a miscalculation of otherwise selfish behaviour. in this paper, we argue that altruism emerges directly from the way in which distinct human decision - making systems learn about rewards. using insights provided by neurobiological accounts of human decision - making, we suggest that reinforcement learning in game - theoretic social interactions (habitisation over either individuals or games) and observational learning (either imitative of inference based) lead to altruistic behaviour. this arises not only as a result of computational efficiency in the face of processing complexity, but as a direct consequence of optimal inference in the face of uncertainty. critically, we argue that the fact that evolutionary pressure acts not over the object of learning (what is learned), but over the learning systems themselves (how things are learned), enables the evolution of altruism despite the direct threat posed by free - riders.
hla antibodies play an important role in transplant rejection and failure and they result after exposure to mismatched hla antigens which can occur after transplantation as well as following blood transfusions or during pregnancy. traditionally, antibodies have been described as specific for hla antigens such as anti - a1, anti - b7, and anti - dr1, or for serologically cross - reacting hla antigens such as the a2-creg and the b7-creg. it has now become apparent that hla antigens carry multiple epitopes which can be defined by molecular structural modeling and amino acid sequence differences between alleles. hlamatchmaker represents an epitope - based approach to assess hla compatibility and to select suitable donors for patients in need of an organ transplant (duquesnoy, 2002, 2006). three recent reviews describe the concept of hlamatchmaker and its usefulness in hla epitope matching for organ transplantation (duquesnoy, 2008a, 2011a ; duquesnoy and marrari, 2009). briefly, hlamatchmaker considers each hla antigen as a string of amino acid configurations as key elements of epitopes that can elicit specific alloantibodies. the original version used triplets, i.e., linear sequences of three residues (duquesnoy, 2002), but the so - called eplet version is based on stereochemical modeling of protein antigen antibody complexes and the contributions of critical amino acid residues that dominate in antigen antibody binding (duquesnoy, 2006). the residues of such patches are within a three ngstrom radius of a non - self residue. each eplet is assigned a position number in the amino acid sequence and the notation system lists only polymorphic residues marked with the standard letter code. hlamatchmaker programs consider class i (duquesnoy, 2006), class ii (duquesnoy and askar, 2007), and mica compatibility and antibody analysis (duquesnoy., 2008a). the www.hlamatchmaker.net website is an information resource and has excel based analysis programs that can be downloaded free of charge. recent developments have increased our understanding of the structural basis of hla antigenicity, i.e., reactivity with specific antibody. hla antibodies are specific for epitopes that can be defined by single eplets or pairs consisting of a non - self eplet presented by the immunizing antigen and a self eplet shared by the antibody producer and the immunizer (duquesnoy., 2005 ; marrari., 2010) the application of hlamatchmaker to the analysis of antibody reactivity may increase our understanding of otherwise unexplained sensitization patterns induced by a given mismatch. for instance, sensitization to certain hla - c mismatches can lead to antibodies reacting with epitopes shared with hla - b antigens (lomago. these findings demonstrate that sensitization induced by an epitope on a hla - c mismatch may cause other class i antigens to become unacceptable mismatches because they share that epitope although the patient may have never been exposed to such antigens. for instance, patients sensitized by a dr2 mismatch have often antibodies reacting with dr1 (marrari and duquesnoy, 2009). conversely, sensitization by a dr1 mismatch can lead to antibodies that react also with dr51 but not with dr2. these findings demonstrate the importance of drb3/4/5 eplets in drb - specific antibody responses of kidney transplant recipients (duquesnoy. hla - dq and hla - dp heterodimers have distinct eplet repertoires and certain dq and dp eplets react often with class ii antibodies (duquesnoy, 2008b ; duquesnoy., 2008b). dq antibodies can also recognize pairs of eplets shared between dqa and dqb chains (tambur., 2010). in the clinical setting of transplantation, it has become apparent that hla epitopes rather than antigens are important for analyzing antibody specificity. the highly sensitized patient represents an enigma for kidney transplantation : not only is it difficult to find a suitably matched donor but subsequent kidney transplants are often less successful. most commonly used is the identification of antibody - defined hla antigens that should be considered unacceptable. this system is designed to identify donors who must be excluded but it does not necessarily mean that all other hla antigens would be compatible for a patient. the other goal is to determine hla antigens that are acceptable mismatches. this strategy represents a direct approach of finding a compatible donor for a sensitized patient (claas., 1989, 2004). hlamatchmaker is a useful tool in the analysis of serum antibody reactivity of sensitized patients and the identification of potential donors with acceptable mismatches (claas., 2004, 2005 ; eurotransplant has incorporated hlamatchmaker in the acceptable mismatch program to identify donors for highly sensitized patients (claas., 2004, 2005 ; this approach shortens the waiting time for a suitable kidney donor and leads to excellent graft survivals comparable to those seen with non - sensitized recipients (claas., hlamatchmaker can be used as a quantitative tool to determine the degree of a mismatch, i.e., the number of mismatched eplets or triplets. a given hla antigen mismatch has an epitope load that is primarily determined by the recipient s hla type representing a repertoire of self epitopes to which no alloantibodies can be made. for some patients, a mismatched antigen might be structurally compatible whereas for other patients it has multiple mismatched epitopes (duquesnoy, 2008a). table 1 shows examples of eplet mismatches of two class i alleles for eight hla phenotypes. for each allele two examples showing how the hla phenotype of the recipient affects the eplet load of a class i allele mismatch (adapted from duquesnoy, 2008b). the incidence of the anti - class i antibody response induced by a transplant or during pregnancy correlates with the number of non - self triplets or eplets on mismatched antigens (dankers. anti - hla - c antibody responses by patients with rejected kidney transplants also correlate with eplet loads of hla - c mismatches (duquesnoy and marrari, 2011). conventional class ii matching criteria consider only hla - dr antigen but this approach is an insufficient reflection of class ii compatibility because antibodies against other class ii mismatches including dp and dq have been shown to diminish transplant success. each dr antigen should be viewed as a package of dr + dq + dp antigens and the overall class ii epitope load depends on the patient s dr, dq, dp type. table 2 illustrates how dr haplotypes have different epitope loads if drb3/4/5, dqb, and dqa are included. let as assume that dr11, 16 corresponds to drb1 11:01, drb3 02:01, dqb1 03:01, dqa1 05:01/drb1 16:01, drb5 02:02, dqb1 05:02, dqa1 01:02 genotype, and that the serological dr antigens correspond to the common dr the dr12 and dr15 mismatches have the lowest eplet loads namely 6 and 9, whereas dr7 and dr9 have 28 and 27 mismatched eplets, respectively. the donor s drb1 16:02 has two eplet differences with the recipient s drb1 16:01. this drb1 15:01 haplotype has no drb1 eplet mismatches but its drb5 01:01 and dqb1 06:02 have 2 and 7 mismatched eplets, respectively. these examples are merely intended to illustrate that high - resolution dr, dq typing can provide detailed class ii matching information at the eplet level. dq haplotype shows differences in class ii eplet loads among serologically defined dr antigen mismatches (adapted from duquesnoy, 2008a). donor - specific, drb1-reactive antibodies are less often detectable than antibodies against other class ii epitopes (duquesnoy., 2008b). in contrast, donor - specific drb3/4/5 mismatches induce more antibody responses and they have higher numbers of incompatible eplets. anti - dq antibodies are rather common and this correlates with more mismatched eplets on dqb and dqa than on drb1 (duquesnoy., 2008b). about one - third of class ii sensitized patients have anti - dp antibodies reactive with a few dpb eplets and an allelic pair of dpa eplets. information about epitope loads of hla mismatches seems clinically useful in the management of transplant patients. epitope loads can be interpreted as risk factors for antibody - mediated rejection in the clinical management of transplant recipients and may eventually lead to new strategies for hla mismatch permissibility to reduce alloimmunization and increase transplant survival. our studies on eplet pairs have always shown that the immunizing allele has one eplet that is non - self whereas as the other is a self eplet shared with the antibody producer (duquesnoy., 2005 ; marrari., 2010 this suggests an autoreactive component of the alloantibody response to an hla mismatch and a recent report has expanded this view to the non - self self paradigm of eplet immunogenicity (duquesnoy, 2011b). this paradigm is based on current concepts about epitope antibody interactions and the development of b - cells specific for autologous proteins. the variable domains of immunoglobulin heavy and light chains determine antibody reactivity and each chain has three complementarity determining regions (cdrs) with hypervariable loops that interact with different parts of an epitope (poljak. the third cdr of the heavy chain (cdr - h3) lies generally in the center of the antigen cdr - h3 has considerable variability in its length (kabat., 1977, 1996 ; shirai., 1999) whereas the other cdrs form limited numbers of so - called canonical structures that stabilize the binding with cdr - h3 (chothia and lesk, 1987 ; kuroda., 2009). antibody complexes have defined a structural epitope as that part of the antigen that makes contact with all cdrs of antibody (davies. a structural epitope has about 1525 surface residues and within it lies the functional epitope consisting of energetic residues that dominate the binding with antibody (getzoff., 1988 ; novotny, 1991 ; laune., 1997 ; van regenmortel, 2002). functional epitopes comprise one patch or a pair of patches of energetic residues separated far enough to be contacted by different cdrs of antibody. in order to be immunogenic, a functional epitope must have at least one non - self residue, i.e., the antibody producer s homologous proteins must have a different residue in the corresponding sequence position. such residues must be on the molecular surface so they can make contact with the specificity - determining cdrs of antibody. the surface of a structural epitope is about 700900 (davies., 1990 ; padlan, 1994 ; maccallum., there is no information about the structure of an hla antigen complexed with alloantibody but ziegler s group has obtained detailed information about a crystallized antigen antibody complex involving a melanoma - associated peptide bound to hla - a1 (hulsmeyer., the structural epitope involves four contact residues of the peptide and 10 contact residues in the helices of hla - a1. what are the possible dimensions of structural hla alloepitopes which have mismatched eplets as functional epitopes ? considering the 700900- surface of a structural epitope one can calculate that surface residues within a 15- radius of a centrally located eplet could make contact with antibody. command of the cn3d structure software program (hogue, 1997) using informative hla molecular models downloaded from http://www.ncbi.nlm.nih.gov/structure. the non - self self paradigm of eplet immunogenicity considers the hypothesis that b - cells carry low - affinity immunoglobulin receptors for self - hla epitopes (duquesnoy, 2011b). their interactions with self - hla will not lead to b - cell activation or antibody production. it seems that the activation of a self - hla specific b - cell by a non - self eplet requires that the remainder of the structural epitope on the immunizing antigen has considerable structural similarity with the corresponding self - hla epitope of the antibody producer. to assess the validity of the non - self self paradigm, we have determined polymorphic residue differences within a 15- radius of an eplet on the immunizer and the alleles of the antibody producer. the goal is to identify the antibody producer s structural epitope that has an identical or very similar residue composition as the immunizing epitope with the mismatched eplet. table 3 summarizes data in three recent publications as experimental support of this hypothesis (duquesnoy, 2011b ; marrari., 2011 ; duquesnoy., submitted). for each antibody response, at least one allele of the antibody producer has no or few differences with the immunizing allele in antibody - accessible positions defined by a 15- radius of the mismatched eplet, the presumed dimension of a structural epitope. most of the data were obtained with human monoclonal antibodies (produced by arend mulder, leiden university medical center) that were specific for eplets or eplet pairs. in each case, the antibody producer had at least one allele which had no or very residue differences with the immunizing allele ; they are highlighted in table 3. two patients had antibodies against the 145r + 82lr pair presented by the immunizing b44:02 ; this allele has no residue differences with the self b13:02 allele except for 145l rather than 145r. it should be noted that both eplets are present on one or more alleles of the antibody producer and, according to hlamatchmaker, they are considered intralocus and interlocus matches which should not induce antibodies. none of the alleles of these antibody producers had however, the combination of these eplets and the non - self self paradigm for hla epitope immunogenicity has offered a ready explanation why the pair of self-145r and self-82lr eplets presented by b44:02 had induced specific antibodies. the b15:01 induced monoclonal 12 is also specific for a pair of self eplets, namely s69tnt + s80nrg (table 3). within the 15- radius of this pair, the b07:02 allele of the antibody producer had no residue differences with b15:01. examples of polymorphic residue differences within 15 of mismatched eplets on antibody - defined epitopes on immunizing alleles and the alleles of the antibody producer. these findings support the concept that hla antibodies originate from b - cells with self - hla immunoglobulin receptors that recognize mismatched eplets as non - self entities on immunizing antigens. it is well known that sensitized patients develop specific antibodies to a restricted number of mismatched epitopes (duquesnoy., 1990 ; rodey., the non - self self paradigm of hla epitope immunogenicity may explain this phenomenon. it is possible that the antibody response to an eplet requires that its structural epitope on the immunizing hla antigen must be structurally very similar to the corresponding self epitope of the antibody producer. any antigen with significant structural epitope differences with patient s self epitopes might prevent b - cell activation and subsequent hla antibody production. the non - self self concept of hla immunogenicity may become clinically useful regarding predicting antibody responses to hla mismatches but it needs of course, experimental verification in the clinical setting. prevention of hla sensitization represents a significant challenge for the non - sensitized transplant candidate. two causes, namely blood transfusions and the transplant itself should be approached as being potentially preventable. perfect hla matching is somewhat impractical because it can be done for small numbers of patients. on the other hand, information about epitope loads and the application of the non - self self paradigm of epitope immunogenicity may useful in assessing risks for antibody - mediated rejection after transplantation and the clinical management of transplant recipients. such permissible mismatching may lead to new strategies to identify suitable donors with minimal risks for allosensitization. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.	hla mismatching is an important risk factor for antibody - mediated rejection and transplant failure. with the realization hla antibodies recognize epitopes rather than antigens, it has become apparent that donor - recipient compatibility should be assessed at the epitope level. recent developments have increased our understanding of the structural basis of hla antigenicity, i.e., the reactivity with specific antibody and, immunogenicity, i.e., the ability to induce an antibody response. hlamatchmaker is a computer algorithm that considers each hla antigen as a series of small configurations of polymorphic residues referred to as eplets as essential components of hla epitopes. this article addresses the relevance of determining epitope - specificities of hla antibodies in the identification of acceptable mismatches for sensitized patients considered for transplantation. permissible mismatching for non - sensitized patients aimed to prevent or reduce hla antibody responses could consider epitope loads of mismatched antigens and the recently developed non - self self paradigm of epitope immunogenicity.
although there is debate about superior vena cava (svc) filtration in patients with upper extremity deep venous thrombosis (dvt), a percutaneous svc filter insertion is now regarded as safe, feasible, and effective in preventing symptomatic pulmonary embolisms due to upper extremity dvt in patients in whom anticoagulation or thrombolysis has failed or is contraindicated (1 - 3). additionally, a retrievable svc filter can be used as a temporary and effective barrier against a symptomatic pulmonary embolism in settings where the risk is transient (e.g., after trauma or during pregnancy). such a device is also useful during aggressive, catheter - directed thrombolysis or mechanical thrombectomy, which can induce thrombus migration to the pulmonary artery (4). the tempofilter ii (b. braun, melsungen, germany) is a kind of temporary caval filter. the filter cone is held in place in the vena cava by a tethering catheter with a subcutaneous anchoring device. most often, the tempofilter ii is used in the inferior vena cava (ivc) for protection against a pulmonary embolism due to lower extremity dvt (5). to our knowledge, there are no printed reports concerning the placement of the tempofilter ii in the svc to prevent a pulmonary embolism in patients with upper extremity dvt. in this article, we present a case of svc filtering using the tempofilter ii in patients with upper extremity dvt. a 67-year - old woman was brought to the emergency unit after a traffic accident. she suffered multiple traumatic injuries including liver laceration, left femoral shaft fracture, pelvic bone fractures, and multiple rib fractures. roughly three weeks later, she developed dyspnea and swelling of the left upper extremity. a contrast - enhanced chest computed tomography (ct) revealed a pulmonary embolism in the right pulmonary artery and thrombosis in the left brachiocephalic vein (fig. ultrasound taken after the diagnosis of pulmonary thromboembolism, a lower extremity dvt was not detected. a left upper extremity venogram revealed complete obstruction of the brachiocephalic vein with lack of collaterals, implying the development of acute upper extremity dvt (fig. to prevent the recurrence of a pulmonary embolism from the left upper extremity venous thrombi, and to reduce her left arm swelling, removal of venous thrombi was necessary. because anticoagulation and thrombolysis were contraindicated due to her recent major trauma history, we decided to perform a thrombus aspiration. to prevent a further possible pulmonary embolism during the procedure, we planned temporary filtration in svc. one day following the diagnosis of upper extremity dvt and pulmonary thromboembolism, the tempofilter ii was placed in the svc percutaneously, via the right femoral vein. after a right femoral vein puncture, a guide wire was threaded through the ivc, right atrium, and up into the svc. the subcutaneous puncture site was enlarged by about 10 mm with an incision and an approximately 20-mm - sized subcutaneous pocket was created by tissue dissection. next, the introducer system (dilator and sheath) was inserted along the guide wire into the svc under fluoroscopic guidance. after the dilator and guide wire were removed, iodinate contrast agent was injected to obtain a superior vena cavogram to clarify the exact location of the thrombus and to confirm the optimal landing zone for a filter in the svc. next, a filter unit was introduced into the svc via the sheath. by pushing and pulling the tethered catheter, after checking the position of the filter, the anchoring device was attached to the tethering catheter just around right femoral vein, and the excess length of the catheter was sectioned immediately above the olive shaped button. after burying the anchoring device into the subcutaneous pocket,, thrombus aspiration was performed through left basilic vein with a 100 cm length 8-fr guiding catheter (guider softip ; boston scientific, natick, ma). thrombus aspiration via a catheter was performed with a 20 cc syringe. during the thrombus aspiration, anticoagulation or thrombolysis was not performed. a follow - up venography performed after thrombus aspiration showed recanalization of the left brachiocephalic vein, regression of collateral veins (fig. 1f), and did n't show any thrombus capture within the unit. a ct angiography obtained two weeks after tempofilter ii placement demonstrated a patent svc with no thrombus around the filter, as well as resolution of the pulmonary embolism in the right pulmonary artery. just one day after a follow up ct angiography, we successfully removed the filter without complication. retrieval of the filter was achieved by making a skin incision around the palpable anchoring device under local anesthesia. the unique design of the tempofilter ii consisting of a long tethering catheter and filter device with no hook is not seen with other filters and because of this, has many advantages. first, the tempofilter ii is a retrievable, temporary filter and this characteristic could make it an effective svc filter. other advantages of the tempofilter ii include a long tethering catheter, which makes filter retrieval easy and rapid due to the simple traction on the tethering catheter. its unique design, which has been mentioned in the above paragraph, makes it possible that the filter possesses a more vertical orientation within the svc (the tethered catheter, which is fixed around inguinal area, acts like a pendulum). the vertical axis and the lack of tilting of the filter within the svc may increase the ability of the filter to capture clots (6). additionally, the tempofilter ii has a decreased incidence of vena cava injury compared to other filter types, because it does not have any hooks on the filter strut. there have been several reports about complications related to hooked temporary svc filters, such as svc perforation, cardiac tamponade, and aortic injuries (7 - 9). the tethered catheter enables the fine correction of filter position by allowing for the movement of the filter to - and - fro within the svc. usually, placement of a filter at the optimal level in the svc is more technically difficult than placement in the ivc, due to the shorter length of the optimal landing zone in the svc (2). the use of the tempofilter ii in the svc also has several potential limitations or drawbacks. first, the tempofilter ii can not be used permanently ; it has been validated for indwelling times of up to six weeks, although the manufacturer states that retrieval is feasible and should require no additional equipment, up to three months after placement (5). therefore, if patients need a permanent svc filter, the tempofilter ii must be replaced with another type of filter device. second, the tempofilter ii should be used in only bed - ridden patients. in mobile patients, the tethering catheter can bend or buckle due to the effect of gravity and the patient 's movement. furthermore, while the lack of hooks on the struts has benefits, this also makes the tempofilter ii filter more prone to migration within the relatively short svc. in cases where the tempofilter ii was used in the ivc, there have been reports of intracardiac migration inducing fatal outcomes (5, 10). a third disadvantage is that the use of the tempofilter ii is also limited by the length of the introducer sheath and the tethered catheter, making it impossible to use the tempofilter ii in tall patients. when the tempofilter ii is inserted into the svc, the tethered catheter is introduced through the common femoral vein and travels through the iliac vein, ivc, and svc. as a result, the length of tethered catheter remaining outside the body may be insufficient to withdraw the introducer sheath up to length of 70 cm. however, if the manufacturer were to provide a longer tethered catheter, this limit could be easily overcome. in conclusion second, they must be removable from any amount of organized and adherent thrombus after anticoagulation therapy has been completed and the indication for caval filtration has ceased (5). based on these requirements and despite its drawbacks and limitations, the tempofilter ii may be one of the good options for the temporary filtration of the svc to prevent pulmonary embolisms in bed - ridden patients with upper extremity dvts.	the tempofilter ii is a widely used temporary vena cava filter. its unique design, which includes a long tethering catheter with a subcutaneous anchor, facilitates the deployment and retrieval of the device. despite this, the tempofilter ii has been used only in the inferior vena cava of patients with lower extremity deep venous thrombosis. in this article, we present a case of superior vena cava filtering using the tempofilter ii in patients with upper extremity deep venous thrombosis.
located on chromosome 15, aromatase is encoded in cyp19a1 gene.1,2 aromatase enzyme is involved in the last step of estrogen biosynthesis that converts testosterones to estrogens via aromatization process. since aromatase has high specificity and is only involved in the last step steroid biosynthesis, inhibition of this enzyme does not affect levels of other biologically critical steroids. in postmenopausal women, aromatization of androgens, which are produced by the adrenal glands, serve as the main source of estrogen production after cessation of ovarian function. as the majority of breast cancers are hormone dependent, agents that target estrogen signaling pathway have been previously investigated as treatments for breast cancer. however, tamoxifen is associated with multiple concerning side effects, particularly risks of endometrial cancer and venous thromboembolism.3,4 this is the result of its partial estrogenic effects in the uterus and vascular system. due to these unfavorable side effects and incomplete blockade of estrogen signaling pathways, an alternative approach to target the ligand production by inhibiting the aromatase enzyme was explored by schwarzel in early 1970s.5,6 currently, there are three aromatase inhibitors (ais) in clinical use, namely anastrozole, letrozole, and exemestane. this review article summarizes classification of ais as well as clinical utilities of ais in various clinical settings in the context of breast cancer. ais can be classified in two distinct subclasses, according to their chemical structures as shown in table 1. an anti - epileptic drug, aminoglutethimide, was initially used for the treatment of breast cancer in the late 1970s.7 this drug suppresses multiple adrenal steroid productions by inhibiting various cytochrome p450 enzymes. due to inhibition of cyp11, cortisol replacement is required for this non - specific inhibitor. later on, it was discovered that the key mechanism of action of aminoglutethimide is inhibition of aromatase enzyme. given that this drug has to be given in combination with steroid replacement, aminoglutethimide is no longer in clinical use. subsequently, specific inhibitors of aromatase were developed. mimicking the structure of androstenedione which is a substrate of aromatase enzyme, type i inhibitors or steroidal ais bind to the substrate - binding site of the aromatase enzyme. its reactive intermediate then binds covalently to the enzyme resulting in an irreversible inactivation of aromatase. these inhibitors are also known as suicidal inhibitor as the enzyme is inactivated by its own function. exemestane is the only oral steroidal ai that is currently in clinical use since formestane requires painful intramuscular injections. in contrast, type ii inhibitors or non - steroidal ais bind non - covalently to the heme moiety of the aromatase enzyme and saturate the binding site. this subclass of ais includes fadrozole, rogletimide, vorozole, anastrozole, and letrozole. since the 1970s, it has been recognized that the majority of breast cancers are hormone dependent. up to three quarters of breast cancers express hormone receptor, namely estrogen receptor (er) and/or progesterone receptor. modulations of either the receptors or its ligand have been proven to be effective strategies not only as treatments for breast cancer but also to prevent breast cancer. as described previously, ais exert their activity by depleting estrogens which are the ligands that activate estrogen signaling pathways. in postmenopausal women, peripheral conversion of androgens to estrogens serves as the sole source of estrogen production after cessation of ovarian function. aromatase is present in multiple tissues, including adipose tissue, brain, blood vessels, skin, bone, normal breast tissue, and breast tumor cells. these locally produced estrogens in the breast tissue have an impact on tumor growth via paracrine or intracrine mechanisms. it has been shown previously that the levels of estrogens in breast tissue do not correlate with circulating estrogen levels in peripheral blood.8 while circulating estrogen levels are commonly low in postmenopausal women, several studies showed strikingly high levels of estrone, estrone sulfate, and estradiol in breast tumor tissue.911 therefore, inhibition of aromatase results in further reduction of estrogens in circulation and local tissue in postmenopausal women. due to a large amount of aromatase substrate in the ovaries of premenopausal women and exquisite sensitivity of ovarian aromatase promoter to gonadotropins, ais are not effective in reducing estrogen production in premenopausal women, unless concurrent ovarian suppression is administered. inhibition of aromatase in premenopausal women initially results in transient reduction of estrogens, which activates hypothalamus and pituitary axis. this results in gonadotropin secretion that stimulates ovaries, causing subsequent rise in estrogen levels.12 based on this observation, ais have also been used in the stimulation protocols for ovulation induction in premenopausal breast cancer survivors to preserve fertility. after a short - term follow - up, the combination of ai and gonadotropins appeared to be safe in breast cancer patients and results in lower peak estradiol levels compared to standard in vitro fertilization. currently, the combination of ai and gonadotropin is considered as a preferred ovarian induction protocol for women with breast cancer.13 given the fact that ais can stimulate ovaries via induction of gonadotropin secretion, the use of ais in perimenopausal women can result in ovarian reactivation which can be detrimental in breast cancer patients. for premenopausal women who became amenorrheic after chemotherapy, smith reported a cohort of 45 women who received adjuvant ais after chemotherapy - induced amenorrhea of 6 months with biochemical evidence of ovarian suppression. alarmingly, 27% of women in this cohort resumed ovarian function ; one woman became pregnant and ten women restarted menstrual period. therefore, careful determination of menopausal status and serial hormone level monitoring, particularly follicle - stimulating hormone and estradiol levels, are critical to ensure the safe use for this class of drug. currently, ais are commonly used as a first line treatment for postmenopausal women with metastatic hormone receptor - positive breast cancer. this is based on the results of several phase iii trials that demonstrated superiority of ais compared to tamoxifen with regards to response rate, median time to progression, and clinical benefit rate.1519 the largest trial in this setting enrolled 916 postmenopausal women and randomized patients to receive either tamoxifen or letrozole. this trial demonstrated a significant improvement in time to progression with letrozole compared to tamoxifen (42 versus [vs ] 23 weeks, hazard ratio [hr ] 0.70, p=0.0001).20 more recently, the strategy to combine ais with selective er down regulator, fulvestrant, was investigated. this was based on the preclinical data that demonstrated more durable tumor control with complete blockade of er signaling pathway, using ai in combination with fulvestrant compared to single agent ai.21 these promising preclinical data have led to the conduction of two phase iii trials, the fulvestrant and anastrozole combination therapy (fact) and southwestern oncology group (swog) s226 trials. the fact trial22 is a phase iii trial that randomized 514 postmenopausal women to receive either a single agent anastrozole or the combination of fulvestrant and anastrozole. disappointingly, this trial showed no difference in median time to progression, 10.8 vs 10.2 months (hr 0.99, p=0.91) and overall survival (os) was 37.8 vs 38.2 months (hr 1.0, p=1.00). in contrast, the swog s0226 trial,23 which enrolled a total of 707 patients and has a similar design, demonstrated not only a significant improvement in progression - free survival (13.5 vs 15 months, hr 0.8, p=0.007) but also os benefit (41.3 vs 47.7 months, hr 0.81, p=0.05). comparing the swog s0226 and fact trials, there were more patients who presented with de novo metastasis without prior adjuvant endocrine therapy in the swog s0226 trial (32.2% vs 59.7%). furthermore, a subset analysis in the swog s0226 trial showed that patients who did not receive prior endocrine therapy appeared to have more benefit from the combination of fulvestrant and anastrozole (hr 0.74, 95% confidence interval [ci ] 0.590.92 vs hr 0.89, ci 0.691.15). in addition, the study of faslodex versus exemestane with / without arimidex (sofea) trial,24 which evaluated the combination of fulvestrant and anastrozole vs fulvestrant alone or exemestane alone in patients with endocrine resistance, did not show the benefit of the combination over single agent fulvestrant or exemestane in this group of patients (median progression - free survival 4.4 vs 4.8 vs 3.4 months, respectively, p=0.98). taken together, these data suggest that the combination of fulvestrant and ai may be beneficial in treatment of nave patients who present with de novo metastasis. there appears to be no significant benefit of combining fulvestrant and ai in patients with endocrine resistance who previously received endocrine therapy. ais are considered as standard of care for adjuvant treatment of postmenopausal women with hormone receptor - positive breast cancer.25 this is based on several phase iii clinical trials that compared the benefit of ais to tamoxifen in the adjuvant setting. several strategies were investigated in these phase iii trials, including upfront ais, switching to ais after 23 years of tamoxifen, and extended ai after completion of tamoxifen for 5 years. across the board, ais were shown to be superior to tamoxifen.6 for the upfront strategy, both anastrozole and letrozole for 5 years have been shown to significantly improve disease - free survival (dfs) compared to 5 years of tamoxifen in the arimidex, tamoxifen, alone or in combination (atac)26 and breast international group (big) 19827 trials, respectively.. the lack of os benefit may be due to crossover in considerable numbers of patients after these trials were reported. nevertheless, ais appeared to be better tolerated with less treatment - related serious adverse events compared to tamoxifen (or 0.57, p 5% risk of breast cancer in 10 years based on the tyrer - cuzick model. after a median follow - up of 5 years, there was significantly less invasive breast cancer in the anastrozole arm compared to the placebo arm (32 vs 64 cases, hr 0.5, p=0.001). with these results, ais are currently considered as one of the chemoprevention options for postmenopausal women with high risk of breast cancer. in general, ais are quite well - tolerated compared to cytotoxic chemotherapy. in contrast to tamoxifen, ais are not associated with increased risk of thromboembolism and endometrial cancer. common side effects of ais include vasomotor symptoms, musculoskeletal symptoms, and bone loss. these medications include venlafaxine, gabapentin, and clonidine.44 musculoskeletal symptoms have been reported in up to 50% of women taking ais and up to 20% of these patients discontinue the treatment due to this side effect.45 in a small pilot study of 29 patients, duloxetine appeared to help alleviate these musculoskeletal symptoms.46 there is an ongoing phase iii trial evaluating duloxetine for treatment of ai - associated musculoskeletal symptoms (swog s1202). for bone loss, currently, denosumab (prolia) is approved by the us food and drug administration specifically for ai - induced bone loss. currently, ais are commonly used as a first line treatment for postmenopausal women with metastatic hormone receptor - positive breast cancer. this is based on the results of several phase iii trials that demonstrated superiority of ais compared to tamoxifen with regards to response rate, median time to progression, and clinical benefit rate.1519 the largest trial in this setting enrolled 916 postmenopausal women and randomized patients to receive either tamoxifen or letrozole. this trial demonstrated a significant improvement in time to progression with letrozole compared to tamoxifen (42 versus [vs ] 23 weeks, hazard ratio [hr ] 0.70, p=0.0001).20 more recently, the strategy to combine ais with selective er down regulator, fulvestrant, was investigated. this was based on the preclinical data that demonstrated more durable tumor control with complete blockade of er signaling pathway, using ai in combination with fulvestrant compared to single agent ai.21 these promising preclinical data have led to the conduction of two phase iii trials, the fulvestrant and anastrozole combination therapy (fact) and southwestern oncology group (swog) s226 trials. the fact trial22 is a phase iii trial that randomized 514 postmenopausal women to receive either a single agent anastrozole or the combination of fulvestrant and anastrozole. disappointingly, this trial showed no difference in median time to progression, 10.8 vs 10.2 months (hr 0.99, p=0.91) and overall survival (os) was 37.8 vs 38.2 months (hr 1.0, p=1.00). in contrast, the swog s0226 trial,23 which enrolled a total of 707 patients and has a similar design, demonstrated not only a significant improvement in progression - free survival (13.5 vs 15 months, hr 0.8, p=0.007) but also os benefit (41.3 vs 47.7 months, hr 0.81, p=0.05). comparing the swog s0226 and fact trials, there were more patients who presented with de novo metastasis without prior adjuvant endocrine therapy in the swog s0226 trial (32.2% vs 59.7%). furthermore, a subset analysis in the swog s0226 trial showed that patients who did not receive prior endocrine therapy appeared to have more benefit from the combination of fulvestrant and anastrozole (hr 0.74, 95% confidence interval [ci ] 0.590.92 vs hr 0.89, ci 0.691.15). in addition, the study of faslodex versus exemestane with / without arimidex (sofea) trial,24 which evaluated the combination of fulvestrant and anastrozole vs fulvestrant alone or exemestane alone in patients with endocrine resistance, did not show the benefit of the combination over single agent fulvestrant or exemestane in this group of patients (median progression - free survival 4.4 vs 4.8 vs 3.4 months, respectively, p=0.98). taken together, these data suggest that the combination of fulvestrant and ai may be beneficial in treatment of nave patients who present with de novo metastasis. there appears to be no significant benefit of combining fulvestrant and ai in patients with endocrine resistance who previously received endocrine therapy. ais are considered as standard of care for adjuvant treatment of postmenopausal women with hormone receptor - positive breast cancer.25 this is based on several phase iii clinical trials that compared the benefit of ais to tamoxifen in the adjuvant setting. several strategies were investigated in these phase iii trials, including upfront ais, switching to ais after 23 years of tamoxifen, and extended ai after completion of tamoxifen for 5 years. across the board, ais were shown to be superior to tamoxifen.6 for the upfront strategy, both anastrozole and letrozole for 5 years have been shown to significantly improve disease - free survival (dfs) compared to 5 years of tamoxifen in the arimidex, tamoxifen, alone or in combination (atac)26 and breast international group (big) 19827 trials, respectively. the lack of os benefit may be due to crossover in considerable numbers of patients after these trials were reported. nevertheless, ais appeared to be better tolerated with less treatment - related serious adverse events compared to tamoxifen (or 0.57, p 5% risk of breast cancer in 10 years based on the tyrer - cuzick model. after a median follow - up of 5 years, there was significantly less invasive breast cancer in the anastrozole arm compared to the placebo arm (32 vs 64 cases, hr 0.5, p=0.001). with these results, ais are currently considered as one of the chemoprevention options for postmenopausal women with high risk of breast cancer. in contrast to tamoxifen, ais are not associated with increased risk of thromboembolism and endometrial cancer. common side effects of ais include vasomotor symptoms, musculoskeletal symptoms, and bone loss. these medications include venlafaxine, gabapentin, and clonidine.44 musculoskeletal symptoms have been reported in up to 50% of women taking ais and up to 20% of these patients discontinue the treatment due to this side effect.45 in a small pilot study of 29 patients, duloxetine appeared to help alleviate these musculoskeletal symptoms.46 there is an ongoing phase iii trial evaluating duloxetine for treatment of ai - associated musculoskeletal symptoms (swog s1202). for bone loss, currently, denosumab (prolia) is approved by the us food and drug administration specifically for ai - induced bone loss. these medications can be used in various spectrums of breast disease ranging from chemopreventative setting, adjuvant setting, to metastatic setting. in general, ais are quite well tolerated with some unique side effects that can often be managed. in particular, long - term use of ais has been shown to be associated with risks of osteoporosis and fracture. since these conditions are preventable, careful monitoring of bone density in patients taking ais and early intervention is critical.	aromatase is an enzyme that converts testosterones to estrogens. inhibition of this enzyme has been shown to have several clinical utilities in breast cancer. currently, there are three aromatase inhibitors (ais) in clinical use, namely anastrozole, letrozole, and exemestane. ais have been used in various clinical settings for breast cancer, ranging from chemoprevention in breast cancer to treating breast cancer in both early stage in the adjuvant setting and metastatic disease. this article reviews mechanism of action, ai classification, and clinical utilities of ais in various clinical settings in the context of breast cancer.
the introduction of indirect composite resins has primarily made to compensate for the drawbacks of direct composite resins. among the proposed advantages are the potential for achieving positive interproximal contacts, less polymerization shrinkage, and better marginal sealing because of the polymerization process that takes place in a laboratory setting. factors which directly affect the physical and mechanical properties of composite resins are chemical composition of the materials, the organic or inorganic portion, type, morphology, and filler content. thickness of the increment inserted into the cavity, intensity, and irradiation time, light spectrum and distance of the tip of the light - curing unit are important factors which affect the polymerization pattern, exposing the composite and adhesive resin to different media and material affect the stability of resin and make them more vulnerable to degradation. in order to improve the mechanical and physical properties of the composites, including hardness, elastic modulus, flexural strength, stiffness, hygroscopic expansion, and solubility, extraoral cure (postcure) extraoral treatment happening under light, heat, and pressure is shown to increase the degree of conversion of dental composites. the findings have supported the alternative curing methods, such as ultraviolet radiation curing, microwave curing, electron - beam curing, and infrared curing methods. they have demonstrated the superiority of microwave curing over the thermal curing. since, restorative materials are exposed to saliva in oral cavity, some researches focus on how aging or water storage affects mechanical properties of direct and indirect composites. the reason behind the chemical degradation of composite resins is mainly because of the diffusion of molecules and ions of nonreacted resin monomers. in fact, once composite resins are put into solutions, the polymer chains will absorb the water, contributing to a swelling which may in turn decrease the bond strength of the polymer chains. the quality and stability of the silane coupling agent are important in minimizing the deterioration of the bond between the filler and polymer and the amount of water sorption. some researches focused on the effects of postcuring (using autoclave and microwave for postcure treatment and postirradiation dry aging at different periods of time that resulted in increased microhardness of composite resin). some articles studied the effect of water storage (storing specimens in distilled water for 24 h and 30 days that caused decreasing the amount of flexural strength and wet - aging of denture base polymers reinforced with short glass fibers that showed less water sorption and solubility of the reinforced denture base polymer) on physical properties of composite resins, while the objective of this study was to evaluate the influence of two postcuring methods and different water storage periods on the microhardness (vhn) strength of direct and indirect composite resins. since, the present study focused on the effect of postcuring and wet - aging on microhardness of composite resin the evaluation of postcuring effect was considered by using both the direct and indirect composite resins. a total of 99 specimens were fabricated as disks in brass mould [(6.5 2.5 mm) [figure 1 ]. direct composite resin (gradia gc / gc corporation, tokyo, japan) (n = 33) and indirect composite resin (gradia gc / gc corporation, tokyo, japan) (n = 66) was inserted into split mold and then a transparent strip (jr - rand corporation, new york, usa) and a glass slide was placed on top surface until the additional amount of composite was released. although all the specimens were early cured (ec) by a halogen light - curing unit (400 - 420 mw / cm, coltolux ii, coltene / whaledent inc. a total of 33 specimens of indirect composite samples were postcured (one by one) under irradiation of 12 fluorescent lamps in laboratory light source (labolite lv - iii gc corp, japan) which scattered light from three directions over specimen, a postcuring unit which is manufactured and recommended by gc corporation, tokyo, japan for their products, and 33 remainder specimens were postcured (one by one) under irradiation of microwaves with power of 540 w for 7 min in microwave unit (mc 2002 jr, lg, korea). then, all of the samples (n = 99) were polished (by the fine diamond bur and the silicon rubber) and were stored in distilled water 37 using incubator (behdad, tehran, iran) and were divided to nine groups as follows : group 1 : ec + 1 day water storage group 2 : ec + 30 days water storage group 3 : ec + 180 days water storage group 4 : ec + microwave 7 min + 1 day water storage group 5 : ec + microwave 7 min + 30 days water storage group 6 : ec + microwave 7 min + 180 days water storage group 7 : ec + labolite 5 min + 1 day water storage group 8 : ec + labolite 5 min + 30 days water storage group 9 : ec + labolite 5 min + 180 days water storage ec = early cured, labolite or labolight = a trade name for laboratory light source the hardness tests vhn (which is reliable for testing microhardness of composite resins) were counted via a wolpert (500 gr/10 s, wolpert, darmstadt, germany) microhardness tester using a vickers diamond indenter. the indentations were performed under a 500 gr load on three random points on the top surface of each specimen. the dimensions of the indentations were measured using the measuring eyepiece of the microscope of the hardness tester, and hardness values were given from standard tables. the data of hardness were analyzed by two - way analysis of variance (anova) and tukey 's post hoc test (t - test) (p < 0.05). the statistical analysis two - way anova revealed curing technique and aging time are two influential factor (p.v. however, to ignore factor of composite type, two - way anova and post hoc tukey 's test was used. two - way anova and tukey 's post hoc test revealed high significant differences of hardness values among postcured indirect composite and ec direct composite (p = 0.0001), microwave curing technique was more effective than the other technique (p = 0.004) [table 1 ]. within this statistical analysis wet - aging decreases surface microhardness of direct and indirect composites : 24-h - stored specimens were significantly harder than 30-day - stored (p = 0.0001) and 180-day - stored (p = 0.0001), and microhardness of 180-day - stored specimens had been reduced by aging rather than 30-day - stored (p = 0.045) [table 2 ]. according to figure 2 and table 3, the micro - hardness amount of specimens post - cured by microwave is more than labolite and early - cured groups. comparison of microhardness (vhn) of all groups with different curing technique comparison of microhardness (vhn) of all groups with different aging time microhardness (vhn) of direct and indirect composite resin after different aging time the microhardness (vhn) amount of each specimen measured after post - curing or early - curing (control group) and aging microhardness test has been chosen for this experiment because it is very common and reliable technique. surface microhardness is believed to be a relevant factor indicating the mechanical strength of a resin and has a substantial correlation to the material 's rigidity. a number of indirect resin restorative system with highly improvements has been presented during the last years. currently, the main concern of modern restorative composites is focused on polymer matrix or fillers modifications to reduce polymerization shrinkage and stress as well as increasing the degree of monomers conversion and improving their general qualities. in the present study, direct and indirect composite specimens, with different curing methods and different time of water storage, these differences probably could be related to the effect of postcuring and filler content on improving hardness of the composite resins. particular postcuring methods have been promoted to increase the polymerization, such as curing in nitrogen atmosphere under heat and pressure or curing in inlay systems proposed by the manufacturer. in most restorative composite resins, postcuring above the glass transition temperature of the resin matrix can enhance the degree of cure and improve the mechanical properties of the composite. lombardo., reported that stove postcuring technique (dry) increases microhardness of composite resin, while autoclave had adverse effect on it, concluding that postcuring technique is very crucial. the postcuring process increases the microhardness, but the polymerization in the autoclave decrease microhardness. hence, exposing to moisture during postcuring probably can decrease the microhardness of the resin. in the present study exposing, the specimens to light (12 fluorescent lamp) in laboratory light source unit which is used in laboratory increased microhardness. we realized that microwave was more effective than high intensity light of laboratory light source unit in increasing the microhardness. poskus., also reported that microwave is a useful technique for postcuring of composite. they stated that microwave postcuring technique was more effectual than oven and autoclave techniques to enhance marginal adaptation and increased microhardness of filtek z250 inlay restoration. sharafeddin and ghahramani study showed that postcuring by microwave and oven techniques increases microhardness of composites resin, which apparently confirms the present study results., study also revealed the same results in which microwave curing is better than thermal curing. in the present study, the 30-day - stored specimens illustrated a significant decrease in the surface microhardness rather than 24-h - stored specimens and 180-day - stored specimens revealed a significant decrease in the surface microhardness in comparison to 24-h and 30-day - stored specimens. therefore, the major cause of decrease in surface microhardness of the composites after water storage may be water sorption. when composite resin is immersed in water, there is a quick separation of uncured monomer or oligomers which presumably completes within several days. most resins entailed 7 days to reach stability and about 4 days to show the preponderance of their expansion. concurrently, water is diffused in to the polymer matrix of composites, swells the polymer, and fills the space between the main chains and crosslinks, as well as occupying the microvoids created during the polymerization phenomenon. time or aging is expected to be a significant factor associated with the amount of water sorption ; moreover, composites which absorb more water, will show more decreased surface microhardness. carreiro., evaluated the effect of aging in distilled water on the hardness and compressive strength of a direct composite resin and three indirect composites. the results showed that aging in water reduced the hardness for all composites, proving the results of present study., study described the effect of 6 months of aging in water on the surface roughness and surface / subsurface hardness of two microhybrid resin composites. eventually, the results revealed that the 6-month period of storage in water presented a significant softening effect on the surfaces of the composites. hahnel., investigated the aging manners of dental composites regarding surface roughness and vickers hardness. this report could also substantiate the results that we obtained in our present investigation. in oliveira they concluded that storage in distilled water had not any considerable effect regarding the evaluated properties. comparing to the results of present study, this difference can be due to the short time of storage, since it has been proved that water sorption is a slow process and more time is needed to measure the effects of this process. within the limitation of this in vitro study, so, postcuring with microwave and labolite must be effective for increasing the degree of conversion which results in long - lasting composite restorations. additional clinical studies are necessary to analyze the success of two different postcuring techniques and aging of restoration in oral cavity. the postcuring process by microwave was more effective than laboratory light source in increasing the surface microhardness.	background : although composite restorations are really valuable for esthetic zones, they have shown less longevity rather than amalgam restorations. since it may be related to the method used for curing the composite, postcuring could increase the degree of conversion and result in more long - lasting composite restorations. this study was planned to evaluate the effect of two different postcuring techniques on microhardness of indirect composite resin after wet - aging and comparing them with the direct type.materials and methods : in this experimental study, 99 composite disk - shaped (6.5 2.5 mm) specimens of composite (gradia gc, japan) were prepared in split mold. the indirect composite specimens were postcured by laboratory light source (labolite lv - iii gc corp, japan) or microwave unit (mc 2002 jr, lg, korea). then, the aging procedure was done for 24 h, 30 and 180 days in distilled water. the vicker 's hardness test (vhn) on surface of specmens was measured by wolpert microhardness tester and the data were analyzed by the two - way analysis of variance (anova) and tukey 's post hoc tests. (p 0.05).results : the statistical analysis revealed that surface microhardness of postcured composite by microwave and laboratory light source was more than that of direct composite (p = 0.0001) and postcuring by microwave was more effective than postcuring by laboratory light source (p = 0.004). the 30 days stored composite demonstrated significant decrease of vhn compared with the 24-h stored samples (p = 0.0001), with a more significant vhn decrease after 180 days of aging (p = 0.045).conclusion : postcuring increased the surface microhardness and aging reduced the surface microhardness of indirect composite.
virus capsids containing hundreds of subunits have evolved to assemble with high fidelity in a short period of time. in many cases, capsid formation is sufficiently robust that, given the right conditions, purified capsid proteins spontaneously assemble into icosahedral virus - like particles. despite a large effort, the geometric control shown in forming a virus capsid far exceeds our ability to control assembly in man - made chemical systems. thus, understanding virus assembly at a molecular level will not only increase our knowledge of a process of great biological and medical importance, but it will also help to develop new self - assembly strategies for materials science. capsid assembly reactions necessarily have a single starting point of bulk subunit and a single ending point of complete capsid. the number of possible intermediates grows combinatorially from both ends of the assembly landscape. however, the concentrations of almost all the intermediates are exceedingly low during assembly. a small number of key intermediates, which may lead to kinetic bottlenecks, are anticipated at both ends of the assembly reaction, due to nucleation at the beginning and capsid closure at the end. the early intermediates are expected to be small fragments of the capsid while the late ones are expected to be almost complete capsids that are missing just a few subunits. recent experiments have begun to identify early intermediates ; however, whether late - assembly bottlenecks exist has remained unknown because it has not been possible to detect and identify them. the assembly of hepatitis b virus (hbv) is of particular interest because it is a devastating pathogen and because it is an attractive target for the development of new assembly - directed antiviral molecules. around 360 million people suffer from chronic hbv infection, which kills around 600,000 people annually. hbv self - assembly is usually studied with the core protein assembly domain, a 149-residue protein, cp149. the building block for the hbv capsid is the cp149 dimer. under the right conditions, the dimers spontaneously assemble to yield icosahedral capsids with 90 dimers (in a t = 3 structure) and 120 dimers (in t = 4). icosahedral capsids are usually described using a triangulation number (t), which gives the number of monomers (60 t) in the capsid. the interdimer association energy for hbv is only about 15 kj / mol at physiological temperature and ionic strength, but this supports formation of globally stable capsids because cp149 dimers are tetravalent. a stronger association energy, which can be contrived by high salt assembly, results in kinetic trapping of intermediates on the assembly pathway because the high frequency of nucleation events and minimal dissociation of partial capsids deplete free subunits. in this work we analyze the trapped intermediates by charge detection mass spectrometry (cdms) and cryo - electron microscopy (cryo - em). these two techniques are complementary : cdms provides information about the number of dimers in the trapped intermediates, and class averages from cryo - em provide information about their structures. over the past few years, mass spectrometry (ms) has emerged as a powerful tool to investigate the composition of biological assemblies including viruses. stockley, ashcroft, and co - workers have used ms to probe the early steps in the assembly of the ms2 bacteriophage capsid, and heck and collaborators have investigated the early stages of hbv capsid assembly. in conventional ms the mass - to - charge ratio (m / z) spectrum is measured, and for large multiply charged ions the charge must be deduced from the charge state ensemble to determine the mass. this approach starts to become problematic for masses around 100 kda. though the m / z peaks are usually still resolved in this range, they broaden and shift due to salt adduction and complex formation. the m / z peaks often do not shift by the same amount, making it difficult to assign the charge state. in some cases, the charge state resolution can be improved by purification, or the mass can be determined because the analyte identity is known. however, as the mass and heterogeneity increase, the problems are compounded until it is no longer possible to determine the mass from the m / z spectrum. here we have used charge detection mass spectrometry (cdms), a single - particle technique where the mass is directly determined from m / z and z measurements for each ion. with this approach, masses can be measured for heterogeneous mixtures of large ions that resist analysis by conventional ms. cdms was first used to measure the masses of micrometer - sized metal particles in 1960. the application of cdms to electrospray ions was described by fuerstenau and benner in 1995, and the use of cdms to measure the masses of intact viruses was reported in 2001. while groundbreaking, the mass resolution achieved in these early studies was rather poor (the peak for rice yellow mottle virus with a mass of 6.5 mda had a full width at half - maximum of 12 mda). we have described a number of key improvements to cdms that have led to large gains in the mass resolution and the limit of detection. without these improvements, mass spectra were measured using a home - built charge detection mass spectrometer that is described in detail elsewhere. they are separated from the ambient gas flow by three differentially pumped regions incorporating an rf ion funnel, an rf hexapole, and an rf quadrupole. ions are extracted from the quadrupole, accelerated through a 100 v potential, and focused into the entrance of a dual hemispherical energy analyzer. the energy - selected ions are then focused into a modified cone trap that contains the charge detection cylinder. ions oscillate in the trap, passing back and forth through the charge detector, for 129 ms. a charge - sensitive preamplifier incorporating a liquid nitrogen - cooled fet detects the image charge. the amplified signal is digitized and transmitted to a computer where the signals are analyzed using a fast fourier transform (fft). the fundamental frequency from the fft is used to derive the m / z of the ion and the magnitude of the fft is used to obtain its charge (z). the masses determined for each ion are then binned to yield the mass spectrum. figure s1 in supporting information (si) shows an example of the charge detection signal and its fft. the loss of charge usually results in a sudden change in the ion s oscillation frequency. in these cases, the measured masses are systematically overestimated by around 1% due to the presence of adducts (the addition of small molecules and counterions to the virus capsid) and imperfect calibration of the m / z and z measurements. the relationship between the oscillation frequency of an ion in the trap and the m / z is determined from simion simulations and the charge is calibrated by introducing test charges, ranging from approximately 150010000 elementary charges (e), through a capacitor. though the calibration is performed with high charges, we have checked that it is valid down to charges as low as 7 e by measuring the m / z spectrum of cytochrome c and ubiquitin with cdms. in this m / z range (100 e) and they are trapped for a long time, the chance of a false positive is essentially zero. assembly of 40 m (1.3 mg / ml) cp149 dimer in 50 mm hepes ph 7.5, was induced by addition of nacl to 1 m final concentration. the resulting solution was allowed to equilibrate for up to 48 h at room temperature. a 200 l aliquot of the assembly reaction was loaded onto a continuous 1040% (w / v) sucrose gradient, containing 300 mm nacl in 50 mm hepes and centrifuged for 5 h at 200000 g. for the cryo - electron microscopy images presented here, the protein layer above the t = 4 particle layer, including intermediate and t = 3 particles, was removed from the gradient, dialyzed into 300 mm nh4oac and concentrated to a final concentration of 3.7 mg / ml protein. for cryo - em specimen preparation, 4 l of purified hbv sample from the sucrose gradient was applied to a glow - discharged carbon - coated 300 cu mesh grid. the sample was left on the carbon film for 25 s, blotted for 4 s, and vitrified by plunge - freezing into liquid ethane using an fei vitrobot. the frozen - hydrated samples were visualized using a jeol-3200 fs electron microscope (em) equipped with a gatan 626 cryotransfer system. the em was operated at 300 kv with an in - column zero - loss energy filter using a slit width of 20 ev to improve image contrast. grids were viewed under low - dose condition (1520 e /) at a nominal magnification of 40000 (corresponding to 3.26 / pixel). images were recorded on a gatan 4k 4k ccd camera at a defocus range of 2.0 to 4.0 m. mass spectrum measured by cdms for hbv capsids assembled under aggressive conditions (1 m nacl) and dialyzed into 300 mm nh4oac. the mass spectrum is a histogram obtained by binning the measured masses for 5893 individual ions with 20 kda bins. after binning, the histograms were smoothed with a five point savitsky - golay algorithm. the red line shows an expanded view of the region between the t = 3 and t = 4 peaks. particles were semi - manually selected using a 200 200 pixel box, centered using eman2, and then normalized and filtered using xmipp. a pool of 5681 particles was subjected to 2-d multireference alignment and classification based on the iterative hierarchical clustering method using cl2d from xmipp. figure 1 shows mass distributions measured by cdms for hbv capsids assembled for 24 h in 1 m nacl and then dialyzed into 300 mm nh4oac for 24 h. samples were dialyzed into nh4oac because this salt is compatible with electrospray. the prominent peaks near 3 and 4 mda are due to the t = 3 and t = 4 capsids. the average number of charges on the t = 3 capsids is + 129 (ranging from 122 to 136 e at fwhm) and the average number of charges on the t = 4 capsids is + 150 (ranging from 143 to 157 e at fwhm). in addition to the t = 3 and t = 4 peaks, there are prominent features at around 3.5 and 3.7 mda due to trapped intermediates. the mass spectrum was measured at regular intervals for a week, and no changes were observed, indicating that the observed intermediates are stable in 300 mm nh4oac. similar measurements were made under milder assembly conditions (0.3 m nacl followed by dialysis into 100 mm nh4oac). these mass spectra also show the peaks at 3.5 mda and 3.7 mda, though with significantly lower abundance. an example is shown in figure s2 of si. with the milder conditions, most of the trapped intermediates annealed over the course of a week, presumably by a process where some species dissociate to provide free subunits for the remainder to be completed. an example of a spectrum dominated by complete t = 3 and t = 4 capsids is shown in figure s3 of si. the points are the measured mass spectrum and the red curve is a least - squares fit to the points using the model described in the text. the blue histogram shows the intensities of the intermediates that provide the best fit to the measured spectrum. the mass resolution achieved in these measurements is insufficient to resolve an intermediate with a specific number of dimers from its immediate neighbors. the peak resulting from a single species is expected to be gaussian with a width dictated by the experimental resolution (which is determined by the uncertainty in the charge and m / z measurements). to obtain better information about the size of the trapped intermediates, a gaussian function was centered and fixed at the mass of all possible intermediates with an integral number of dimers. initially, the intensities of all the intermediates were set to the same value, and then they were optimized by monte carlo sampling to obtain the best fit to the measured spectrum using a least - squares criterion. the points are the measured spectrum, and the red curve shows the best fit. the blue histogram in figure 2 shows the optimized gaussian intensities for each intermediate. note that many of the possible intermediates have intensities that are near zero. it is possible to draw a number of conclusions from the fit shown in figure 2. first, the peak at around 3.5 mda is mainly due to trapped intermediates of 104 and 105 dimers. second, the 3.7 mda peak is attributed mainly to intermediates of 110 and 111 dimers. third, the simulations shows that there is a low mass tail on the t = 4 peak, which corresponds mainly to an intermediate with 117 dimers with a smaller component at 118. other spectra that we have analyzed lead to similar results, though in some of them the 118-dimer intensity is larger. in addition to the 104/105, 110/111, and 117/118 features discussed above, there are peaks in the histogram at 107 and 114 dimers. these are not associated with clear peaks in the mass distribution ; thus, their assignments are not as reliable as the features discussed above. in some of the other spectra we have analyzed, the peak at 107 dimers shifts to 108 ; however, the 114 peak does not move. (a) hbv capsids separated on a sucrose gradient showing the extracted fraction (red brace) used for cryo - em analysis. (b) a representative cryo - em micrograph of a frozen - hydrated hbv specimen. (c) selected class averages show a t = 3 average, a t = 4 average, and three averages with defects, respectively. the averages in panel (c) correspond to classes 3, 7, 20, 8, and 1, respectively, in figure s2 of si. in addition to the main features discussed above, there is a small signal 10 dimer units above the t = 4 peak that may result from off - pathway assembly into metastable, non - icosahedral structures beyond the t = 4 capsid. these species were not detected in the cryo - em studies discussed below (their abundance is quite small, and they probably have low symmetry, and thus, their absence is not surprising). hbv capsids were further investigated by cryo - em, which allows visualization of particles undistorted by staining and drying artifacts. images were analyzed for evidence of incomplete or irregular particles by hierarchical class averaging. to minimize confusion of authentic t = 4 particles with intermediates during em analysis, the sample was harvested from a sucrose gradient fraction including the t = 3 band and extending to, but not including, the t = 4 band (red brace in figure 3a). this way, all intermediate species leading up to the t = 4 particles are imaged, with a minimal number of obscuring t = 4 particles. in a typical micrograph (figure 3b and figure s4 of si), along with numerous t = 3 and t = 4 particles, we observe apparently incomplete particles, arcs, and ellipsoids, as well as gemini structures suggestive of two partial capsids. to overcome the noise inherent in cryo - em we subjected 5681 images (excluding arcs) to hierarchical class averaging. we started with four classes (figure s5a of si) that were progressively split to provide greater definition of the characteristics of a given group of images. a hallmark of a good clustering structure is that, as classes are iteratively refined, the number of images switching between classes decreases. at 4, 8, and 16 classes, classification was very stable. at the final level of classification (28 classes, figure s5b of si), however, a large portion of images repeatedly switched classes during iteration, indicating that some classes were redundant and that few additional features would be observed by increasing the number of classes. fifteen of the 28 final classes (3119 images) correspond to t = 3 particles based on their 32 nm diameter. six of 28 classes (1293 images) appear to be intact t = 4 particles. both the t = 3 and t = 4 averages have well - defined internal features. the remaining seven classes (1269 images) show evidence of significant defects. they all show irregularities in the density of the protein ring outlining the averaged particle, suggesting a missing wedge of protein (see the red dashed lines in figure 3c). they are slightly elliptical and inconsistent with an icosahedral particle (see figure s5b of si, numbers 0, 1, 15, 18, and 20). we did not attempt further analysis to obtain more detailed structural information. the broad distribution of species present in the mass range below t = 4 and the paucity of particles in any one class would make particle selection and reconstruction very challenging. in a separate experiment we selected a sucrose gradient fraction with less mass than the t = 3, but we did not find any incomplete t = 3 capsids. this is consistent with the cdms results where only incomplete t = 4 capsids were found. a control 2-d cryo - em experiment verified that capsids with missing density were a function of assembly conditions and not an artifact of the classification scheme. hbv capsids were assembled for 24 h in a low salt buffer (0.15 m nacl) to produce full capsids. focusing on t = 4 capsids to identify incomplete particles, we selected 8064 images and used the classification scheme described above. the resulting class averages are consistent with intact t = 4 capsids viewed from different orientations (figure s6 of si). the cdms and cryo - em results presented here all indicate that high - molecular weight intermediates can be trapped by assembly in high salt conditions where cp149-cp149 association is strong ; a strong association energy is predicted by theory to trap intermediates because the high frequency of nucleation events depletes free subunits that are required to complete a nascent particle. the prominent trapped intermediates observed in the cdms spectra must persist because these species are resistant to dimer addition and loss. the most likely explanation for this behavior is that they have a lower free energy than their neighbors. the cryo - em measurements support this view. if we had observed aberrant structures in the cryo - em class averages, they would probably be off the assembly pathway. however, we found incomplete t = 4 capsids, which indicates that they are on - pathway. many paths and intermediates are possible ; it is surprising we see so few intermediates persist. the masses of the partial capsids along with the missing density in the cryo - em images provide a basis for developing structural models for the prominent trapped intermediates. theory suggests constraints consistent with the cryo - em data and with persistent, on - path, metastable intermediates ; their structures should be an incomplete capsid with one contiguous hole, edges of this hole should have dimers that are in high - affinity sites, and addition of dimers to this hole should be to low - affinity contacts. these rules provide the basis for a non - icosahedral particle that is more stable than complexes with one more or one less dimer. the model structures shown in the figure are based on coordinates for hbv t = 4 from ref (46) and are displayed with ucsf chimera. a plausible 117-mer (figure 4a) lacks a trimer of dimers, such as the three dimers central to a facet, leading to a roughly circular hole satisfying the requirements described above ; the dimers at the edge of the hole are bound to three other dimers, and if another dimer is added, it can only bind to two dimers. a likely metastable 111-mer (figure 4b) has a roughly triangular hole corresponding to the loss of a full facet. a possible 110-mer (figure 4c) is missing the five dimers around a five - fold axis plus the five surrounding dimers resulting in a roughly circular hole. a metastable 104-mer complex can be modeled by extending the hole in the 111-mer to two icosahedral facets (figure 4e). viewed from the side, these models appear slightly elliptical and could easily result in cryo - em projections that have relative weaknesses in density (e.g., figure 4f). structures that satisfy the stability rules outlined above can only be found for some of the possible intermediates, in particular, for the 104-mer and above ; structures with all the dimers in high affinity sites can only be found for 104, 106, 108, 110, 111, 113, 115, and 117. of these we unambiguously detect 104, 110, 111, and 117, which are notably symmetric. a structure with 108 dimers that satisfies the stability rules is missing the six dimers around a two - fold axis, plus the surrounding six dimers. this structure (figure 4d), is closely related to the 104-mer. while we do not see a strong peak for the 108-mer in figure 3, there are peaks in the histogram at 107/108 which could be due to this species. the stability rules are based on counting the number of dimer contacts while keeping the capsid rigid. rigorous optimization of the energies of possible intermediates may better explain why some are apparently preferred and others are not. the most prominent trapped intermediates for the t = 4 capsid appear to be associated with missing icosahedral facets. the intermediates with 104, 111, and 117 dimers are consistent with one assembly path with multiple metastable pause points. however, the 110-mer, missing a pentamer of dimers and its surrounding five dimers, appears to be on a different path. models of possible structures for the trapped intermediates in hbv t = 4 assembly. t = 4 capsids missing (a) 3 ; (b) 9 ; (c) 10 ; (d) 12 ; and (e) 16 cp149 dimers. (f) shows a side view of the t = 4 capsid missing 16 dimers, illustrating its slightly elliptical appearance. there are two classes of dimers in a hbv t = 4 capsid : blue dimers extend from pentameric to hexameric vertices, and purple dimers extend between hexameric vertices. in all of the proposed structures, the dimers surrounding the hole are in contact with at least three other dimers. finally, trapped intermediates were not observed for the t = 3 capsid in either the mass spectra or the cryo - em measurements. the different geometries of the t = 3 and t = 4 capsids result in differences in the species that satisfy the stability rules. however, we have not been able to deduce an unequivocal explanation for the lack of t = 3 intermediates on the basis of the currently available literature. late intermediates for the assembly of the hbv t = 4 capsid are kinetically trapped by assembly under high salt conditions. cryo - em measurements indicate that the intermediates are incomplete t = 4 capsids. the detection of incomplete capsids rather than aberrant structures suggests that they are on - path intermediates. from their stability and kinetic accessibility the prominent intermediate with 104 dimers is assigned to an icosahedron missing a two neighboring facets. the intermediate with 111 dimers is attributed to an icosahedron missing a single facet, and the 117-dimer species is assigned to a capsid missing a ring of three dimers in the center of a facet. though assembly conditions used in our experiments are not physiological, we detected intermediates that are kinetically accessible, thermodynamically stable and that may help define the paths of hbv assembly.	the assembly of hundreds of identical proteins into an icosahedral virus capsid is a remarkable feat of molecular engineering. how this occurs is poorly understood. key intermediates have been anticipated at the end of the assembly reaction, but it has not been possible to detect them. in this work we have used charge detection mass spectrometry to identify trapped intermediates from late in the assembly of the hepatitis b virus t = 4 capsid, a complex of 120 protein dimers. prominent intermediates are found with 104/105, 110/111, and 117/118 dimers. cryo - em observations indicate the intermediates are incomplete capsids and, hence, on the assembly pathway. on the basis of their stability and kinetic accessibility we have proposed plausible structures. the prominent trapped intermediate with 104 dimers is attributed to an icosahedron missing two neighboring facets, the 111-dimer species is assigned to an icosahedron missing a single facet, and the intermediate with 117 dimers is assigned to a capsid missing a ring of three dimers in the center of a facet.
a 60-year - old man was referred to the emergency department with a 2-day history of febrile sensation, nausea, and emesis. he also complained of occasional, tolerable, left - lower quadrant abdominal pain in the past three months. he had suffered from bacterial coxitis about 10 years ago and it was aggravated to severe ankylosis in both hip joints that had not been treated. plain film radiography showed an 18.1 mm calcification in the right ureteral course, an 18.5 mm calcification in the left ureteral course, and bilateral hip joint ankylosis (fig. the patient 's initial serum creatinine level was 5.1 mg / dl and a nonenhanced computed tomography scan was checked to rule out other causes of obstructive nephropathy. the computed tomography scan revealed both ureteral stones to be in the area of both upper ureters with significant hydronephrosis (fig. 2). percutaneous nephrostomy tubes were inserted into the dilated pelvocaliceal system of both kidneys for the immediate relief of the obstructive nephropathy. after this intervention and adequate intravenous antibiotics, the patient 's symptoms subsided and his serum creatinine level was normalized. when the patient 's condition became stable, we decided to perform elective surgical management to remove the bilateral ureteral stones rather than swl because of the relatively large size of the stones. however, the patient could not be placed in the dorsal lithotomy or low lithotomy position to perform a retrograde ureteroscopy because of the severe hip joint ankylosis. in this situation, we decided to perform antegrade flexible ureteroscopy through a percutaneous trans - renal route to approach the ureteral stones. before the start of the procedure, while the patient was under general anesthesia, the patient was placed in the prone position with appropriate padding and prepared and draped in a sterile fashion. the tract was dilated by using coaxial telescopic dilators to 14 f and a second 0.035 inch safety guidewire was placed. a 12/14 f ureteral access sheath (applied medical, rancho santa margarita, ca, usa) was placed to allow for optimal visualization and safe introduction of the flexible ureteroscope (fig. we used a holmium laser machine set at an energy of 1 j and a rate of 15 hz. the fragmented stones were passed down to the bladder by irrigation and a flexible ureteroscope was advanced to the ureteral orifice to confirm that all stone fragments were removed from the ureter. following lithotripsy, a double - j stent was placed through an antegrade route and the same procedure was performed on the contralateral side simultaneously. stone fragments in the bladder were evacuated by manual bladder irrigation through a 20 f foley catheter. both double - j stents were removed through the retrograde approach by using a flexible cystoscope 2 weeks after the procedure. over the past 15 years, rigid ureteroscopy has been established as a minimally invasive modality for the treatment of ureteral calculi, and it has proven to be equivalent to swl for proximal ureteral stones and superior to swl for distal ureteral stones. recently, advances in endoscopic instruments, especially the new generation flexible ureteroscope and holmium laser lithotripter, have enabled retrograde ureteroscopy to become a first - line option for most ureteral stones and even small intrarenal stones. the combination of these instruments allows for an excellent stone - free rate with a low postoperative complication rate in the treatment of urinary calculi. however, there are some limitations of the retrograde ureteroscope in certain situations. it is hard to approach to the upper urinary tract in cases of a transplanted kidney, urinary diversion, and urethral or ureteral stenosis. in these cases, this modality enables a reliable access to the kidney and an opportunity to flush fragments down into the bladder rather than having to remove bits of stone ureteroscopically or wait for them to pass spontaneously. for these reasons, the percutaneous antegrade approach has been used in the treatment of ureteral calculi in selected cases. several studies have reported that the antegrade approach in the treatment of large impacted stones in the upper ureter is an alternative instead of retrograde ureteroscopy. antegrade removal of ureteral stones is also used when swl has failed and the retrograde approach is not suitable, especially in urinary diversions or renal transplants [2 - 4 ]. in previous studies, the conventional instruments such as a nephroscope or rigid or semirigid ureteroscope with tract dilation have mostly been used through the antegrade approach. in our case, flexible ureteroscopy allowed us to easily approach the stone through a 12/14 f ureteral access sheath with minimal tract dilation. former studies have demonstrated that the antegrade approach is a safe and effective treatment option for ureteral calculi in selected patients. however, for large or impacted stones, the ureteral injury rate and ureteral stricture rate of the antegrade approach exceed those of retrograde ureteroscopy. to prevent these complications, we need to use a smaller caliber ureteroscope and avoid ureteral mucosal injury during lithotripsy. a flexible ureteroscope, which usually has a smaller caliber than a rigid or semirigid ureteroscope, and a ureteral access sheath can reduce friction between the ureteral mucosa and ureteroscope. a laser lithotriptor, if direct thermal injury is avoided, can also reduce the risk of mechanical mucosal damage by stone fragments compared with other lithotripters. no perioperative complications were observed in our case, which was treated with a flexible ureteroscope and laser lithotripter through a ureteral access sheath. antegrade flexible ureteroscopy with laser lithotripsy is an attractive treatment option for patients who are unsuitable for retrograde ureteroscopy. the most important evolutions in our case were the use of an antegrade flexible ureteroscope with a ureteral access sheath percutaneously.	in the past several decades there has been a remarkable development of small - caliber, flexible ureteroscopes and various ancillary instruments for stone manipulation and retrieval. percutaneous antegrade ureteroscopy can be substituted in select cases for retrograde ureteroscopy. we report a case of a 60-year - old man with severe ankylosis in both hip joints who was diagnosed with bilateral ureteral stones. the patient underwent antegrade flexible ureteroscopy and laser lithotripsy. this case illustrates the role of antegrade flexible ureteroscopy combined with the holmium : yag laser as a minimally invasive, safe, and effective technique for the management of stones in a patient who can not undergo a retrograde approach.
hepler and strand defined pharmaceutical care as the responsible provision of drug therapy for the purpose of achieving definite outcomes that improve the patients quality of life by preventing and resolving drug - related problems.1 improving the quality of a patient s life is the primary objective of every health organization in the world. diagnosis and prescribing medication to patients is the role of the physicians and compounding and dispensing medication was the pharmacist s role.2 the function of the pharmacist has now changed, and they play a direct role in patients care.2,3 a better interaction between physicians and pharmacists has led to safer, more effective, and less costly drug therapies.4,5 several studies on pharmacist physician collaborations have proven that direct patient care is still exclusively in the hands of the physician and that pharmacist input in managing drug therapy is dependent on the physician.612 volume have advocated pharmaceutical care to increase patients satisfaction with pharmacist s activities, which may increase patients expectation that pharmacists will work on their behalf to assist them with their health care needs. while much is known about physician pharmacist interactions, little information has been published in the middle east region, and there are no reports originating from qatar. accordingly, this study was conducted to determine physicians perceptions and expectations of their experience with hospital pharmacists, involvement in direct patient care. this study will evaluate physicians comfort, expectations, and experience with pharmacists in a middle eastern hospital setting. pharmacist interactions, little information has been published in the middle east region, and there are no reports originating from qatar. accordingly, this study was conducted to determine physicians perceptions and expectations of their experience with hospital pharmacists, involvement in direct patient care. this study will evaluate physicians comfort, expectations, and experience with pharmacists in a middle eastern hospital setting. a cross - sectional study was conducted at hamad medical corporation (hmc) in qatar during january and march 2006. the questionnaire used was developed by smith and validated in california.13 the data collected using this questionnaire included respondent characteristics, perceptions, expectations, and experience of physicians with pharmacists at hmc. demographic information including age, gender, nationality, place of work, current position, current area of practice, country where qualification was obtained, year of qualification obtained, and interaction with pharmacists, including the purpose, was collected. hmc comprises five public hospitals (hamad general hospital [hgh ], women s hospital [wh ], rumaila hospital [rh ], al - amal hospital [aah ], and al khor hospital [akh ]) and 21 public primary health centers (phcs). a total of 500 questionnaires were distributed to the physician offices of all public hospitals and phcs in qatar. administrative assistants were requested to distribute the questionnaires to all the physicians who agreed to participate in the study during the week. the completed questionnaires were retrieved from the respective offices / centers by the administrative assistants. levels of comfort were self - reported as uncomfortable, moderately comfortable, and comfortable for each item and were numerically coded as 1, 1, and 2, respectively, for calculating the comfort index by adding all items for each individual. items on expectation and experience were self - reported on a five - point likert scale (strongly disagree, disagree, neutral, agree, and strongly agree) and numerically coded as 2, 1, 0, 1, and 2, respectively, to calculate the index on the expectations and experience of physicians with pharmacists to show the overall impact of all the items on their expectations and experiences. descriptive statistics were calculated for all the variables included in the study. the spss statistical package (version 14.0 ; spss, chicago, il) was used to analyze the results. student s t - test was applied to obtain the significant difference in mean level of indices between gender and nationality. a p value 0.05) and nationality (p > 0.05). in the past few years, efforts worldwide have increasingly turned into extending the role of the pharmacists beyond the dispensary and into a more active role of dealing with patients drug - related problems.14 this study described physicians comfort with pharmacists being involved in patient education, the use of nonprescribed medications, detecting and preventing prescription errors, and designing and monitoring pharmacotherapeutic plans, which have been similarly explored in a study done in kuwait in 2005.15 more than half of the physicians in our sample (55%) were uncomfortable with pharmacists prescribing and almost half of the participants (48%) were uncomfortable with pharmacists treating minor illnesses in our study. similar results on treating minor illness were shown in other studies.1517 a possible explanation for this finding is that general practitioners mostly believe that pharmacists do not have sufficient medical training to be able to correctly interpret a diagnosis or to participate in the clinical decision process.1821 fewer physicians in jordan were reluctant to allow pharmacists to treat minor illnesses (34%),22 which shows an increase in this trend in our study. most physicians in the study expected pharmacists to be an expert in drug therapy and act as an educator for the safe and appropriate use of medications. this is promising in the light of the evolving extended roles of the pharmacist and the emergence of concepts like pharmaceutical care and good pharmacy practice. these include educating, monitoring, and caring for patients in collaboration with other health care professionals. accordingly, pharmacists will be required to provide high - quality drug information, which leads to an increase for the need for reliable, accurate, trustworthy, and up - to - date information.23 more physicians were comfortable or moderately comfortable with pharmacists designing drug therapy and assisting patients in selecting appropriate nonprescription medications than those who were not. this is reassuring, considering the increasing number of medicines that have been and are being deregulated to over - the - counter status worldwide, which would help in reducing costs as well as physicians time.2426 moreover, this deregulation is also believed to be a step forward to strengthen the advisory role of the community pharmacist. physicians experience with pharmacists providing more clinical services, such as informing physicians about more cost - effective alternatives, problems their patients are experiencing with their medications, or providing clarification of drug - therapy objectives, was poor, which indicates that pharmacists need to be trained differently to be able to provide such services. the curricula in universities from which qatar pharmacists graduate need to be reviewed and modified toward pharmaceutical care in order to graduate more patient - oriented pharmacists. we share this study with the college of pharmacy, qatar university, and with the pharmacy technician program, college of the north atlantic - qatar. there is a need for qatar pharmacists to work more closely with physicians, thereby providing the physician with an opportunity to observe pharmacists performing clinical responsibility, which builds the physicians confidence in the pharmacists. physicians working with pharmacists have developed a collaborative approach to health care, but a lot of improvement is still required. physicians show good comfort and expectations of pharmacists in performing their duties, but this expectation was not met by their experiences. however, greater effort needs to be directed toward increasing the awareness of physicians about the importance of collaboration among health care professionals and what benefits can be reflected from this on patient quality of life and health care. less than one - half of potential respondents were included in the data, which therefore does not reflect the actual experiences of physicians and pharmacists in the hospitals. we also need to investigate pharmacists experiences and expectations of physicians to cover both parts of the relationship. less than one - half of potential respondents were included in the data, which therefore does not reflect the actual experiences of physicians and pharmacists in the hospitals. we also need to investigate pharmacists experiences and expectations of physicians to cover both parts of the relationship.	objectives : the purpose of this study was to investigate the physicians perceptions, and expectations of their experiences with the pharmacists at hamad medical corporation (hmc) in qatar.method:a cross - sectional study was conducted at hmc between january and march 2006 using a validated questionnaire. the self - administered questionnaire was distributed to 500 physicians who were working at hmc comprising hamad general hospital, women s hospital, rumaila hospital, al - amal hospital, al khor hospital, and primary health centers. the questionnaire was composed of four parts, investigating the physicians expectations, experiences, and perceptions of the pharmacists.results:a total of 205 questionnaires were completed (response rate 41%). a total of 183 physicians (89%) expected the pharmacist to educate patients about safe and appropriate use of drugs, whereas 118 (57%) expected the pharmacist to be available for health - care team consultation during bedside rounds. the indices of physicians showing how comfortable they were with pharmacists, and their expectations of pharmacists, were 61% and 65%, respectively, whereas the index on experience of physicians with pharmacists was lower (15%).conclusions : physicians were comfortable with pharmacists and had high expectations of pharmacists in performing their duties. however, physicians reported a poor experience with pharmacists, who infrequently informed them about the effectiveness of alternative drugs, patients experiencing problems with prescribed medications, and who took personal responsibility to resolve any drug - related problem.
we present the first reported case of gastropleural fistula with intramural extension into the wall of descending thoracic aorta. an 88-year - old male presented to the emergency room with several weeks of cough and fatigue as well as recent onset of melena. he had been treated unsuccessfully as an outpatient with oral antibiotics for a presumed pneumonia. past medical history was notable for endovascular prior stent - graft placement in a thoracoabdominal aneurysm, 2 years before current presentation [figure 1 ]. initial laboratory evaluation demonstrated mild leukocytosis, and initial chest radiograph demonstrated left lower lobe opacities with pleural effusion. a noncontrast computed tomography (ct) of the chest was obtained, which demonstrated air and soft tissue thickening abutting the descending thoracic aorta, wall thickening of the adjacent stomach, as well as a heterogeneous, thick - walled left pleural air and fluid containing collection with layering intermediate to high - density material [figure 2 ]. a repeat ct scan of the chest and abdomen was obtained before and after administration of intravenous (iv) contrast and after an 80 s delay. the pre - iv contrast image confirmed a large gastropleural fistula as the cause for patient 's empyema [figure 3 ] with a clearly visualized diaphragmatic defect. however, the oral contrast extended to the level of the patient 's vascular graft struts, concerning for intramural involvement [figure 4 ]. the post - iv contrast images demonstrated the extensive periaortic soft tissue thickening extending to the infrarenal abdominal aorta and displacing it anteriorly [figure 5 ]. there was also evidence of pseudoaneurysm formation below the ostium of the right renal artery and below the level of patient 's vascular stent [figure 5 ] suggestive of an infectious pseudoaneurysm formation. (a) axial noncontrast computed tomography image of the chest demonstrates air and soft tissue thickening (arrow) at the level of the aorta, in proximity to the adjacent stomach. there appears to be continuity between the gastric fundus and pleural space, with no intervening diaphragm. (b) axial computed tomography image demonstrates a heterogeneous left pleural collection (arrow) with thick - wall, air, fluid, and high - attenuation material. (a) axial computed tomography image of the chest and abdomen after administration of dilute water - soluble oral contrast demonstrating clear extravasation of oral contrast from the stomach into the left pleural space through a large defect in the gastric wall and diaphragm (arrow). (b) axial computed tomography image of the chest after administration of dilute water - soluble oral contrast demonstrating extension of the contrast from the stomach, through the pleural space, and between the struts of the aortic vascular stent (arrow). (a) axial computed tomography image of the chest and abdomen after the administration of intravenous contrast demonstrates extension of the periaortic soft tissue thickening into the abdomen (arrow). (b) axial computed tomography image of the abdomen demonstrating anterior displacement of the aorta (arrowhead). (c) axial computed tomography image of the abdomen demonstrating the contained extravasation of contrast (arrow) from the right lateral wall of the abdominal aorta. gastropleural fistulas are rare conditions that are typically seen in the setting of trauma and diaphragmatic injury or gastric perforation secondary to peptic ulcer disease and malignancy. patients who have a prior history of thoracic or upper abdominal surgery, as well as local radiation therapy, are also at risk. our patient had no known risk factors for fistula formation. however, initial noncontrast imaging demonstrated wall thickening of the gastric fundus in close approximation to a left pleural empyema, raising suspicion for fistula formation. diagnosis of a gastropleural fistula can be made with an upper gi examination, preferably with water - soluble contrast. analysis of the pleural fluid can demonstrate gastric contents or bile, which would also confirm the diagnosis. alternatively, rapid diagnosis in the acute setting can be obtained with ct after administration of dilute water - soluble oral contrast. pleural air and fluid alone can suggest the diagnosis in the appropriate clinical scenario ; these findings are less specific and are seen in the setting of an isolated empyema although in this scenario, the diaphragm is intact and the perigastric fat planes are preserved. additional imaging findings that can be found in the presence of a gastropleural fistula include hydropneumothorax and tension pneumothorax. our patient 's initial noncontrast ct demonstrated extensive air and soft tissue thickening surrounding the distal descending thoracic aorta with loss of fat planes and subtle thinning of the aortic wall. these findings are concerning for but not diagnostic of aortoenteric communication and may be found in patients who have aortitis or infection without fistula formation. these cases can be separated into primary aortoenteric fistulas versus secondary aortoenteric fistulas, which occur in patients who have undergone aortic repair and have underlying perigraft infection. this blurs the imaging distinction between infections with associated fistula versus infections without fistula. in patients with suspected aortic fistulas, precontrast and arterial phase imaging should also be obtained to evaluate for endoleak, in patients with prior aneurysm repair. oral contrast is not routinely administered given the potential for obscuration of iv contrast extravasation into the bowel lumen. in our patient, high - attenuation material within the pleural space on the initial noncontrast ct may have represented blood products or gastric contents. however, performing an additional scan with oral contrast clearly identified this to be gastric material. ideally, when the diagnosis is uncertain but an aortoenteric fistula is suspected, a triphasic ct should be obtained : initially noncontrast, followed by arterial phase iv contrast and then a late phase after administering oral contrast. imaging features that more strongly correlate with the presence of an aortoenteric fistula include extravasation of contrast into the bowel lumen, ectopic perigraft gas, and focal disruption of the aortic contour. in this case, there was soft tissue thickening and air surrounding the aorta. in addition, oral contrast could be seen extending between the struts of the aortic stent confirming the intramural involvement. other imaging findings such as perigraft fluid, loss of the intervening fat plane between bowel and aorta, and pseudoaneurysm formation can be seen in cases of infectious aortitis or perigraft infection without fistula formation. our patient did have infection and inflammation extending along the aorta, with pseudoaneurysm formation at a location distal to the fistula. the presence of these abnormalities in the setting of gi bleeding should raise suspicion for a possible aortoenteric fistula. while there have been reported cases of gastropleural and aortoenteric or aortogastric fistulas, to our knowledge, this is the first reported case of a gastropleural fistula with extension into the aortic wall. the discontinuous posterior gastric wall suggested a perforated gastric ulcer leading to gastropleural fistula formation. this was not diagnosed in the early stages, with infection and inflammation consequently spreading into the mediastinum and aortic wall at the level of the patient 's aortic graft. this unique case demonstrates that corrosive contents of a gastric perforation can erode through an intact diaphragm with spillage into the pleural space. if left untreated, these corrosive gastric contents in the pleural space can further erode into adjacent mediastinal structures such as the descending aorta, as in this case. this stresses the importance of prompt diagnosis and treatment in these life - threatening cases. on initial imaging, the fact that the fistula potentially involved three separate compartments vascular, pleural, and gi complicated the subsequent ct protocol that was utilized. to correctly identify such fistulas and their associated complications the initial contrast - enhanced phase can be determined by the suspected source of the fistula on the noncontrast imaging : if enteropleural, then oral contrast should be administered first while if aortopleural or aortoenteric, then iv contrast without oral contrast should be administered [table 1 ]. gastropleural and aortoenteric fistulas are rare complications that require prompt diagnosis and clinical intervention. understanding the ct imaging features of both of these entities is essential to pursue the appropriate ct protocol based on the patient 's prior imaging findings or presenting clinical scenario.	gastropleural fistula is a relatively rare complication that can be seen as a result of traumatic, nontraumatic, benign, and neoplastic etiologies. most commonly, these are found in patients with diaphragmatic herniation or prior thoracic surgery. aortoenteric fistulas are rare communications typically between the abdominal aorta and bowel. we present a rare case of an 88-year - old male who developed a gastropleural fistula with erosions into the wall of the descending thoracic aorta. computed tomography (ct) is a leading modality in evaluation of suspected gastropleural or aortoenteric fistulas given the quick scan time and widespread availability. prompt diagnosis is essential and requires an understanding of appropriate ct protocols and ct imaging appearance.
internal jugular phlebectasia (ijp) is a congenital fusiform dilatation of the internal jugular vein that appears as a soft, compressible mass in the neck during straining or is triggered by the valsalva maneuver. the possible differential diagnosis for the swelling could include a laryngocele, branchial cyst, cystic hygroma, cavernous hemangioma, and superior mediastinal cysts. this can affect any neck vein, especially internal jugular, external jugular, anterior jugular, superficial communicus in decreasing order. color doppler imaging confirms the diagnosis and is the gold standard. surgical treatment, usually with esthetic purposes, consists of the excision of the dilated portion of the vein or a unilateral excision of the vein. exploration and wrapping the dilated segment in an 8-mm - diameter polytetrafluoroethylene (ptfe) tube graft was done. our approach offers another surgical option to the treatment of the jugular vein phlebectasia with excellent results as it cures the patient of the swelling and at the same time does not hamper the venous drainage of the brain. a 12-year - old boy presented to us with history of a swelling appearing only on the right side of the neck on straining and coughing for the last 6 months. it was not associated with any other features like pain, change of voice, facial congestion, and difficulty in swallowing or breathing. on clinical examination, there was a soft cystic swelling over lower one - third of right side of the neck, apparent only on straining, coughing, or performing valsalva maneuver, being completely undetected otherwise [figure 1 ]. local temperature was not raised, the swelling was nontender, and it was not possible to get below the swelling. x - ray neck revealed no widening or air at the region of the mass, thus excluding laryngocele. color doppler was done which revealed internal jugular vein dilatation upon valsalva maneuver and confirmed the diagnosis of ijp. since the swelling had increased to a noticeable size upon minimal exertion, which made his parents worried, decision of surgery exploration and wrapping the dilated segment in an 8-mm - diameter ptfe tube graft was planned. under general anesthesia, an oblique incision over the anterior margin of the right sternocleidomastoid was made and the whole of the internal jugular vein was dissected out of the carotid sheath from its origin at the base of the skull to its drainage in subclavian vein [figure 2 ]. the tributaries of the internal jugular vein were ligated and divided ; the ptfe graft was cut open longitudinally and wrapped around the vein in its entire extent. head high position was given causing the vein to collapse and the cut edges of the graft were sutured to each other using 4 - 0 silk sutures on an atraumatic needle [figure 3 ]. care was taken to prevent inadvertent damage to the jugular vein or the contents of the carotid sheath. this reinforcement prevented the vein from dilating and at the same time preserved its function. during valsalva maneuver, apparent swelling seen on the right side of the neck intraoperative photograph showing dilated internal jugular vein right internal jugular vein wrapped in an 8-mm ptfe tube graft postoperative period was uneventful ; there were no surgical site complications. color doppler done after 2 weeks of surgery showed patency of the vascular channel with no evidence of thrombus. the term phlebectasia indicates abnormal outward dilatation of the vein without tortuosity and differs from the term varix, which implies dilatation plus tortuosity. it was first described by harris in 1928 and characterized by gerwig in 1952 as a fusiform or saccular dilated segment of a vein. this can affect any neck vein, especially internal jugular, external jugular, anterior jugular, superficial communicus in decreasing order of frequency. more than 100 cases of phlebectasia involving the neck veins including anterior and external jugular veins have been reported in the world literature. because there have been only sporadic reports of venous ectasia in the neck, the possible causes are gross anatomic abnormality, mechanical compression or trauma, congenital structural defects in the vein wall, or may be idiopathic. this is a benign and usually asymptomatic condition which is more common in boys ; males are twice more commonly affected than females. jugular vein phlebectasia commonly presents in childhood as a localized swelling in the anterior neck. these masses are nontender and compressible, appearing during situations in which there is an increase in intrathoracic pressure. the differential diagnosis of a neck mass is broad, but a swelling that occurs only on straining reduces the possibilities to laryngocele, inflation of pulmonary apical bullae, superior mediastinal mass, and phlebectasias. of these, the diagnosis of laryngocele is the most common. laryngoscopy directly rules out the possibility of a laryngocele, and a thoracic computed tomography (ct) scan rules out the possibility of a mediastinal cyst or tumor. ultrasonography during a valsalva maneuver easily establishes the diagnosis of jugular vein phlebectasia and this should be used as a first - line imaging test. during a valsalva maneuver, the diameter of the affected vein may increase up to 2.2 times compared with its measurements at rest. color doppler imaging confirms the presence or absence of blood flow and thrombus formation in the lumen of the vein and is the gold standard for diagnosis of jugular vein phlebectasia. generally, histopathologic studies show normal pattern. in some cases, there is a loss or disordered arrangement in the smooth muscle cells, elastic fibers, and connective tissue. in literature, those in favor of conservative management state that as it is a benign swelling and there have been no instances of serious complications, conservative approach should be adopted with close follow - up. also, ligation of the jugular vein may produce effects of venous congestion in a small subset of patients, resulting in cerebral edema. those in favor of surgical approach state that since hemodynamics in the dilated vein are markedly changed, it is prone to form intramural thrombus. secondly, the involved vein has a potential to rupture as most patients are children with poor ability of self - control and protection. also, a small cervical scar is better than a big cervical mass, and long - term emotional trauma faced by the patient goes in favor of surgical management. in most surgical cases reported, the internal jugular vein and associated veins have been ligated, with the loss of the normal venous drainage pattern on that side. whereas our approach offers another surgical option to the treatment of the jugular vein phlebectasia with excellent results, as it cured the patient of the swelling and at the same time did not hamper the venous drainage of the brain.	internal jugular phlebectasia (ijp) is a rare disease in which there is a fusiform dilatation of internal jugular vein, usually presenting as a neck mass in children. accurate diagnosis from careful history, physical examination, and radiological study can be made. we report a 12-year - old boy with history of swelling appearing on the right side of the neck only on straining, coughing, or during a valsalva maneuver. diagnosis of right ijp was made. exploration and wrapping the dilated segment in an 8-mm - diameter polytetrafluoroethylene tube graft was done. because of its rarity, this entity is frequently ignored or misdiagnosed. this case report intends to stress the importance of keeping ijp as differential diagnosis while dealing with such a swelling to avoid invasive investigations and inappropriate treatment.
nine stallions were chosen as historically important ancestors for the japanese thoroughbred breeding, according to yoshizawa. their names, birth years and birth countries are listed in table 1table 1.names, birth years and birth countries of nine stallions, whose genetic contributions were assessed by gene dropping simulationnamebirth yearbirth countryst. simon1881englandtourbillon1928francehyperion1930englandnearco1935italynasrullah1940englandroyal charger1942englandnative dancer1950usanorthern dancer1961canadasunday silence1986usa. gene dropping simulation was applied to the foal populations in 1978 and 2005. for the stallion sunday silence born in 1986, only the foal population in 2005 was analyzed. in the simulation, unique alleles were assigned to the nine stallions, and the genotypes of all descendants along the actual pedigree were generated through monte carlo simulation following mendelian segregation rules. by replicating 10,000 times of this process, the distribution of frequencies of alleles from each of the stallions was estimated. theoretically, the mean of the distribution of allele frequencies for a stallion gives the genetic contribution of the stallion to the foal population [1, 5 ]. the probability of extinction of alleles originated from a given stallion was calculated as the proportion of replicates in which both alleles from the stallion had not existed in the foal population. 1.distributions of allele frequencies of nine stallions in the foal populations of the japanese thoroughbred in 1978 and 2006. shows the distributions of allele frequencies of the nine stallions in the foal populations in 1978 and 2005. the shapes of the distributions can be classified into two typical types : one is the shape seen in st. simon, tourbillon and royal charger, in which the peak is observed at a position close to the lower boundary of allele frequency. a large part of alleles originated from these stallions have been already extinct from the foal population, or even if they are still segregating, the frequencies will be critically low. this type of distribution has the peak at a relatively high allele frequency, and the probability of allele extinction, i.e., the ordinate at the allele frequency of zero, is zero or close to zero. also historical stallions such as nasrullah were included in this type of distribution shape, meaning that the later stallions are connected to the current population through many genealogical pathways, and their genes are surely transmitted along the pathways. distributions of allele frequencies of nine stallions in the foal populations of the japanese thoroughbred in 1978 and 2006. for the four stallions, st. simon, tourbillon, nearco and royal charger, the distributions of allele frequencies shifted to the lower boundary of allele frequencies from 1978 to 2005, suggesting that most of the alleles carried by these stallions have been extinct from the japanese thoroughbred population during the past three decades. a drastic change with the opposite direction was observed in the distribution for northern dancer. this reflects the fact that the active use of his progeny for breeding in japan was in 1980s. the distribution for northern dancer in 2005 showed the peak at the allele frequency around 0.03. a peak at the same position was also observed in the distribution for native dancer. since this stallion is the maternal grandsire of northern dancer, the peak is due to alleles transmitted from native dancer to northern dancer. the distribution for native dancer in 2005 had another higher peak at the lower boundary of allele frequency, which is not observed in this peak indicates that most of the genetic contribution of native dancer to the japanese thoroughbred population is thorough his grandson northern dancer. in other words, the original contribution of native dancer, that is, the frequency of allele carried by native dancer but not transmitted to northern dancer, is negligibly low, although this stallion has been considered as an important contributor to the japanese thoroughbred population. table 2table 2.genetic contributions and probabilities (pr(lost)) of extinction of nine stallions alleles in the foal populations in 1978 and 2005.stalliongenetic contributionpr (lost)1978200519782005st. simon0.01430.01560.01730.0401tourbillon0.00460.00510.00240.0110hyperion0.01600.024000nearco0.02550.035100nasrullah0.02210.027900royal charger0.00260.00810.00040.0119native dancer0.00480.02350.00120.0001northern dancer0.00100.04580.03150sunday silence0.034701) because this stallion was born in 1986, only the foal population in 2005 was analyzed. shows the genetic contributions and the probabilities of extinction of the nine stallions alleles in the foal populations in 1978 and 2005. a notable point is that the probability of allele extinction for some stallions such as st. royal charger has increased, although the genetic contributions retained an essentially same value or even showed an increase during the period from 1978 to 2005. the increased probabilities of allele extinction suggest that the lineages connecting these ancestors to the japanese thoroughbred population had been limited during this period, and the genes transmitted from them had passed through a bottleneck of lineages. on the other hand, the probabilities of allele extinction for the three stallions, hyperion, nearco and nasrullah, were estimated to be zero in 1978 and 2005, meaning that the japanese thoroughbred population is connected to these ancestors without sever bottleneck of lineages. 1) because this stallion was born in 1986, only the foal population in 2005 was analyzed. to assess the genetic diversity of the foal population, unique alleles were assigned to all the ancestors (base animals) in the base population, and the gene dropping simulation with 10,000 replicates was carried out for the foal population in each year. average number of alleles surviving in the foal population was calculated for each year. the number of surviving alleles is a measure of genetic diversity relative to the base population. 2.the number of surviving alleles (allelic diversity) in the foal populations from 1978 to 2005, when unique alleles are assigned to the base animals. presents the number of surviving alleles (allelic diversity) in the foal population in each year. although total numbers of alleles assigned to the base animals were varied among years of the foal populations, they were within range from 4,800 to 5,500. the number of surviving alleles in the foal population in 1978 was 946.8 on average over 10,000 replicates, but the number decreased to 426.6 in 2005, which is only 8.0% of the total number of assigned alleles to the base animals. after the initial decline in 19781984, there was a period in which the number of surviving alleles reached a plateau. the later period of decline agrees with the period in which the demographic estimate of the effective population size decreased by the intensive use of a limited number of stallions for breeding. these results strongly suggest that the genetic diversity in the japanese thoroughbred population has reduced after early 1990s. in fact, analysis of microsatellite markers has indicated a progressive decline of genetic diversity in the recent japanese thoroughbred population. more detail pedigree analysis for the assessment of genetic diversity will be carried out in our succeeding studies. the number of surviving alleles (allelic diversity) in the foal populations from 1978 to 2005, when unique alleles are assigned to the base animals. throughout the present study, application of the similar analysis to the population in each country will clarify the geographical distribution of genetic resource in the entire breed.	genetic contributions of nine historically important ancestors and allelic diversity in the japanese thoroughbred population were examined by applying the gene dropping simulation to the foals produced from 1978 to 2005. full pedigree records traced to ancestors (base animals) born around 1890 were used for the simulation. alleles originated from some of the historically important ancestors were found to be at risk of future extinction, although their genetic contributions to the foal population have increased during the last three decades. the proportion of surviving alleles to the total alleles assigned to the base animals was 8.0% in the foal population in 2005, suggesting that a large part of genetic variability contained in the base animals is extinct in the current population.
the therapeutic efficacy of herbal formula originates from the combination of two or more herbal medicines and is exerted by the interaction between them. several types of herbal combinations have been used for the purpose of increasing or enhancing the therapeutic effect, reducing toxicity or the adverse effect, weakening the action of another combined herbal medicine, or causing toxicity or an adverse effect, expressed as seven types of herbal combination. their combinations are expressed as mutual reinforcement, mutual assistance, mutual restraint, mutual suppression, mutual inhibition, and single effect. besides single effect, mutual reinforcement and mutual assistance have been considered as desirable (and positive) for exhibiting the therapeutic effect (synergistic), while mutual suppression and mutual inhibition have been used due to their aggravating or negative effect (antagonistic). most reports in the literature have dealt with synergic interactions of herbal medicine such as astragali radix with rehmanniae radix to promote diabetic wound healing and glycyrrhiza glabra with solanum xanthocarpum and adhatoda vasica to stabilize mast cells. there are no reports of mutual inhibition or mutual suppression. the bud of syzygium aromaticum (sa, syn. eugenia caryophyllata) is a representative herbal medicine of mutual suppression when combined with the radix of curcuma aromatica (ca). sa has been used to treat symptoms such as vomiting, hiccough, abdominal pain, diarrhoea, lack of appetite, vaginal discharge, and weakness of legs for thousands of years in korea and china. among its efficacies, reducing vomiting or hiccough and alleviating abdominal pain or diarrhoea are known to be antagonized by the addition of ca. in other words, the therapeutic efficacy of herbal medicine can be reduced or eliminated by combining certain kind of herbal medicine, and hence chemical components of therapeutic efficacy can be also influenced at the levels of the contents or composition. the pharmacological effects of sa have been reported as antioxidant properties, antifungal activity, hypoglycaemic effect, bone - preserving effect, chemopreventive potential for lung cancer, regulation of the immune response, and antiobesity effect. these effects have mostly been investigated with its extracted essential oil. owing to the volatile characteristics of its constituents, most of chemical components in sa have been analysed for its essential oil using gc - ms. high concentrations of the volatile compounds such as eugenol, eugenol acetate, - and -caryophyllene, and 1-ethyl-3-nitrobenzene [1215 ] were detected. most quantitative analyses by hplc have focused on the determination of eugenol [1618 ] and other compounds, including gallic acid, ellagic acid, and quercetin glycoside. there is little to be found in the literature on the validated quantitative analysis of those compounds. in the present study, to evaluate the effect of ca on the constituents of sa, we quantified and then compared the amounts of three reference compounds (gallic acid, ellagic acid, and eugenol) and investigated the variance of the relative peak areas between sa and then sa with ca (sac) including the three reference compounds. regression analysis was carried out to determine whether sa and sac exhibit linear regression and the influence of the addition of ca to sa. methanol, acetonitrile, and water (hplc grade) were purchased from jt baker inc. gallic acid, ellagic acid, and eugenol were purchased from sigma - aldrich (st louis, mo, usa). the purity of all standard compounds was > 98% except for ellagic acid (95%). the herbal medicines were purchased from the herbal medicine company kwangmyungdang medicinal herbs (ulsan, korea). a voucher specimen (2012-ebm88 - 90) has been deposited in the basic herbal medicine research group of the korea institute of oriental medicine. a 100 g quantity of dried powder of each sample (sa, ca and sac) was extracted by 2 h reflux with 1000 ml distilled water at 100c. an extract was centrifuged at 3000 rpm for 10 min and the supernatant filtered through a paper filter, and then it was lyophilized to create a powder. accurately weighed sa, ca and sac samples (20 mg) were dissolved separately in 20 ml distilled water and filtered through a 0.2 m syringe filter (smartpor, woongki science, seoul, korea) prior to the hplc injections. the standard compounds were accurately weighed and then dissolved in methanol to prepare stock solutions in concentrations of 1000 g / ml. the stock solutions containing the standard compounds were diluted to produce working solutions, which were used to construct calibration curves. the hplc system comprised a shimadzu lc-20a (kyoto, japan) equipped with a solvent delivery unit (lc-20at), autosampler (sil-20ac), column oven (cto-20a), degasser (dgu-20a3), and photodiode array detector (spd - m20a). separation was performed on a gemini c18 column (4.6 250 mm, 5 m ; phenomenex, torrance, ca, usa) maintained at 40c. the mobile phase consisted of water (a) and acetonitrile (b), both containing 1% acetic acid. gradient elution of the mobile phase was applied : 570% (b) over 030 min, 70100% (b) over 3035 min, held for 5 min. the flow rate was 1.0 ml / min and the injection volume was set to 10 l. the detection wavelengths were optimized according to the maximum absorption wavelengths of standard compounds : gallic acid at 280 nm, ellagic acid at 254 nm, and eugenol at 280 nm. the within - day (intraday) and between - day (interday) precisions were calculated by analysing sample extracts added by three different concentration levels of reference compounds (low, medium, and high) with values determined as rsd. the accuracy of the method used was investigated by means of a recovery test, which involved adding three different concentration levels of reference compounds (low, medium, and high) to the samples, followed by extraction using the methods described above. the recovery was calculated as follows : recovery (%) = ((detected concentration original concentration)/spiked concentration) 100. the repeatability was evaluated by five replicates of sa solutions and the stability was determined by sa solution prepared over five consecutive days. the peaks that showed differences between sa and sac were chosen and their relative areas to total area were calculated. student 's paired t - test was used to test whether the mean of each peak differed significantly. regression analysis on the peaks that influenced the difference between samples was performed using open - source software stock solution containing the reference compounds was diluted to eight levels of concentration to construct calibration curves. the correlation coefficient (r) of compounds ranged from 0.9996 to 0.9998, showing good linearity. the values of lods and loqs calculated by the concentrations of each compound at signal - to - noise ratios of 3 and 10 were as follows : lod = 0.010.11 g / ml and loq = 0.030.36 g / ml (table 1). all the reference compounds were well detected and separated on chromatograms at their maximum absorption wavelengths using the methods described in figure 2(a). the precisions of the reference compounds were represented as rsd values, calculated as the percentage of standard deviation divided by the mean value. the rsd values of the intra - day and inter - day precisions ranged from 0.12% to 2.57% and 0.08% to 2.26%, both being 1 means the peak areas detected in sac extract were affected by the combination of ca and sa. the ratios of gallic acid, eugenol, and peak 3 were > 1.0, whereas those of 18 peaks (including ellagic acid) were 0.2, and cook 's distance was close to 0.5 for gallic acid and > 1 for ellagic acid, and eugenol, which means that these compounds could be considered as outliers (e.g., figure 6(d)). when putting those four plots together, we observed little normality and the three reference compounds (gallic acid, ellagic acid, and eugenol) were determined as possible outliers responsible for violating the constant variance. however as gallic acid, ellagic acid, and eugenol were evaluated as the most influential factors in the regression model and were abundant in samples, excluding them does not benefit to investigate the effects of adding ca on the extraction efficiency of the component in sa. the components in sa could be influenced by the combination thereof with ca, with most showing decreasing trends in their amounts, indicating that the components in ca can interfere with the extraction of components from sa. we verified the antagonistic effect of ca on sa by quantitative determination and regression analysis by means of chemical analysis.	this study was designed to perform simultaneous determination of three reference compounds in syzygium aromaticum (sa), gallic acid, ellagic acid, and eugenol, and to investigate the chemical antagonistic effect when combining curcuma aromatica (ca) with sa, based on chromatographic analysis. the values of lods and loqs were 0.010.11 g / ml and 0.030.36 g / ml, respectively. the intraday and interday precisions were 0.87, indicating a linear relationship between sa and sac. these results demonstrate that the components contained in ca could affect the extraction of components of sa mainly in a decreasing manner. the antagonistic effect of ca on sa was verified by chemical analysis.
dna, which carries the genetic information, is liable to alterations by various agents of endogenous and environmental origin. in order to prevent the deleterious effects yielded by such dna lesions and to maintain the integrity of the genome and cellular functions, cells are equipped with several dna repair systems, such as base excision repair (ber), nucleotide excision repair (ner), and mismatch repair (for review, see). ber is one of the most versatile repair pathways that can deal with base lesions resulting mainly from alkylation, oxidation, deamination, and replication errors. this pathway is initiated by damage - specific dna glycosylases that release the altered or inappropriate bases by cleavage of the n - glycosylic bond from the phosphate - sugar backbone, thereby resulting in production of the apurinic / apyrimidinic (ap) sites (reviewed in [2, 3 ]). a class of dna glycosylases, designated bifunctional dna glycosylases, possess additional ap lyase activity that further cleaves the phosphodiester bond 3 to the ap site generated by its own glycosylase activity. when ber is initiated by such bifunctional dna glycosylases, ap endonuclease removes 3-unsaturated aldehyde left behind by the ap lyase activity. as a consequence of either pathways, unlike ber that mainly repairs base lesions, ner deals with a wide variety of helix - distorting lesions, including ultraviolet (uv) light - induced pyrimidine photodimers as well as intrastrand crosslinks and bulky adducts induced by numerous chemical compounds. xeroderma pigmentosum (xp) is one of the autosomal recessive disorders that are associated with defective ner and clinically characterized by severe photosensitivity against uv exposure and a high risk of skin cancer. to date, seven ner - deficient xp genetic complementation groups have been identified (xp - a through -g), for each of which the responsible gene has been cloned. unlike most of the other xp groups, xp - c patients show defects only in one of the two ner subpathways, that is, global genome ner that covers the entire genome. the other subpathway is referred to as transcription - coupled ner, which repairs transcription - blocking lesions on the transcribed strand of active genes. the xpc protein, the responsible gene product for the xp group c, plays an essential role in dna damage recognition and the following initiation of global genome ner [57 ]. it forms in vivo a stable heterotrimeric complex with either rad23a or rad23b, one of the two mammalian homologues of saccharomyces cerevisiae rad23p, and centrin 2, which is known also as a component of the centrosome [810 ]. rad23 stabilizes the xpc protein in vivo [11, 12 ], and the xpc - rad23 heterodimer is sufficient to reconstitute the cell - free ner reaction [13, 14 ], whereas centrin 2 appears to potentiate the damage - specific dna binding activity of the xpc complex. biochemical and structural analyses revealed that this complex recognizes a specific dna secondary structure containing a junction between double - stranded dna and a single - stranded 3-overhang [1618 ]. after the dna binding by xpc, atpase / helicase activities of tfiih (exerted by the xpb and xpd subunits) open dna duplex and demarcate damage, probably with the aid of xpg, xpa, and replication protein a (rpa). the damaged strand is then cleaved in both sides of the lesion by two structure - specific ner endonucleases, ercc1-xpf and xpg, and the resulting gap is refilled with the dna repair synthesis. we have previously shown that xpc interacts directly with one of the dna glycosylases, thymine dna glycosylase (tdg). tdg removes thymine or uracil residues from g / t or g / u mismatches, which are mainly derived from hydrolytic deamination of 5-methylcytosines or cytosines, respectively (for review, see). tdg is unique in that it can not dissociate from the substrate dna by itself after accomplishing its enzymatic activity [22, 23 ]. crystal structure of the e. coli homolog of tdg, the mismatch - specific uracil dna glycosylase (ecmug), demonstrated that the inability to turnover is due to its tight interaction with the guanine opposite the ap site [24, 25 ]. extensive biochemical analyses have suggested that this feature may be applicable also to the human tdg [22, 23, 26 ]. since continuous attachment of tdg to ap sites must interfere with the following ber process, it has been conceivable that there should be certain factors that promote dissociation of tdg from the processed dna. one candidate for such factors relieving tdg of the product inhibition is ap endonuclease 1 (ape1), which acts in the ber pathway immediately after the dna glycosylases [22, 2729 ]. another factor would be sumoylation of tdg, which occurs at a single specific site (lys330 in human tdg). the x - ray crystal structure of sumoylated tdg revealed that this modification induces a significant conformational change in the c - terminal domain of tdg, which seems to sterically prevent its interaction with dna. thus, it has been proposed that the sumoylation following base excision may promote the enzymatic turnover of tdg, although precise timing and regulation of the modification in vivo still remain to be elucidated. in addition, we have previously shown that xpc - rad23b forms a ternary complex with tdg bound to the substrate dna and promote dissociation of tdg from the ap site. like tdg and ape1, xpc can recognize and bind to ap sites, although the observed affinity is relatively low if opposite base is guanine. therefore, two notions have been conceivable concerning molecular mechanisms underlying the stimulation of tdg turnover by xpc : direct physical interaction and competition for the ap site. more recently, it has been reported that xpc interacts with and stimulates the activity of ogg1, a major dna glycosylase that removes various oxidative base lesions including 8-oxoguanine. here we further investigate possible involvement of the xpc protein complex in ber by using various dna glycosylases as well as a sumoylated form of tdg, which gives further insights to the molecular mechanisms for the tdg stimulation. purification of recombinant human xpc and his - tagged rad23b (rad23b - his) was carried out [34, 35 ], and the xpc - rad23b - his heterodimer was reconstituted in vitro as described previously. his - tdg, gst - tdg, gst, and his - smug1 were also purified as described elsewhere [20, 36 ]. ecmug and ung2, both fused to the n - terminal his - tag, were expressed in e. coli strain bl21 (de3) using the pet-28a vector (novagen). expression of his - ecmug was induced with 1 mm isopropyl--d - thiogalactopyranoside (iptg) at 37c for 3 hours. the bacterial cell pellets were sonicated in buffer a [25 mm sodium phosphate (ph 7.5), 1 mm edta, 25 mm nacl, 0.01% nonidet p-40, 1 mm dithiothreitol (dtt), 0.25 mm phenylmethylsulfonyl fluoride (pmsf), protease inhibitor cocktail (complete ; roche diagnostics) ] and centrifuged for 30 minutes at 100,000 g. the supernatant was loaded onto a phosphocellulose column (p-11 ; whatman) equilibrated with buffer b [20 mm sodium phosphate (ph 7.5), 10% glycerol, 0.01% triton x-100, 0.25 mm pmsf ] containing 0.1 m nacl. after the resin was washed thoroughly with the same buffer, bound proteins were eluted stepwise with buffer b containing 0.2 and 1 m nacl. proteins recovered in the 1 m nacl fraction were then applied to a column packed with talon resin (clontech) equilibrated with buffer c [20 mm tris - hcl (ph 7.5), 10% glycerol, 0.1 m nacl, 0.01% triton x-100, 0.25 mm pmsf ] containing 5 mm imidazole. the column was then washed extensively with the same buffer, followed by stepwise elution of the bound proteins with buffer c containing 20, 100, and 250 mm imidazole. finally, the 100 mm imidazole fraction containing his - ecmug was dialyzed against buffer d [20 mm sodium phosphate (ph 7.5), 1 mm edta, 10% glycerol, 0.01% triton x-100, 1 mm dtt, 0.25 mm pmsf ] containing 0.2 m nacl. expression of his - ung2 was induced with 1 mm iptg at 30c for 3 hours. the bacterial cell extract was prepared as described above and loaded onto a hiload 16/10 sp sepharose hp column (ge healthcare biosciences) equilibrated with buffer b containing 0.1 m nacl. after washing the resin with the same buffer, bound proteins were eluted stepwise with buffer b containing 0.5 and 1 m nacl. his - ung2 recovered in the 0.5 m nacl fraction was further purified with talon resin, exactly as described above for his - ecmug. the 250 mm imidazole fraction was then loaded onto a mono s pc 1.6/5 column equilibrated with buffer d containing 0.1 m nacl. the column was developed with a linear gradient of 0.10.7 m nacl in buffer d and his - ung2 was eluted around 0.4 m nacl. finally, the sample was subjected to gel filtration chromatography using a superdex 75 pc 3.2/30 column (ge healthcare biosciences) equilibrated with buffer d containing 0.2 m nacl. sumoylation of his - tdg was conducted in e. coli strain bl21 (de3) as described. after induction of protein expression with 1 mm iptg at 30c for 3 hours, bacterial cell extract was prepared and fractionated on sp sepharose and talon columns exactly as described above for his - ung2. the 250 mm imidazole fraction from the talon column containing sumo-1-his - tdg was subsequently loaded onto a mono q hr 5/5 column (ge healthcare biosciences) equilibrated with buffer e [20 mm tris - hcl (ph 7.5), 1 mm edta, 10% glycerol, 0.01% triton x-100, 1 mm dtt, 0.25 mm pmsf ] containing 0.1 m nacl. the column was developed with a linear gradient of 0.10.5 m nacl in buffer e and sumo-1-his - tdg was eluted around 0.2 m nacl. finally, proteins were loaded onto a mono s pc 1.6/5 column equilibrated with buffer d containing 0.1 m nacl and then eluted with a linear gradient of 0.10.5 m nacl in buffer d. sumo-1-his - tdg was collected from the fractions around 0.2 m nacl. gst - fusion proteins were expressed in e. coli strain bl21 (de3) using the expression vector pgex4t3 and purified through two successive chromatography procedures using a gstrap column (ge healthcare biosciences) and then a mono s pc 1.6/5 column. flag - tagged mbd4 (flag - mbd4) was expressed in insect cells using the bac - to - bac baculovirus expression system (invitrogen). high five cells were infected with the recombinant baculovirus, and proteins were extracted from the cells with buffer f [25 mm tris - hcl (ph 8.0), 1 mm edta, 0.3 m nacl, 10% glycerol, 1% nonidet p-40, 1 mm dtt, 0.25 mm pmsf, protease inhibitor cocktail (complete) ] and dialyzed against buffer d containing 0.1 m nacl. samples were then loaded onto a hiload 16/10 sp sepharose hp column (ge healthcare biosciences) equilibrated with buffer d containing 0.1 m nacl. after washing the column with the same buffer, bound proteins were eluted stepwise with buffer d containing 0.5 and 1 m nacl. the 0.5 m nacl fraction was subsequently loaded to an anti - flag m2-agarose column (sigma - aldrich) and eluted with buffer d plus 1 m nacl and 0.1 mg / ml 3 x flag - peptide (sigma - aldrich). the eluate was adjusted at 0.3 m nacl by dilution with buffer b and then loaded to a hitrap heparin hp column (ge healthcare biosciences) equilibrated with buffer d containing 0.3 m nacl. bound proteins were eluted stepwise with buffer d containing 0.5 and 1 m nacl, and flag - mbd4 appeared in the 1 m nacl fraction. finally, the fraction was diluted by buffer d in order to decrease the nacl concentration to 0.3 m again and subjected to a mono s pc 1.6/5 column equilibrated with buffer d containing 0.3 m nacl. the elution of the bound protein was conducted with a linear gradient of 0.31 m nacl in buffer d, where flag - mbd4 was collected from the fractions around 0.5 m nacl. the enzymatic activities of dna glycosylases in the presence or absence of xpc - rad23b were measured as described previously. substrates used (g / t, g / u, and ss - u) are shown in figure 1. glutathione - sepharose 4 fast flow (ge healthcare biosciences : 20 l) was mixed with recombinant proteins as indicated in 100 l of the binding buffer 20 mm sodium phosphate (ph 7.4), 1 mm edta, 150 mm nacl, 1 mm dtt, 5% glycerol, 0.01% triton x-100, 100 g / ml bovine serum albumin. after incubation on ice for 1 hour with occasional agitation, the beads were washed eight times with 500 l of the binding buffer and the proteins retained on the beads were eluted using 20 l of the binding buffer containing 10 mm glutathione. since purified xpc has a tendency to aggregate when boiled in the presence of sds, each eluate was mixed before denaturation with the whole cell extract (0.4 g protein) from xp4pasv cells that do not express endogenous xpc. an aliquote of each eluate mixed with xp4pasv cell extract was subjected to 8% sds - page and analyzed by immunoblotting using polyclonal anti - xpc or anti - gst antibodies. the polyclonal antibodies against xpc and tdg were raised as described previously [20, 35 ]. the polyclonal anti - gst (ge healthcare biosciences) and anti - sumo-1 (santa cruz biotechnology) antibodies were purchased. we first examined whether xpc can stimulate the enzymatic activity of tdg that removes uracils from the g / u mismatches by the conventional dna glycosylase assay involving the substrate shown in figure 1(a). we have previously shown by using the dna substrate containing a single g / t mismatch that xpc - rad23b stimulates the activity of tdg by promoting dissociation of the enzyme from the ap site. although tdg has been shown to exhibit significantly higher activity with g / u than g / t mismatches, the severe product inhibition has been also observed with the g / u substrate. in this point of view, the effect of xpc on tdg may be similar regardless of which substrate is used, because the ap site opposite guanine is in any way produced as a result of the enzymatic action. compared with the reaction kinetics of tdg alone, addition of xpc - rad23b stimulated the activity of tdg on g / u mismatches up to 5-fold in a dose - dependent manner (figure 2), exactly as expected. in order to test the significance of protein - protein interaction between xpc and tdg for the stimulation, we purified an e. coli homolog of tdg, the mismatch - specific uracil - dna glycosylase (ecmug). unlike tdg, which is involved in correction of both g / t and g / u mismatches, ecmug is primarily g / u - specific, with very low activity toward g / t mismatches. however, ecmug resembles human tdg in that, after cleavage of the particular bases, it remains tightly bound to the ap site generated after exhibiting the glycosylase activity [2225 ]. we first tested the presence or absence of physical interaction between xpc and ecmug fused to glutathione s - transferase (gst - ecmug). ten nanomolar each of gst - ecmug, gst - tdg as a positive control, or gst alone as a negative control was mixed with the equal concentration of xpc - rad23b, and proteins were pulled down with glutathione - sepharose beads. immunoblot analyses revealed that a significant amount of xpc was bound to gst - tdg whereas only little amount of xpc was bound to gst alone as expected (figure 3, compare lanes 1 and 2 ; see also). it should be noted that the amount of gst - tdg recovered in the bound fraction was only ~20% of gst. meanwhile, the binding between xpc and ecmug was as much as the background level (figure 3, compare lanes 1 to 3), indicating that xpc - rad23b does not interact with ecmug in vitro under the conditions tested. we next added xpc into the nicking assay using the g / u substrate in order to see whether it influences the activity of ecmug. although significant physical interaction could not be detected (figure 3), it was possible that xpc - rad23b may still stimulate the activity of ecmug by displacing the glycosylase from the ap site, since xpc itself also seems to have a binding affinity toward the ap site. as shown in figure 4, however, xpc hardly affected the enzymatic activity of ecmug ; while 0.8 nm ecmug processed about 6% of the substrate dna (1.6 nm) within 120 minutes, ~9% of the uracil was removed from the g / u mismatched - dna in the presence of 4 nm xpc - rad23b. although there might be thus a very weak stimulation, the effect of xpc on ecmug activity was much less pronounced than that on tdg. a covalent modification of tdg by sumo-1 was shown to dramatically affect its dna binding properties [30, 32 ]. consequently, tdg appears to lose its activity on g / t mismatches, whereas the excision of uracils from g / u substrates is even enhanced upon sumoylation. the stimulation of the uracil cleavage seemed to be due to relief of tdg from the product inhibition via sumo-1 conjugation. given that the sumoylated tdg may not be bound to the ap site so stably, it was of our great interest to know whether xpc can interact with and stimulate the activity of the modified enzyme. to test this idea, we prepared sumo-1-conjugated tdg by expressing e1 (aos1-uba2), e2 (ubc9), sumo-1, and tdg (his- or gst - tagged) in e. coli simultaneously, which enables efficient sumoylation of tdg within bacteria. the sumoylated his - tdg (figure 5(a)) and gst - tdg (data not shown) were purified to near homogeneity. we confirmed that the purified protein specifically reacts with both anti - tdg and anti - sumo-1 antibodies (figure 5(b)). analysis of the protease - digested fragments with mass spectrometry and peptide sequencing further demonstrated that sumo-1 is conjugated exactly to the predicted site (lys330 of tdg ; data not shown). at first, we tested a protein - protein interaction between xpc and the sumoylated tdg by gst pull - down assay. as shown in figure 3, a significant amount of xpc was coprecipitated with sumo-1-gst - tdg if compared to gst alone, and approximately 2-fold more xpc appeared to bind to sumo-1-gst - tdg than to nonmodified gst - tdg. this indicates that xpc can bind to tdg regardless of the presence or absence of modification by sumo-1. as reported by hardeland., the purified recombinant sumo-1-tdg had only marginal activity for g / t mismatches (data not shown). on the other hand, sumo-1-tdg showed nearly linear kinetics of uracil removal from g / u mismatches up to 120-minute incubation, suggesting that the product inhibition was much less pronounced as described previously (figure 5(c) ;). interestingly, this activity of sumo-1-tdg was further stimulated by the addition of xpc in a dose - dependent manner (figure 5(c)). incubation of 4 nm xpc - rad23b resulted in ~5-fold processing of the g / u mismatch in 120 minutes, and this ratio was comparable to the stimulation of nonmodified tdg cleaving the same substrate (figure 5(c)). thus, these results demonstrate that xpc can upregulate the activity of not only nonmodified tdg but also sumo-1-modified tdg in vitro. since it has been reported that xpc stimulates ogg1 in addition to tdg, we next investigated the effect of xpc on various dna glycosylases, especially the monofunctional dna glycosylases that are involved in g / t and/or g / u mismatch repair, including ung2, mbd4 (also referred to as med1), and smug1. ung2 and smug1 mainly remove uracils not only from double - stranded dna but also from single - stranded dna, whereas mbd4 as well as tdg is more or less specialized toward correction of g / t and g / u mismatches particularly in the cpg context. as shown in figure 6, xpc showed no obvious effect on the activity of ung2 against g / u mismatch (figure 6(a)), that of mbd4 against g / t mismatch (figure 6(b)), or that of mbd4 against g / u mismatch (data not shown). although the activities of both ung2 and mbd4 were somewhat reduced in the presence of xpc, this may be due to the nonspecific binding of xpc to dna. on the contrary, xpc showed a striking stimulation on the activity of smug1 against g / u mismatch in a dose - dependent manner (figure 6(c)). in the absence of xpc, 2.5 pm smug1 processed about 5% of 1.6 nm g / u - mismatched dna in 120 minutes under the conditions tested. meanwhile, the presence of 2 nm xpc - rad23b boosted the smug1 activity up to 4-fold under the equivalent conditions. this stimulation was also observed when a single - stranded 30-mer oligonucleotide containing a single uracil residue was used as a substrate (figures 1 and 6(d)). while 62.4 pm smug1 cleaved about 2% of the substrate (1.6 nm) in 120 minutes, addition of 1 nm xpc - rad23b stimulated the cleavage up to 8% under the same conditions. although smug1 is known to remove uracils more efficiently from single - stranded dna than from double - stranded dna, our results showed the opposite pattern. this might be due to the difference in length of the substrate dna between g / u - mismatched double - stranded dna (60 bp) and single - stranded dna (30 mer). these results suggest that xpc functionally interacts not only with tdg but also with smug1. since our results indicate that xpc could stimulate the activity of smug1 in vitro, we tested the existence of the protein - protein interaction between xpc and smug1 by using the gst - pull down assay (figure 7). ten nanomolar each of gst or gst - fused smug1 was incubated with an equimolar amount of xpc - rad23b, and then bound to the glutathione - sepharose beads. after eluting the bound protein with glutathione, the presence of gst and gst - smug1 or xpc was visualized by immunoblotting using anti - gst or anti - xpc antibody, respectively. while equivalent molar amounts of gst and gst - smug1 were observed within the eluates, only little amount of xpc was bound to the control gst. on the other hand, although this protein - protein interaction seems to be relatively weak (only ~0.4% of the input was bound to xpc), these results indicate presence of a direct interaction between xpc and smug1. in this study, we have further examined the molecular mechanisms of interaction between xpc and tdg that facilitate the enzymatic turnover of tdg. in our previous studies, xpc appeared to promote dissociation of tdg from the ap site that is generated by its own glycosylase activity on g / t mismatch. at least two possible mechanisms have been considered, which could contribute to the displacement of tdg from the abasic reaction product : (1) protein - protein interaction between xpc and tdg weakens the binding affinity of tdg to the substrate dna, and (2) xpc and tdg compete for the same ap site, and thus tdg is pushed out. in order to examine which factor is more critical for the enhanced enzymatic turnover, ecmug, an e. coli homolog of mammalian tdg, was utilized as a model that can bind to the ap site as well as tdg [24, 25 ] but can not interact with xpc (figure 3). our results indicate that the stimulation of ecmug by xpc is almost negligible when compared to human tdg (figure 4). although there might be very weak stimulation of ecmug, which could be due to competitive displacement of the enzyme from the ap site by xpc - rad23b, these results point to importance of the protein - protein interaction for the stimulation of tdg by xpc. in agreement with this idea, we show here that xpc also stimulates the activities of sumoylated tdg and smug1, both of which interact physically with xpc (figures 3 and 7). our present data also indicate that very stable association of dna glycosylase with the produced ap site is not a prerequisite for stimulation by xpc. it has been reported that modification of tdg by sumo alters its structure in the c - terminal domain, which leads to the release of tdg from the ap site and therefore induces accelerated enzymatic turnover [3032 ]. in fact, in contrast to the immediately saturating reaction patterns of unmodified tdg, the sumoylated enzyme exhibited a nearly linear kinetics of uracil removal from g / u mismatches. even under these conditions it is possible that, like the reported case for ogg1, xpc may stimulate loading of certain dna glycosylases to substrate dna. although competition with xpc for the ap site may not be sufficient by itself to displace the tightly bound dna glycosylase, our data do not exclude that the binding affinity of xpc to the ap site may still contribute to promotion of the enzymatic turnover. considering the fact that xpc and ape1 stimulate the activity of tdg in an additive fashion and that ape1 also further stimulates the activity of sumo-1-tdg, xpc, sumoylation, and ape1 might be collaborating to control the repair of g / t and g / u mismatches positively in vivo. to understand the relationship between these three factors in detail, in vivo regulation of the tdg sumoylation should be clarified in the future experiments. in addition, it has been shown that xpc undergoes in vivo posttranslational modifications, such as ubiquitylation [40, 41 ], sumoylation, and phosphorylation [42, 43 ]. although the observed stimulation of dna glycosylases seemed to occur in the absence of these modifications, it is still possible that one or more of these modifications may affect interaction with and/or stimulation of some dna glycosylases. mammalian cells contain at least four nuclear dna glycosylases that can correct g / u and/or g / t mismatches, that is, tdg, ung2, mbd4, and smug1. although precise niche for each of these dna glycosylases remains to be clarified, their biological roles have been suggested through observations of in vivo behaviors and through interacting partners. ung2 seems to be specialized in counteracting u : a base pairs formed by misincorporation of dutp during dna replication. among the uracil - dna glycosylases, only ung2 specifically accumulates in the replication foci during the s phase, thereby colocalizing with proliferating cell nuclear antigen (pcna) and rpa. on the other hand, smug1 was first identified by in vitro expression cloning and also independently found to be a backup enzyme for ung2 [45, 46 ]. unlike ung2, smug1 may account for the repair of replication - independent premutagenic g / u mispairs resulting from cytosine deamination in vivo, since it is expressed similarly in nonproliferating and proliferating cells and it does not colocalize with pcna during the s phase. tdg and mbd4 apparently have very similar substrate specificities : g / t and g / u mispairs that are spontaneously generated by deamination of cytosines in cpg dinucleotides. besides its function in ber, tdg is also known to interact with and regulate several transcription factors [4851 ]. mbd4 interacts with a mismatch repair protein mlh1, which may be involved in regulation of some dna damage response pathways. interestingly, human ung, tdg, and smug1 genes are closely located within the long arm of chromosome 12, and these genes are believed to be the derivatives from an identical ancestral gene. in fact, structural studies revealed that they contain several common folds despite rather low (~10%) amino acid sequence homology [55, 56 ]. in these points of view, tdg and smug1 might still possess the common surface that might be the remnant of the ancestor to interact with xpc. it has been reported that tdg is able to process certain oxidative pyrimidine lesions, such as thymine glycol, 5-hydroxyuracil, and 5-hydroxymethyluracil, when paired with guanine. in addition, smug1 was shown to be a primary repair enzyme for a subset of oxidized uracil derivatives such as 5-formyluracil, 5-hydroxymethyluracil, and 5-hydroxyuracil in both single- and double - stranded dna [57, 58 ]. taken together with the recent report showing that xpc stimulates the activity of ogg1, these findings suggest that xpc might be involved in repair of diverse oxidative dna lesions. in fact, lack of endogenous xpc seems to result in inefficient repair of spontaneous oxidative dna damage. interestingly, while a significant amount of h2o2 is produced upon uv irradiation in most of the complementation groups of the xp cells including xp - c compared to normal cells, plasmid reactivation assays revealed that only xp - c cells exhibited markedly reduced repair capacity for some singlet oxygen - induced dna lesions. furthermore, extensive mutational analyses of the trp53 of uvb - induced skin tumors in xpc mice revealed a mutational hotspot at a nondipyrimidinic cpg site in codon 122 [61, 62 ]. at this site, c to t transition has been frequently detected specifically in xpc mice (not in xpa or csa mice), which is plausibly explained by reduced tdg activity in the absence of functional xpc protein. however, the tandem mutations ac > tt and ac > ct have been also found at the same site, which can not be attributed either to uv - induced dipyrimidinic photolesions or to deamination of the cytosine alone. in addition, it has been reported that age - dependent spontaneous mutations significantly accumulate in xpc mice even without uv exposure, and p53 gene mutations were identified in internal tumors of xp - c patients. many of these mutations are less likely to be the result of defects in correction of g / t and/or g / u mismatches. it could be possible that impaired repair of certain types of oxidative damage may be involved in occurrence of these mutations. here we show that xpc stimulates activities of a certain spectrum of dna glycosylases, for which protein - protein interactions seem to be important, rather than competition for ap sites. in order to clarify the significance of interaction between xpc and tdg or smug1 in vivo, it should be necessary to understand the precise physiological roles of the different thymine- and uracil - dna glycosylases. further studies would reveal novel and important aspects in understanding the mutagenesis and carcinogenesis in xp - c patients.	we showed that xpc complex, which is a dna damage detector for nucleotide excision repair, stimulates activity of thymine dna glycosylase (tdg) that initiates base excision repair. xpc appeared to facilitate the enzymatic turnover of tdg by promoting displacement from its own product abasic site, although the precise mechanism underlying this stimulation has not been clarified. here we show that xpc has only marginal effects on the activity of e. coli tdg homolog (ecmug), which remains bound to the abasic site like human tdg but does not significantly interacts with xpc. on the contrary, xpc significantly stimulates the activities of sumoylated tdg and smug1, both of which exhibit quite different enzymatic kinetics from unmodified tdg but interact with xpc. these results point to importance of physical interactions for stimulation of dna glycosylases by xpc and have implications in the molecular mechanisms underlying mutagenesis and carcinogenesis in xp - c patients.
in southern brazil, incidence of ot ranges from 9.8% to 22% (24). in 1985, as part of a study to determine prevalence of toxoplasmic retinochoroidal lesions among a random population, 270 archived eyes submitted to the so paulo eye bank consecutively were examined for macroscopic chorioretinal lesions or histologic evidence of retinochoroiditis (r. belfort, jr. eighteen (6.7%) eyes had chorioretinitis and 9 (3.3%) possessed cicatrial lesions ; total prevalence was 10% (27/270). of these 27 eyes, 8 (3.0%) had mononuclear cells, and 1 (0.4%) had acute, inflammatory infiltrate consistent with ot (figure 1). representative retinochoroidal lesions depicted for 3 of 18 archived eyes examined by optical microscopy for evidence of toxoplasma gondii infection ; 270 single eyes were examined, each from a different donor, obtained during 1985 and 2009 in so paulo, so paulo state, brazil. original magnification (left to right) : 20, to determine updated infection prevalence rates, we obtained 114 eyes collected during 2009 from eye banks in 2 disease - endemic regions of brazil with different ot incidence rates, the south (joinville, n = 15) and southeast (so paulo, n = 42) (figure 2). the eyes were collected consecutively from deceased persons who donated to eye banks, 15 in joinville and 42 in so paulo. all eyes were from patients who died of natural causes or in an accident and who had no history or symptoms of ophthalmologic disease. the average age of donors was 54 years (range 2076 years) ; exclusion criteria included a history of lymphoma, leukemia, leptospirosis, hepatitis b or c, hiv, rabies, or bacteria associated with endocarditis. matched serum samples for each eye were not available. cities where retinochoroidal scars consistent with toxoplasma gondii infection were found on eyes donated to eye banks in brazil during 1985 and 2009 : so paulo, so paulo state, and its relative position in southern brazil to joinville, santa catarina state ; 522 km separates the 2 cities. retinal dna was extracted by using a qiaamp dna blood midi kit (qiagen, valencia, ca, usa). multilocus pcr dna sequencing was performed by using b1, nts2, and gra7 loci, as described (5,6). positive (type i rh dna) and negative (water only) controls were run alongside each pcr. cleveland, oh, usa) before dna sequencing (rml genomics, hamilton, mt, usa). specimens from 3 (7.1%) of 42 donors from so paulo tested positive at nts2 (technical appendix table), similar to the incidence rate determined among the eyes examined macroscopically in 1985 and consistent with previously published work (7). our results show that no significant change in relative frequency of toxoplasma infection occurred in eyes donated in so paulo over a 25-year period. dna sequence analysis of the 3 positive samples identified an archetypal allele shared by types ii and iii strains at nts2. 37) was positive at b1 (sensitivity 33% relative to nts2 ; b1 is a standard diagnostic marker). the 15 donors from joinville, 13 (87%) tested positive for toxoplasma dna at nts2. six samples possessed type i alleles, 1 had a unique allele sharing phylogenetic ancestry with type i, 2 had drifted type ii or type iii alleles, and 4 possessed a mixture of 2 allelic types, indicative of mixed strain co - infection. the detection of 5, 3, and 3 distinct and mostly novel alleles at nts2, b1, and gra7, respectively, argue against the possibility that the pcr results were due to a false - negative amplification. a high incidence of mixed infection has been observed previously in australian marsupials (8), but it is considered rare in human infection (9). in contrast, only 6 samples were positive at b1 (sensitivity 46% relative to nts2) ; 1 sample had a type three samples were positive at gra7 (sensitivity 23% relative to nts2) ; a type i allele, and 2 alleles sharing phylogenetic ancestry with type i and type iii, respectively (technical appendix table). southern brazil experiences different incidence rates and severity of ot, although overall toxoplasmosis prevalence rates are similar. studies in metropolitan so paulo identified toxoplasma antibodies in samples from pregnant patients that ranged from 59% to 65% across 2 decades, but no data on frequency of ot was reported (10,11). one 2004 study showed that 10% of the metropolitan sao paulo population has ot (7). seroprevalence in so jose do rio preto, another city in so paulo state, is 74.5%, and the incidence of ot in 20092010 was 20.3% (2). seropositivity in erechim, a southern city in rio grande do sul, is 88% ; ot occurs frequently (18%) and is severe (12). our results reveal differences in the ability of parasites to infect eyes collected from the general population in different geographic regions of brazil. the difference in eye infection prevalence rates suggests that, although most of the so paulo population is seropositive, only 7% of the population harbor toxoplasma parasites capable of migrating to the eye. however, 87% of eyes from joinville tested positive for parasite dna (a 1:1 correlation with seroprevalence), which may indicate that no apparent barrier to parasite entry into the eye exists in this geographic region. although not all patients bearing parasite infections in their eyes develop ot, similar to the finding that only 30%50% of aids patients experience recrudescent disease (13), our results may explain the increased incidence of ot in southern brazil and help clarify possible factors controlling the disease. our findings also show that the 110 gene - copy nts2 marker proved more sensitive than b1, a marker traditionally used to detect infection (5). in addition, nts2 polymorphisms enabled mixed strain co - infections to be detected in the eyes of donor no. 5. multilocus typing established that atypical strains common to brazil caused most eye infections and identified different toxoplasma genotypes circulating between the 2 locations ; e.g., strains bearing alleles that share phylogenetic ancestry with type i were only identified in joinville. previous studies suggested a link between parasite genotype and development of ot (14). our work using serologic typing has established that the parasite genotype is associated with more serious congenital and ocular disease among patients in the united states and europe (6,15). ultimately, to determine whether increased eye infection rates in joinville is dependent on parasite genotype, or an associated host genetic risk factor, prospective sampling should be done on eyes donated to eye banks. the serostatus of the donor, whether retinal scars are present, and the geographic origin of the donor can be recorded to determine factors associated with parasite infection and whether such donated eyes should be precluded from transplantation. polymorphisms in the nts2, b1, and gra7 genes by direct pcr on toxoplasma gondii infected eyes identified in eye banks from joinville and so paulo city, brazil.	we found significantly higher incidence of toxoplasma gondii dna in eye bank specimens from joinville in southern brazil (13/15, 87%) than in so paulo (3/42, 7% ; p = 2.1 10e8). pcr dna sequence analysis was more sensitive at locus nts2 than at locus b1 ; a high frequency of mixed co - infections was detected.
heme oxygenase-1 (ho-1), also known as heat shock protein 32 (hsp32), is an inducible enzyme that catalyzes the breakdown of heme, producing carbon monoxide, iron, and biliverdin. it is often upregulated in tumor tissues, and its inhibition is considered as a means of sensitizing the tumors to anticancer drugs. although ho-1 expression is increased in malignant prostate tissues, its expression in prostate cancer cell line, pc-3, is low. induction of ho-1 expression by hemin in pc-3 cells resulted in decreased cell proliferation and migration. overexpression of ho-1 also led to nuclear location and was associated with downregulation of matrix metalloprotease 9 (mmp9), which plays an important role in tumor cell invasion and angiogenesis. ho-1 expression can be induced by many inducers, and many regulatory pathways have been proposed [6, 7 ]. a number of antioxidant response element (are)-like motifs are present in the promoter of ho-1 gene. six of these sites were found as clusters at e1 (3928 bp) and e2 (9069 bp) regions of the human ho-1 promoter ; they are termed stre1 through stre6. besides these stre sites, other response elements, such as hse, srebp binding site, and an intronic sp1 enhancer in addition, an egr-1 binding site in mouse ho-1 promoter that is inducible by zinc protoporphyrin ix (znpp) has also been reported. znpp, a naturally occurring molecule formed in iron deficiency or lead poisoning, is a potent competitive inhibitor of ho-1. inhibition of ho-1 by znpp led to suppression of tumor cell growth and znpp has been suggested to be a useful agent for antitumor therapy. however, znpp has also been shown to regulate expression of ho-1 at the transcriptional level, and the effect of znpp on ho-1 expression is controversial. for example, it is shown to induce ho-1 expression in hamster fibroblast (ha-1) cells but not in neuro-2a mouse neuroblastoma cells and primary cultures of rat cortical neurons. in fact, it even suppressed the induction of ho-1 by statins or lipopolysaccharide. in our earlier study, znpp was found to induce ho-1 expression in human prostate adenocarcinoma pc-3 and breast adenocarcinoma mcf-7 cells. it is a much stronger inducer of ho-1 than atorvastatin, one of the statins. in this study, we used prostate cancer pc-3 cells to investigate the mechanism of action of znpp. we herein report that znpp upregulates ho-1 in pc-3 cells via the antioxidant response pathway. n - acetyl cysteine (nac) was product of sigma - aldrich (st. louis, mo, usa). znpp and protein kinase inhibitors were purchased through emd chemicals inc. (gibbstown, nj, usa). antibodies against human -actin and ho-1 were purchased from cell signaling technology (danvers, ma, usa) and enzo life sciences (plymouth meeting, pa, usa), respectively. antibodies against keap1 and phospho - nrf2 (ps40) were products of proteintech group, inc. human prostate adenocarcinoma pc-3 cell line was purchased from the american type culture collection (manassas, va, usa). these cells were maintained as monolayer cultures in dmem / f12 medium (invitrogen, carlsbad, ca, usa) supplemented with 10% fetal bovine serum, 100 units / ml of penicillin, 100 g / ml of streptomycin, and 0.25 g / ml of amphotericin b (complete medium) and were kept at 37c in a humidified atmosphere containing 5% co2. the enhancer - luciferase reporter plasmids were constructed by inserting sequences of various synthetic response elements into the filled - in nhei / bglii sites of pgl3-promoter vector (promega, madison, wi, usa) or ecorv site of pgl4-promoter vector via blunt - end ligation as described in our earlier study. internal control plasmid, pgl4.74[hrluc / tk ], was purchased from promega (madison, wi, usa). pc-3 cells were grown to 80% confluence in t25 flasks and treated with 10 m znpp or equal amount of dmso (vehicle) for various time intervals up to 48 h. total rna was extracted using nucleospin nucleic acid purification kits (clontech, palo alto, ca, usa). first - strand cdna was synthesized from 5 g of total rna using thermoscript (invitrogen, carlsbad, ca, usa) in a volume of 20 l. pcr was done for 30 cycles (denaturation at 94c for 30 sec, annealing at 59c for 30 sec, and extension at 72c for 60 sec) using 1 l of the first - strand cdna, 10 pmol of gene specific primers and 2.5 units of jumpstart taq dna polymerase (sigma - aldrich, st. primers for -actin : 5-cctcgcctttgccgatcc-3 and 5-ggatcttcatgaggtagtcagtc-3. primers for ho-1 : 5-agaagagctgcaccgcaagg-3 and 5-cctctgaagtttaggccattgc-3. real - time pcr (rt - qpcr) was performed with stepone real - time pcr system (applied biosystems, foster city, ca, usa) using taqman gene expression master mix and ready - made human hmox1 (hs 01110250_m1), actb (hs 99999903_m1), nqo1 (hs00168547_m1), gstp1 (hs00943351_g1), bach1 (hs00230917_m1), nfe2l2 (hs00975960_m1), and keap1 (hs00202227_m1) gene expression assays (applied biosystems, foster city, ca, usa). briefly, cells grown to 90% confluence in 24-well plates were cotransfected in triplicates with 250 ng of enhancer - luciferase reporter plasmid and 25 ng of pgl4.74[hrluc / tk ] internal control plasmid, using lipofectamine 2000 (invitrogen, carlsbad, ca, usa). six hours after transfection, the medium was replaced with fresh one containing 10 m znpp or same amount of dmso (vehicle). at 3048 h after - transfection, the growth medium was removed, and the cells were rinsed twice with ice - cold phosphate buffered saline and were lysed by shaking for 15 min at 25c with 100 l of passive lysis reagent (promega, madison, wi, usa). aliquots of 20 l of the cell lysates were assayed for firefly and renilla luciferase activities using a 20/20 luminometer (turner biosystems, sunnyvale, ca, usa) and dual - luciferase reporter assay system (promega, madison, wi, usa). the results were expressed as relative luciferase activity (a ratio of the activities of firefly luciferase / renilla luciferase). cells were seeded in triplicates at 0.51 10 cells / well in 48-well plate in complete medium. at about 25% confluence, cells were treated with various concentrations of znpp or vehicle (dmso) for 48 h. cell survival was determined using celltiter 96 nonradioactive cell proliferation assay (promega, madison, wi, usa) according to the protocol provided by the manufacturer. pc-3 cells grown to 80% confluence in t25 flasks were treated with 10 m znpp or vehicle for various time intervals up to 48 h. cells were lysed with 0.5 ml of 1x laemmli sample buffer containing 1% halt protease inhibitor and phosphatase inhibitor cocktails (thermo scientific, rockford, il, usa), sonicated for 2 15 sec, and centrifuged at 10,000 rpm for 15 min at 4c. aliquots of 50 g protein extract were analyzed on 10% sds - polyacrylamide gel and transferred to pvdf membranes. the blots were analyzed by western blot according to the procedure provided by westerndot 625 kit (invitrogen, carlsbad, ca, usa). briefly, the blots were incubated in 8 ml of blocking buffer in a small plastic dish for 1 h at room temperature with gentle agitation. then they were incubated with the diluted primary antibody (1 : 1000 dilution) at 4c overnight. after washing 3 times with 50 ml of 1x wash buffer, 5 min each, blots were incubated with 8 ml of biotin - xx - goat anti - rabbit antibody (1 : 2000 dilution) in blocking buffer for 1 h. they were washed 3 times with 50 ml of 1x wash buffer for 5 min each, and then incubated with 8 ml of qdot 625 streptavidin conjugate (1 : 2000 dilution) in blocking buffer for 60 min at room temperature. finally, the blots were washed 3 times with 50 ml of 1x wash buffer for 5 min each, and once with 20 ml of ultrapure water. the wet blots were placed on a uv trans - illuminator and pictures were taken with a polaroid camera and orange filter. statistically significant differences between data points of two groups were determined by student 's t - test. by convention, in fact, it induced significant cell proliferation at a concentration of 0.610 m, and only suppressed cell growth above 10 m (figure 1(a)). therefore, 10 m znpp was used for all subsequent experiments. basal expression level of ho-1 protein in pc-3 was undetectable. znpp induced ho-1 protein expression in a dose - dependent manner, with the highest induction level at 10 m (figure 1(b)). for a time course study, incubation of the cells with 10 m znpp for 4 h showed only a slight induction of ho-1 protein, but the induction was high after 16 h and was maintained through 48 h of incubation (figure 2(a)). the ho-1 mrna level as determined by rt - pcr also showed similar profile (figure 2(b)). of all the known responsive elements in the ho-1 promoter, znpp activated mainly the are - like elements (stres). as shown in figure 3, stre3 showed the highest (6.6-fold) induction level by znpp, although these elements had different basal expression levels of relative luciferase activities due to different copy number of the response elements present in the luciferase - reporter constructs. znpp did not activate the hse, srebp, and sp1 elements (figure 3). a number of protein kinases are known to be involved with the activation of antioxidant response element. to investigate the effect of various protein kinase inhibitors and antioxidant on the activation of stre by znpp, cells transfected with stre3-pgl3 were pretreated for 2 h with sb203580 (p38-mapk inhibitor), ly294002 (phosphatidylinositol 3-kinase inhibitor), u0126 (mek inhibitor), sp600125 (jnk inhibitor), ipa-3 (p21-activated kinase inhibitor iii), nac (antioxidant), rottlerin (pkc- inhibitor), ro 31 - 8220 (pan pkc inhibitor), or ro 32 - 0432 (pkc- inhibitor) prior to treatment with znpp for 24 h. as shown in figure 4, sp600125 and ro 32 - 0432 had little effect on the activation of stre3 by znpp. sb203580, nac, and ipa-3 reduced the activation of stre3 by znpp to 72.9%, 62.4%, and 83.6%, respectively, of the level by znpp alone. however, ly294002, u0126, and ro 31 - 8220 attenuated the activation to 39.4%, 40.2%, and 41.5%, respectively, of the znpp - alone control. on the other hand, rottlerin activated stre3 element by itself and had a synergistic effect with znpp (2-fold over the level by znpp alone). to confirm the effect of ly294002, u0126, ro 31 - 8220, and rottlerin on znpp - activation of stre3 element, ho-1 mrna levels were determined by real - time pcr and protein levels by western blot analyses. for real - time pcr, the relative levels of ho-1 mrna were determined in cells pretreated with ly294002, u0126, ro 31 - 8220, or rottlerin for 1 h prior to znpp treatment for 3 h. the results showed that ly294002 and u0126 did not attenuate znpp - induction of ho-1 mrna, but ro 31 - 8220 completely suppressed the effect of znpp (figure 5). on the other hand, real - time pcr also confirmed the synergistic effect of rottlerin with znpp ; znpp alone upregulated ho-1 by 8.2-fold over vehicle - treated control and rottlerin plus znpp upregulated ho-1 by 36.0-fold (figure 5). ro 31 - 8220 significantly suppressed the upregulation of ho-1 protein by znpp, while ly294002, u0126, and ro 32 - 0432 had little effect on znpp - induction of ho-1 (figure 6(a)). however, the synergistic effect of rottlerin with znpp was not evident due to the high level of upregulation of ho-1 by znpp alone. western blot analysis also showed that sb203580 and ipa-3 had no effect on znpp - induction of ho-1 expression (data not shown). since ro 31 - 8220 is a pan pkc inhibitor, this suggests that pkc may be involved in znpp - upregulation of ho-1. there are many pkc isoforms, but the involvement of pkc- can be excluded by the lack of suppression of ro 32 - 0432 (pkc- inhibitor) on znpp - upregulation of ho-1 protein (figure 6(a), lane 7). to determine if other pkc isoforms may be involved in znpp - activation of ho-1, pc-3 cells were treated with myristoylated pseudosubstrates of pkc-, pkc- and pkc-, or pkc- inhibitor prior to znpp treatment. western blot analysis showed no effect of these inhibitors on ho-1 protein level (figure 6(b)). hence, involvement of pkc-, pkc- and pkc-, and pkc- can also be excluded. since bach1, nrf2 and keap1 proteins have been shown to interact with antioxidant response element, the effect of znpp on the mrna and protein levels of these proteins were investigated by real - time pcr and western blot analyses. real - time pcr analyses showed that there were no drastic changes in the expression of bach1, nfe2l2 (nrf2), and keap1 genes when pc-3 cells were treated with znpp. the expression levels of bach1, nfe2l2, and keap1 in cells treated with 10 m znpp for 3 h were 73.5, 81.8, and 98.8%, respectively, of those in control cells. western blot analysis showed no change in phospho - nrf2(ps40) protein but a gradual decrease in keap1 protein in cells treated with znpp for 224 h (figure 7). only a faint band of bach1 protein was detected in pc-3 control cells, but none at all in cells treated with znpp, despite antibodies from two companies were used. to investigate the effect of znpp on the expression of other antioxidant responsive genes, the expression levels of nqo1 and gstp1 in cells treated with 10 m znpp for 3 h the results showed that the expression levels of nqo1 and gstp1 were 89.8 and 77.7%, respectively, of those in control cells. to determine if rottlerin and znpp can act as prooxidant, cells were pretreated with 10 mm nac prior to incubation with 10 m znpp or 2 m rottlerin, and relative levels of ho-1 mrna were determined by real - time pcr. the results showed that nac, even at 10 mm, reduced znpp - induction of ho-1 expression by only 43% (figure 8). rottlerin by itself did not induce ho-1 mrna expression after 5 h incubation (figure 8), and only a faint band of ho-1 protein was detected on western blot after 24 h incubation with rottlerin (data not shown). in this study, we demonstrated that znpp upregulated ho-1 expression in pc-3 cells in a dose - dependent manner, and that upregulation was done mainly through activation of the are - like response elements (stres) of ho-1 promoter. although similar response elements are present in the promoter of other antioxidant responsive genes, such as nqo1 and gstp1 [17, 18 ], real - time pcr analysis showed no upregulation of these genes by znpp. it should be noted that the induction of ho-1 protein is much stronger than that of ho-1 mrna, suggesting znpp may also affect other pathways that stabilize the ho-1 protein level. we also demonstrated that preincubation of the cells with ro 31 - 8220, a pan inhibitor of pkc, abrogated znpp - induction of ho-1 expression, suggesting that activation of ho-1 gene by znpp may involve pkc. it is intriguing to note that induction of ho-1 expression in pc-3 cells by hemin inhibited cell proliferation, whereas our results showed that although znpp also induced ho-1 expression in pc-3 cells, proliferation was not inhibited until znpp concentration exceeded 10 m (figure 1(a)). this may be due to the fact that znpp is also a well - known ho-1 inhibitor. ho-1 enzymatic activity may be required for the inhibition of cell proliferation. when znpp level was increased to above 10 m the ability of znpp to activate the are - like response elements (stres) of ho-1 promoter suggests that znpp upregulates ho-1expression via the nrf2-are signaling pathway. phosphorylation of nrf2 and/or modification of cysteine residues of keap1 results in decreased nrf2 ubiquitination and degradation, and hence activation of the are signaling pathway. phosphorylation of nrf2 at ser residue by pkc results in dissociation of nrf2 from the nrf2-keap1 complex, translocation of nrf2 into nucleus, and activation of are - like element of genes responsive to oxidative stress [20, 21 ]. our results showed no change in phospho - nrf2 (ps40) level in znpp - treated cells as compared to control (figure 7). however, our results did show a progressive decrease in keap1 level in znpp - treated cells (figure 7). the significance of the decrease in keap1 level remains to be studied. on the other hand, however, bach1 is unlikely involved in the upregulation of ho-1 by znpp for two reasons. first, basal expression level of bach1 in pc-3 cells is very low ; it was barely detectable in control cells by western blot (figure 7), and no downregulation of bach1 mrna induced by znpp was detected by real - time pcr. second, preincubation of the cells with 10 mm nac prior to znpp treatment did not completely abrogate the znpp - induction of ho-1 (figure 8). since upregulation of ho-1 by znpp can be suppressed by ro 31 - 8220, znpp - induction may be mediated by pkc. the involvement of pkc- in the upregulation of ho-1 by many phyto - chemicals has been demonstrated, as rottlerin and pkc- small interfering rna were able to attenuate ho-1 induction by these compounds [2327 ]. it should be noted that although rottlerin is not an efficient pkc- inhibitor, the involvement of pkc- was confirmed by the use of pkc- small interfering rna in these studies. on the other hand, involvement of pkc- and yet our results showed that preincubation of pc-3 cells with ro 32 - 0432, pkc- inhibitor, rottlerin, and pseudosubstrates of pkc-, pkc-, and pkc- did not suppress znpp - induction of ho-1 expression, and hence the involvement of,,,,, and isoforms of pkc can be ruled out. pc-3 cells are known to express pkc,,,, and isoforms as determined by nuclease protection assay and,,, and as determined by western blot. involvement of pkc,, and isoforms has not been tested in this study, because specific inhibitors for these isoforms are not commercially available. the pkc isoform involved in znpp - induction of ho-1 remains to be determined., rottlerin had a synergistic effect with znpp, and this was confirmed at the mrna level (figure 5). there is at least one report showing that rottlerin was able to upregulate ho-1 through reactive oxygen species (ros) dependent and pkc--independent pathway in human colon cancer ht29 cells, as its induction was abrogated by antioxidant nac but not by suppression of pkc- expression by small interfering rna technology. in pc-3 cells, rottlerin did not induce ho-1 to any significant level by itself, but had a synergistic effect with znpp. ro 31 - 8220, a pan inhibitor of pkc, has been reported to induce apoptosis independent of pkc activity. however, our results showed that ro 31 - 8220 was able to suppress znpp - induction of ho-1 mrna after only 3 h of incubation (figure 5). furthermore, pc-3 cells treated with 5 m ro 31 - 8220 for 24 h retained 93% viability (data not shown). these suggest that the suppression of znpp - induction of ho-1 by ro 31 - 8220 was not due to induction of apoptosis. on the other hand, ro 31 - 8220 has been shown to inhibit other kinases, such as mapkap kinase-1 (also known as rsk-2) and p70 s6 kinase. involvement of these kinases in znpp - activation has not been ruled out. in conclusion,	zinc protoporphyrin ix (znpp), a naturally occurring molecule formed in iron deficiency or lead poisoning, is a potent competitive inhibitor of heme oxygenase-1 (ho-1). it also regulates expression of ho-1 at the transcriptional level. however, the effect of znpp on ho-1 expression is controversial. it was shown to induce ho-1 expression in some cells, but suppress it in others. the objective of this study is to investigate the effect of znpp on ho-1 expression in prostate cancer pc-3 cells. incubation of pc-3 cells with 10 m znpp for 4 h showed only a slight induction of ho-1 mrna and protein, but the induction was high after 16 h and was maintained through 48 h of incubation. of all the known responsive elements in the ho-1 promoter, znpp activated mainly the stress response elements. of the various protein kinase inhibitors and antioxidant tested, only ro 31 - 8220 abrogated znpp - induced ho-1 expression, suggesting that activation of ho-1 gene by znpp may involve protein kinase c (pkc). the involvement of pkc,,,,, and isoforms was ruled out by the use of specific inhibitors. the isoform of pkc involved and participation of other transcription factors remain to be studied.
a 21-year - old male presented with a painless decrease in vision of the left eye for 1 day. he had fever along with maculopapular eruption on hands and foot and ulceration on the lower labial mucosa [fig. c ] for 5 days and he had already been diagnosed by a dermatologist as a case of hfd. on presentation, his va was 20/20 and 20/30 in the right and left eyes, respectively. fundus evaluation of right eye was normal, and in the left eye, there were multiple intraretinal hemorrhages (irhs) within the temporal arcade and serous elevation of the macula [fig. (e) fundus autofluorescence shows generalized reduction at macula and some dense areas of hypo - autofluorescence corresponding to intraretinal hemorrhage. (f) optical coherence tomography showing bifocal serous elevation of the macula. (g and h) combined fluorescein angiography and indocyanine green angiography in the early and late phase, respectively. early phase fluorescein angiography showing blocked fluorescence corresponding to intraretinal hemorrhage and indocyanine green angiography showing moth - eaten appearance. late - phase fluorescein angiography shows diffuse leak and indocyanine green angiography showing diffuse hypercyanescence the systemic workup including complete blood counts, peripheral blood smear, and c - reactive protein was wnl and he was negative for human immunodeficiency virus. the fundus autofluorescence (faf) showed a generalized reduction of faf from macula with some areas of dense hypo autofluorescence corresponding to irh [fig. 1e ], and the optical coherence tomography (oct) (heidelberg, germany, version 1.9.20.0) showed bifocal serous elevation of the macula [fig. he also underwent a combined fluorescein angiography (fa) and indocyanine green angiography (icg) which, in the early phase, showed blocked fluorescence in the areas of irh on fa and moth - eaten appearance of the choroidal vasculature (cv) on icg [fig. 1 g ], and in the late phase, fa showed diffuse leak suggestive of a breach in the outer blood - retinal barrier and hypercyanescence on icg suggestive of the inflammatory pathology of the cv [fig. 1h ], the right eye being normal in all investigations. based on the clinical findings and other investigations, the patient was started on oral prednisolone (1 mg / kg body weight) in tapering dose after the clearance from an internist and was reviewed after a week. next week, his va had dropped to 20/40 in the left eye and the right eye was normal. his general clinical evaluation showed complete resolution of the maculopapular rash on palms and soles and his oral ulcers had healed. 2b ] and oct showed resolution of serous detachment with debris collection at the inner segment - outer segment junction (is / os) with intact external limiting membrane [fig. 2c ]. his next follow - up at 1 month showed improvement in va (20/20) and anatomical improvement corroborated by fundus evaluation, faf and oct [fig. his last follow - up at 2 months showed va in both eyes 20/20, and he was symptomatically normal. the anterior segment was wnl, and the fundus showed normal foveal reflex in both eyes. the faf was nearly normal with some stippling, probably due to recovering retinal pigment epithelial function, and the foveal contour on oct was normalized with the reformation of is / os junction [fig. (b) fundus autofluorescence showing reduction of the area of hypo - autofluorescence and resolving intraretinal hemorrhage. (c) optical coherence tomography showing complete resolution of serous detachment at macula with intact external limiting membrane and debris at inner segment / outer segment junction. (f) optical coherence tomography showing reformation of the inner segment / outer segment junction and resolution of the debris. (i) normalized foveal contour with resolved debris at inner segment / outer segment junction hfd commonly affects children and immunocompromised adults, and only a few cases of immunocompetent adults have been described in literature. the mechanism of eye involvement has been proposed to be either direct viral infection by hematogenous spread or autoimmune process. the ocular involvement was first described by yannuzzi. as unilateral acute idiopathic maculopathy (uaim) in nine patients with preceding viral prodrome. the condition is associated with ca16 or e71 infection, but a number of other serotypes have also been described. reported that the most common association of uaim was viral fever as a prodromal symptom (50%) and hfd was in 25% cases with a positive titer of coxsackievirus in 50% cases. they also described the moth - eaten appearance of cv on icg, but late leak or areas of hypercyanescence was not seen in any patient, which is in contrast to our case where the leak in the late phase was present. the neurosensory detachment is a common finding and has been described in the reported literature. the use of systemic steroids in hfd is a controversial issue as the spontaneous resolution of the disease has been reported in about 3 months. two reports on the use of steroids in hfd have been described previously. in this case, we used steroids, and the patient had anatomical and functional normalization within 2 months which goes in conjunction with the previous reports of steroid usage, and the recovery was faster. the difference between this case and a previous case where steroids were used is that in our case, there was complete recovery of retinal architecture as shown by normalization of oct and reformation of is / os junction. the present case and the previous two cases did not have any ocular or systemic side effect with steroids usage. the occurrence of hm in association with hfd in an immunocompetent adult is an uncommon event, and spontaneous resolution of the disease is known but the use of steroids can shorten the duration of ocular morbidity, and faster visual rehabilitation can be achieved.	hand, foot, and mouth disease (hfd) is a common systemic infection occurring in childhood or immunocompromised adults caused by enteroviruses, the most common being coxsackievirus a16 and enterovirus 71. it is characterized by maculopapular eruptions over the hands and feet and ulcerative stomatitis. ocular involvement is a rare complication and commonly manifests as inflammatory macular pathology. we report a case of hfd in an immunocompetent adult male with unilateral ocular involvement presenting as hemorrhagic maculopathy and its management with complete anatomical and functional recovery.
a 69-year - old woman was diagnosed with an intracardiac tumor during a preoperative work - up for sigmoid colon cancer. echocardiography demonstrated that there was a 2.52.6 cm mass in the left atrium, which had a relatively broad base at the interatrial septum. the mass was not highly mobile and did not result in any valvular dysfunction or hemodynamic compromise. cardiac magnetic resonance imaging (mri) also revealed a 2.4 cm mass located at the interatrial septum (fig. 1). the initial diagnosis was a myxoma or a bronchogenic cyst in the left atrium. only antegrade cardioplegia was used because the tumor was located very close to the coronary sinus. the tumor was not visualized from outside the heart. during palpation from the inferior side of the heart, a solid mass after aortic cross - clamping, a right atriotomy was performed and extended to the left atrial roof. the tumor had cystic features, and the diagnostic impression from the operative finding was a bronchogenic cyst. after excision of the mass, the empty interatrial space was filled with tisseel (baxter healthcare co., westlake village, ca, usa) glue. the interatrial septum, left atrial roof, and right atrium were then anatomically reconstructed. 2). the largest cystic nodule measured 2.01.51.2 cm and had a serous component. a microscopic section showed typical biphasic architecture with antoni a (hypercellular) and b (hypocellular) patterns (fig. immunohistochemically, s-100 protein staining was strongly positive in most cells, which is consistent with schwannoma (fig. currently, fewer than 15 cases of atrial schwannoma can be found in a pubmed search. further, to the best of our knowledge, there have been no reports on cardiac schwannoma located in the interatrial space. according to the previous case reports, patients with intracardiac schwannoma had various presentations, such as new - onset atrial fibrillation, recent shortness of breath, pleuritic chest discomfort, and chronic cough [25 ]. in our case, the diagnosis was made during a preoperative work - up for colon cancer surgery. even benign cardiac tumors should be completely excised, irrespective of the symptoms, because the tumors may grow and compress the cardiac chambers and cause thromboembolism, arrhythmia, or even sudden death. we think that the initial misdiagnosis could be attributed to the unusual anatomic location of the tumor. in fact, the tumor was located inside the heart but outside the cardiac chambers ; therefore, accurate preoperative characterization of the tumor was difficult, even with echocardiography and cardiac mri. although imaging studies did not suggest the possibility of metastatic cancer, one of our initial differential diagnoses was a metastatic tumor from the patient s colon cancer. in particular, the dissection of the inferior pole of the tumor was relatively challenging because the margin was immediately above the coronary sinus. we shaved the intramuscular portion of the coronary sinus wall. in conclusion, this case report describes a rare presentation of intracardiac schwannoma in the interatrial space. cardiovascular surgeons should consider schwannoma in the differential diagnosis of a low - mobility mass close to the interatrial septum.	we report the case of a 69-year - old woman who was diagnosed with intracardiac schwannoma without symptoms. preoperative echocardiography and cardiac magnetic resonance imaging showed a mass attached to the interatrial septum. the initial diagnosis was a myxoma or a bronchogenic cyst. the tumor was successfully excised under cardiopulmonary bypass. however, the pathology of the excised tumor was consistent with schwannoma. we suggest that cardiovascular surgeons consider schwannoma to be a possible differential diagnosis for a mass close to the interatrial septum.
the genus gasterophilus (diptera : gasterophilidae) includes nine species, six of which are reported to cause gastrointestinal myiasis in equid hosts (wall and shearer 2001). all species were originally restricted to the palearctic and afrotropical regions (colwell. 2007), but three major species g. nasalis, g. intestinalis, g. haemorrhoidalis have been distributed worldwide (zumpt 1965). the adult flies are not parasitic and do not feed (wall and shearer 2001). they are large, 1115 mm in length and their body is covered with yellowish hairs. the eggs are usually deposited on the hair of the host in a particular body region. after hatching, the larvae burrow into the tissue of the host, the first stage larvae hatch and moult to l2, which can be present in different regions of the gastrointestinal tract, and in l3 remains attached to the mucosa for 810 months. the rounded body, hooked mouth - parts and spines enable the larvae to live in the gastrointestinal tract. species can be distinguished based on pattern of spines on the body (wall and shearer 2001). in g. intestinalis, the third instar larvae are relatively large, between 1.27 to 1.91 cm long and spines arranged in two rows (zumpt 1965, sweeney 1990). here we report a type of gastric myiasis caused by g. intestinalis in an old lion in a zoo in sistan, southeast iran. the present case aimed to report the finding of g. intestinalis into the stomach of an old lion in a zoo in sistan, southeast iran. larvae were collected during a post - mortem examination of the digestive tract of lion for parasitological purposes (fig. twelve larvae were collected from the stomach and preserved in 70% alcohol with 5% glycerin. samples were taken to the laboratory of parasitology, unit of the veterinary medicine, university of zabol, for morphological identification. all the collected larvae were identified under a stereomicroscope using the identification key (zumpt 1965). the isolated larvae were creamy - white in color, narrow at their anterior end and wide at their posterior end. gasterphilus larvae on the stomach mucosal membrane of a lion gasterophilus intestinalis collected from the stomach of a lion larvae of most gasterophilidae causing equine gastric myiasis have been recorded in southern italy (otranto. gasterophilus intestinalis, g. nasalis, g. hemorrhoidalis, g. inermis, g. nigricornis and g. pecorum were detected in turkey (gkcen. 2008). khalifa. (2005) obtained g. haemorrohidalis from the stomach of egyptian equines. distribution of species of this genus also has been investigated in brazil and g. intestinalis reported to be responsible for the majority of myiasis cases (felix. iranian equine myasis associated with gasterophilus spp., have been reported on a few occasions, three species of g. nasalis, g. intestinalis and g. inermis were identified by tavassoli and bakht (2012). aside from the higher incidence of infection by gasterophilus larvae in equid hosts, a few reports of infections in pigs, dogs and rabbits are provided by hall and wall (1995) and sayn ipek (2012). besides veterinary importance, gasterophilidae larvae can be found in humans with at least three cases reported (james 1947, harwood and james 1979, royce. 1999). in wild animals has been reported rarely. only, kumar. gasterophilus involvement in the gastric myiasis of lion has not been reported, so the present study is the first report. further studies are recommended to complete the lack of data on parasite fauna of wild animals.	myiasis is the infection caused by a variety of dipterous (fly) larvae in vertebrate s tissue (man and domestic or wild animals). species of gasterophilus are obligate parasite of horses, donkeys, zebras, elephants and rhinoceroses. there are records worldwide, but mostly, in tropical and subtropical regions. this case report describes a type of gastric myiasis caused by g. intestinalis in an old lion in a zoo in sistan, southeast iran. myiasis in lions is rarely reported and this is the first report of gastric myiasis in lion.
iatrogenic spinal cord injury resulting from elective cervical spine surgery is a rare and devastating adverse event. the incidence of iatrogenic spinal cord injury following cervical spine operations is challenging to determine. flynn reviewed 82 114 anterior cervical spine operations and documented a postoperative neurological injury rate of 0.3%. lee examined 1445 anterior cervical spine surgery patients and reported a rate of 0.1% spinal cord injury with neurological deficit. some instances of postoperative neurological deficit following cervical spine surgery can be predicted by neuromonitoring changes during the procedure, or due to an obvious intraoperative event leading to injury of the spinal cord, while others may only be recognized when the patient emerges from anesthesia. given the low incidence, it is not surprising that clearly defined protocols to manage interoperative spinal cord injury during elective cervical spine surgery have not been developed. most spine surgeons will encounter this adverse event once or twice, if at all, in an entire career. given these small numbers, appropriate practices must be determined more based on consensus rather than data. a review of cases of iatrogenic spinal cord injury might serve to inform development of a plan for response to such events. although the incidence is rare, the impact of iatrogenic spinal cord injury resulting from cervical spine surgery is substantial and has potential for serious patient, surgeon, institutional, and medicolegal ramifications. the purpose of this investigation is to examine the rate of iatrogenic spinal cord injury associated with cervical spine surgery and to report patient and surgical factors associated with these injuries. this study aimed to evaluate the incidence and factors associated with iatrogenic spinal cord injury during elective cervical spine surgery. we conducted a retrospective multicenter case series study involving 21 high - volume spine surgical centers from the aospine north america clinical research network, selected for their clinical research infrastructure and experience. medical records for 17 625 patients who received cervical spine surgery (levels from c2 to c7) between january 1, 2005, and december 31, 2011, were reviewed to identify occurrence of 21 predefined treatment adverse events. adverse events examined included reintubation requiring evacuation, esophageal perforation, epidural hematoma, c5 palsy, recurrent laryngeal nerve palsy, superior laryngeal nerve palsy, hypoglossal or glossopharyngeal nerve palsy, dural tear, brachial plexopathy, blindness, graft extrusion, misplaced screws requiring reoperation, anterior cervical infection, carotid artery injury or cerebrovascular accident, vertebral artery injuries, horner s syndrome, thoracic duct injury, quadriplegia, intraoperative death, revision of arthroplasty, and pseudomeningocele. trained research staff at each site abstracted the data from medical records, surgical charts, radiologic imaging, narratives, and other source documents for the patients who experienced one or more of the adverse events from the list. copies of case report forms were transferred to the aospine north america clinical research network methodological core for processing, cleaning, and data entry. of the 3 patients suffering iatrogenic spinal cord injury, 2 were male and 1 was female. the mean age was 57.3 years, with an average hospital length of stay of 12 days. the diagnosis and reason for surgery was myelopathy for 2 patients and degenerative disk disease for 1 patient. one patient underwent anterior surgery only with 2-level cervical corpectomy (c5, c6), one underwent posterior surgery only, and one underwent circumferential surgery (anterior and posterior) including cervical corpectomy. two patients underwent surgery from c3 to c7, while one patient underwent surgery from c4 to c7. poor baseline neuromonitoring signals were noted in one patient, no baseline motor response was noted in another, with data unknown from the third patient. in patient 1, a 67-year - old patient who underwent 2-level anterior corpectomy of c5 and c6, a dural defect was identified during resection of ossification of the posterior longitudinal ligament (pll) with subsequent neuromonitoring change following its removal. the dural defect was covered then with a duragen patch, followed by graft placement. the patient had a partial recovery but had residual upper and lower extremity weakness at follow - up. patient 2 was a 36-year - old patient who underwent both transcranial motor evoked potential and somatosensory evoked potential monitoring. at the outset of the case, the patient had no motor response on monitoring. the patient underwent anterior corpectomy surgery, and on awaking from anesthesia was found to be quadriplegic. the patient was taken for emergent magnetic resonance imaging (mri), following which the posterior component of the surgery was completed. patient 3 was a 69-year - old patient who underwent c3 - 7 posterior decompression and fusion. the patient awoke from anesthesia with quadriplegia, and did not recover function at the time of discharge. iatrogenic spinal cord injury is a devastating and rare adverse event following cervical spine surgery, with previously reported rates between 0.1% and 0.3%. in this series, incidence rates of the participating centers ranged from 0.0% to 0.24% of cases. injury to the spinal cord may result in a range of clinical severity from incomplete injury with mild motor or sensory deficit to complete quadriplegia with loss of sensation and bowel / bladder function (american spinal injury association [asia ] a spinal cord injury). this study only examined quadriplegia, and thus may not include patients that had less severe spinal cord or nerve root injuries. there are multiple potential etiologies of spinal cord injury during cervical spine surgery, including aggravation of preexisting spinal stenosis during positioning or surgical approach, malpositioned instrumentation or bone graft penetrating or compressing the cord, mechanical blunt trauma to the spinal cord, and vascular injury due to hypotension or arterial interruption. it is also possible that some cases of iatrogenic spinal cord injury occur due to a combination of these factors. this adverse event is too rare to accurately calculate a formal incidence, although a rate between 0.1% and 0.3% appears to be a reasonable estimate across all types and indications for cervical spine surgery. it is expected that this rate will depend on the nature and severity of the spinal pathology being addressed. for example, surgical correction of complex cervical deformity would be expected to have higher rates of spinal cord injury than anterior cervical discectomy and fusion or posterior foraminotomy, with neurological deficits reported in up to 13.5% of deformity patients. neuromonitoring utilizing somatosensory evoked potentials and transcranial motor evoked potentials (tcmeps) is frequently used in cervical spine surgery and may help surgeons intervene to reverse the immediate cause of intraoperative spinal cord injury. for procedures performed in the prone position, obtaining potentials with the neck in a neutral posture (prior to prone positioning) may be beneficial in some cases to provide baseline neurophysiologic data. potentials can then be repeated in the prone surgical position to help identify cervical positioning related neuromonitoring alterations. direct correlation between cerebrospinal fluid (csf) oxygenation and tcmeps has been shown in a pig model with clamping of spinal radicular arteries, with reversal of these neuromonitoring changes following unclamping of the vessels. in a canine study, multiple bilateral spinal radicular vessel ligation was required to create irreversible neurological deficit. in human studies examining neuromonitoring changes during scoliosis surgery, neuromonitoring changes associated with hypotension are often reversible with mean arterial pressure (map) elevation, and do not lead to permanent postoperative neurological deficit in the majority of situations. in cases of neuromonitoring changes without an obvious reversible surgical explanation, evaluation of blood pressure and correction of hypotension if possible should be undertaken. an investigation by clark retrospectively reviewed 140 patients with cervicothoracic spondylotic myelopathy undergoing spine surgery, of which 16 (11%) had intraoperative deceases in tcmeps. in total, there were 8 patients from this group who awoke with neurological deficits : 5 with c5 palsy and 2 with paraparesis. a significant correlation (p 85 mm hg has been shown to improve motor function and bowel / bladder recovery following traumatic spinal cord injury, and may be performed for up to 7 days, although some centers perform only 48 to 72 hours of map elevation. optimizing spinal cord oxygenation and avoiding hypotension are important interventions in optimizing outcomes following iatrogenic spinal cord injury. the neurological sequelae of traumatic spinal cord injury occurs due to an initial traumatic mechanical injury followed by secondary insult stemming from ischemia, reperfusion, ionic dysregulation, cellular excitotoxicity, swelling, and free - radical mediated peroxidation. numerous prospective human studies have been performed to investigate pharmacologic interventions to reverse the deleterious inflammatory response and neurological deficits from traumatic spinal cord injury, although unfortunately none have proven dramatically successful thus far. therefore at this time, no strong recommendations regarding steroids or other investigational medications can be made to provide to patients who suffer iatrogenic spinal cord injury resulting from cervical spine surgery. is commonly performed to decrease csf pressure in an attempt to prevent spinal cord injury during thoracoabdominal aorta surgery ; no data currently exist to examine whether this may be beneficial in cases of iatrogenic spinal cord injury during cervical spine surgery. this review demonstrates a similar lack of a protocol - based approach to discovery of a new postoperative neurological deficit. as case numbers will be too small to develop such a protocol based on data, a consensus - based approach appears appropriate. postoperative institutional safety improvement review of protocols and procedures are imperative following serious adverse events such as iatrogenic spinal cord injury and were likely performed in each of the cases presented in this investigation. unfortunately, details of individual institution safety improvement initiatives were not included in our data set. neuromonitoring utilizing somatosensory evoked potentials and transcranial motor evoked potentials (tcmeps) is frequently used in cervical spine surgery and may help surgeons intervene to reverse the immediate cause of intraoperative spinal cord injury. for procedures performed in the prone position, obtaining potentials with the neck in a neutral posture (prior to prone positioning) may be beneficial in some cases to provide baseline neurophysiologic data. potentials can then be repeated in the prone surgical position to help identify cervical positioning related neuromonitoring alterations. direct correlation between cerebrospinal fluid (csf) oxygenation and tcmeps has been shown in a pig model with clamping of spinal radicular arteries, with reversal of these neuromonitoring changes following unclamping of the vessels. in a canine study, multiple bilateral spinal radicular vessel ligation was required to create irreversible neurological deficit. in human studies examining neuromonitoring changes during scoliosis surgery, neuromonitoring changes associated with hypotension are often reversible with mean arterial pressure (map) elevation, and do not lead to permanent postoperative neurological deficit in the majority of situations. in cases of neuromonitoring changes without an obvious reversible surgical explanation, evaluation of blood pressure and correction of hypotension if possible should be undertaken. an investigation by clark retrospectively reviewed 140 patients with cervicothoracic spondylotic myelopathy undergoing spine surgery, of which 16 (11%) had intraoperative deceases in tcmeps. in total, there were 8 patients from this group who awoke with neurological deficits : 5 with c5 palsy and 2 with paraparesis. a significant correlation (p 85 mm hg has been shown to improve motor function and bowel / bladder recovery following traumatic spinal cord injury, and may be performed for up to 7 days, although some centers perform only 48 to 72 hours of map elevation. optimizing spinal cord oxygenation and avoiding hypotension are important interventions in optimizing outcomes following iatrogenic spinal cord injury. the neurological sequelae of traumatic spinal cord injury occurs due to an initial traumatic mechanical injury followed by secondary insult stemming from ischemia, reperfusion, ionic dysregulation, cellular excitotoxicity, swelling, and free - radical mediated peroxidation. numerous prospective human studies have been performed to investigate pharmacologic interventions to reverse the deleterious inflammatory response and neurological deficits from traumatic spinal cord injury, although unfortunately none have proven dramatically successful thus far. therefore at this time, no strong recommendations regarding steroids or other investigational medications can be made to provide to patients who suffer iatrogenic spinal cord injury resulting from cervical spine surgery. is commonly performed to decrease csf pressure in an attempt to prevent spinal cord injury during thoracoabdominal aorta surgery ; no data currently exist to examine whether this may be beneficial in cases of iatrogenic spinal cord injury during cervical spine surgery. this review demonstrates a similar lack of a protocol - based approach to discovery of a new postoperative neurological deficit. as case numbers will be too small to develop such a protocol based on data, a consensus - based approach appears appropriate. postoperative institutional safety improvement review of protocols and procedures are imperative following serious adverse events such as iatrogenic spinal cord injury and were likely performed in each of the cases presented in this investigation. unfortunately, details of individual institution safety improvement initiatives were not included in our data set. iatrogenic spinal cord injury following elective cervical spine surgery is a rare and devastating adverse event occurring in up to 0.24% of cases in this multicenter cohort. this study was limited in its ability thoroughly assess risk factors and outcomes of this adverse event due to the rarity of the event and the small number of cases encountered. no standard protocol exists that can guarantee prevention of this complication, and there is a lack of consensus regarding evaluation and treatment when it does occur. utilization of ionm and response to interoperative alerts should be standardized based on surgeon consensus. emergent imaging with mri or ct myelography to evaluate for compressive etiology or malpositioned instrumentation, appropriate surgical correction when appropriate, and maintenance of adequate mean arterial blood pressure should generally be performed in cases of postoperative spinal cord injury.	study design : retrospective cohort study of prospectively collected data.objective:to examine the incidence of iatrogenic spinal cord injury following elective cervical spine surgery.methods:a retrospective multicenter case series study involving 21 high - volume surgical centers from the aospine north america clinical research network was conducted. medical records for 17 625 patients who received cervical spine surgery (levels from c2 to c7) between january 1, 2005, and december 31, 2011, were reviewed to identify occurrence of iatrogenic spinal cord injury.results:in total, 3 cases of iatrogenic spinal cord injury following cervical spine surgery were identified. institutional incidence rates ranged from 0.0% to 0.24%. of the 3 patients with quadriplegia, one underwent anterior - only surgery with 2-level cervical corpectomy, one underwent anterior surgery with corpectomy in addition to posterior surgery, and one underwent posterior decompression and fusion surgery alone. one patient had complete neurologic recovery, one partially recovered, and one did not recover motor function.conclusion:iatrogenic spinal cord injury following cervical spine surgery is a rare and devastating adverse event. no standard protocol exists that can guarantee prevention of this complication, and there is a lack of consensus regarding evaluation and treatment when it does occur. emergent imaging with magnetic resonance imaging or computed tomography myelography to evaluate for compressive etiology or malpositioned instrumentation and avoidance of hypotension should be performed in cases of intraoperative and postoperative spinal cord injury.
rapid sequence induction of anesthesia is a specific technique originally used by anesthetists, described formally in 1970 by stept and safar, who showed it to reduce the risk of aspiration of gastric contents. with the advent of critical care, this procedure was increasingly used for the rapid control and protection of the airway, and for ventilation. safe, effective airway management in critically ill or injured patients is the cornerstone of resuscitation. it is defined as the virtual simultaneous administration of a potent sedative agent and neuromuscular blocking drug to facilitate tracheal intubation. by providing unparalleled access to the airway, and superior protection against adverse effects such as aspiration, rsi is a protocol predicated on the successful administration of several medications in a specific sequence. the medications fall into one of several categories of agents : pretreatment, induction, and paralytic. the purpose of this survey was to find out which specialty is performing rsis in the emergency departments and to determine drug preferences of emergency physicians during rsi in turkey. university and state hospitals with ongoing emergency medicine residency programs were included in the study. hospitals within the specified regions were selected with the only inclusion criteria being that the hospital had an emergency medicine department. the chief of the program or department was contacted via e - mail and requested to answer a survey consisting of six questions. the first question was to determine the specialties that perform the rsi in the emergency department. the following questions were about the drug preferences during premedication, defasciculation, induction, and neuromuscular blocking. from the total of 41 emergency medicine residency programs, 35 emergency departments (28 university hospitals and 7 state hospitals) were successfully contacted during the period from january - february 2008. 1rapid sequence intubation performing specialtytable 1frequently preferred drugs during rapid sequence intubation n%n%premedicationdefasciculationfentanyl2256.4vecuronium1548.4lidocaine717.9succinylcholine722.6atropine615.4midazolam619.3midazolam25.1rocuronium39.7oxygen12.6inductionhaloperidol12.6etomidate1441.2maintenance of sedationpropofol926.5midozolam1645.8fentanyl617.6propofol1234.3midazolam514.7etomidate411.4nmbdiazepam25.7succinylcholine2271.0norcuron12.8vecuronium516.1rocuronium39.7atracurium13.2abbreviations : n, number of answers in total ; nmb, neuro - muscular blocking agent rapid sequence intubation performing specialty frequently preferred drugs during rapid sequence intubation abbreviations : n, number of answers in total ; nmb, neuro - muscular blocking agent recent literature has indicated that rapid sequence intubation (rsi) with neuromuscular blocking agents has become the most common method emergency physicians use to achieve tracheal intubation. this survey is to be the first to determine the specialties that perform rsi in the emergency department and drug preferences during rsi in turkey. according to the responses, frequently preferred premedications were fentanyl, the defasciculating agent vecuronium, the induction agent etomidate, and the neuromuscular blocking agent (nmba) was succinylcholine. still, there is no consensus on the ideal induction agent for emergency rsi. a number of pharmacologically distinct medications are presently used for sedation, dissociation, hypnosis, or induction. likewise, the muscle paralysis achieved by the nmba is undoubtedly responsible for most of the improvement in ease of intubation using rsi techniques. the ideal (induction and nmba) agent would smoothly and quickly render the patient unconscious, unresponsive, and amnestic in one arm / heart / brain circulation time. such an agent would also provide analgesia, maintain stable cerebral perfusion pressure and cardiovascular hemodynamics, be immediately reversible, and have few, if any, adverse side effects, in other words, have rapid onset, brief duration, and no side effects. this may be because of misunderstanding of the responders or the fault of the authors of this article that happened during construction of the survey. in some questions the responders were allowed to answer more than one option, but in some questions they were not. this may have resulted in confusion during answering the survey. the concerns with using the nondepolarizing agents for intubation are their slow onset and long duration of action. from a pharmacodynamic standpoint, succinylcholine s extremely rapid onset of action and its short duration of action make it the ideal nmba for rsi. unfortunately, succinylcholine can cause such adverse effects as fasciculation, increased intracranial pressure, and increased intra - ocular pressure. while many of these side effects are of little clinical significance, succinylcholine also can have some potentially life - threatening effects. despite these side effects, succinylcholine was the most preferred nmba. in our survey, de - fasciculation after the patient has been successfully intubated, consideration of long - term sedation and muscle relaxation must be made. patients frequently can be managed with sedation alone, but neuromuscular blockade also is required at times. propofol, ketamine, benzodiazepines, and opioids all are appropriate choices for sedation, with any of the nondepolarizing agents useful for paralysis. the drugs used have the potential to turn an urgent airway problem into a life - threatening situation. emergency medicine in turkey is in its infancy and needs to develop official guidelines in this field. limitations of the survey the responders were not requested to define the reason for their drug preferences during rsi. different trainers could perform rsi with different drugs ; however, the survey requested only one response from each ed. in the majority of the emergency departments in turkey, emergency medicine physicians perform the rsi, with anesthetists performing it only in a few departments. the frequently preferred premedication was fentanyl, the defasciculating agent vecuronium, the induction agent etomidate, and nmba was succinylcholine. multiregional analysis of current activity to be completed by the chief of the emergency medicine department (or the responsible of the education program) name of the instutition........................................... which specialty is performing rapid sequence intubation (rsi) in your emergency department?emergency medicineanesthesiaother (please specify)..................................... in unexceptional circumstances, which one of the following medications do you prefer for premedication during rsi?lidocaineatropinefentanylother (please specify)..................................... in unexceptional circumstances, which one of the following medications do you prefer for defasciculation during rsi?vecuroniumrocuroniumatracuriumhaloperidolmidazolamother (please specify)..................................... in unexceptional circumstances, which one of the following medications do you prefer for induction during rsi?thiopentalketamineetomidatepropofolfentanylother (please specify)..................................... which one of the following drugs do you prefer for paralysis during rsi?succinylcholinevecuroniumrocuroniumatracuriumother (please specify)..................................... for the maintenance of sedation, which agent(s) do you prefer ? (please specify).............................................................................................................................. which specialty is performing rapid sequence intubation (rsi) in your emergency department ?.................................... in unexceptional circumstances, which one of the following medications do you prefer for premedication during rsi ?.................................... in unexceptional circumstances, which one of the following medications do you prefer for defasciculation during rsi ?.................................... in unexceptional circumstances, which one of the following medications do you prefer for induction during rsi ? other (please specify)..................................... which one of the following drugs do you prefer for paralysis during rsi ?	aimto determine which specialty was performing rapid sequence intubation (rsi) in the emergency departments and to determine drug preferences of emergency physicians during rsi in turkey.methodall emergency departments were contacted via e - mail, and the chiefs of the departments were requested to answer a survey consisting of six questions. hospitals within the specified regions were selected with the only inclusion criteria being that the hospital had an emergency medicine department. we determined that there were 32 university and 9 state hospital emergency medicine residency programs.resultsthirty-five emergency departments responded. in 31 (73%) departments emergency medicine physicians, in 4 (10%) departments anesthetists, and in 7 (17%) departments physicians of either specialty were routinely performing rsi. the most commonly preferred drugs were fentanyl for premedication, vecuronium for defasciculation, etomidate for induction, and succinylcholine for neuromuscular blocking.conclusionin the majority of the emergency departments in turkey, emergency medicine physicians perform the rsi ; the anesthetists perform it in only a few departments.
heavy metal investigations are very important scientific issue in poland. that is because of the fact that manufacturing causes the contamination of the different environmental components. moreover, research on metals are a challenge for scientist because of its complexity concerning methods of their determination, speciation analysis, toxicity for living organisms, transformation and migration in the environment, methods of monitoring, and remediation strategies (szyczewski. especially the bukowno region is an area contaminated with heavy metals, mainly zinc (zn), lead (pb), and cadmium (cd), but also thallium (tl) and arsenic (as). this is due to the exploitation and processing of zn pb ores since the eleventh century, as well as movements of pollutants from ore outcrops of triassic dolomites in the olkusz vicinity. podzol and podzolic soils, limestone soil, and fawn dominate in the studied area. the presence of primary and secondary minerals of zn, iron (fe), and pb are found in these soils (gruszczyski. 1990 ; trafas. 1990 ; cabaa 2009 ; gralski 2005 ; urbaski 2008 ; kiciska 2009). one of the major pollution sources in the area is the flotation waste site arising from the processing of zn pb ores in the mining and metallurgical plant zgh bolesaw in bukowno. in the landfill, 1.51.6 million mg of flotation wastes containing 0.921.32 % zn and 0.060.72 % pb are deposited annually. the estimated content of the accumulated metals is 250.5 gg of zn, 117.7 gg of pb, 13 gg of as, and 2 gg of cd (grecka. 1994 ; sikora. 1996). since the beginning of the waste deposition in 1973, 38 tg of wastes have been deposited (szuwarzyski and kryza 1993 ; pajor 2005). due to the mineralogical, chemical, and texture characteristics, the study of metal concentrations in surface soils in the vicinity of the waste site made by cabaa and sutkowska (2006), gruszecka and helios - rybicka (2006), and kiciska (2011) showed medium and heavy contamination of zn, pb, and cd on the basis of classification developed by the institute of soil science and plant cultivation iung in puawy, poland (kabata - pendias. 1993). in addition, contents of as and tl indicated by helios - rybicka. (2004), adamiec and helios - rybicka (2004), and kiciska (2009) exceeds the local geochemical background value (kabata - pendias and pendias 2001). the effect of zn pb ore extraction, processing, and disposal of emerging wastes in the silesia cracow region of poland is contaminating topsoils with pb, zn, cd, as, and tl at considerable distances (cabaa. 2012). due to the high metal content in the environment, preliminary assessments of the health risk to local populations were carried out (gruszecka and helios - rybicka 2009 ; gruszecka 2010). the results lead to the necessity for further analyses of metals doses taken in various routes of exposure. metal contamination of the soil surface layer suggested an investigation into the migration of heavy metals in soil profiles was necessary to determine the potential contamination of the lower soil horizons and groundwater. soil profiles were taken on the east side of the flotation waste site of the mining and metallurgical plants zgh bolesaw in bukowno, due to the fact that during previous research (gruszecka 2011) the contamination of the surface soil with metals was found. the schematic locations of the six 140 cm soil profiles (i vi) are shown in fig. 1. profiles were sampled on podzol soils that take the form of loose sand overgrown with pines (profile i, iv, and v), birches (profile ii) and both pines and birches (profile iii and vi). 1location of soil profiles i vi (based on the topographic map in the 1992 reference system, in scale 1:10,000, sheets : m-34 - 64-a - a-1, m-34 - 64-a - a-2, m-34 - 64-a - a-3, and m-34 - 64-a - a-4) location of soil profiles i vi (based on the topographic map in the 1992 reference system, in scale 1:10,000, sheets : m-34 - 64-a - a-1, m-34 - 64-a - a-2, m-34 - 64-a - a-3, and m-34 - 64-a - a-4) from the separated soil horizons, a total of 25 samples were extracted, in which values of eh and ph in h2o and 1 mol / dm kcl (according to pn - iso 10390 1997) were measured. concentrations of as, cd, pb, tl, and zn using an atomic absorption spectrophotometer with a graphite cuvette unica ice 3500 and icp - ms apparatus (mass spectrometer with inductively coupled plasma elan drc - e perkin elmer) in solutions obtained by the bcr three - stage sequential extraction procedure (quevauviller. analysis was conducted in the environmental analysis laboratory in the department of geology, geophysics and environmental protection of the agh university of science and technology in krakow, poland (fig. 2laboratory procedure scheme laboratory procedure scheme the mineral composition of the soil horizon samples was determined to verify that the mineral composition had a significant effect on the accumulation of heavy metals. soil samples were subjected to observation of the mineral composition in the transmitted light under a polarizing microscope (jenalab karl zeiss jena) using zooms 2.5 0.05 10 0.25 50 0.08. the horizon samples were analyzed with x - ray diffraction (xrd) and scanning electron microscopy with energy dispersed spectroscopy (sem - eds). sem - eds analysis was conducted using a microscope with field emission (jeol 5200), equipped with an energy dispersed analysis of x - rays system for the microprobe analysis, as well as a back scattered electron detector (yag bse). qualitative analysis of mineral composition was conducted by the x - ray powder method of debye - scherrer. patterns of xrd of all samples were recorded using an x - ray diffractometer (dron-3.0) with the following parameters : cu k radiation, ni filter, 35 kv lamp voltage, lamp current of 30 ma, the registration step by step : step 2 = 0.05, counting time 1 s / step. to eliminate the influence of apparatus on the intensity of the analytical line, each sample io (initial intensity) values of interlayer distances obtained from xrd distance patterns were used to identify mineral phases occurring in tested samples, based on the information contained in the directory international centre for diffraction data (icdd) and the computer program xrayan. for the interpretation of mineral phases, the computer program claylab ver. 3. among the six soil profiles taken in the research area, three had a 1020-cm organic (o) horizon. the thickness of the humus horizon (a) was between a few and 10 cm. the level eluvial horizon (e) had a thickness from several to 30 cm. the thickness of the illuvial horizon (b) varied from 60 to 90 cm. 3formation of the soil profiles on the east side of the postflotation waste site zgh bolesaw in bukowno formation of the soil profiles on the east side of the postflotation waste site zgh bolesaw in bukowno the average results of the mineralogical analysis in transmitted light observation, xrd and sem - eds investigations are summarized in table 1. the mineral composition of each soil horizon mainly differed in the quantitative composition of the various minerals. the mineralogical analysis showed that the main components of each soil horizon were quartz, k - feldspar (microcline), and plagioclases. in the analysis in transmitted light, there was also evidence that, with decreasing depth, the amount of om decreased and was absent in the c horizon. in the o, a, e, and b horizons, ore minerals were observed, often zn- and pb - bearing minerals such as pyrite and sphalerite and carbonate minerals such as calcite, dolomite, and ankerite as well as micas such as sericite. however, in the a, e, b, and c horizons, grains of goethite were also present. each of the horizons, except the c horizon, had a small amount of clay minerals. in the o horizon, there were also fragments of rock (sandstone) and the e horizon had additional iron oxides.table 1average mineral composition for individual soil horizonssoil horizontransmitted lightxrd and sem analysisoquartz (places regenerative quartz), k - feldspars (often kaolinitization)microcline, plagioclases, organic matter, ore minerals, chalcedony, sericite, individual carbonates, clay minerals (e.g., glauconite), fragments of rocks (sandstones)quartz, fe - dolomite / ankerite, plagioclases, k - feldspars, apatiteaquartz (places regenerative quartz), k - feldspars (places kaolinitization)microcline, goethite, individual carbonates, organic matter, ore minerals, clay minerals, sericitequartz, fe - dolomite, calcite, pyrite, probably in trace amount barite, k - feldspar, plagioclases, carbonatesequartz (regenerative quartz), k - feldspars (places sericitization and kaolinitization)microcline, iron oxides, plagioclases (often illitization and diagenesis of grain is observed), single grains of chalcedony, goethite, muscovite (occasionally), sericite, organic matter (occasionally), single grains of glauconite, single carbonates, colophane, iron oxidesquartz, carbonates (fe - dolomite), k - feldspars, plagioclases, gypsum, sphalerite, pyrite, fe - dolomiteblarger grain of quartz (regenerative quartz), single k - feldspars (places sericitization and kaolinitization), carbonates, fine crystalline clay - iron matter, sericite with organic matter, goethite, plagioclases (places illitization), ore minerals, organic matterquartz, k - feldspars, plagioclases, carbonates (calcite), fe - dolomite, gypsum, pyrite, baritecquartz (places regenerative quartz), k - feldspars (places sericitization and kaolinitization), glauconite, chalcedony, goethitequartz, k - feldspars, plagioclases average mineral composition for individual soil horizons in the xrd analysis (fig. 4), the patterns for the a, e, and c horizons were very close, indicating the presence of minerals such as quartz and small amounts of carbonates such as calcite, dolomite, and ankerite and k - feldspars. slightly different curves represented the values for the a and b horizons, where the dominant minerals were quartz and carbonates (calcite, dolomite, and ankerite). in addition, a small proportion of feldspar was also seen, as well as the presence of barite and gypsum.fig. 4xrd diffraction patterns of soil horizons on the east side of the postflotation waste site in bukowno xrd diffraction patterns of soil horizons on the east side of the postflotation waste site in bukowno in the sem - eds observation (fig. 5), silicate minerals and quartz were observed, and ore minerals such as sphalerite and galena were present. 5examples of sem microphotographs of soil horizons on the east site of the postflotation waste site in bukowno examples of sem microphotographs of soil horizons on the east site of the postflotation waste site in bukowno both the potential (6.68.0) and active (6.08.0) ph values (table 2), determined the soil ph as slightly acidic through neutral to alkaline. the om ranged from 2 to 80 %.table 2physical properties and total metal concentration in soil profilessoil horizonph (kcl)ph (h2o)eh (mv)om (%) total concentration (mg / kg d.m.)ascdpbtlznprofile ioa8.07.7786013.613.31,1031.90883e7.16.927103.81.1430.79136b7.17.056102.11.23070.28145c7.16.92320.40.3370.0232profile iioa7.06.8213018.510.44541.10831e7.27.249102.60.8350.1469b7.17.133203.02.21730.26167c7.17.13820.41.4360.0133profile iiio6.66.721801.53.26250.03298a6.67.132411.87.32860.15390e7.06.819100.97.1480.2278b6.96.835102.627.023540.101,352c7.37.03020.40.3370.0136profile ivo7.98.0344010.78.81,0441.441,474a7.36.823344.876.94,1814.505,254e6.86.01071.014.93062.51995b7.67.03110100.040.92,5646.533,329c7.27.45421.31.9370.05114profile voa7.07.537201.01.9480.07205e6.77.545201.10.1210.0253b6.57.620200.86.01240.2290c7.17.65820.40.3360.0141profile vio7.07.157605.45.91090.55415a7.07.347501.81.5470.07120e7.27.037100.040.9230.0365b7.17.352204.61.0220.2366c7.17.35110.50.5110.01276() lack of horizon physical properties and total metal concentration in soil profiles the total contents of heavy metals (table 2) in surface soils (025 cm) were compared (table 3) with the permissible soil concentrations in poland (table 4). because of the fact that the research area is populous and the common is that residents grow vegetables for their own nutritional needs, analyzed metal contents in soil profiles were also compared with permissible levels for agricultural soils. permissible values were according to the regulation of the minister of the environment on the soil quality standards and ground quality standards (gj no. 1359) and the chemical quality of soil classification recommended for agricultural use of soil developed by the institute of soil science and plant cultivation iung (kabata - pendias. 1993, 1995).table 3total contents of metals in the topsoil and their permissible levels by polish lawascdpbtlznsoil profileiiiiiiivvviiiiiiiivvviiiiiiiivvviiiiiiiivvviiiiiiiivvvicontent9.29.34.011.71.41.94.23.31.63.01.01.020218413315120391.60.80.11.00.10.236837215760917079g.b.20,21813000,32050i170100ii2100200iii3500700iv52,5001,500v>5>2,500>1,500a20150100b204100300c60156001,000 g.b. geochemical background (kabata - pendias and pendias 2001), a group a (rme 2002), b group b (rme 2002), c group c (rme 2002), () not determinedtable 4permissible concentrations of metals in soils by polish guidelinespermissible metals concentration classificationsascdpbtlznmg / kg d.m.geochemical background (kabata - pendias and pendias 2001)natural element concentration20.218130chemical quality grades of soil by iung (kabata - pendias. 1995)0soil not contaminated, the natural content of heavy metals0,32050isoils with elevated concentrations of heavy metals170100iiweakly contaminated soils2100200iiimedium contaminated soils3500700ivheavily contaminated soils52,5001,500vvery heavily contaminated soils>5>2,500>1,500types of ground regulation of the minister of the environment (gj no. 1359)group aprotected areas based on the water law act and protection of the nature act 20150100group bagricultural land, forest land and trees, woodland and shrubs204100300group cindustrial areas, minerals, communication areas60156001,000() not determined total contents of metals in the topsoil and their permissible levels by polish law g.b. geochemical background (kabata - pendias and pendias 2001), a group a (rme 2002), b group b (rme 2002), c group c (rme 2002), () not determined permissible concentrations of metals in soils by polish guidelines the total as concentration in topsoil of two soil profiles was at the geochemical background level (2 mg / kg). in the remaining four profiles, the geochemical background level was exceeded four to six times. the permissible as concentration for protected areas (group a) according to the regulation of the minister of the environment the permissible concentration of as for agricultural soils in the iung classification of chemical quality of soil degrees was not specified. the concentration of cd in all analyzed profiles exceeded the geochemical background content (0.2 mg / kg). according to the iung classification, three samples were heavily cd contaminated (grade iv), and three samples were weakly cd contaminated (grade ii). profile i also exceeded the permissible content for group b soils (agricultural soils and urban areas) according to the regulation of the minister of the environment. in all six profiles, the pb content in topsoil exceeded the geochemical background level (18 mg / kg). in four samples, pb content classified them as third grade (medium contaminated) of the iung chemical quality classification and also exceeded the permissible content for group b soils according to the regulation of the minister of the environment. two samples were classified as grade i (elevated concentration of heavy metals) for pb concentration. three soil profile topsoils were classified as grade iii of the iung chemical quality classification (medium contaminated soils) for zn concentration. two samples exceeded the permissible concentration for group b soils according to the regulation of the minister of the environment, and five samples exceeded the permissible concentration for group a soils (protected areas). according to the iung chemical quality classification, two soil samples had low zn contamination (grade ii), and one had elevated zn concentrations (grade i). soil tl content is not in the iung chemical quality classification or the regulation of the minister of the environment. measured tl concentrations in three profiles were at the geochemical background level (1 mg / kg) and below the geochemical background level in three profiles. 6), the highest total as content was observed in the o, a, and b horizons. arsenic was associated mainly with sulfides and om (stage iii of the bcr extraction). it was observed that the higher the om content in the horizon, the more as was related to the oxidized fraction. 6binding forms of metals as a percentage of the total metal content in soil profiles binding forms of metals as a percentage of the total metal content in soil profiles the highest total cd concentration was in the o, a, and b horizons. approximately 90 % of the total cd in all soil horizons was bound to replaceable positions or associated with carbonates (bcr extraction stage i). the total pb content was highest in the o, a, and b horizons. approximately 60 % of the total pb concentration was present in mobile forms, and in the o, a, and b horizons, this constituted > 80 %, while concentration of mobile forms in the e and c horizons dropped to 5>2,500>1,500a20150100b204100300c60156001,000 g.b. geochemical background (kabata - pendias and pendias 2001), a group a (rme 2002), b group b (rme 2002), c group c (rme 2002), () not determinedtable 4permissible concentrations of metals in soils by polish guidelinespermissible metals concentration classificationsascdpbtlznmg / kg d.m.geochemical background (kabata - pendias and pendias 2001)natural element concentration20.218130chemical quality grades of soil by iung (kabata - pendias. 1995)0soil not contaminated, the natural content of heavy metals0,32050isoils with elevated concentrations of heavy metals170100iiweakly contaminated soils2100200iiimedium contaminated soils3500700ivheavily contaminated soils52,5001,500vvery heavily contaminated soils>5>2,500>1,500types of ground regulation of the minister of the environment (gj no. 1359)group aprotected areas based on the water law act and protection of the nature act 20150100group bagricultural land, forest land and trees, woodland and shrubs204100300group cindustrial areas, minerals, communication areas60156001,000() not determined total contents of metals in the topsoil and their permissible levels by polish law g.b. geochemical background (kabata - pendias and pendias 2001), a group a (rme 2002), b group b (rme 2002), c group c (rme 2002), () not determined permissible concentrations of metals in soils by polish guidelines the total as concentration in topsoil of two soil profiles was at the geochemical background level (2 mg / kg). in the remaining four profiles, the geochemical background level was exceeded four to six times. the permissible as concentration for protected areas (group a) according to the regulation of the minister of the environment the permissible concentration of as for agricultural soils in the iung classification of chemical quality of soil degrees was not specified. the concentration of cd in all analyzed profiles exceeded the geochemical background content (0.2 mg / kg). according to the iung classification, three samples were heavily cd contaminated (grade iv), and three samples were weakly cd contaminated (grade ii). profile i also exceeded the permissible content for group b soils (agricultural soils and urban areas) according to the regulation of the minister of the environment. in all six profiles, the pb content in topsoil exceeded the geochemical background level (18 mg / kg). in four samples, pb content classified them as third grade (medium contaminated) of the iung chemical quality classification and also exceeded the permissible content for group b soils according to the regulation of the minister of the environment. two samples were classified as grade i (elevated concentration of heavy metals) for pb concentration. three soil profile topsoils were classified as grade iii of the iung chemical quality classification (medium contaminated soils) for zn concentration. two samples exceeded the permissible concentration for group b soils according to the regulation of the minister of the environment, and five samples exceeded the permissible concentration for group a soils (protected areas). according to the iung chemical quality classification, two soil samples had low zn contamination (grade ii), and one had elevated zn concentrations (grade i). soil tl content is not in the iung chemical quality classification or the regulation of the minister of the environment. measured tl concentrations in three profiles were at the geochemical background level (1 mg / kg) and below the geochemical background level in three profiles. considering the metal binding forms in individual soil horizons (fig. 6), the highest total as content was observed in the o, a, and b horizons. arsenic was associated mainly with sulfides and om (stage iii of the bcr extraction). it was observed that the higher the om content in the horizon, the more as was related to the oxidized fraction. 6binding forms of metals as a percentage of the total metal content in soil profiles binding forms of metals as a percentage of the total metal content in soil profiles the highest total cd concentration was in the o, a, and b horizons. approximately 90 % of the total cd in all soil horizons was bound to replaceable positions or associated with carbonates (bcr extraction stage i). the total pb content was highest in the o, a, and b horizons. approximately 60 % of the total pb concentration was present in mobile forms, and in the o, a, and b horizons, this constituted > 80 %, while concentration of mobile forms in the e and c horizons dropped to < 50 % with the exception of the vi profile, where the trend was reversed. with increasing depth, the content of the reduced forms of pb increased. of all analyzed metals, the lowest concentration of oxidized forms was observed for pb. the total tl content was highest in the o, a, and b horizons. about 50 % of the total tl concentration was extracted at stage i of the procedure. the lower the soil horizon, the higher the content of reducible and oxidizable tl forms. the distribution of total zn concentration between the different extraction stages is much the same as for cd. approximately 80 % of zn was related to the water- and acid - soluble forms. increasing depth in the profile increased the share of the reduced and oxidized zn forms. the investigation showed that the soils on the eastern side of the postflotation waste site were mostly loose sands. the soil profiles were formed mainly from sand and bedrock (sand) was found at 120 cm. the highest concentrations of metals were measured in the o, a, and b horizons. this may be primarily associated with the presence of om, with which metals can be combined to form complex compounds. it can also be caused by the accumulation of contaminants on the surface and the gradual migration with depth in the soil profile (except of eluvial horizon e). in the study area, this migration occurred to a depth of 6090 cm. such results also indicate that mineral composition had an insignificant influence on the accumulation of heavy metals in individual soil horizons. the lack of such dependence can be also conditioned to low differences in mineral composition of soil horizons. perhaps in other types of soil (with more minerals in the horizons), the influence of each soil horizon on the accumulation of heavy metals could be better observed. in the bcr sequential extraction procedure for this reason, more specific tests defining concentrations of metals in the residue should be made. the reported water table level in the research area is at a depth of 23 m (cieko 2011), so currently, there is no risk of groundwater contamination associated with the migration of metals in the soil profiles. however, further studies including infiltration of rainwater into the soil profile with the loads of pollutants carried with them should be made. it is not the total metal concentration that determines its harmfulness, but their form. the study of metal binding forms in soils using the bcr three - stage sequential extraction showed, that in the case of cd, zn, and pb, mobile forms, i.e., those that are released into the environment through water and even weak acid leaching, dominated. in addition, the depth of the absorption layer in the root zone for typical crops reached 80 cm (baszkowski 2012). assuming that the total contents of 90 % cd, 60 % zn, and 80 % pb occurred as mobile forms, concentrations of 10 mg / kg cd, 694 mg / kg zn, and 922 mg / kg pb could potentially be taken up in the root zone and moved to higher levels of the food chain. in the case of tl, 60 % of its concentration occurred in mobile forms, which corresponded to < 1 mg / kg. up to 20 % was in exchangeable positions or bound with carbonates, which corresponded to < 5 mg / kg in bioavailable forms. it appears advisable to conduct further studies to determine the actual amount of metal taken up by the crops in the study area. by comparing the total metal concentrations in the soil horizons with the polish legal limits in force, it is found that the tested soils exceeded the permissible levels for agricultural soils. because of the widespread vegetable cultivation in home gardens by the local population, it is advisable to determine the actual uptake of metals by cultivated crops on soils of such metal pollution. uptake of as and tl, are not governed by polish law or the chemical quality classification for agricultural soils but should also be considered. it seems to be necessary in this situation to determine the relationship between the content of as and tl in the soils and the dose taken in the pathways of exposure associated with agricultural use of this type of soil.	the lead zinc industry in the bukowno region of southern poland has polluted the surface layer of the surrounding soils mainly with lead (pb), cadmium (cd), zinc (zn), arsenic (as), and thallium (tl). analysis of six soil profiles, taken on the east side of the postflotation waste site of the mining and metallurgical plants zgh " bolesaw " in bukowno, showed that they were podzol soils, taking form of loose sands with neutral ph and reducing conditions. concentration of organic matter in the horizons ranged from 2 to 80 %. the main components of the mineral soil were quartz, carbonates, k - feldspars, plagioclases, and micas (sericite). the highest total concentrations of metals were found in the o, a, and b horizons. over 90 % of the cd content, 80 % of the pb content, 60 % of the zn content, 60 % of the tl content, and 20 % of the as content occurred as mobile forms. the corresponding total concentrations were 10 mg / kg cd, 922 mg / kg pb, 694 mg / kg zn, < 1 mg / kg tl, and < 5 mg / kg as. this can potentially be taken up from the soil and transported in the trophic chain. comparing the total metal content with the legal limits in poland, it is observed, that the investigated soils exceeded the permissible levels of cd, pb, and zn for agricultural soils. arsenic and tl are not reflected in the chemical quality of soil classifications.
dirofilariosis, caused by dirofilaria immitis, is found world - wide, but the most endemic areas are those with high temperatures and appropriate mosquito vector populations. this vector - borne parasite can cause patent infections in dogs, cats and wild canidae adult heartworms may cause clinical signs ranging from mild cough to congestive heart failure, intravascular hemolysis and pulmonary thromboembolism which are often fatal if untreated (soulsby 1986). dirofilaria immitis in dogs can be diagnosed through careful morphological examination of circulating microfilariae, detection of circulating antigens, histochemical or immuno - histochemical staining of circulating microfilariae or, more recently, through molecular approaches. morphological identification of circulating microfilariae, however, is not always easy and is potentially misleading (rishniw. the adult worms are observed mainly in the subcutaneous tissue of dogs, and produce microfilariae that circulate in the blood stream of infected dogs. dignosis of it can be done by blood smear evaluation for the presence of microfilariae, serologic detection antigen or antibodies and detection of microfilarial dna by pcr (lee. 2004). dirofilaria immitis is transmitted by several culicid mosquito species belonging to a wide range of genera, including culex, aedes, ochlerotatus, anopheles, armigeres and mansonia (cancrini. 1995). aedes vexans and culex pipiens were detected as the potential vectors of d. immitis in turkey (yildirim. for the first time, cytochrome c oxidase i (coi) sequences were obtained from iranian specimens of an. only culex theileri were found naturally infected with third - stage (infective) larvae of d. immitis (azari - hamidian. dna - based diagnostic tests for d. immitis and d. repens infections have been shown to overcome some deficiencies of parasitological and serological diagnosis, and specific and sensitive polymerase chain reaction (pcr)-based assays have been reported (mar. the usefulness of different pcr methods for the identification of dirofilaria spp microfilaria in dog blood (the definitive host) has been reported in recent publications (gioia. dirofilaria immitis is one of the several species of parasitic nematodes that hold the obligate symbiont bacteria wolbachia spp. large colonies of wolbachia live in the subdermal lateral cords of both female and male nematodes, as well as in the reproductive structures of females (mchaffie 2012). the aim of the current study was to performe a single - step multiplex pcr to detect and differentiate d. immitis and d. repens on genomic dna isolated from dog blood and also detect the seroprevalance of d. immitis by elisa. the amplification was performed using a set of primers designed on a portion of the small subunit ribosomal rna gene of the mitochondrion (12s rdna). the other aim of this work was to pcr detection of the wolbachia which is play an important role in d. immitis biology and contributes to the inflammatory pathology of the heart - worm. a total of 161 whole blood and sera samples were obtained from stray dogs in elazig province of eastern turkey within 2010. these dogs had been captured from suburbs by the local authorized for the aim of spaying and during this procedure the blood samples were acquired under anesthesia. the blood and sera samples were stored in 20 c untill use and age, breed and genders were recorded. the blood samples were removed from freezer and waited at room temperature untill thawed. then 1 ml blood sample was putted into an eppendorf tube and centrifuged during 5 min by 5000 rpm for sink to the bottom of possible microfilaria. supernatant was removed and prior to gdna isolation pellet was digested overnight at 56 c with 600 l lysis buffer of the kit to which 20 l proteinase - k (20 mg / ml) (sigma, usa) were added. the tubes were incubated at 56 c for overnight and the kit procedure was followed and at the last step the pellet was resuspended in 80 l sterile distilled water, and the gdna samples were stored at 20 c until use. the multiplex - pcr reactions for d. immitis and d. repens were performed using two sets of primer in the same reaction. general primer pairs 12sf (5-gttccagaataatcggcta-3) and 12srdeg (5-attgacggatg(ag)tttgtacc-3) were used previously designed on the 12s rdna region (casiraghi. 2004). besides we used a specific forward primer for d. immitis (12sf2b 5-tttttacttttttggtaatg-3) and a specific reverse primer for d. repens (12sr2 5-aaaagcaacacaaataa (ca)a-3) previously designed by gioia. the pcr reactions were carried out in a total volume of 50 l containing 5 l of genomic dna for each sample amplification, 5 l of mgcl2, 1.25 mm of each dntp, 5 l 10 x pcr buffer, 0.5 iu taq dna polymerase and 20 pmol of each primers. the thermal profile used was 92 c for 1 min ; 40 cycles of 92 c for 30 s, 52 c for 45 s, 72 c for 1 min and final elongation step at 72 c for 10 min. the amplified products were separated by electrophoresis in 2% agarose gel with a tris - boric acid edta (tbe, ph 8.3) buffer at 90 v for 45 min. following electrophoresis, the amplified products were visualized with ethidium bromide (0,5 g / ml) staining for 45 min at room temperature. dirofilaria immitis genomic dna positive control sample was extracted from microfilariae present in the blood of infected dogs (gifted from another research group) (yildirim. another gdna control sample was extracted from an adult d. repens parasite (this worm was gifted by luigi venco (veterinary hospital citt`a di pavia, viale cremona pavia, italy). extracted dna was also tested for the presence of wolbachia using a pcr - based assay and the gene primer wsp. a specific primer sets (forward 5-tggtccaataa gtgatgaag aaactagcta-3, the pcr mixtures were composed of 5 l of 10x pcr buffer, 5 l of mgcl2, 125 m of each dntps, 20 pmol of each primers, 0.2 l (5 iu) taq - dna polymerase and 5 l of genomic dna was used for each pcr reaction. the reactions were performed on a pcr thermal cycler (thermo electron corporation, waltham, ma, usa) under the following conditions : 94 c for 3 min, 40 cycles of 94 c for 1 min, 52 c for 1 min and 72 c for 1 min with a final extension at 72 c for 5 min. pcr products were analyzed on 1.4% agarose gels stained by ethidium bromide and visualized under ultra - violet light. randomly selected six dirofilaria and two wolbachia samples were sequenced for confirmation of the pcr results. filarcheck (agrolab, italy) kit was used for working the dog sera for serological analysis. microplate wells were coated with a monoclonal antibody against the circulating antigen of d. immitis. the data were evaluated by spss 15.0 programme using of 2x2 fischer 's exact test and pearson 's chi square test. a total of 161 whole blood and sera samples were obtained from stray dogs in elazig province of eastern turkey within 2010. these dogs had been captured from suburbs by the local authorized for the aim of spaying and during this procedure the blood samples were acquired under anesthesia. the blood and sera samples were stored in 20 c untill use and age, breed and genders were recorded. the blood samples were removed from freezer and waited at room temperature untill thawed. then 1 ml blood sample was putted into an eppendorf tube and centrifuged during 5 min by 5000 rpm for sink to the bottom of possible microfilaria. supernatant was removed and prior to gdna isolation pellet was digested overnight at 56 c with 600 l lysis buffer of the kit to which 20 l proteinase - k (20 mg / ml) (sigma, usa) were added. the tubes were incubated at 56 c for overnight and the kit procedure was followed and at the last step the pellet was resuspended in 80 l sterile distilled water, and the gdna samples were stored at 20 c until use. the multiplex - pcr reactions for d. immitis and d. repens were performed using two sets of primer in the same reaction. general primer pairs 12sf (5-gttccagaataatcggcta-3) and 12srdeg (5-attgacggatg(ag)tttgtacc-3) were used previously designed on the 12s rdna region (casiraghi. 2004). besides we used a specific forward primer for d. immitis (12sf2b 5-tttttacttttttggtaatg-3) and a specific reverse primer for d. repens (12sr2 5-aaaagcaacacaaataa (ca)a-3) previously designed by gioia. the pcr reactions were carried out in a total volume of 50 l containing 5 l of genomic dna for each sample amplification, 5 l of mgcl2, 1.25 mm of each dntp, 5 l 10 x pcr buffer, 0.5 iu taq dna polymerase and 20 pmol of each primers. the thermal profile used was 92 c for 1 min ; 40 cycles of 92 c for 30 s, 52 c for 45 s, 72 c for 1 min and final elongation step at 72 c for 10 min. the amplified products were separated by electrophoresis in 2% agarose gel with a tris - boric acid edta (tbe, ph 8.3) buffer at 90 v for 45 min. following electrophoresis, the amplified products were visualized with ethidium bromide (0,5 g / ml) staining for 45 min at room temperature. dirofilaria immitis genomic dna positive control sample was extracted from microfilariae present in the blood of infected dogs (gifted from another research group) (yildirim. another gdna control sample was extracted from an adult d. repens parasite (this worm was gifted by luigi venco (veterinary hospital citt`a di pavia, viale cremona pavia, italy). extracted dna was also tested for the presence of wolbachia using a pcr - based assay and the gene primer wsp. a specific primer sets (forward 5-tggtccaataa gtgatgaag aaactagcta-3, the pcr mixtures were composed of 5 l of 10x pcr buffer, 5 l of mgcl2, 125 m of each dntps, 20 pmol of each primers, 0.2 l (5 iu) taq - dna polymerase and 5 l of genomic dna was used for each pcr reaction. the reactions were performed on a pcr thermal cycler (thermo electron corporation, waltham, ma, usa) under the following conditions : 94 c for 3 min, 40 cycles of 94 c for 1 min, 52 c for 1 min and 72 c for 1 min with a final extension at 72 c for 5 min. pcr products were analyzed on 1.4% agarose gels stained by ethidium bromide and visualized under ultra - violet light. randomly selected six dirofilaria and two wolbachia samples were sequenced for confirmation of the pcr results. filarcheck (agrolab, italy) kit was used for working the dog sera for serological analysis. microplate wells were coated with a monoclonal antibody against the circulating antigen of d. immitis. the data were evaluated by spss 15.0 programme using of 2x2 fischer 's exact test and pearson 's chi square test. multiplex - pcr reaction showed the expected amplification products of approximately 500 bp for the genus dirofilaria, 327 bp for d. repens and of 204 bp for d. immitis. m : molecular weight marker (100 bp), 1 : positive control of mix infection (500 bp, 327 bp and 204 bp), 2 : positive control of dirofilaria repens (500 bp and 327 bp), 3 : positive control of dirofilaria immitis (500 bp and 204 bp), 4 : only d. repens detected sample, 5, 6, 7 : only d. immitis detected samples, 8, 9, 10 : mix infected samples. m : molecular weight marker (100 bp), 1, 2 : positive samples (630 bp), 3 : positive control the results of the pcr assay according to the ages and gender of filarial agents and wolbachia are shown in table 1. thirty five male dogs were examined by multiplex - pcr and the prevalance values were 5.7% for d. immitis, 2.8% for d. repens and 2.8% for mixed infection (both d. immitis and d. repens). on the other hand, 126 female dogs were examined by pcr and only one case was d. immitis (0.8%) and two cases were d. repens (1.5%). positivity of filarial agents and wolbachia according to ages and gender among the 161 samples screened by the elisa, 6 samples (3.7%) tested positive for the d. immitis. there was no significant difference in the number of positive d. immitis infection among female dogs (4 out of 126, 3.2%) and male dogs (2 out of 35, 5.7%). only 2 out of the 69 dogs belonging to the 01 yrs old group were positive (2.9%), while 4 out of 71 dogs belonging to the 24 yrs old group were positive (5.6%). a total of 21 dogs belonging to the > 4 yrs old group showed no seropositivity of d. immitis infection. adult d. immitis, inhabit the right ventricle of the heart the pulmonary arteries where they cause canine heartworm disease while the adults d. repens usually inhabit the subcutaneous tissue. in addition, it is well - known that d. immitis and d. repens produce microfilariae that circulate in the blood of dogs (soulsby 1986). dirofilaria immitis occurs worldwide in tropical, sub - tropical and temperate climates however d. repens occurs in the oldworld, in particular, throughout the mediterranian sub - region, south asia and sub - saharan africa (cringoli. vector borne pathogens are sensitive to climatic condition, and there are some evidence that climate change may increase the incidence and dentensity of the diseases transmission (purse. climate change has significant potential to intensify the vector borne diseases (khasnis and nettleman 2005). dirofilaria immitis vectors are mosquitoes of culicidae family with nearly 70 species susceptible for developing of parasite and thus considered potential vectors (vezzani and carbajo 2006). aedes albopictus is reported as the primary potential vector of d. immitis in italy (cancrini. theileri was detected as a vector of d. immitis in portugal (santa - ana. pipiens are the main potential vectors for d. immitis in central turkey. in the current study several studies have been published regarding the distribution and prevalence of d. immitis in dogs in turkey. it was first reported in a dog the year of 1951 in turkey (guralp 1981). tasan (1983) detected micro - filaraemia in 53/283 (18.7%) stray dogs in elazig. the prevalence was recorded as 1.52% in istanbul (oncel and vural 2005), 9.6% in kayseri, (yildirim. 2007), 8.1% in erzurum (simsek. 2011) besides 12.3%, 18.3%, 10.5% and 14.8% in sakarya, kocaeli, mersin and ankara, respectively (simsek. 2008). the prevalence of d. immitis in dogs has been determined traditionally by postmortem inspection, detection of micro - filariae and serological testing. in addition, some antiparasitic treatments such as macrolides may render an infected dog amicrofilaraemic for 69 months (hoover. thus, serological and microfilarial examinations should be applied together for screening d. immitis in dogs. dna based diagnostic tests for d. immitis infections have been shown to overcome some deficiencies of parasitological and serological diagnosis, and specific and sensitive polymerase chain reaction (pcr)-based assays have been reported (rishniw. the current study describes a quick and accurate molecular method for the simultaneous detection of the d. immitis and d. repens for the first time in turkey. although there have been some records about variability between age and dirofilariosis prevalence (montoya. 2003), some authors (rowley 1981, martin and collins 1985) have reported that age has no effect on dirofilariosis and it can be occur in all ages dog. while the others (song. 2001) have stressed that ages and positivity, have positive relation and especially 37 ages group have higher risk than the others. (2001) found the lowest prevalence in 13 ages (6.3%) and following 36 ages (14.1%) while the highest rates has been found in up to 6 ages (23.7%). (2008) determined the highest percent 35 ages dog (17.5%) while there was no positivity in up to 6 ages. in the current study, all dogs that were defined only d. immitis by pcr were in 24 ages group (4.2%). similarly, the elisa seropositivity was 2.9% in 01 ages and 5.6% in 24 ages dog. a possible explanation for higher seroprevalence of d. immitis infection in older dogs might be due to their longer exposure to the risk factor like mosquito (fan. (1980) also indicated that the age of dogs was an important risk factor and determined by time of exposure in the endemic area. in the present study, elisa and pcr positivity were higher in male than female dogs. 1998) indicated that heartworm infection was more common in male dogs than female dogs, and the generally higher infection rate in male dogs had been postulated to be due to their stronger attraction to mosquitoes. however, simsek. (2008), reported 10.7% for males and 14.4% for females. more male dogs live in the outdoor, due to their use in defence of property. they are, therefore, more likely to be bitten by mosquitoes (montoya. however, all studied stray dogs in this work were living in out - door. we believe that, some individual parameters like hormonal changes and immune deficiencies in female dogs may more tend to dirofilariosis. canine heartworm disease is generally diagnosed by antigen testing for d. immitis, and/or identification of microfilariae in the blood of infected dogs. however, some other filariae, including dipetalonema reconditum, d. repens and approximately 1% of d. immitis infestations, can produce persistent microfilaremias with negative heartworm antigen tests (rishniw. thus, serological and molecular techniques should be use as combined. in this study, the sero - prevalence with elisa was 3.7% while the positivity was 1.8% by pcr. this differences might be related with some possible cross reactions with the other nematodes, single - sex adults and/or possible treatment of microfilaria by macrolids. the adult parasites are observed in the subcutaneous tissues of dogs and produce microfilariae that circulate in the perifer blood of infected dogs. it is less remarkable than d. immitis due to the lower pathogenicity (soulsby 1982). tasan (1984), necropsied 120 dogs and found the occurence of d. repens as 2.5% in elazig province of turkey. whereas, yildirim (2004) examined a total of 300 dog blood by modified knott and membrane filtration tests and no detected any d. repens micofilaria in ankara. in the current study, one of the examined dogs (0.6%) was infected with only d. repens by pcr and three of them (1.8%) were infected with both d. immitis and d. repens. microfilariae of d. repens are difficult to discriminate from d. immitis, since they have similar morphology. staining of microfilaria was widely used for discriminate the both species. in the last years, pcr analysis was reported to be quite sensitive and specific for the differentiate the species (lee. (2010) designed a single - step multiplex pcr was based on the amplification of a partial 12s rrna gene of the mitochondrion with a mix of general and species - specific filarial primers in a single reaction. we also used the same primers for the amplification of d. immitis and d. repens 12s rrna genes by pcr in a single tube. thus, the simultaneous detection of both d. immitis and d. repens in naturally infected dogs has been achived for the first time in turkey. wolbachia is an intracellular alphaproteo - bacteria endosymbionts sheltered in a broad range of insects and nematodes (pfarr and hoerauf 2007). according to reports based on dna amplification, one in five of the arthropods are infected with wolbachia, rendering this bacterium the most ubiquitous intracellular symbiont yet described (bourtzis 2008). we amplified the wsp (wolbachia surface protein) gene by pcr and detected 6.2% (10/161) positivity in the current study. (2009) collected 150 dogs blood from izmir and aydn provinces and detected the prevalences as 12.3% for both d. immitis and wolbachia sp. in the current work, we determined the wolbachia in 6 samples together with d. immitis and d. repens and in 4 samples without dirofilaria spp as well. in this instance, either those 4 samples were infected with any dirofilaria species and the pcr could not detect or those dogs had another residence for wolbachia in the dogs. it is widely accepted that wolbachia is released into the tissues of the infected host following worm death and that bacteria derived molecules provoke innate inflammatory responses (saint andre. thus, doxycycline treatment may reduce wolbachia levels in adultworms and less severe pathology as well. this is the first study on the detection of dirofilaria species using of multiplex - pcr in turkey. besides, it was attentioned to neglected filarial nematod which is d. repens in turkey and obtained actual prevalence data about d. repens, d. immitis and wolbachia, as well. besides those results have been shown that canine dirofilariosis still prevalent and there is no effective reduction yet.	background : dirofilaria immitis and dirofilaria repens are the most common species of filarial nematodes described in the dogs. a single - step multiplex pcr was applied to detect and differentiate simultaneously and unequivocally d. immitis and d. repens on dna extracted from canine peripheral blood and besides to detect the seroprevalance of d. immitis by elisa in elazig province, turkey. a pcr detection of the wolbachia, which plays an important role in d. immitis biology and contributes to the inflammatory pathology of the heartworm, was also applied for the first time in turkey.methods:a total of 161 whole blood and sera samples were collected from stray dogs and stored at 20 c until used. after dna extraction, all samples were processed with dirofilaria primers by multiplex - pcr and wolbachia primers by conventional pcr besides elisa for serology. the amplification was performed using a set of primers designed on a portion of the small subunit ribosomal rna gene of the mitochondrion (12s rdna).results : three of the examined dogs (1.8%) were found to be infected with only d. immitis, one (0.6%) with d. repens and three (1.8%) with both parasites. besides, 10 out of 161 dogs (6.2%) were found infected with wolbachia sp. finaly, the seroprevalence of dirofilariosis in the examined dogs was found to be 3.7% (6/161).conclusion : although dirofilariosis is not a serious problem in the region, the stray dogs still continue to be a source of infection.
the literature on smoking has underscored that tobacco consumption is potentially fatal and that nicotine in particular is highly addictive. () successful smoking cessation requires a structured intervention, this type of intervention being more effective in helping smokers to quit. () the literature shows a relationship between psychological morbidity (depression, anxiety, and stress) and the use of substances, including nicotine. () it is a relationship between smoking and the presence of symptoms related to depression, () anxiety, () and stress. () in terms of quality of life, the literature shows that smoking is a risk behavior that has a major impact on health. () in contrast, not smoking is associated with better physical and mental quality of life. () many smokers do not regard smoking as a health problem and minimize their nicotine dependence ; that is, they harbor misconceptions about smoking. () according to the literature, the way individuals perceive their health and disease has important implications for their behavior, () as is the case with smoking. smoking representations are categorized into cognitive representations (which evaluate the perceived health effects of smoking, the duration of smoking, control, the effectiveness of smoking cessation treatment, and how smokers identify smoking - related symptoms) ; emotional representations (which evaluate smoking - related concerns and how smokers feel emotionally affected) ; and comprehensibility (i.e., individuals ' level of understanding of their smoking behavior). threatening cognitive representations, emotional representations, and comprehensibility indicate an increased perception that smoking is harmful to health and bring into question the quality of life of smokers. () regarding sociocognitive variables, smoking is a behavior in which intention is influenced by the social dimension. intention is a key aspect of the theory of planned behavior (tpb) and is an important predictor of smoking behavior. (,) the tpb () developed from the theory of reasoned action, to which the concept of perceived behavioral control was added. the tpb proposes that an individual 's intention to engage in a certain behavior is the key determinant of that behavior, because it reflects the individual 's level of motivation and readiness to make efforts to engage in the behavior. therefore, according to the tpb, behavioral intentions are influenced by three aspects : attitudes toward the behavior ; subjective norms ; and perceived behavioral control. the tpb states that increasingly favorable attitudes and subjective norms regarding a certain behavior translate to a higher intention to engage in that behavior, as does higher perceived behavioral control. () the intention to quit smoking is important, moderating smoking cessation interventions and the effect of medical advice. () therefore, according to the tpb, the most important proximal determinant of behavior is the intention that results from the combination of attitudes toward the behavior, subjective norms, and perceived behavioral control. beliefs are a central element in the tpb and are considered solid cognitive and affective foundations for the construction of attitudes, subjective norms, and perceived behavioral control. according to the stress - coping model, () in the presence of a stressor (as is the case of smoking), smokers evaluate their characteristics and how they can influence their external or internal resources. these resources will enable smokers to organize appropriate coping strategies to obtain a result, such as quitting smoking. the objective of the present study was to analyze psychological morbidity as a moderator of the relationship between smoking representations and quality of life in smokers and former smokers, as well as to determine which psychological variables discriminate between smokers with and without the intention to quit smoking. our study sample was a convenience sample of individuals recruited from among those in the braga region, in northern portugal. the participants were recruited during medical consultations (in a central hospital and a private company), in which they were informed of the nature and purpose of the study by the physicians. in the university, the professors were contacted and were asked to inform their students about the study. data were collected over a one - year period, and all of the data collection instruments were completed at a single time point. participation was voluntary, volunteers consenting to participate after having received information on the scope and purpose of the study. the inclusion criteria were being over 18 years of age, being a daily smoker, and having been a former smoker for at least three months. there were no conflicts of interest, and the study was approved by the research ethics committees of the data collection sites. with the objective of analyzing the moderating effect of psychological morbidity on the relationship between smoking representations and quality of life in smokers and former smokers, we used the regression model proposed by baron and kenny () and simple slopes analysis in order to determine the significance of the interaction among (t) variables. () in order to determine which psychological variables discriminate between smokers with and without the intention to quit smoking, we used discriminant analysis. () we used the following instruments : a sociodemographic questionnaire to obtain information to characterize the study sample in terms of group, marital status, gender, level of education, age, age at smoking onset, attempts to quit smoking, type of treatment, and smoking status of the partner.the brief illness perception questionnaire, (,) an instrument composed of nine items, which are used in order to assess emotional representations (five items), cognitive representations (two items), and the comprehensibility of the disease (one item)the instrument scores the items on a scale of zero to ten (with the exception of item 9). in terms of internal consistency, given that each scale is composed of only one item and that it is a revised version of the original instrument, it is impossible to calculate cronbach 's alpha coefficient based on factor analysis. the original version refers only to test - retest reliability, which is high. in our study sample, the subscales were moderately correlated with one another, as occurred in the original version. regarding the scales of cognitive representations, emotional representations, and comprehensibility, high scores indicate threatening cognitive and emotional representations and poorer comprehensibility of the disease.the partner interaction questionnaire, (,) an instrument composed of one positive scale (eight items) and one negative scale (eleven items), which assess the support that smokers received in the last three months when attempting to quit smoking. this adapted version was developed for a sample of smokers and former smokers, who completed the same questionnaire items. a higher score translates to greater (positive or negative) support from partners. the depression, anxiety, and stress scale, (,) an instrument composed of 21 items organized into three subscales (i.e., anxiety, depression, and stress), each subscale comprising seven items. a higher score translates to greater psychological morbiditymedical outcomes study 36-item short - form health survey, (,) an instrument composed of eight dimensions grouped into two general components (i.e., physical quality of life, comprising 21 items, and mental quality of life, comprising 14 items). a higher score translates to a better perceived quality of life. rather than an overall scale score, two summary scores are obtained, corresponding to physical quality of life and mental quality of life. a tpb questionnaire to assess the intention to quit smoking, () which was constructed on the basis of the guidelines proposed by ajzen () and francis. () the adapted version of the questionnaire is composed of seven subscales action and coping planning ; behavioral beliefs ; attitudes toward the behavior ; perceived behavioral control ; subjective norms ; normative / control beliefs ; and intention. a high score on the questionnaire indicates a higher value on each scale a sociodemographic questionnaire to obtain information to characterize the study sample in terms of group, marital status, gender, level of education, age, age at smoking onset, attempts to quit smoking, type of treatment, and smoking status of the partner. the brief illness perception questionnaire, (,) an instrument composed of nine items, which are used in order to assess emotional representations (five items), cognitive representations (two items), and the comprehensibility of the disease (one item) the instrument scores the items on a scale of zero to ten (with the exception of item 9). in terms of internal consistency, given that each scale is composed of only one item and that it is a revised version of the original instrument, it is impossible to calculate cronbach 's alpha coefficient based on factor analysis. the original version refers only to test - retest reliability, which is high. in our study sample, the subscales were moderately correlated with one another, as occurred in the original version. regarding the scales of cognitive representations, emotional representations, and comprehensibility, high scores indicate threatening cognitive and emotional representations and poorer comprehensibility of the disease. the partner interaction questionnaire, (,) an instrument composed of one positive scale (eight items) and one negative scale (eleven items), which assess the support that smokers received in the last three months when attempting to quit smoking. this adapted version was developed for a sample of smokers and former smokers, who completed the same questionnaire items. a higher score translates to greater (positive or negative) support from partners. the depression, anxiety, and stress scale, (,) an instrument composed of 21 items organized into three subscales (i.e., anxiety, depression, and stress), each subscale comprising seven items. a higher score translates to greater psychological morbidity medical outcomes study 36-item short - form health survey, (,) an instrument composed of eight dimensions grouped into two general components (i.e., physical quality of life, comprising 21 items, and mental quality of life, comprising 14 items). a higher score translates to a better perceived quality of life. rather than an overall scale score, two summary scores are obtained, corresponding to physical quality of life and mental quality of life. a tpb questionnaire to assess the intention to quit smoking, () which was constructed on the basis of the guidelines proposed by ajzen () and francis. () the adapted version of the questionnaire is composed of seven subscales action and coping planning ; behavioral beliefs ; attitudes toward the behavior ; perceived behavioral control ; subjective norms ; normative / control beliefs ; and intention. a high score on the questionnaire indicates a higher value on each scale we recruited 224 daily smokers (52.7% of whom were males) and 169 former smokers who had quit smoking at least three months prior (68.6% of whom were males). of the smokers, 49.1% were single and 61.6% had attended the third year of high school. the mean age of the smokers was 28.58 8.69 years (range, 18 - 58 years). most of the smokers had been smoking for more than three years, and the age at smoking onset was 14 - 18 years. of the smokers, 60% had made at least one attempt to quit smoking, 96% had never undergone any smoking cessation treatment, and 56.7% had a partner who did not smoke. of the former smokers, 72.8% were married and 39% had attended high school. the mean age was 43.10 13.02 years (range, 19 - 63 years). most of the former smokers had started smoking at the age of 12 - 18 years. all former smokers had made at least one attempt to quit smoking, 32% having sought smoking cessation treatment. in the group of smokers, comprehensibility had a positive predictive effect on mental quality of life (= 0.207 ; p 0.001). the interactions between psychological morbidity and emotional representations (= 0.129 ; p 0.001), between psychological morbidity and cognitive representations (= 0.281 ; p 0.001), and between psychological morbidity and comprehensibility (= 0.446 ; p 0.050) were significant. our results showed that psychological morbidity had a moderating effect on the relationship between mental quality of life and emotional representations (t = 2.348 ; p = 0.019) and on the relationship between mental quality of life and cognitive representations (t = 1.875 ; p < 0.001), the relationship being negative when psychological morbidity is low. psychological morbidity also had a moderating effect on the relationship between mental quality of life and comprehensibility (t = 4.57 ; p 0.001), the relationship being positive when psychological morbidity is high (figure 1). figure 1 moderating effect of psychological morbidity (pm) on the relationship between mental quality of life and and smoking representations in smokers : emotional representations (a), cognitive representations (b), and comprehensibility (c). in the group of former smokers, comprehensibility had a positive predictive effect on mental quality of life (= 0.207 ; p 0.001). the interaction between psychological morbidity and cognitive representations was significant (= 0.333 ; p 0.001). our results showed that psychological morbidity had a moderating effect on the relationship between mental quality of life and cognitive representations (t = 1.255 ; p = 0.001), the relationship being negative when psychological morbidity is low (figure 2). psychological morbidity was found to have no moderating effect on the relationship between mental quality of life and emotional representations or on the relationship between mental quality of life and comprehensibility. figure 2 moderating effect of psychological morbidity (pm) on the relationship between mental quality of life and cognitive representations in former smokers. in the group of smokers, emotional representations had a negative predictive effect on physical quality of life (= 0.263 ; p 0.001), and the interaction between psychological morbidity and emotional representations was significant (= 0.215 ; p 0.001). our results showed that psychological morbidity had a moderating effect on the relationship between physical quality of life and emotional representations (t = 1.956 ; p = 0.005), the relationship being negative when psychological morbidity is low (figure 3). psychological morbidity was found to have no moderating effect on the relationship between physical quality of life and cognitive representations or on the relationship between physical quality of life and comprehensibility. figure 3 moderating effect of psychological morbidity (pm) on the relationship between physical quality of life and emotional representations in smokers. in the group of former smokers, cognitive representations had a negative predictive effect on physical quality of life (= 0.128 ; p 0.050), and the interaction between psychological morbidity and cognitive representations was significant (= 0.329 ; p 0.001). psychological morbidity had a moderating effect on the relationship between physical quality of life and cognitive representations (t = 1.875 ; p = 0.001), the relationship being negative when psychological morbidity is low (figure 4). psychological morbidity was found to have no moderating effect on the relationship between physical quality of life and emotional representations or on the relationship between physical quality of life and comprehensibility. figure 4 moderating effect of psychological morbidity (pm) on the relationship between physical quality of life and cognitive representations in former smokers. the results of the discriminant analysis showed that 59.8% of the smokers had the intention to quit smoking, whereas 40.1% did not. regarding the variables discriminating between the two groups, the results showed that the smokers who had the intention to quit smoking more often presented with low comprehensibility, threatening emotional representations, behavioral beliefs, and perceived behavioral control, as well as with normative / control beliefs, than did those who did not. the relationship between the discriminant function and the original variable, in the case of intention to quit smoking, allowed us to see the contribution of each variable to group discrimination and showed that the most important variable is perceived behavioral control, followed by emotional representations. the discriminant function analysis allowed us to obtain a canonical discriminant variable, with results of significance (wilk 's lambda = 0.495 ; chi - square = 71.778 ; p 0.001) and a canonical correlation of 0.715. these results showed that the discriminatory power of the function is significant and that the two groups (smokers with and without the intention to quit smoking) are significantly different on the basis of the study variables. the results of the functional classification matrix for smokers with and without the intention to quit smoking showed that 77% of cases are correctly classified, and the proportion of unexplained variance was 49.5%. the negative relationship between mental quality of life and threatening emotional and cognitive representations of smoking indicates that perceived quality of life decreased as the perceived threat posed by smoking increased. these results can be explained by the literature, which shows that threatening representations are associated with poorer perceived quality of life. () by relating this information to the results of our study, we found that threatening (cognitive and emotional) representations of smoking translate to an increased perception that smoking is harmful to health and, consequently, decreased mental quality of life. the literature shows how psychological morbidity is related to smoking behavior, () psychological morbidity having physical consequences at the level of decreased mental quality of life. (,) according to the stress - coping model, () psychological morbidity can be viewed as an emotional response that can have a direct impact on the outcome measure, i.e., mental quality of life. also according to the stress - coping model, smoking can be viewed as a stressor that has a direct effect of increasing psychological morbidity and the perceived threat posed by smoking. therefore, smokers, in the presence of lower psychological morbidity associated with smoking, more often present with low mental quality of life and threatening emotional and cognitive representations ; that is, mental quality of life decreases as the perception that smoking is harmful to health increases. this indicates that, in the presence of higher psychological morbidity, a poorer comprehensibility of the reasons for smoking translates to better mental quality of life as perceived by smokers. comprehensibility refers to smokers knowing why they smoke and what the symptoms related to smoking are. therefore, psychological morbidity can have a protective effect that will probably maintain smoking behavior in order to protect smokers, giving them the perception of good mental quality of life. for the former smokers in the present study, psychological morbidity had a moderating effect (which was similar to that observed in the smokers) on the relationship between mental quality of life and cognitive representations. this indicates that, in the presence of lower psychological morbidity, the former smokers had shown poorer perceived mental quality of life and threatening cognitive representations when they were smokers. the relationship between physical quality of life and the emotional and cognitive representations of smoking was found to be negative in the smokers and former smokers investigated in the present study ; that is, in the presence of fewer symptoms of psychological morbidity in smokers and former smokers, physical quality of life is lower when cognitive and emotional representations of smoking are more threatening. therefore, threatening cognitive and emotional representations of smoking are associated with lower perceived quality of life, which is in agreement with the literature. () the discriminant analysis showed that the smokers who had the intention to quit smoking more often presented with low comprehensibility, threatening emotional representations, behavioral beliefs, and perceived behavioral control, as well as with normative / control beliefs, than did those who did not. according to the literature, () smokers who have the intention to quit smoking are in a stage of preparation for quitting smoking. in our study, those who had the intention to quit smoking had a poorer understanding of the reasons for smoking (comprehensibility), showed greater concern with the fact that they smoked, and felt emotionally affected by the threat that smoking posed to their health (emotional representations). these results suggest that smokers in whom the perception that smoking poses a health threat is increased are more motivated to quit smoking. according to the tpb, () smokers who have the intention to quit smoking are aware of their control over quitting (perceived behavioral control), take into account what others expect from them (i.e., more often present with normative / control beliefs), and consider the likely outcomes of quitting (i.e., more often present with behavioral beliefs). in fact, the tpb states that beliefs are important in the construction of attitudes, subjective norms, and perceived behavioral control, beliefs being implicated in behavioral intention (to quit smoking). () the sociocognitive variables (perceived behavioral control, behavioral beliefs, and normative / control beliefs) are important in the context of devising a smoking cessation plan. therefore, threatening emotional representations of smoking and a poorer understanding of smoking lead smokers to believe that smoking is harmful to their health ; to show a poorer understanding of the reasons for smoking ; and to believe that quitting smoking can be important to their health. () the sociocognitive variables in the present study show that perceived behavioral control, normative / control beliefs, and behavioral beliefs play an important role in the intention to quit smoking. () the fact that the study sample was a convenience sample of individuals recruited exclusively from among those in northern portugal limits the generalization of the results to other regions of the country, given that the social and cultural characteristics of each region should be taken into consideration. future studies should examine the role of sociocognitive variables, particularly in intention, in samples that are more diverse, as well as evaluating the role of family coping as a moderating variable in the relationship between smoking representations and the intention to quit smoking. the present study underscored the importance of the moderating effect of psychological morbidity on mental and physical quality of life in smokers and former smokers, as well as underscoring the importance of sociocognitive variables in the intention to quit smoking. the results obtained contribute to informing smoking cessation programs regarding a reduction in psychological morbidity and the inclusion of sociocognitive variables (tpb). analisar a morbidade psicolgica como um moderador na relao entre as representaes do tabaco e a qualidade de vida em fumantes e ex - fumantes, assim como conhecer as variveis psicolgicas que discriminam os fumantes com e sem inteno para deixar de fumar. estudo quantitativo, correlacional e transversal com uma amostra de convenincia constituda por 224 fumantes e 169 ex - fumantes. verificou - se um efeito moderador da morbidade psicolgica na relao entre a qualidade de vida (fsica e mental) e as representaes do tabaco (representaes cognitivas e emocionais e compreenso) nos fumantes e nos ex - fumantes. os fumantes com inteno para deixar de fumar apresentavam menor compreenso, representaes emocionais mais ameaadoras, mais crenas de comportamento, maior controle comportamental percebido e mais crenas normativas / controle do que aqueles sem essa inteno. os resultados deste estudo enfatizam a importncia da morbidade psicolgica como moderadora, bem como das variveis sociocognitivas, junto dos fumantes que querem deixar de fumar. a literatura tem vindo a salientar que o consumo de tabaco potencialmente mortal para o fumante e que a nicotina, em particular, altamente aditiva. () por sua vez, a cessao tabgica requer uma interveno estruturada para que o sucesso seja assegurado e mais eficaz. () a literatura nos mostra uma relao entre a morbidade psicolgica (depresso, ansiedade e estresse) e o consumo de substncias, em que se inclui a nicotina. () se trata de uma relao entre o consumo de tabaco e a presena de sintomas relacionados com depresso, () ansiedade () e estresse. () em termos de qualidade de vida, a literatura destaca o hbito tabgico como um comportamento de risco, com grande impacto na sade do fumante. () por sua vez, o no consumo de tabaco relaciona - se com uma melhor qualidade de vida ao nvel fsico e mental. () muitos fumantes no consideram ter um problema de sade pelo fato de fumarem e minimizam a sua dependncia tabgica (i.e., apresentam representaes desajustadas acerca de fumar). () de acordo com a literatura, o modo como as pessoas pensam acerca da sua sade e doena tem implicaes importantes no seu comportamento, () como o caso do consumo de tabaco. as representaes categorizam - se em cognitivas (avaliam a percepo das consequncias para a sade do fumante, a durao do hbito tabgico, o controle que o fumante tem, se o tratamento para deixar de fumar eficaz e como o fumante identifica os sintomas relacionados com fumar) ; emocionais (avaliam a preocupao associada a fumar e quanto o fumante se sente afetado emocionalmente) ; e de compreenso (contempla o quanto o indivduo compreende o seu comportamento de fumar). representaes cognitivas, emocionais e de compreenso mais ameaadoras indicam uma maior percepo de que fumar faz mal sade, colocando assim em causa a qualidade de vida do fumante. () no que respeita s variveis sociocognitivas, fumar apresenta - se como um comportamento no qual a inteno influenciada pela dimenso social. a inteno um aspeto chave na teoria do comportamento planejado (tcp) e se revela como um preditor importante do comportamento de fumar. (,) a tcp () se desenvolveu a partir da teoria da ao refletida, qual foi adicionado o conceito de compreenso de controle do comportamento. a tcp prope que a inteno de uma pessoa para executar um comportamento o determinante fundamental desse comportamento, porque reflete o nvel de motivao da pessoa e a prontido para implementar esforos no desempenho do comportamento. desse modo, para a tcp, as intenes para a implementao de um comportamento envolvem a influncia de trs aspetos : as atitudes face ao comportamento ; as normas subjetivas ; e o controle comportamental percebido. essa teoria indica que quanto mais favorveis forem as atitudes e as normas subjetivas em relao a um comportamento, assim como quanto maior for a percepo de controle desse comportamento, mais forte ser a inteno da pessoa para realizar esse comportamento. () a inteno para deixar de fumar revela - se importante, moderando as intervenes para a cessao tabgica e o efeito do aconselhamento do mdico. () assim, para a tcp, o determinante proximal mais relevante para o comportamento a inteno que resulta da combinao das atitudes face ao comportamento, s normas subjetivas e ao controle percebido. as crenas, por sua vez, so um elemento central na tcp e so consideradas bases slidas, ao nvel cognitivo e afetivo, para a construo das atitudes, normas subjetivas e controle comportamental percebido. o modelo de estresse / enfrentamento () preconiza que, perante um agente estressor, como o caso do consumo de tabaco, o fumante avalia as suas caractersticas e como essas podem influenciar os recursos externos ou internos do fumante. estes recursos vo permitir que o fumante organize estratgias de enfrentamento adequadas com vista a um resultado, como por exemplo, deixar de fumar. o presente trabalho teve por objetivo analisar a morbidade psicolgica como um moderador na relao entre as representaes e a qualidade de vida em fumantes e ex - fumantes, bem como determinar quais so as variveis psicolgicas que discriminam os fumantes com ou sem inteno para deixar de fumar. tratou - se de uma amostra de convenincia recrutada na zona de braga, no norte de portugal. os participantes foram recrutados durante consultas mdicas (em um hospital central e uma empresa privada), nas quais os mdicos informaram os indivduos sobre a natureza e a finalidade do estudo. na universidade, os docentes foram contatados e solicitados a informar os seus alunos acerca do estudo. o perodo de coleta dos dados teve a durao de um ano, e o preenchimento dos instrumentos efetuou - se num momento nico. a participao foi voluntria, sendo o seu consentimento de resposta precedido de informao sobre o mbito e a finalidade do estudo. os critrios de incluso para a amostra foram ter mais de 18 anos e ser fumante dirio ou ser ex - fumante h pelo menos trs meses. sem conflitos de interesse, a pesquisa foi aprovada pelos comits de tica de cada local de coleta dos dados. com o objetivo de analisar o efeito moderador da morbidade psicolgica na relao entre as representaes e a qualidade de vida em fumantes e ex - fumantes, recorreu - se ao uso da regresso usando o modelo de baron e kenny () e a anlise de simple slopes para determinar o valor da significncia da interao entre variveis (t). () por sua vez, para determinar quais as variveis psicolgicas que discriminam os fumantes com ou sem inteno para deixar de fumar, recorreu - se ao uso de anlise discriminante. () foram utilizados os seguintes instrumentos : questionrio sociodemogrfico para a obteno de informaes para caracterizar os participantes da amostra ao nvel de grupo, estado civil, sexo, habilitaes literrias, idade, incio do consumo tabgico, tentativas para deixar de fumar, tipo de tratamento e status tabgico do parceiroquestionrio de crenas sobre a doena, verso breve(10,18) : esse instrumento constitudo por nove itens que permitem avaliar as representaes emocionais (cinco itens), cognitivas (dois itens) e de compreenso da doena (um item). o instrumento cota os itens numa escala de zero a dez (exceo do item 9). em termos de consistncia interna, visto que cada escala s composta por um item, sendo uma verso revista do instrumento original, com base na anlise fatorial, no possvel calcular um coeficiente alfa de cronbach. a verso original apenas se refere fidelidade teste - reteste, sendo essa elevada. na presente amostra, tal como na verso original, as subescalas se correlacionaram entre si de forma moderada. em relao s escalas de representaes cognitivas, emocionais e de compreenso, um valor elevado indica representaes cognitivas e emocionais mais ameaadoras e menor compreenso da doena partner interaction questionnaire,): esse instrumento constitudo por uma escala positiva (oito itens) e uma negativa (onze itens) que avaliam o apoio recebido pelo fumante para deixar de fumar nos ltimos trs meses. essa verso adaptada foi elaborada para uma amostra de fumantes e ex - fumantes, que responderam aos mesmos itens do questionrio. um resultado elevado significa maior apoio, positivo ou negativo, por parte do parceiro escala de depresso, ansiedade e estresse,): esse instrumento constitudo por 21 itens, organizados em trs subescalas (ansiedade, depresso e estresse), cada uma com sete itens. quanto mais elevada a pontuao, maior o ndice de morbidade psicolgicamedical outcomes study 36-item short - form health survey (,): esse instrumento constitudo por oito dimenses que se agrupam em dois componentes gerais (qualidade de vida fsica, com 21 itens, e qualidade de vida mental, com 14 itens). quanto mais elevada for a pontuao, melhor ser a percepo de qualidade de vida. ao invs de um valor total da escala, obtm - se dois valores sumrios correspondentes qualidade de vida fsica e qualidade de vida mentalquestionrio do comportamento planejado para deixar de fumar (): esse questionrio foi construdo com base nas diretrizes propostas por ajzen () e por francis. planejamento do enfrentamento e ao ; crenas de comportamento ; atitudes face ao comportamento ; controle comportamental percebido ; normas subjetivas ; crenas normativas / controle ; e inteno. um resultado elevado no questionrio indica um valor mais intenso em cada escala questionrio sociodemogrfico para a obteno de informaes para caracterizar os participantes da amostra ao nvel de grupo, estado civil, sexo, habilitaes literrias, idade, incio do consumo tabgico, tentativas para deixar de fumar, tipo de tratamento e status tabgico do parceiro questionrio de crenas sobre a doena, verso breve(10,18) : esse instrumento constitudo por nove itens que permitem avaliar as representaes emocionais (cinco itens), cognitivas (dois itens) e de compreenso da doena (um item). o instrumento cota os itens numa escala de zero a dez (exceo do item 9). em termos de consistncia interna, visto que cada escala s composta por um item, sendo uma verso revista do instrumento original, com base na anlise fatorial, no possvel calcular um coeficiente alfa de cronbach. a verso original apenas se refere fidelidade teste - reteste, sendo essa elevada. na presente amostra, tal como na verso original, as subescalas se correlacionaram entre si de forma moderada. em relao s escalas de representaes cognitivas, emocionais e de compreenso, um valor elevado indica representaes cognitivas e emocionais mais ameaadoras e menor compreenso da doena partner interaction questionnaire,): esse instrumento constitudo por uma escala positiva (oito itens) e uma negativa (onze itens) que avaliam o apoio recebido pelo fumante para deixar de fumar nos ltimos trs meses. essa verso adaptada foi elaborada para uma amostra de fumantes e ex - fumantes, que responderam aos mesmos itens do questionrio. um resultado elevado significa maior apoio, positivo ou negativo, por parte do parceiro escala de depresso, ansiedade e estresse,): esse instrumento constitudo por 21 itens, organizados em trs subescalas (ansiedade, depresso e estresse), cada uma com sete itens. quanto mais elevada a pontuao, maior o ndice de morbidade psicolgica medical outcomes study 36-item short - form health survey (,): esse instrumento constitudo por oito dimenses que se agrupam em dois componentes gerais (qualidade de vida fsica, com 21 itens, e qualidade de vida mental, com 14 itens). quanto mais elevada for a pontuao, melhor ser a percepo de qualidade de vida. ao invs de um valor total da escala, obtm - se dois valores sumrios correspondentes qualidade de vida fsica e qualidade de vida mental questionrio do comportamento planejado para deixar de fumar (): esse questionrio foi construdo com base nas diretrizes propostas por ajzen () e por francis. planejamento do enfrentamento e ao ; crenas de comportamento ; atitudes face ao comportamento ; controle comportamental percebido ; normas subjetivas ; crenas normativas / controle ; e inteno. foram recrutados 224 fumantes dirios (52,7% homens) e 169 ex - fumantes h pelo menos trs meses (68,6% homens). dos fumantes, 49,1% eram solteiros e 61,6% tinham frequentado a mdia de idade dos fumantes foi de 28,58 8,69 anos (variao, 18 - 58 anos). a maioria dos fumantes fumava h mais de trs anos e a idade de incio situou - se entre 14 e 18 anos. dos fumantes, 60% efetuaram, pelo menos, uma tentativa para deixar de fumar, 96% nunca realizaram qualquer tratamento para deixar de fumar, e 56,7% tinham um parceiro / a que no fumava. em relao aos ex - fumantes, 72,8% eram casados, 39% tinham frequentado o ensino mdio, e a mdia de idade foi de 43,10 13,02 anos (variao, 19 - 63 anos). a maioria dos ex - fumantes comeara a fumar entre 12 e 18 anos. os ex - fumantes efetuaram pelo menos uma tentativa para deixar de fumar, sendo que 32% tinham procurado tratamento para deixar de fumar. no grupo dos fumantes, a compreenso exerceu um efeito preditor positivo na qualidade de vida mental (= 0,207 ; p 0,001). a interao entre a morbidade psicolgica e as representaes emocionais (= 0,129 ; p 0,001), entre a morbidade psicolgica e as representaes cognitivas (= 0,281 ; p 0,001) e entre a morbidade psicolgica e a compreenso (= 0,446 ; p 0,050) foi significativa. assim, de acordo com os resultados, a morbidade psicolgica exerceu um efeito moderador na relao entre a qualidade de vida mental e as representaes emocionais (t = 2,348 ; p = 0,019) e cognitivas (t = 1,875 ; p < 0,001), sendo essa relao negativa quando a morbidade psicolgica baixa. por sua vez, a morbidade psicolgica exerceu um efeito moderador na relao entre a qualidade de vida mental e a compreenso (t = 4,57 ; p 0,001), sendo a relao positiva quando a morbidade psicolgica alta (figura 1). figura 1 efeito moderador da morbidade psicolgica (mp) na relao entre qualidade de vida mental e as representaes relacionadas ao tabaco nos fumantes : representaes emocionais (a), representaes cognitivas (b) e compreenso (c). no grupo dos ex - fumantes, a compreenso exerceu um efeito preditor positivo na qualidade de vida mental (= 0,207 ; p 0,001). a interao entre a morbidade psicolgica e as representaes cognitivas foi significativa (= 0,333 ; p 0,001). de acordo com os resultados, a morbidade psicolgica exerceu um efeito moderador na relao entre a qualidade de vida mental e as representaes cognitivas (t = 1,255 ; p = 0,001), sendo essa relao negativa quando a morbidade psicolgica baixa (figura 2). no se verificou um efeito moderador da morbidade psicolgica na relao entre a qualidade de vida mental e as representaes emocionais e de compreenso. figura 2 efeito moderador da morbidade psicolgica (mp) na relao entre qualidade de vida mental e as representaes cognitivas nos ex - fumantes. em relao qualidade de vida fsica, no grupo dos fumantes, as representaes emocionais exerceram um efeito preditivo negativo na qualidade de vida fsica (= 0,263 ; p 0,001), e a interao entre a morbidade psicolgica e as representaes emocionais foi significativa (= 0,215 ; p 0,001). de acordo com os resultados, a morbidade psicolgica exerceu um efeito moderador na relao entre a qualidade de vida fsica e as representaes emocionais (t = 1,956 ; p = 0,005), sendo essa relao negativa quando a morbidade psicolgica baixa (figura 3). no se verificou um efeito moderador da morbidade psicolgica na relao entre a qualidade de vida fsica e as representaes cognitivas e de compreenso. figura 3 efeito moderador da morbidade psicolgica (mp) na relao entre qualidade de vida fsica e as representaes emocionais nos fumantes. no grupo dos ex - fumantes, as representaes cognitivas exerceram um efeito preditivo negativo na qualidade de vida fsica (= 0,128 ; p 0,050), e a interao entre a morbidade psicolgica e as representaes cognitivas foi significativa (= 0,329 ; p 0,001). assim, a morbidade psicolgica exerceu um efeito moderador na relao entre a qualidade de vida fsica e as representaes cognitivas (t = 1,875 ; p = 0,001), sendo essa relao negativa quando a morbidade psicolgica baixa (figura 4). no se verificou um efeito moderador da morbidade psicolgica na relao entre a qualidade de vida fsica e as representaes emocionais e de compreenso. figura 4 efeito moderador da morbidade psicolgica (mp) na relao entre qualidade de vida fsica e as representaes cognitivas nos ex - fumantes. os resultados da anlise discriminante identificaram 59,8% de fumantes com inteno para deixar de fumar e 40,1% de fumantes sem inteno para deixar de fumar. relativamente s variveis que discriminam os grupos, os resultados mostraram que os fumantes com inteno para deixar de fumar apresentaram menor compreenso, representaes emocionais mais ameaadoras, maiores crenas de comportamento, maiores crenas normativas / controle e maior controle comportamental percebido. a relao entre a funo discriminante e a varivel original, no caso da inteno para deixar de fumar, permitiu ver a contribuio de cada varivel para a discriminao dos grupos e mostrou que a varivel que assume maior importncia o controle comportamental percebido, seguida das representaes emocionais. a anlise da funo discriminante permitiu a obteno de uma varivel cannica discriminante, com resultados de significncia (wilk 's lambda = 0,495 ; qui - quadrado = 71,778 ; p 0,001) e uma correlao cannica de 0,715. esses resultados revelaram que o poder discriminatrio da funo significativo e os dois grupos, fumantes com e sem inteno para deixar de fumar, so significativamente diferentes com base nas variveis estudadas. os resultados da matriz de classificao da funo para os fumantes com e sem inteno para deixar de fumar indicaram que 77% dos casos so corretamente a relao negativa entre a qualidade de vida mental e as representaes emocionais e cognitivas mais ameaadoras do tabaco indica que os fumantes perceberam a sua qualidade de vida mental diminuir medida que aumentava a percepo de ameaa relacionada com o consumo de tabaco. esses resultados podem ser explicados luz da literatura, que nos revela que representaes mais ameaadoras esto associadas a uma pior percepo da qualidade de vida. () relacionando essa informao com os resultados do nosso estudo, verifica - se que quanto mais ameaadoras as representaes (cognitivas e emocionais) associadas ao tabaco, mais o fumante percebe o tabaco como prejudicial sua sade, vendo diminuda a sua qualidade de vida mental. no que diz respeito morbidade psicolgica, a literatura mostra - nos como se relaciona com o comportamento tabgico, () apresentando consequncias fsicas ao nvel da diminuio da qualidade de vida mental. (,) de acordo com o modelo de estresse / enfrentamento, () a morbidade psicolgica pode ser encarada como uma resposta emocional que pode ter um impacto direto ao nvel da varivel de resultado, i.e., na qualidade de vida mental do indivduo. ainda de acordo com o modelo, o consumo de tabaco pode ser visto como um estressor com efeito direto sobre o aumento da morbidade psicolgica e da ameaa percebida em relao ao tabaco. assim, os fumantes, na presena de menor morbidade psicolgica associada ao consumo de tabaco, apresentam menor qualidade de vida mental e representaes emocionais e cognitivas mais ameaadoras, i.e., a sua qualidade de vida mental diminui medida que aumenta a percepo dos perigos do tabaco para a sua sade. curiosamente, a relao entre a qualidade de vida mental e a compreenso positiva, indicando que, na presena de maior morbidade psicolgica, quanto menos os fumantes compreendem porque fumam, eles tm uma percepo de melhor qualidade de vida mental. entende - se por compreenso o fato de os fumantes perceberem porque fumam e quais so os sintomas associados ao consumo de tabaco. assim, a morbidade psicolgica pode ter um efeito protetor que provavelmente poder manter o comportamento tabgico no sentido de proteger os fumantes, dando - lhes a percepo de uma boa qualidade de vida mental. no caso dos ex - fumantes, a morbidade psicolgica teve um efeito moderador, semelhante ao dos fumantes, na relao entre a qualidade de vida mental e as representaes cognitivas. a relao entre a qualidade de vida e as representaes cognitivas foi negativa, indicando que na presena de menor morbidade psicolgica, os ex - fumantes, quando fumavam, percebiam uma menor qualidade de vida mental e apresentavam representaes cognitivas mais ameaadoras. a relao entre a qualidade de vida fsica e as representaes emocionais e cognitivas relacionadas ao tabaco mostrou - se negativa nos fumantes e nos ex - fumantes, i.e., na presena de menos sintomas de morbidade psicolgica pelos fumantes e ex - fumantes, a qualidade de vida fsica menor quando as representaes do tabaco (cognitivas e emocionais) so mais ameaadoras. assim, as representaes cognitivas e emocionais mais ameaadoras relacionadas ao tabaco esto associadas a uma pior percepo da qualidade de vida, o que est de acordo com a literatura. () a anlise discriminante revelou que os fumantes com inteno para deixar de fumar apresentaram menor compreenso, representaes emocionais mais ameaadoras, mais crenas de comportamento, mais controle comportamental percebido e mais crenas normativas / controle. de acordo com a literatura, () os fumantes com inteno para deixar de fumar encontram - se em uma fase de preparao para iniciar um processo para deixar de fumar. no nosso estudo, quem teve inteno para deixar de fumar compreendia menos porque fumava (compreenso), apresentou maior preocupao associada com o fato de fumar e sentiu - se afetado emocionalmente pela ameaa que o tabaco representava para a sua sade (representaes emocionais). esses resultados podem indicar que os fumantes, ao terem uma percepo mais ameaadora do tabaco para a sua sade, podero estar mais motivados para deixar de fumar. de acordo com a tcp, () os fumantes com inteno para deixar de fumar tm noo do seu controle em relao a deixar de fumar (controle comportamental percebido), tm em conta o que os outros esperam que faam (mais crenas normativas / controle) e consideram os provveis resultados de deixar de fumar (mais crenas de comportamento). de fato, a tcp refere que as crenas so importantes na construo de atitudes, normas subjetivas e controle comportamental percebido, estando envolvidas na inteno do comportamento (deixar de fumar). () as variveis sociocognitivas (controle comportamental percebido, crenas de comportamento e crenas normativas / controle) mostram - se importantes no sentido da construo de um plano de cessao tabgica. assim, representaes emocionais do tabaco mais ameaadoras e menor compreenso do tabaco levam os fumantes a considerar que fumar faz mal sua sade, compreendem menos porque fumam e consideram que deixar de fumar pode ser importante para a sua sade. () as variveis sociocognitivas do presente estudo indicam a importncia do controle comportamental percebido, das crenas normativas / controle e das crenas de comportamento na inteno para deixar de fumar. () o fato de a amostra ser de convenincia, recolhida apenas na zona norte de portugal, limita a generalizao dos resultados para outras regies do nosso pas, dado que se deve ter em considerao as caractersticas sociais e culturais de cada regio. investigaes futuras devero analisar o papel das variveis sociocognitivas particularmente em relao inteno, em amostras mais diversificadas, bem como avaliar o papel do enfrentamento familiar como varivel moderadora na relao entre as representaes do tabaco e a inteno para deixar de fumar. o presente estudo sublinhou a importncia do efeito moderador da morbidade psicolgica na qualidade de vida mental e fsica de fumantes e ex - fumantes, bem como a importncia das variveis sociocognitivas na inteno para deixar de fumar nos fumantes. os resultados contribuem para a importncia de informar os programas de interveno na cessao tabgica em relao diminuio da morbidade psicolgica, bem como incluso das variveis sociocognitivas (tcp).	objective : to analyze psychological morbidity as a moderator of the relationship between smoking representations and quality of life in smokers and former smokers, as well as to determine which psychological variables discriminate between smokers with and without the intention to quit smoking. methods : this was a quantitative, correlational cross - sectional study involving a convenience sample of 224 smokers and 169 former smokers. results : in smokers and former smokers, psychological morbidity had a moderating effect on the relationship between mental / physical quality of life and smoking representations (cognitive representations, emotional representations, and comprehensibility). smokers with the intention to quit smoking more often presented with low comprehensibility, threatening emotional representations, behavioral beliefs, and perceived behavioral control, as well as with normative / control beliefs, than did those without the intention to quit. conclusions : the results of this study underscore the importance of the moderating effect exerted by psychological morbidity, as well as that of sociocognitive variables, among smokers who have the intention to quit smoking.
the snail lymnaea acuminata lamarck (lymnaeidae) is the vector of liver flukes, fasciola gigantica cobbold (fascioliodae) and fasciola hepatica linnaeus (fascioliodae) which are responsible for endemic fascioliasis in cattle population of northern india [1, 2 ]. one way to reduce the risk of fascioliasis is to delink the life cycle of the flukes by killing the vector snail [3, 4 ]. recently, it has been reported that myristica fragrans (myristicaceae) seed (nutmeg) and aril (mace) have potent molluscicidal activity against lymnaea acuminata. the present study is an extension of our previous study aimed at elucidating the effect of the active moieties on the different enzymes namely, acetylcholinesterase (ache), acid phosphatase (acp), and alkaline phosphatase (alp) in the nervous tissue of snail lymnaea acuminata. adult lymnaea acuminata (length, 2.25 0.20 cm) were collected locally from ramgarh lake located almost adjacent to this university campus. twenty experimental snails were kept in a glass aquarium containing 3 l of dechlorinated tap water at 2224c. each set of experimental snails was exposed to sublethal concentrations 40 and 80% of 24 h and 96 h lc50 of trimyristin (24 h- 6.21, 12.43 mg l ; 96 hours- 2.80, 5.60 mg l) and myristicin (24 hours- 0.60, 1.20 mg l ; 96 hours 0.06, 0.12 mg these concentrations were based on 24 and 96 hours lc50 values earlier reported by us. control animals were kept in an equal volume of dechlorinated water under similar conditions without any treatment. after 24 and 96 hours of treatment snails were removed from the aquaria and rinsed with water. nervous tissue, present around the buccal mass of the snail, was taken out for the measurement of ache, acp, and alp activities in the treated and control groups of snails. in vitro experiments were performed by dissolving the molluscicides in ether and an appropriate volume containing 3, 5, 7, and 9 g of trimyristin and 0.3, 0.5, 0.7, and 1.0 g of myristicin (0.04, 0.07, 0.09, 0.1, and 0.15 mm of trimyristin and 0.01, 0.02, 0.03, 0.05, and 0.09 mm of myristicin) was added to 10 mm path length cuvette separately. molluscicides were preincubated for 15 minutes at 25c with an enzyme source and then enzyme activity was determined. the michaelis - menten constant (km) and maximum velocity (vmax) of different enzyme inhibitions were calculated by nonlinear regression. lineweaver - burk plots for the hydrolysis of different concentrations of substrate by the treated (0.09 mm trimyristin and 0.03 mm myristicin) and untreated enzymes were plotted to observe mode of enzyme inhibition. ic50 values of myristicin and trimyristin were calculated in between the negative log concentration of inhibitor versus relative activity between inhibited and uninhibited enzymes. acetylcholinesterase (ache) activity was measured by the method of ellman. as modified by singh and agarwal. 50 mg of nervous tissue of lymnaea acuminata taken around the buccal mass was homogenized in 1.0 ml of 0.1 m phosphate buffer ph 8.0 for 5 minutes in an ice bath and centrifuged at 1000 g for 30 minutes at 4c. enzyme activity was measured in a 10 mm path length cuvette using an incubation mixture consisting of 0.1 ml of enzyme source, 2.9 ml of 0.1 m buffer ph 8, 0.1 ml of chromogenic agent dtnb (5,5-dithio - bis-2-nitrobenzoic acid), and 0.02 ml of freshly prepared atchi (aetylthiocholine iodide) solution in distilled water. the change in optical density at 412 nm was recorded for 3 minutes after every 30 s interval at 25c. hydrolyzed min mg protein. for the estimation of kinetic constants of ache, in vitro inhibition of the enzyme was carried out at different concentrations (3.0 10 m, 5.0 10 m, 7.0 10 m, and 1.0 10 m) of the substrate acetylthiocholine iodide. acid phosphatase (acp) activity in the nervous tissue of lymnaea acuminata was measured by the method of bergmeyer as modified by singh and agarwal. / v) was prepared in ice cold 0.9% nacl and centrifuged at 5000 g for 15 minutes at 4c.. 0.2 ml of enzyme source was added to 1.0 ml of acid buffer substrate (0.41 g citric acid, 1.125 g sodium citrate, and 165 mg 4-nitrophenyl phosphate sodium salt to 100 ml of double distilled water) preincubated at 37c for 10 minutes. the yellow colour, developed due to the formation of 4-nitrophenol, was determined colorimetrically at 420 nm. for the determination of kinetic constants of acid phosphatase, in vitro inhibition of the enzyme was carried out at different concentrations (1.25 10 m, 1.8 10 m, 3.0 10 m, and 5.4 10 m) of the substrate 4-nitrophenyl phosphate. alkaline phosphatase (alp) activity in the nervous tissue of lymnaea acuminata was measured by the method of bergmeyer as modified by singh and agarwal. tissue homogenate (2%, w / v) was prepared in ice cold 0.9% nacl and centrifuged at 5000 g for 15 minutes at 4c. 0.1 ml of enzyme source was added to 1.0 ml of alkaline buffer substrate (375 mg glycine, 10 mg mgcl2.6h2o, 165 mg 4-nitrophenyl phosphate disodium salt in 42 ml of 0.1 n naoh and a mixture was made up to 100 ml with double distilled water). 10 ml of 0.02 n naoh was then added to the incubation mixture. the yellow colour, developed due to the formation of 4-nitrophenol, was determined colorimetrically at 420 nm. for the determination of kinetic constants of alkaline phosphatase, in vitro inhibition of the enzyme was carried out at different concentrations (1.2 10 m, 1.8 10 m, 3.0 10 m, and 5.4 10 m) of the substrate 4-nitrophenyl phosphate. protein estimation was carried out by the method of lowry. using bovine serum as a standard. each experiment was replicated at least six times and results were expressed as mean se of six replicates. student 's t - test was applied between control and treated groups to locate significant (p <.05) variations. table 1 shows that ache activity in the nervous tissue of lymnaea acuminata of control group was 0.73 mole sh hydrolyzed min mg protein. in vivo treatment of 40 and 80% of 24 and 96 h lc50 of trimyristin and myristicin caused significant (p <.05) inhibition in ache activity in the nervous tissue of lymnaea acuminata. maximum inhibition of ache activity (49% of control) was observed in snails exposed to 80% of 96 h lc50 of myristicin (table 1). the acid phosphatase activity in the nervous tissue of lymnaea acuminata of control group was 32.3 mole substrate hydrolyzed 30 min mg protein (table 1). in vivo treatment of 40 and 80% of 24 and 96 hours lc50 of trimyristin and myristicin caused significant (p <.05) inhibition in acp activity in the nervous tissue of lymnaea acuminata. maximum inhibition of acp activity (44% of control) was observed in snails exposed to 80% of 96 hours lc50 of myristicin (table 1). the alkaline phosphatase activity in the nervous tissue of lymnaea acuminata of control group was 30.0 mole substrate hydrolyzed 30 min mg protein (table 1). in vivo treatment of 40 and 80% of 24 and 96 h lc50 of trimyristin and myristicin caused significant (p <.05) inhibition in alp activity in the nervous tissue of lymnaea acuminata. maximum inhibition of alp activity (52% of control) was observed in snails exposed to 80% of 96 h lc50 of myristicin (table 1). in vitro preincubation of 0.04, 0.07, 0.09, 0.1, and 0.15 mm of trimyristin and 0.01, 0.02, 0.03, 0.05, and 0.09 mm of myristicin caused significant (p <.05) dose dependent inhibition in ache, acp, and alp activities (table 2). ic50 values of trimyristin / myristicin against ache, acp, and alp were 0.11, 0.16 and 0.18 / 0.03, 0.07 and 0.6 mm, respectively (table 2). the km and vmax values of uninhibited ache were 3.17 10 m and 1.25 mole km, of trimyristin (figure 1(a)) and myristicin (figure 1(b)) inhibited ache were 5.55 10 m and 3.84 10 m, respectively. vmax values of trimyristin and myristicin inhibited ache were 1.0 and 0.95 mole km and vmax values of uninhibited acp were 1.37 10 m and 50.00 mole substrate hydrolyzed 30 min mg protein, respectively (table 3). km values of trimyristin (figure 2(a)) and myristicin (figure 2(b)) inhibited acp were 1.09 10 m and 2.12 10 m, respectively. vmax values of trimyristin and myristicin inhibited acp were 38.46 and 50 mole substrate hydrolyzed 30 min mg protein, respectively. km and vmax values of uninhibited alp were 2.0 10 m and 62.50 mole substrate hydrolyzed 30 min mg protein, respectively (table 3). km values of trimyristin (figure 3(a)) and myristicin (figure 3(b)) inhibited alp were 2.27 10 m and 1.66 10 m, respectively. vmax values of trimyristin and myristicin inhibited alp were 55.5 and 50 mole substrate hydrolyzed 30 min mg protein, respectively (table 3). in vivo and in vitro sublethal treatments of trimyristin and myristicin caused a significant inhibition of ache, acp and alp activities in the nervous tissue of lymnaea acuminata. in in vitro condition, the activity of the enzyme is in the presence of drug. extent of enzyme inhibition in in vivo and in vitro conditions is the almost same. in in vivo condition titer of the drug at action site may be low, yet it inhibited the enzyme up to the same extent as in in vitro condition. ic50 values of trimyricitin and myristicin clearly indicate that both are more potent inhibitors of ache than acp and alp. ache inhibition results in accumulation of acetylcholine at the nerve synapses, so that the postsynaptic membrane is in a state of permanent stimulation producing paralysis, ataxia, general lack of coordination in neuromuscular system, and eventual death. it has been reported that n - hexane extract of m. fragrans seeds significantly inhibited ache activity in brain of swiss albino mice and in in vitro, hydroalcoholic extracts of m. fragrans inhibited 50% of ache activity at concentration of 100150 g / ml using ache obtained from bovine erythrocytes. acid phosphatase, a lysosomal enzyme, plays an important role in catabolism, pathological necrosis, autolysis, and phagocytosis. alkaline phosphatase plays a critical role in protein synthesis, shell formation, other secretary activities, and transport of metabolites in gastropods. the kinetic study clearly indicates that the inhibition of ache by trimyristin and myristicin is competitive noncompetitive, as km and vmax values of uninhibited and inhibited enzymes were different and slopes of inhibited and uninhibited ache were also changed ; both were not parallel to each other. it is evident from the lineweaver - burk plots that the slopes of trimyristin inhibited acp, myristicin inhibited alp and uninhibited acp / alp were not changed ; both were parallel to each other, whereas the intercepts of inhibited and uninhibited acp / alp were changed. inhibition of acp by myristicin is competitive, as the km values of the uninhibited and inhibited myristicin enzymes were different and vmax of both were same, as evident from same intercept (1/vmax) on the y axis of lineweaver - burk plots. inhibition of ache, acp, and alp by trimyristin and myristicin indicate, different types of inhibition kinetics. it seems that the molluscicidal components of myristica fragrans kill the snails by inhibiting these enzymes in different ways.	this study was designed to investigate the effects of molluscicidal components of myristica fragrans houtt. (myristicaceae) on certain enzymes in the nervous tissue of freshwater snail lymnaea acuminata lamarck (lymnaeidae). in vivo and in vitro treatments of trimyristin and myristicin (active molluscicidal components of myristica fragrans houtt.) significantly inhibited the acetylcholinesterase (ache), acid and alkaline phosphatase (acp / alp) activities in the nervous tissue of lymnaea acuminata. the inhibition kinetics of these enzymes indicates that both the trimyristin and myristicin caused competitive noncompetitive inhibition of ache. trimyristin caused uncompetitive and competitive / noncompetitive inhibitions of acp and alp, respectively whereas the myristicin caused competitive and uncompetitive inhibition of acp and alp, respectively. thus results from the present study suggest that inhibition of ache, acp, and alp by trimyristin and myristicin in the snail lymnaea acuminata may be the cause of the molluscicidal activity of myristica fragrans.
systemic sclerosis (ssc), also known as scleroderma, is a connective tissue disease of unknown etiology that is characterized by fibroproliferative changes in multiple organs, as well as microvascular and immunologic dysregulation. one of the most morbid conditions associated with ssc is interstitial lung disease (ild), which occurs in 2590% of ssc patients, depending on the detection methods used and the demographics of the population being studied [1, 2 ]. the pathologic mechanisms responsible for the initiation and maintenance of ssc ild remain poorly characterized. approximately 42% of patients with ssc ild will die of disease progression within 10 years of diagnosis, and currently no curative therapies exist to combat this morbid complication. much of the research literature on ssc - associated fibrosis has focused on the roles of fibroblasts and myofibroblasts, the effector cells that are ultimately involved in the production of collagen and other extracellular matrix (ecm) proteins. however, the development of fibrosis in ssc is indeed a complex process involving crosstalk amongst multiple cell types, including epithelial, endothelial, immune, and mesenchymal cell types. in idiopathic pulmonary fibrosis (ipf), a progressive fibrosing lung disease that has a median survival of between two and three years, the principle defect is thought to be recurrent epithelial injury with resultant epithelial cell senescence and/or apoptosis. epithelial injury can lead to the recruitment and activation of fibroblasts, which can be derived from resident fibroblasts, circulating fibrocytes, or the differentiation of epithelial cells, endothelial cells, or pericytes into fibroblasts. the best characterized of these changes in cell differentiation involves epithelial cells and has been termed epithelial - to - mesenchymal transition (emt). alveolar type ii epithelial cell (at2) injury has long been observed in lung biopsies from patients with ild, and recent animal data suggests a causal relationship between at2 injury and fibrosis. sisson. recently demonstrated that targeted deletion of at2 cells, using diphtheria toxin driven by a specific lung epithelial cell promoter leads directly to lung fibrosis. the most convincing evidence for the contribution of emt to lung fibrosis came from studies by kim., who used genetic fate - mapping methods to demonstrate the capacity of alveolar epithelial cells to undergo emt in an established mouse model of lung fibrosis. based on these data and others, injured alveolar epithelial cells are viewed as potential drivers of pathologic pulmonary fibrosis. wells. measured the speed of clearance of technetium - labeled diethylene - triamine - pentaacetate (tc - dtpa) from the lungs in 53 patients with ssc ild and found that rapid clearance, which suggested breach of epithelial barrier function, serum levels of the mucin - like glycoprotein kl-6, which is produced exclusively by lung epithelial cells and is associated with lung epithelial cell damage, are increased in ild associated with connective tissue diseases. recurrent lung epithelial injury via chronic microaspiration has been proposed as a mechanism contributing to lung fibrosis. after the skin, the most commonly affected organ system in ssc is the gastrointestinal tract, affecting approximately 5090% of all patients [911 ]. the esophagus is the most frequently involved site of the gi tract, leading to gastroesophageal reflux (ger). in a rodent model, chronic gastric fluid aspiration leads to a lymphocytic and obliterative bronchiolitis as well as parenchymal fibrosis, with increased tgf levels in bronchoalveolar lavage fluid.. the bile acid chenodeoxycholic acid stimulates tgf production in human airway epithelial cells and induces fibroblast proliferation in vitro in a tgf-dependent manner. correlative data support a relationship between chronic microaspiration and ssc ild as well as other fibrotic lung diseases such as ipf [14, 15 ]. a strong association between ger and ipf has been recently reported in several studies, with an estimated prevalence of 6788% for distal esophageal reflux and 3071% for proximal esophageal reflux based on 24-hour esophageal ph monitoring. interestingly, symptoms of reflux were poor predictors for the diagnosis of ger, implying a significant component of silent microaspiration [1618 ]. besides microaspiration, other mechanisms leading to lung fibrosis could also be at play in ssc ild, involving not only epithelial cells but also endothelial, mesenchymal, and immune cell types. however, the hypothesis that microaspiration leads to ssc pulmonary fibrosis via recurrent epithelial injury is certainly an important one that needs to be strongly considered, especially given the prevalence of ger in ssc. tgf is a pleiotropic cytokine that affects cell proliferation, differentiation, and apoptosis and is involved in a multitude of homeostatic functions. importantly, tgf is regarded as the master switch of fibrosis in many tissues, including the lung. the major effects of tgf include inhibition of epithelial cell proliferation, induction of fibroblast proliferation and the expression of genes encoding components of the ecm, and inhibition of the expression of metalloproteinase genes. tgf can stimulate fibroblast conversion into contractile myofibroblasts, which actively produce collagen and other ecm proteins, and may serve as an inducer of emt, leading to fibrosis. mice that possess a gain of function mutation in the tgf pathway develop progressive fibrosis in multiple organs resembling ssc. global deletion of smad3, a critical mediator of tgf signaling, or specific deletion of the tgf receptor ii from lung epithelial cells affords resistance to bleomycin - induced lung fibrosis [22, 23 ]. increased expression of tgf1 or tgf2 is seen in early skin lesions and in lung tissue from patients with ssc ild [25, 26 ], and tgf1 was significantly elevated in bronchoalveolar lavage fluid from ssc patients with pulmonary fibrosis. a critical role for tgf in ssc has been highlighted by dna microarray studies of ssc skin and fibroblasts. recently, sargent. generated a tgf-responsive signature in dermal fibroblasts comprised of 894 responsive genes. analysis of these genes in ssc skin biopsies revealed that this tgf-responsive signature occurred exclusively in a subset of skin biopsies from patients with diffuse ssc, and in particular, those who had a higher incidence of lung fibrosis. importantly, these data suggest that a subset of ssc patients has disease that is predominantly driven by tgf. there are three isoforms of tgf in mammals which are all bind to the same heteromeric receptor, leading to activation of the canonical pathway via phosphorylation of smad proteins. in addition, noncanonical pathways are activated by tgf receptors, including several protein kinases (p38, jnk, erk, c - abl, tgf--activated kinase) and the lipid kinase pi3 kinase and its downstream target akt. however, the phenotypes of mice lacking the different tgf isoforms are disparate, which could be explained by differences in isoform expression patterns or differential regulation of non - canonical signaling pathways. mice deficient in tgf1 exhibit uncontrolled tissue inflammation, autoimmunity, and premature death, demonstrating a critical role for tgf1 in immune homeostasis [29, 30 ]. these data suggest that general blockade of tgf should be approached with caution. a clinical trial of ssc patients utilizing an antibody directed against tgf1 showed no appreciable therapeutic effect, although the potency of this antibody has been questioned. given its pleiotropic effects, tgf inhibition using strategies targeted to specific regions involved in fibrosis might be a better alternative. most other approaches currently under consideration for targeting tgf block either tgf receptors or tgf itself. these approaches might lead to unwanted side effects by interfering with important homeostatic effects of tgf at sites outside the organs affected by tissue fibrosis. although mice lacking v6 do have mild inflammation in the lungs and skin, these effects are much less severe than those seen in mice lacking even a single tgf isoform. additionally, the v6 integrin is highly upregulated in diseased tissue providing a mechanism for injury - induced tgf activation as compared to homeostatic control of tgf activity. by inhibiting only a subset of tgf activation, particularly in injured epithelial organs, targeting v6 could allow treatment of tissue fibrosis with substantially reduced risk of disrupting beneficial homeostatic control of inflammation and immunity. the regulation of tgf activity involves multiple interactions of various proteins with the tgf cytokine. tgf is normally secreted as a complex which includes the bioactive peptide of tgf1, an amino terminal fragment of the tgf1 gene product called the latency - associated peptide (lap), and the latent tgf-binding protein (ltbp). the tgf gene product is cleaved within the endoplasmic reticulum by the endopeptidase, furin, and it is assembled as a complex of two disulfide - linked homodimers formed from the shorter carboxy - terminal fragment (the active cytokine) and the longer amino - terminal fragment, lap. these two homodimers associate noncovalently to form the small latent complex, which is unable to activate the tgf receptor because lap shields the mature tgf homodimer from interaction with its receptor. in most cells, this small latent complex becomes disulfide linked to one of the latent tgf-binding proteins (ltbp). this large complex is secreted and attaches to components of the extracellular matrix and is covalently cross - linked to ecm proteins via the action of extracellular tissue transglutaminase. this preformed latent tgf complex exists at a high concentration in the ecm of most organs with little evidence of tgf activation. given the diverse and potent effects of tgf, its activity must be tightly regulated in a spatially specific manner. integrins are cell surface molecules comprised of alpha and beta chain heterodimers that regulate cell adhesion, survival, proliferation, and migration. the 31 and 64 integrins recognize the epithelial basement protein, laminin 5 and play an important role in maintenance of epithelial integrity [3538 ]. the other 6-lung epithelial integrins recognize ligands that are not present at baseline but are components of the provisional matrix that are upregulated in response to injury or inflammatory stimuli. the v6 integrin is the only integrin that is restricted in its expression to epithelial cells. this integrin, minimally expressed in healthy airway and alveolar epithelial cells at baseline, gets rapidly induced at these sites in response to a variety of insults, including lung injury. notably, and of possible relevance to the skin fibrosis of ssc, v6 is also upregulated on keratinocytes in the setting of wound healing but is minimally expressed at baseline. in vitro, the v6 integrin binds to a number of ligands, including fibronectin, tenascin - c, and osteopontin via interactions with an arginine - glycine - aspartic acid (rgd) tripeptide sequence, a sequence also recognized by several other integrins including those that share the v subunit. however, the in vivo relevance of v6 interactions with these ligands remains uncertain. mice completely lacking the 6 integrin subunit, which pairs exclusively with the v subunit, were viable with a near - normal life expectancy, but developed low - grade inflammation of the skin and lungs and late - onset emphysema [43, 44 ]. following intratracheal delivery of bleomycin, a drug used to induce pulmonary fibrosis, 6 deficient mice developed exaggerated inflammation in the lung but were remarkably protected from the subsequent development of pulmonary fibrosis. these phenotypic findings suggested a role for the v6 integrin in regulating tgf, a key negative regulator of inflammation but a positive regulator of fibrosis. amino acid sequence analysis revealed the presence of an rgd - binding sequence in the latency - associated peptide (lap) of tgf1 and 3, and lap1 and 3 were demonstrated to be bona fide ligands for v6 [47, 48 ]. cells expressing the v6 integrin were shown to generate tgf activity that could be detected by an in vitro tgf reporter assay, and this activity was dependent upon cell - cell contact and could be specifically blocked with antibodies to v6. microarray analysis of lungs from mice treated with bleomycin revealed a large group of tgf-inducible genes that were induced at much lower levels in the 6 knockout mice compared with wild - type mice. collectively, these data provide strong evidence that the v6 integrin on lung epithelial cells is an important regulator of tgf activation. activation could be inhibited by blocking actin polymerization and by inhibitors of rho kinase, suggesting a role for force generation by the actin cytoskeleton which presumably alters the conformation of latent complexes tethered to the extracellular matrix by matrix - bound ltbp, allowing for exposure of the active tgf cytokine and its interaction with tgf receptors. regulation of tgf activity in the lung was found to play an important role in the maintenance of alveolar homeostasis. low - grade inflammation in the lungs of the 6 knockout mice was characterized by increased numbers of alveolar macrophages, neutrophils, lymphocytes, and eosinophils, and this inflammation was reversed by transgenic overexpression of constitutively active tgf. microarray analysis of 6 deficient lungs showed more than a 20-fold increase in the expression of matrix metalloproteinase 12 (mmp12). this protease, which is predominantly expressed by macrophages, preferentially degrades elastin, and has been implicated in the pathogenesis of emphysema. emphysema was noted in older 6 deficient mice, and crossing the 6 deficient mice with mice lacking mmp12 completely rescued this phenotype. expression of a wild - type form of the 6 integrin prevented emphysema development, while expression of a mutant 6 integrin subunit unable to support tgf activation did not prevent emphysema development. studies have shown that the development of emphysema in 6 deficient mice correlates tightly with the upregulation of mmp12, suggesting that mmp12 could serve as a surrogate biomarker to assess for this particular consequence. ssc ild can be histopathologically classified as nonspecific interstitial pneumonia (nsip) or usual interstitial pneumonia (uip) [5154 ]. nsip is the more commonly encountered histopathologic subtype, comprised of varying degrees of inflammation and fibrosis, with some forms being predominantly inflammatory (cellular nsip) and others primarily fibrotic (fibrotic nsip). it remains unclear whether cellular nsip and fibrotic nsip represent a progression of one underlying disease process or rather two separate disease phenotypes, which in some cases can coexist within the same patient. uip is the pathologic pattern observed in idiopathic pulmonary fibrosis (ipf) and can also be seen in ssc ild. uip consists of interstitial fibrosis in a patchy pattern, honeycomb changes (both macroscopic and microscopic), and foci of fibroblastic proliferation. although the uip pattern in ssc is less commonly encountered clinically, it can be seen with increased frequency in patients with more severe fibrotic lung disease, including those with end - stage ssc ild requiring lung transplant. currently, there are no highly effective agents for the treatment of fibrotic lung diseases. several studies using anti - tgf agents have demonstrated protection from lung fibrosis in disease models [46, 56, 57 ]. given the homeostatic roles of tgf in inflammation, immune regulation, and carcinogenesis, perhaps a better strategy for tgf inhibition would be to specifically target tissue - restricted activators of tgf such as the v6 integrin. in patients with ipf and ssc ild with a uip pattern, the v6 integrin is highly upregulated on lung epithelium, implicating this pathway in tgf activation. in the only published report to date, upregulation of v6 was found on lung epithelium in seven out of seven ssc patients with uip and in a single patient with ssc ild who had fibrotic nsip, but not in patients with cellular nsip, however, the numbers of patients with nsip analyzed were too small to draw meaningful conclusions. it would therefore be important to better characterize whether upregulation of v6 specifically segregates with the uip and fibrotic nsip subsets of ssc ild, and what role, if any, this integrin plays in the cellular nsip subset. anecdotal evidence and case series suggest that immunomodulators might more effectively target the cellular nsip subset of ssc ild, whereas the fibrotic nsip and uip subsets are thought to be more recalcitrant to currently available therapies. of particular interest, a mouse model of radiation - induced lung fibrosis identified a sharp upregulation of v6 expression by immunohistochemical analysis at 18 weeks following radiation challenge, with staining seen only in regions of fibrosis and similar upregulation in fibrotic regions was found in lungs of ipf patients. it thus appears that the induction of v6 correlates closely with fibrosis and that this integrin is often present at high concentrations in regions where active tgf could be contributing to disease progression. a highly potent - blocking antibody to the v6 integrin was developed and shown to prevent fibrosis in mouse models of bleomycin- and radiation - induced lung fibrosis [46, 56 ]. in these studies, near maximal effects on collagen production were obtained at 1 mg / kg weekly dosing of the antibody. importantly, a treatment (as opposed to prophylaxis) trial was performed in mice by giving the v6-blocking antibody at day 15 following intratracheal bleomycin administration, and decreased fibrosis at day 60 was observed using the hydroxyproline assay to measure lung collagen content. given the finding of low - grade inflammation in the lungs of the 6 deficient mice as well as their late stage development of emphysema, a process that was dependent on mmp12, a concerted effort was made to characterize whether a similar inflammatory phenotype with elevated mmp12 levels was observed in mice receiving the v6-blocking antibody. transcript profiling of the lungs of mice treated with high doses (10 mg / kg) of the v6 blocking antibody paralleled the changes seen in 6 integrin knockout mice, including upregulation of mmp12 levels. importantly, at lower doses of the v6 blocking antibody (1 mg / kg or 3 mg / kg), mmp12 induction was greatly diminished, and bal cell counts and inflammatory cytokines were not different than in saline - treated mice [46, 56 ]. at these lower doses of blocking antibody, significant inhibition of collagen production was still observed, as assessed by an in vivo collagen luciferase reporter system, suggesting that the antifibrotic effect of v6 inhibition could be uncoupled from the proinflammatory effect. induction of tgf activation by bleomycin, as measured by phospho - smad levels in lung lysates, was completely blocked at the 3 mg / kg but not by the 1 mg / kg dose of v6 blocking antibody suggesting that complete blockade of tgf signaling is not required to achieve antifibrotic efficacy and inhibition of tgf-induced fibrosis can be achieved without excessively perturbing the homeostatic functions of tgf. treatment of healthy, unchallenged mice with high doses of the v6 blocking antibody has been shown to lead to mixed cellular infiltrates (macrophages, lymphocytes, neutrophils) in lung tissue, not dissimilar to the inflammation seen in the 6 knockout mice. however, long - term treatment of healthy primates with a humanized form of the same v6 blocking antibody leads to a minimal to mild increase in lung macrophages, which resolves completely following discontinuation of treatment, with no increase in mixed cellular inflammation (unpublished observations). these findings have suggested that inhibition of v6 does not induce the same degree of inflammation in primates as seen in mice. additionally, no evidence of emphysema has been observed after 6 months of weekly dosing with high doses of v6 antibody in mice or primates and there has been no evidence of elevated mmp-12 expression in primates with v6 antibody treatment as observed in mice. inhibition of v6 as a means of locally dampening tgf activation by epithelial cells provides a rational therapeutic approach for conditions such as lung fibrosis. importantly, the antifibrotic effect of v6 inhibition can be achieved at a dose that is uncoupled from its proinflammatory effect in mice [46, 56 ]. a phase ii trial using a humanized v6 blocking antibody (stx-100) in ipf patients will soon be underway, and these results should be of considerable interest to the ssc community. evaluation of the utility of inhibition of v6-mediated tgf activation in ssc ild, particularly the uip and fibrotic nsip subgroups, may be worth considering, especially if these early studies in ipf prove promising. in addition, recent data implicate an important role for epidermal keratinocytes in ssc skin fibrosis. v6 is induced on injured keratinocytes in other settings, so the expression of v6 should be more closely evaluated in skin samples from ssc patients to determine whether a subset of these patients might also benefit from v6 blockade for treatment of skin fibrosis. given the known heterogeneity of ssc within and beyond the limited and diffuse subsets [60, 61 ], the inhibition of epithelial v6-mediated tgf activation may not address some of the other manifestations of ssc, in particular the vascular complications in which endothelial injury has been posited as an initiating mechanism. in fact, it is unlikely that any single treatment strategy will effectively combat the various pathologic manifestations of ssc. whether the mechanisms leading to fibrosis of the skin and other internal organs in ssc are dependent upon v6-mediated tgf activation remains to be determined. additional mechanisms involved in tgf activation, such as the integrins v3, v5, and v8, could be playing a contributory role, but discussion of this is beyond the scope of the current paper. importantly, when considering strategies that target tgf activity, potential side effects should be carefully monitored, such as the development of aberrant inflammation or cancer. however, in light of the morbidity and mortality associated with fibrotic lung diseases, especially ipf or the more fibrotic phenotypes of ssc ild (uip and fibrotic nsip), perhaps these treatment risks can be justified given the lack of alternatives short of lung transplantation in some cases. achilles heel of pulmonary fibrosis, and the ability to locally inhibit its activity presents an attractive strategy that may likely be met with clinical success.	interstitial lung disease (ild) is a commonly encountered complication of systemic sclerosis (ssc) and accounts for a significant proportion of ssc - associated morbidity and mortality. its pathogenesis remains poorly understood, and therapies that treat ssc ild are suboptimal, at best. ssc ild pathogenesis may share some common mechanisms with other fibrotic lung diseases, in which dysregulation of lung epithelium can contribute to pathologic fibrosis via recruitment or in situ generation and activation of fibroblasts. tgf, a master regulator of fibrosis, is tightly regulated in the lung by the integrin v6, which is expressed at low levels on healthy alveolar epithelial cells but is highly induced in the setting of lung injury or fibrosis. here we discuss the biology of v6 and present this integrin as a potentially attractive target for inhibition in the setting of ssc ild.
glucose6phosphate (g6pd) deficiency is the most common human enzyme defect which affects more than an estimated 400 million people worldwide 1, 2, 3. the most frequent clinical manifestations of g6pd deficiency are neonatal jaundice and acute hemolytic anemia, often triggered by oxidative stress 1, 3 from infection and exposure to medication and certain foods (e.g., fava beans) 3, 4. some g6pddeficient variants cause chronic hemolysis, leading to congenital nonspherocytic anemia 1, 3. the g6pd enzyme catalyzes the first step in the pentose phosphate pathway, oxidizing glucose6phosphate to 6phosphogluconolactone and reducing nicotinamide adenine dinucleotide phosphate (nadp) to nadph 1, 3. nadph is crucial in producing reduced glutathione in the erythrocyte cytoplasm for protecting hemoglobin against oxidative damage. in case of g6pd deficiency, the presence of oxidizing agents leads to oxidation of the sulfhydryl bridges between parts of the hemoglobin molecule, thereby decreasing the solubility of hemoglobin which causes hemolysis 2. the inheritance of g6pd deficiency shows a typical xlinked pattern 1, 3, 4, in which mainly men are affected, and females, in case being heterozygous, have less severe clinical manifestations 1, 2, 3. here, we report two girls with compound heterozygote g6pd deficiency, one of them presented with a severe hemolytic anemia. we discuss the etiology of g6pd deficiency in females and emphasize the importance to consider g6pd deficiency in acute hemolytic anemia, not only in males, but also in females. a healthy 5yearold girl was referred to our hospital for jaundice and fatigue after eating fava beans suggestive of g6pd deficiency. the family history revealed a paternal aunt who was not allowed to eat fava beans. complete blood count showed a picture of a hemolytic anemia (hb 5.4 g / dl, mcv 82 fl, reticulocytes 104/1000 rbc, schistocytes, and bite cells) with elevated ldh (912 u / l, normal 313618 u / l) and total bilirubin (1.4 mg / dl, normal 0.21.3 mg / dl), and lowered haptoglobin (10.4 mg / dl, normal 26185 mg / dl). spectrophotometric analysis revealed deficiency of g6pd (0.1 u / g hb) indicating a severe g6pd deficiency (class ii according to the world health organization classification). dna analysis showed two mutations : the g6pd mediterranean variant (c.563c > t (p.ser188phe)) and the g6pd chatham variant (c.1003g > a (p.ala335thr)), both in compound heterozygous state. although her father and her 6yearold sister never had symptoms of hemolysis, family screening with spectrophotometric analysis revealed that both were severely affected (respectively, 0.1 u / g hb and 0.0 u / g hb, normal range on this age 4.17.9 u / g hb). enzyme deficiency was not confirmed in her mother and her brother (respectively, 6.0 u / g hb and 8.7 u / g hb). the father was hemizygous for the c.563c > t (p.ser188phe) mutation, while the mother was heterozygous for the c.1003g > a (p.ala335thr) mutation, but more surprisingly, her sister turned out to have the same mutations in compound heterozygosity. father : g6pd mediterranean variant (c.563c > t (p.ser188phe)) in hemizygosity. mother : g6pd chatham variant (c.1003g > a (p.ala335thr)) in heterozygosity males are hemizygous for the g6pd gene and can have normal gene expression or be g6pd deficient. females, who have two copies of the g6pd gene on each x chromosome (xq28), can have normal gene expression, be heterozygous or rarely homozygous for a mutation or compound heterozygous for two mutations on the g6pd gene 1. heterozygous females have, in general, less severe clinical manifestations than g6pddeficient males 1, 2, 3, but the mean red blood cell enzyme activity depends on the degree of inactivation of one of the x chromosomes (lyonization) 4, and may be normal, mildly to moderate reduced, and grossly deficient. although very rare, in some populations, in which the frequency of the g6pddeficient allele is very high 1, mainly in (sub) tropical regions 3, females can be homozygous 1 or compound heterozygous. almost all of the variants are missense point mutations causing single amino acid substitutions 3. the world health organization has categorized the mutant enzymes into five classes according to the degree of enzyme deficiency and hemolysis. the g6pda is the most common variant, associated with mild to moderate hemolysis, while the g6pd mediterranean variant is the most abnormal variant found in caucasians and is classically associated with favism (hemolysis after ingestion of broad beans) 5. + 2pq + q2 = 1 (2pq = heterozygous and q2 = homozygous state in the female population) 7. according to this formula, the prevalence of heterozygous g6pddeficient iraqi females would be 11.5% versus 0.37% for homozygous females. compound heterozygosity, in which two different heterozygous mutations on the g6pd gene are found, each on another chromosome, is even rarer. in baghdad, iraq, three polymorphic variants constituted more than 80% of g6pddeficient variants among males, namely : the mediterranean (74.3%), chatham (5%), and a (2%) 8. the sisters discussed are compound heterozygous for two class ii variants, mediterranean, and chatham, which can cause acute severe hemolysis. diagnosing g6pd deficiency can be challenging as normal results of a peripheral blood smear and spectrophotometric analysis do n't exclude g6pd deficiency. in the acute setting of a hemolytic anemia, the presence of red blood cell anomalies, such as spherocytes and bite cells on a peripheral blood smear (fig. several screening tests for g6pd deficiency are available, for example, the fluorescent spot test, the ascorbate cyanide test, and the dye reduction test. the definitive test requires quantitation of the enzyme and can be performed by spectrophotometric analysis, which directly measures the rate of nadph formation (proportional to the g6pd activity) 9 through its characteristic absorption peak at wavelength of 340 nm 10. the red cell activity is expressed in international units (iu) per gram of hemoglobin. the standardized normal reference value of the g6pd activity is established by taking the median g6pd activity value of a group of healthy male controls. in our laboratory, the reference for normal values at room temperature is in the range of 4.17.9 u / g hb (newborns 50% higher). however, after severe hemolysis, young red blood cells and reticulocytes have much higher g6pd activity (up to 5x) ; the analysis can be false negative. in those cases or after red blood cell transfusion, tests need to be repeated after approximately 23 months when the red blood cell mass is repopulated with cells of all ages. carrier state in females is difficult to determine, because of the normal enzyme activity in the unaffected x chromosome. in those cases, it is sometimes necessary to perform polymerase chain reaction (pcr) tests to reveal the genetic abnormality. this xlinked hereditary deficiency is mainly affecting males, but should also be considered in women presenting with a hemolytic anemia, even when family history is negative and the g6pd level is normal at presentation.	key clinical messageglucose6phosphate (g6pd) deficiency is the most common human enzyme defect, often presenting with neonatal jaundice and/or acute hemolytic anemia, triggered by oxidizing agents. g6pd deficiency is an xlinked, hereditary disease, mainly affecting men, but should also be considered in females with an oxidative hemolysis.
schmincke has described an undifferentiated nasopharyngeal carcinoma showing a diffuse pattern mimicking lymphoma.1 this has been designated as lymphoepithelial carcinoma (lymphoepithelioma) or undifferentiated carcinoma with lymphocytic stroma. similar tumors occurring in extrapharyngeal sites have been referred to as lymphoepithelioma - like carcinoma (lelc). lelc has been reported in the lung, salivary gland, thymus, stomach, urinary bladder, uterus, vagina, bile duct, and gallbladder.2345678 only two cases of pure type lelc involving the gallbladder have been described in the literature.78 in mixed type of lelc in the urinary bladder, invasive urothelial carcinoma, squamous cell carcinoma, adenocarcinoma, adenourothelial carcinoma, or in situ carcinoma of each type has been associated with lelc.9 epstein - barr virus (ebv) infection has been implicated in the oncogenesis of almost all cases of lymphoepithelial carcinoma of the nasopharynx.10 however, ebv is only detected in lelc of the stomach, esophagus, hepatobiliary tract, lung, thymus, and the salivary gland, but not in lelc of the breast, skin, or urinary bladder.91112 herein, we report the first case of mixed type lelc of the gallbladder not associated with ebv. an 83-year - old korean man was referred from a local clinic under the suspicion of gallbladder cancer following an abdominal computed tomography (ct) scan. fluorodeoxyglucose (fdg)-positron emission tomography (pet)-ct revealed a hypermetabolic lesion in the gallbladder (fig. the patient had epigastric pain for three days prior to his visit to the clinic. no peritoneal or liver metastasis was observed and the cystic duct margin was free of tumor. pathological examination of surgical specimens revealed that the tumor infiltrated diffusely from the mucosa to the subserosa with multiple erosions. the majority (> 95% of tumor volume) of the tumor consisted of nests and individual cells of undifferentiated carcinoma admixed with prominent lymphoplasmocytic infiltrates. the epithelial cells had large vesicular oval - shaped nuclei with occasional large prominent nucleoli (fig. 2). immunohistochemical staining results were positive for ae1/ae3 and cytokeratin 7 but negative for cytokeratin 20, carcinoembryonic antigen, or ebv. the remaining part of the tumor (< 5%) consisted of adenocarcinoma confined to the mucosa. the glandular component was characterized as a well differentiated adenocarcinoma partially overlying the lelc. a transition from adenocarcinoma to lelc lymphoid stroma, especially those surrounding the lelc, was composed of a mixture of cd3-positive t lymphocytes, cd20-positive b lymphocytes, and plasma cells with predominantly cd3-positive t lymphocytes and plasma cells (fig., a diagnosis of non - ebv - associated mixed type lelc of the gallbladder was made. (4 months after the surgery), the patient has no sign of recurrence. lymphoepithelioma was originally described in the nasopharynx of asian patients.1113 it is closely associated with ebv.1113 there is also a close pathogenetic relationship between lelc and ebv.11 cases in individuals of asian ethnicity have predominated literature reports of biliary lelc.6 lelc is classified as pure type when 100% of the tumor demonstrates an lelc pattern or mixed type when the tumor is associated with usual urothelial carcinoma, squamous cell carcinoma, or adenocarcinoma.9 reported cases of lelc of the urinary bladder have been classified as the pure type in 57% of cases and the mixed type in 43% of cases.9 according to some studies, pure or predominant lelc has a better prognosis than focal lelc.111314 however, tamas.9 have reported that there is no significant difference in prognosis between pure type and mixed type of lelc, although only a few cases of pure type lelc are treated with chemotherapy. some cases of lelc are associated with ebv, but some cases are not associated with ebv. there is no obvious histopathological difference between ebv - positive and ebv - negative lelcs in each organ.67912 lelc can occur at various anatomic sites. only two cases of pure type lelc of the gallbladder have been reported.78 ebv is frequently identified in lelc in the lungs, stomach, thymus, and salivary gland, but not in other organs.6789 the two cases of lelc of the gallbladder78 and one case of common bile duct lelc6 reported in the literature are negative for ebv. therefore, the involvement of ebv genome integration in lelc oncogenesis might depend on the site of origin of lelc. the overlying mucosa in mixed type lelc can show dysplasia, carcinoma in situ, or invasive carcinoma.9 in the present case, dysplasia and adenocarcinoma of the overlying mucosa were observed. the mixed component was minor (< 5%) and confined to the mucosa only. lelc is usually associated with a more favorable prognosis compared to conventional carcinoma.9 the presence or absence of ebv has no prognostic significance in lelc of various organs.15 in biliary lelc, differences between the prognoses of ebv - positive and ebv - negative cases have not been established yet due to the limited number of cases reported to date. in conclusion, we report a case of non - ebv - associated mixed type lelc of the gallbladder. due to the rarity of this disease, additional case studies are needed to establish clinicopathological findings of lelc of the gallbladder.	lymphoepithelioma - like carcinoma (lelc), an undifferentiated carcinoma with intense lymphoplasmacytic infiltrates, is commonly reported in the nasopharynx and occasionally in other organs. pure type of lelc has previously been reported in the gallbladder. mixed type could be reportable in comparison with other organs. here we present a case of an 83-year - old man with mixed lelc and adenocarcinoma in the gallbladder. to the best of our knowledge, this is the first case of mixed lelc and adenocarcinoma in the gallbladder.
the liver plays a critical role in regulating metabolic and immune responses after severe burn 1, 2. severe burn causes liver damage and dysfunction, demonstrated by increased hepatic apoptosis and decreased liver derived albumin up to 2 weeks post - burn in a murine model 3. recently, we found that elevated intracellular calcium and endoplasmic reticulum (er) stress play critical roles in mediating post - burn hepatic damage 4. after translation, unfolded polypeptide chains dissociate from ribosome and enter the er for post - translational modification. misfolded and unfolded protein accumulation in the er disrupts its function and causes er stress followed by numerous subsequent metabolic responses 5, 6. interestingly, er stress has been shown to inhibit protein synthesis by regulating its translational processes 7 - 9. we, therefore, hypothesize that er stress plays a key role in mediating protein synthesis in the liver post - burn. in the current study, we treated hepg2 cells with thapsigargin (tg), a serca pump inhibitor and evaluated the effects of er stress on protein synthesis. the aims of the present study are : 1) to establish an in vitro model to isotopically measure hepatic protein synthesis and 2) to evaluate protein fractional synthetic rate (fsr) in response to er stress by using this model. determination of protein synthesis using the tracer incorporation technique involves measuring the rate of incorporation of a tracer amino acid into the product protein over time 10. stable isotopes are superior to radioactive isotopes because they are free from the potential risk of radioactive contamination and cell damage 11., we used 1,2-c2-glycine and l-[ring - c6]phenylalanine as tracers simultaneously ; both are stable isotopes commonly used for protein synthesis in basic and clinical research 12, 13. this would enable us to compare protein synthesis rates calculated from different tracers. also, we measured intracellular free amino acid enrichments as an alternative precursor for calculating protein fsr, and compared it with the plateau enrichment of protein - bound amino acids. the above methodological considerations served to establish hepg2 in vitro model for protein synthesis measurement. were cultured in dulbecco 's modified eagle medium (dmem) containing 10% fetal bovine serum, 1% penicillin / streptomycin, 2 mm l - glutamine and were incubated at 37c in a humidified atmosphere of ambient air with 5% co2. two stable isotope labeled amino acids, including 1,2-c2-glycine (1.02) and l-[ring - c6]phenylalanine (0.31) from cambridge isotopelaboratories, ma (numbers in parentheses are mass ratios of labeled to unlabeled substrates), were supplemented and mixed well in dmem culture medium. this isotope - labeled culture medium was stored at 4c and used throughout the entire experiment. when cells reached confluence at day 4, a portion of cells was subcultured for an additional 3 passages (to day 14) using the same isotope tracer - enriched culture medium. from day 0 to day 14, cells were harvested at each designated time point and stored at -80c for further processing and analysis. in prior to examination for hepatic protein synthesis in response to er stress, hepg2 cells were treated the typical er stress inducer tg from 1 nm to 1 m over a 24-hour period to determine the dose and time response. the treated cells were transferred to the isotope - labeled culture medium and harvested followed by the procedure described as above. antibodies against human glucose - regulated protein (grp78/bip) and phosphor - inositol requiring enzyme 1 (p - ire-1) were purchased from abcam inc. (cambridge, ma) ; glyceraldehyde 3-phosphate dehydrogenase (gapdh) were purchased from cell signaling tech, inc. (danvers, ma) ; dimethyl sulfoxide (dmso) was from sigma - aldrich, inc. (san louis, mo) ; supersignal west pico chemiluminescent substrate was purchased from thermo scientific inc. cells were labeled with fura 2-acetoxymethyl ester at 25c in 107 mm nacl, 7.2 mm kcl, 1.2 mm mgcl2, 1 mm cacl2, 11.5 mm glucose, 0.1% bovine serum albumin, and 20 mm hepes (ph 7.2), and calcium imaging was done as described 14 by using metafluor data acquisition and analysis software (molecular devices, inc. hepg2 cells were collected and homogenized in 150 mm nacl, 50 mm tris - hcl, ph 7.8, 1% (w / v) triton x-100, 1 mm edta, 0.5 mm phenylmethanesulfonyl fluoride, 1x complete protease inhibitor mixture (roche molecular biochemicals, indianapolis, in) and a phosphatase inhibitor cocktail (sigma - aldrich, san louis, mo). the homogenate was centrifuged at 20,000 xg for 30 minutes at 4c and the pellet discarded. twenty micrograms of each protein sample was subsequently analyzed by sds - page and western blotting. amino acids from the cell supernatant and pellet hydrolysate were isolated using a cation exchange column (dowex ag 50w - x8, bio - rad laboratories, hercules, ca) and dried in a rotary vacuum evaporator. the intracellular free amino acids and protein hydrolysates were derivatized with n - methyl - n-(t - butyldimethylsilyl) trifluoroacetamide at 100c for 1 hour for 1,2-c2-glycine and l-[ring - c6]phenylalanine enrichment measurements. the enrichments of intracellular amino acids and protein - bound amino acids were measured by gas chromatography - mass spectrometry (gc - ms). the abundance of ions was monitored at the mass to charge ratio (m / z) 246 and 248 (m+0, m+2) for glycine, and at m / z 234 and 240(m+0, m+6) for phenylalanine. fsr for protein synthesis were calculated as : where et2 - et1 is the change in product enrichment (protein) between time points t2 and t1, and ep is the mean precursor enrichment over the time (t2 - t1). as described in greater detail in the results section, it was necessary to define the appropriate time course for linear change of product enrichment, and delineate product plateau enrichment as well. the linear change of product enrichment was validated to calculate the fsr. the true precursor enrichment for calculation of the fsr of protein is represented by the product plateau enrichment. in hepg2 cells, protein - bound enrichments arose and reached plateaus after 7 days of cell culture (glycine ttr=18.530.61% and phenylalanine ttr=26.021.15%) (figure 1a). linear regression analysis showed that the r values were 0.99 for both 1,2-c2-glycine and l-[ring - c6]phenylalanine enrichments in the protein - bound amino acid pool from 0 to 72 hours (figure 1b). because of the linear increase in product enrichment during this period, we speculate that the tracer recycling from protein breakdown is negligible, and the equation for fsr calculation is valid in the current study. using the product plateau enrichment as the true precursor enrichment, we found that fsr were 0.970.02 and 0.990.05%/hr calculated from 1,2-c2-glycine and l-[ring - c6]phenylalanine, respectively. intracellular free amino acid enrichments reached constant values immediately, which were stable throughout the period of experiment (average intracellular free glycine ttr= 20.220.51% and free phenylalanine ttr=26.211.51%). no significant difference was found between the average of the intracellular ttr and plateau ttr measured with either glycine or phenylalanine. when the corresponding intracellular free amino acid enrichments were used, fsr was 0.930.04%/hr with glycine labeling and 0.990.01%/hr with phenylalanine, which were not significantly different (p = 0.08) (table 1). we found that the response to ionomycin diminished as the tg dose increased ; further, there was no response to increasing doses of tg to 100 nm and 1 m, indicating calcium was completely depleted from er storage with a higher dose of 24-hour tg treatment figure 2a and b). we found that a dose of 100 nm tg led to the expression of two prominent er stress markers, glucose - regulated protein (grp78/bip) and phosphor - inositol requiring enzyme 1 (p - ire-1) (figure 3a). furthermore, we treated cells with 100 nm of tg over the time period and observed that the expression of grp78/bip and p - ire-1 were upregulated at 6 hours and the expression of p - ire-1 increased significantly at the 24-hour time point when compared to the dmso treatment (figure 3b). based the above cellular response, we thus chose the condition of 24 hours, 100 nm of tg treatment to conduct the next experiment of protein synthesis measurement. following 100 nm of tg 24-hour treatment, the cells were transferred to the isotope - labeled culture medium and harvested followed by the procedure described in the materials and methods section as above. r values were 0.96 and 0.98 for glycine and phenylalanine enrichments, respectively in the protein - bound pool from 0 to 72 hours. fsr significantly (p<0.05) decreased to 0.680.03 and 0.610.06%/hr, calculated using the plateau enrichments of glycine and phenylalanine as precursor enrichments, respectively (p<0.05), demonstrating inhibition of protein synthesis in response to tg - induced er stress (figure 4). in hepg2 cells, protein - bound enrichments arose and reached plateaus after 7 days of cell culture (glycine ttr=18.530.61% and phenylalanine ttr=26.021.15%) (figure 1a). linear regression analysis showed that the r values were 0.99 for both 1,2-c2-glycine and l-[ring - c6]phenylalanine enrichments in the protein - bound amino acid pool from 0 to 72 hours (figure 1b). because of the linear increase in product enrichment during this period, we speculate that the tracer recycling from protein breakdown is negligible, and the equation for fsr calculation is valid in the current study. using the product plateau enrichment as the true precursor enrichment, we found that fsr were 0.970.02 and 0.990.05%/hr calculated from 1,2-c2-glycine and l-[ring - c6]phenylalanine, respectively. intracellular free amino acid enrichments reached constant values immediately, which were stable throughout the period of experiment (average intracellular free glycine ttr= 20.220.51% and free phenylalanine ttr=26.211.51%). no significant difference was found between the average of the intracellular ttr and plateau ttr measured with either glycine or phenylalanine. when the corresponding intracellular free amino acid enrichments were used, fsr was 0.930.04%/hr with glycine labeling and 0.990.01%/hr with phenylalanine, which were not significantly different (p = 0.08) (table 1). er is the major intracellular place of abundant calcium storage. we examined cytosolic calcium alterations in hepg2 cells by using an ionophore, inonomycin. we found that the response to ionomycin diminished as the tg dose increased ; further, there was no response to increasing doses of tg to 100 nm and 1 m, indicating calcium was completely depleted from er storage with a higher dose of 24-hour tg treatment figure 2a and b). we found that a dose of 100 nm tg led to the expression of two prominent er stress markers, glucose - regulated protein (grp78/bip) and phosphor - inositol requiring enzyme 1 (p - ire-1) (figure 3a). furthermore, we treated cells with 100 nm of tg over the time period and observed that the expression of grp78/bip and p - ire-1 were upregulated at 6 hours and the expression of p - ire-1 increased significantly at the 24-hour time point when compared to the dmso treatment (figure 3b). based the above cellular response, we thus chose the condition of 24 hours, 100 nm of tg treatment to conduct the next experiment of protein synthesis measurement. following 100 nm of tg 24-hour treatment, the cells were transferred to the isotope - labeled culture medium and harvested followed by the procedure described in the materials and methods section as above. r values were 0.96 and 0.98 for glycine and phenylalanine enrichments, respectively in the protein - bound pool from 0 to 72 hours. fsr significantly (p<0.05) decreased to 0.680.03 and 0.610.06%/hr, calculated using the plateau enrichments of glycine and phenylalanine as precursor enrichments, respectively (p<0.05), demonstrating inhibition of protein synthesis in response to tg - induced er stress (figure 4). in the current study, we found that protein - bound amino acid enrichments linearly increased from 0 to 72 hours of cell culture, and reached a plateau after 7 days of cell culture. by using two different stable isotope labeled amino acids simultaneously, we established an in vitro model to measure protein synthesis in hepg2 cells. we chose one non - essential amino acid glycine and one essential amino acid phenylalanine as tracers to conduct the current study because they are easily available and been widely applied in the many areas of research. linear change of enrichment for both tracers within 72 hours indicates that intracellular amino acid recycle is negligible and fsr equation is valid. the values of fsr were comparable between the glycine and phenylalanine tracers, indicating the consistency and reproducibility of the current model. aminoacyl - trna pool enrichment has been thought to be the true precursor of protein synthesis 16. true precursor enrichment can be determined by continuing the tracer infusion until a plateau is reached in the product enrichment theoretically 10. martini 17 found that the protein - bound enrichment increased linearly for the first 3 days and essentially reached the plateau enrichment by day 5 in fibroblast and myocyte. they concluded that intracellular free amino acid enrichment is an optimal surrogate for precursor enrichment. in our study, the ttrs of glycine and phenylalanine are 1.03 and 0.31 in the medium, which are higher than the intracellular levels. however, the intracellular enrichment of both amino acids are consistent with the plateau in product enrichment, and there was no significant difference of fsr value when calculated by product plateau or intracellular free amino acid enrichment in hepatocytes. thus, if necessary the intracellular free amino acid enrichments are accepted as precursor enrichments for fsr calculation. intracellular protein breakdown may affect the accuracy of fsr calculation through amino acid recycling intracellularly, meaning that the amino acids released from proteolysis are re - synthesized into protein. this amino acids recycling may underestimate protein fsr because the recycling tracer is not included in fsr calculation. thus, a correction has to be made to the equation if the recycling accounts for a significant amount. in this experiment, we assume that the protein breakdown is negligible within 72 hours of cell culture. this is because hepg2 cells grow from 50% to 90%, from day 0 to day 4 in culture dishes. in addition, a linear increase of protein - bound amino acid enrichment from 0 to 72 hours was observed. it blocks sarco / er ca atpase (serca) activity to deplete calcium from er storage and subsequently induces er stress 18, 19. tg was found to serve as a rapid, potent, and efficacious inhibitor of amino acid incorporation in cultured mammalian cells 20 - 22. in the present study, we demonstrated that tg induced calcium depletion from er and er stress activation in both a dose- and time - dependent manner. in addition, we found that tg induced er stress in other hepatocyte cell lines such as human primary hepatocyte and huh7, and also observed the similar results compared to the current study (unpublished data). further, protein fsr decreased as expected with 100 nm of tg treatment for 24 hours in hepg2 cells. with both glycine and phenylalanine isotope tracers, we found that the protein - bound amino acid enrichments also increased linearly, and fsr values, which were calculated using the intracellular free amino acid enrichments as precursor enrichment, were also close to those calculated using the plateau product enrichment as the precursor enrichment, indicating the reproducibility of the current methodology, even in response to external stress. in addition, we found that tg induced er stress in other hepatocyte cell lines such as human primary hepatocyte and huh7, and also observed the similar results compared to the current study (unpublished data). we did not explore that molecular signal pathway of er stress modulating protein synthesis in this study. er stress was shown to regulate protein synthesis at the initiation and elongation steps 23 - 25. er stress inhibits protein synthesis via activated perk dependent eif2 (eukaryotic translation initiation factor 2) phosphorylation 26. hypoxic injury attenuated protein synthesis via elongation factor 2 (eef2) in cardiomyocytes, which is associated with amp - activated protein kinase (ampk) activation 8. in the current study, we confirmed that tg - induced er stress inhibits hepatic protein synthesis with decreased protein fractional synthetic rate. the molecular mechanism of the underlying event will be further investigated. in conclusion, we used stable isotope tracer incorporation technique to measure protein synthesis in the current hepg2 cell model. protein synthesis rates calculated from intracellular free amino acid enrichments were close to the true values calculated from product plateau in hepatocytes. furthermore, by using the current established model, we found that hepatic protein fsr decreased in response to tg - induced er stress. the measurement of liver protein synthesis can be used to assess liver impairment and function recovery as well as to test the effects of different stimuli and pathophysiological conditions on protein metabolism. this model may provide a powerful tool to further investigate the mechanism of cellular signaling regulation in hepatic protein synthesis.	severe burn - induced liver damage and dysfunction is associated with endoplasmic reticulum (er) stress. er stress has been shown to regulate global protein synthesis. in the current study, we induced er stress in vitro and estimated the effect of er stress on hepatic protein synthesis. the aim was two - fold : (1) to establish an in vitro model to isotopically measure hepatic protein synthesis and (2) to evaluate protein fractional synthetic rate (fsr) in response to er stress. human hepatocellular carcinoma cells (hepg2) were cultured in medium supplemented with stable isotopes 1,2 - 13c2-glycine and l-[ring-13c6]phenylalanine. er stress was induced by exposing the cells to 100 nm of thapsigargin (tg). cell content was collected from day 0 to 14. alterations in cytosolic calcium were measured by calcium imaging and er stress markers were confirmed by western blotting. the precursor and product enrichments were detected by gc - ms analysis for fsr calculation. we found that the hepatic protein fsr were 0.970.02 and 0.990.05%/hr calculated from 1,2 - 13c2-glycine and l-[ring-13c6]phenylalanine, respectively. tg depleted er calcium stores and induced er stress by upregulating p - ire-1 and bip. fsr dramatically decreased to 0.680.03 and 0.600.06%/hr in the tg treatment group (p<0.05, vs. control). tg - induced er stress inhibited hepatic protein synthesis. the stable isotope tracer incorporation technique is a useful method for studying the effects of er stress on hepatic protein synthesis.
lung cancer causes more deaths in the united states each year than breast, colon, pancreatic, and prostate cancer combined, approximately 157,300 deaths estimated in 2010. cigarette smoking is the most significant risk factor for developing lung cancer and contributes to 8090% of these deaths [2, 3 ]. over the past four to five decades, significant progress has been made to elucidate the carcinogenic mechanisms of tobacco smoking. using animal models, it has been shown that among over 60 established carcinogens in cigarette smoke, 20 can cause lung tumors. it has been proposed that these carcinogens, when metabolized, form dna - adducts which may directly cause genetic alterations if not repaired. when these genetic alterations affect tumor suppressor genes or tumor oncogenes, they can promote cell proliferation and malignant transformation. studies in lung cancer patients clearly suggest that cigarette smoking can lead to acquisition of genetic mutations in p53 and ras oncogene [6, 7 ]. in addition, cigarette smoke is proposed to cause immunosuppression, which provides an environment for tumor progression [8, 9 ]. recently, dna hypermethylation has been recognized as an alternative, epigenetic mechanism for gene silencing in lung cancer, in addition to genetic mutation. several environmental exposures are thought to cause aberrant dna methylation, including dietary factors, chemotherapeutic agents, and heavy metals. tobacco smoke exposure has been associated with increased expression of dna methyltransferases [1114 ]. consistent with this observation, lung cancers arising in heavy smokers show increased hypermethylation of various genes, especially cdkn2a (p16) and rassf1, compared with lighter smokers or nonsmokers [1527 ]. however, these results do not reveal whether dna hypermethylation occurs early or late in carcinogenesis. early changes in carcinogenesis (especially those related to smoking) are hypothesized to occur somewhat diffusely in the lung and may therefore be detectable in noncancerous lung tissue, as well as in any cancers which arise [2830 ]. for example, frequent hypermethylation of cdkn2a, rassf1, cdh13, and other genes has been observed in sputum samples from cancer - free smokers, suggesting that they may be hypermethylated early [3134 ]. in contrast, late changes in carcinogenesis are thought to arise mainly in overtly malignant tissues. we recently analyzed matched cancerous and noncancerous lung tissues from patients with nonsmall cell lung cancer (nsclc). we observed that in the 27 genes tested, most dna methylation changes were tumor - specific and therefore might be considered late changes in carcinogenesis. however, in these nsclc patients, a small number of genes, including ccnd2, apc, cdh1, and rarb (table 1), were also hypermethylated in a portion of noncancerous lung tissues, suggesting that one or more of these genes might become hypermethylated as an early precancerous change. we hypothesized that early changes in dna methylation, if present, might be associated with exposure to cigarette smoke. furthermore, because smoking - related lung tumors and emphysema are known to disproportionately affect the upper lobes of the lungs [36, 37 ], we hypothesized that methylation changes related to smoking would similarly be more frequent in the upper lobes, compared with the lower lobes. all procedures were conducted in accordance with institutional review board and human subjects committee approval. subjects were retrospectively enrolled who had undergone lung surgeries (lung volume reduction, lung transplant, wedge biopsy, or lobectomy) for nonmalignant diseases including emphysema, chronic bronchitis, bronchiectasis, granulomatous disease, various infectious diseases, and cystic or pulmonary fibrosis, at the university of washington medical center (uwmc) between july 1st 1995 and july 1st 2005. all specimens were reviewed by an expert pathologist (cdj) to confirm that they represented noncancerous lung tissue. all clinical data were gathered from subjects ' uwmc medical records, including smoking history and primary pulmonary diagnosis. subjects were excluded for the following reasons : previous diagnosis of lung cancer, insufficient lung tissue for methylation analysis, or unknown pack years of smoking. in total, 372 nonmalignant lung tissue samples from 159 subjects were identified for dna methylation analysis. from each block, six 20-m sections were cut and deparaffined by xylene extraction. proteinase k was used to digest the resulting tissue pellets overnight, at 48c. finally, dna was purified using a qiaamp dna minicolumn (qiagen) according to the manufacturer 's instructions. as previously described in detail, in vitro fully methylated dna (methylated dna control) and human sperm dna (unmethylated dna control) were converted with clinical samples. briefly, 1 g dna was modified by 5 mol / l sodium bisulfite, desulfonated with naoh, and then purified and resuspended in 80 l elution buffer (eb ; 10 mmol / l tris - hcl, ph 8.0). all primers and probes for methylight assays were designed specifically for bisulfite - converted fully methylated dna. amplification of bisulfite converted beta - actin (actb) dna was used to normalize for the quantity of input dna. samples negative for actb were excluded from methylation analysis. of 372 identified samples, 56 (15%) were excluded because they were negative for actb. the percentage of samples excluded after bisulfite conversion was similar in smokers (15%) and nonsmokers (16%). a plasmid containing bisulfite converted actb gene of known concentration was diluted and used as a standard curve for quantification. the assay for a given set of samples was only considered valid if the converted unmethylated human sperm dna was not amplified, whereas the converted fully methylated dna was amplified. for each locus, the percentage methylated reference (pmr) was calculated by dividing the gene / reference ratio of a sample by the gene / reference ratio of fully methylated dna control. genes were considered to be positive for any hypermethylation at pmr > 0%. for comparisons between groups, to provide independent observations, we randomly selected one tissue block per subject to represent each subject 's hypermethylation profile. to evaluate potential differences in gene methylation by site of the lung, paired upper, and lower lobe tissue samples from within subjects were compared using mcnemar 's test. to assess the univariate and multivariate relationships between gene methylation and independent variables (smoking, age, gender, lobe of lung, pack years, and years since quitting), we included all available tissue samples from each subject and employed generalized estimating equations (gee). this method enables the analysis of data with repeated measurements (multiple tissue samples per subject from different lobes) and accounts for within - subject correlations. in selecting a model, a logit link was used and we assumed an exchangeable working correlation structure to account for intrasubject correlation. parameter estimates were exponentiated to provide odds ratios (or) and 95% confidence intervals (ci). a 2-sided 0.05 test level determined statistical significance for all analyses. all analyses were conducted using sas version 9.1 (sas institute inc., cary, nc). we retrospectively enrolled 151 subjects who contributed a total of 316 available pathology blocks (table 2). at the time of their surgery, 121 subjects were current or former smokers (ever - smokers), while 30 reported no smoking history (never - smokers). among the never - smokers, none had any history of cancer, either prior to or after the surgery that yielded the tissue used in this study. of the ever - smokers, 10 had a history of prior cancer other than lung (1 breast, 2 cervical, 2 colon, 2 prostate, 1 testicular, and 2 uterine) and all were cancer free at surgery. four of the ever - smokers developed a cancer subsequent to the surgery that yielded the cancer - free lung tissue (1 bladder, 1 colon, and 2 nsclcs, one at 2 years after, one at 5 years after). the clinical data show that never - smokers and ever - smokers who had undergone lung surgery comprised two distinct populations. ever - smokers were significantly older than never - smokers (61 years versus 44 years). further, of ever - smokers who contributed specimens from lung surgery, 71% had a diagnosis of emphysema, compared to only 10% of never - smokers. we analyzed a total of 316 available pathology blocks from these 151 subjects, including 177 upper lobe samples, 105 lower lobe samples, 30 middle lobe or lingula samples, and 4 whose lobe of origin was unclear. multiple blocks were available for 98 (81%) of ever - smokers and 13 (43%) of never - smokers ; from the 121 ever - smokers, 269 samples were tested, while from the 30 never - smokers, 47 samples were tested. sample sites varied substantially in ever - smokers and never - smokers as 50% of ever - smokers, compared to 17% of never - smokers, contributed only samples from the upper lobes. this difference arose because many ever - smokers in our sample underwent lung volume reduction surgery for emphysema, which predominantly affects the upper lung zones when induced by smoking. considering one random tissue block per subject, only apc (39%), ccnd2 (21%), cdh1 (7%), and rarb (4%) were hypermethylated in more than 2% of subjects (figure 1). all 15 remaining genes (bves, cdh13, cdkn2a (p16), cdkn2b, dapk1, igfbp3, igsf4, kcnh5, kcnh8, mgmt, opcml, pcsk6, rassf1, runx, and tms1) were hypermethylated in less than 2% of subjects. ccnd2 was hypermethylated significantly more frequently in ever - smokers compared to never - smokers (26% versus 3%, p <.001). apc was hypermethylated somewhat more frequently in ever - smokers (41% versus 30%), but this did not achieve statistical significance (p =.3). apc and ccnd2 were often hypermethylated in the same samples ; 179 (57%) samples were negative for both genes, 16 (5%) were positive for hypermethylation of ccnd2, but not apc, 68 (22%) were positive for hypermethylation of apc but not ccnd2, and 53 (17%) samples were positive for both genes. ccnd2 hypermethylation was significantly correlated with apc hypermethylation in all subjects (or = 7.3, 95% ci = 3.913.8) and in smokers only (or = 7.4, 95% ci = 3.914.0). in nonsmokers, 31 (66%) samples were negative for both genes, 14 (30%) were positive for apc only, and 2 (4%) samples were positive for both apc and ccnd2. in univariate gee analyses of all specimens (table 3), ccnd2 hypermethylation was significantly associated with a positive smoking history, increasing age, and sample origin from the upper versus lower lobe of the lung. apc hypermethylation was significantly less frequent among females and moderately more frequent in upper lobes compared to lower lobes but was not significantly associated with a positive smoking history. in a multivariate model simultaneously assessing smoking history, age, gender, and location of the sample (upper versus lower lobe) in all subjects, hypermethylation of ccnd2 remained significantly associated with increased age (or = 1.7, 95% ci = 1.22.4 for each 10 years of age) and upper lobe location (or = 2.0, 95% ci = 1.03.8). ccnd2 hypermethylation was somewhat associated with a positive smoking history (or = 2.8, 95% ci = 0.612.1) but this did not achieve statistical significance. within the subset of 269 samples from 121 ever - smokers (table 4), apc hypermethylation was not related to pack - years of cigarette smoking or years since quitting smoking. in univariate gee analysis, ccnd2 hypermethylation was significantly associated with greater pack years but was not related to years since quitting. however, in a multivariate gee analysis simultaneously assessing pack years, years since quitting, age, gender, and location of the sample (upper versus lower lobe), ccnd2 hypermethylation was no longer associated with pack years (or = 1.0, 95% ci = 0.91.2 per 10 pack years). smoking - related lung tumors and emphysema are known to disproportionately affect the upper lobes of the lungs. thus, if hypermethylation of a gene is associated with smoking, we might expect to find more hypermethylation in upper lobe samples compared to lower lobe samples, among ever - smokers. examining all 269 samples from ever - smokers (table 4), in univariate gee analysis, both apc (or = 2.0, 95% ci = 1.13.5) and ccnd2 (or = 1.9, 95% ci = 1.03.5) hypermethylation were more common in upper compared to lower lobes. in multivariate analysis including pack - years, years since quitting, age, gender, and upper versus lower lobe, apc hypermethylation remained significantly associated with upper - lobe sample location (or = 2.1, 95% ci = 1.14.0), while ccnd2 's positive association with upper lobes was reduced to slightly below the level of statistical significance (or = 1.7, 95% ci = 0.93.4). among the 121 ever - smokers in our cohort, 30 had both upper and lower lobe samples available, and 12 of 30 pairs had discordant hypermethylation status (1 positive and 1 negative), of which 8 of 12 displayed apc hypermethylation in an upper lobe but not a lower lobe sample (p =.25). for ccnd2, only 7 of 30 pairs had discordant hypermethylation status, of which only 3 of 7 were hypermethylated in the upper but not the lower lobe (p =.7). thus, too few subjects had discordant hypermethylation in upper and lower lobe samples to yield statistically meaningful results in within - subjects comparisons.. however, it is currently unknown whether changes in dna methylation are early events, occurring in previously normal lung tissue or whether they are late changes that occur only in overt tumor cells. to attempt to answer these questions, we tested dna hypermethylation in lung tissues from subjects without cancer both smokers and nonsmokers using a panel of 19 genes which we had previously found to be hypermethylated in some nonsmall cell lung cancers [35, 44 ]. this unique study design allowed us, for the first time, to characterize the dna hypermethylation profile of nonsmokers ' lung tissues and to compare this profile to that of smoke - exposed lung. importantly, we observed that ccnd2, which is known to be frequently hypermethylated in lung cancer tissue [35, 4447 ], was hypermethylated more frequently in ever - smokers (26%) than in never - smokers (3%). also, as predicted, in ever - smokers, ccnd2 was hypermethylated more frequently in samples from the upper lobes, which are known to suffer far more negative effects from cigarette smoke, such as lung cancer and emphysema [36, 37 ]. these findings support the conclusion that ccnd2 reflects an early, precancerous change in the lung, caused by cigarette smoke. ccnd2 encodes cyclin d2, a protein involved in cell cycle progression that is thought to act as a regulator of cyclin dependent kinase 4 and cyclin dependent kinase 6 in the transition from g1 to s - phase. ccnd2 hypermethylation appears to be common in many cancers. in breast cancer, where it has been studied most extensively, ccnd2 hypermethylation is detected frequently, though it appears to be rarely detected in normal breast tissue [4854 ]. interestingly, while ccnd2 hypermethylation (and therefore low ccnd2 protein expression) has been associated with poor prognosis in epithelial ovarian cell cancer and recurrence of hepatocellular carcinoma, increased ccnd2 expression has been associated with poor prognosis in diffuse large b - cell lymphoma. in the lung, ccnd2 hypermethylation has been found in 4056% of nsclcs [35, 44, 45, 47 ]. in noncancerous lung tissue, whereas virmani. found ccnd2 hypermethylation in 0 of 18 samples, our previous investigation found ccnd2 hypermethylation in 24% of noncancerous lung tissues from patients with nsclc. this closely matches the rate observed in the present study, in cancer - free ever - smokers (26%). possibly, our group observed a higher rate of ccnd2 hypermethylation in both cancerous and cancer - free lung tissues because we used methylight assays instead of methylation - specific pcr (msp), which was used by virmani. thus, we may have detected low levels of hypermethylated genes in cancer - free tissues which were not detected by msp. discrepancies may also be due to the somewhat different primers and probes used in analyses, which indicate different sequence regions investigated. in addition, kubo. did not observe any ccnd2 hypermethylation in 30 matched noncancerous lung tissues but it should be noted that in this study, 70% of subjects were nonsmokers who would not be expected to have significant rates of ccnd2 hypermethylation. combined, these results reveal a progression in the rate of ccnd2 hypermethylation in the lung, corresponding with the risk for developing lung cancer. while ccnd2 hypermethylation was very infrequent (3%) in our current study 's low - risk group of 30 never - smokers, it was more frequent in a high - risk group of ever - smokers (2426% in our current and previous studies), and most frequent in overt nsclc tissue (4056%). this risk - stratified progression in lung tissues suggests that ccnd2 hypermethylation may truly reflect an early precancerous change in the lung, en route to overt cancer, which may be due to the effects of smoking. still, our findings regarding ccnd2 should be regarded as preliminary at this time, for several reasons. in multivariate analysis, the effect of smoking status on ccnd2 hypermethylation was reduced to trend - level significance after taking into account the effects of sample location (upper versus lower lobe) and subject age. this likely occurred because in our sample, the majority of smokers underwent lung surgery for emphysema and represented a significantly older group, more likely to contribute samples from upper lobes (where emphysema is most prominent). in contrast, nonsmokers were younger and underwent lung resection for a variety of diseases. with such significant correlation of these factors, multivariate analysis may not have reliably separated each factor 's relative contribution to gene hypermethylation. thus, observed differences in the rate of ccnd2 hypermethylation could be attributable to any of these factors or others that differed between ever and never - smokers. emphysema, for example, made ccnd2 hypermethylation more likely although significant rates of ccnd2 hypermethylation were also found in smokers with other diagnoses. while ccnd2 hypermethylation could be part of the unique pathophysiology of emphysema, it more likely arose because emphysema reflects severe smoking - induced lung damage. the effect of age on ccnd2 hypermethylation has not been studied previously in noncancerous lung, although several genes have been reported to undergo increased rates of hypermethylation with age, in various body tissues, including cdh1 and dapk1 in the lung. in noncancerous breast epithelium and in peripheral blood samples from cancer - free subjects, advanced age thus, the relationship between age and ccnd2 hypermethylation remains unknown at this time. in weighing the relative contributions of age, sample location, and emphysema status on ccnd2 hypermethylation, it is worth noting that smoking history was by far the strongest single predictor of ccnd2 hypermethylation in univariate analysis (or = 6.9, 95% ci = 1.629.8). one limitation of the present study was that despite our overall large number of 151 subjects, only 30 were never - smokers. this occurred because never - smokers far less frequently undergo lung resections which produce tissue. this may have been part of the reason why in multivariate analysis, we observed only trend - level significance for smoking 's effect on ccnd2 hypermethylation. we were able to improve our statistical power somewhat by using generalized estimating equations (gee) for our univariate and multivariate analyses, allowing us to enter multiple tissue blocks per subject when available (multiple observations), without biasing the results. however, future studies should seek to verify the low rate of ccnd2 hypermethylation we observed in never - smokers. an additional limitation of our study design was that all subjects had an underlying non - cancer pulmonary diagnosis that necessitated lung surgery. thus, while observed gene hypermethylation was unrelated to cancer, it can not definitely be said to represent healthy lung. finally, due to our study design, we only provide indirect evidence of interaction between smoking and ccnd2 hypermethylation. future studies utilizing animal models may be useful to elucidate the potential causal relationship between smoking and ccnd2 hypermethylation. in our current and previous studies, cdkn2a (p16) was hypermethylated in 26% of cancer tissues but was rarely hypermethylated in noncancerous lung tissues, regardless of smoking status. however, cdkn2a hypermethylation has previously been characterized as an early event in lung carcinogenesis [2830 ], and hypermethylation of cdkn2a has been commonly detected in sputum samples from heavy smokers without lung cancer [32, 61 ]. overall, a very wide range of hypermethylation rates for cdkn2a has been reported in the literature, for noncancerous lung tissues. along with other researchers who observed low rates of cdkn2a hypermethylation in noncancerous lung tissues, our results suggest that cdkn2a hypermethylation may actually represent a later change in carcinogenesis [6266 ]. however, the surprisingly large discrepancies between studies may be related to differences in assay methodology (including pcr primers and specific cpg islands) or patient populations. ccnd2 hypermethylation likely represents an early, smoking - induced, precancerous change in the lung ; it is very infrequent in the lung tissue of never - smokers, more frequent among smokers, and most frequent in overt nsclc tissue. in addition, this study supports the conclusions of our previous investigation, that although they are hypermethylated in many nsclc tumor tissues, rassf1, dapk1, bves, cdh13, mgmt, kcnh5, and to some extent cdh1 and rarb, are rarely hypermethylated in the cancer - free lung, even after significant tobacco exposure. in addition, if hypermethylation of ccnd2 or other genes represents an early precancerous change, it is possible that drugs aimed at reversing dna methylation could be used to prevent smoking - related carcinogenesis.	it remains unknown whether tobacco smoke induces dna hypermethylation as an early event in carcinogenesis or as a late event, specific to overt cancer tissue. using methylight assays, we analyzed 316 lung tissue samples from 151 cancer - free subjects (121 ever - smokers and 30 never - smokers) for hypermethylation of 19 genes previously observed to be hypermethylated in nonsmall cell lung cancers. only apc (39%), ccnd2 (21%), cdh1 (7%), and rarb (4%) were hypermethylated in > 2% of these cancer - free subjects. ccnd2 was hypermethylated more frequently in ever - smokers (26%) than in never - smokers (3%). ccnd2 hypermethylation was also associated with increased age and upper lobe sample location. apc was frequently hypermethylated in both ever - smokers (41%) and never - smokers (30%). bves, cdh13, cdkn2a (p16), cdkn2b, dapk1, igfbp3, igsf4, kcnh5, kcnh8, mgmt, opcml, pcsk6, rassf1, runx, and tms1 were rarely hypermethylated (< 2%) in all subjects. hypermethylation of ccnd2 may reflect a smoking - induced precancerous change in the lung.
type 1 diabetes mellitus (t1d) is a chronic disease that carries a great risk of morbidity and mortality, as a result of the microvascular and macrovascular complications that reduce an affected individual 's quality of life and life expectancy. progress in diabetes management in recent decades has improved the survival rates among t1d patients, although life expectancy remains lower for these individuals compared to nondiabetic subjects of equal age. diabetes has emerged as a major health problem in societies in which noncommunicable diseases are the most common causes of disability and death [1, 2 ]. furthermore, diabetes treatment has become a large financial burden because of the increased associated direct and indirect costs. the occurrence of hypertension in t1d patients is directly correlated with the presence of microvascular complications, primarily nephropathy and retinopathy and the progression of these chronic complications [4, 5 ]. there is strong evidence, relating to the efficacy and cost effectiveness of treatment, to support blood pressure control in t1d and t2d patients, as well as the nondiabetic population, for reducing levels of morbidity and mortality [68 ]. target blood pressure levels have been described in many guidelines, including the american diabetes association (ada), american heart association (aha), and brazilian diabetes society (bds). however, a large gap remains between the recommendations for blood pressure control and the values that have been described in most observational t1d [1214 ] and t2d studies. previous studies on the prevalence, awareness, treatment type, and control of hypertension have examined nondiabetic populations or t2d patients [16, 17 ] but rarely t1d patients. the coronary artery calcification in type 1 diabetes study (cact1) demonstrated a higher rate of hypertension among t1d (43%) patients compared to nondiabetic subjects (15%) but observed a similar rate of hypertension awareness between t1d subjects (53%) and controls (45%). furthermore, the eurodiab study demonstrated a hypertension prevalence of 24% among t1d patients and less than half of these patients were aware of this condition. only 42.2% of the t1d patients in this study received treatment, and only 26.7% of the treated t1d patients attained the established blood pressure targets. the results of these studies emphasize the difficulties associated with the treatment of hypertensive t1d patients in routine clinical care and the need for improved treatment quality. the absence of national data on the prevalence, awareness, type of treatment, and control of hypertension in t1d patients led the brazilian type 1 diabetes study group (brazdiab1sg) to conduct this study, seeking to provide current and reliable data on the topic with regard to the ada guidelines. this was a multicenter, cross - sectional, and observational study that was conducted between december 2008 and december 2010 in 28 public secondary (ambulatory outpatient clinics) and tertiary care level clinics (ambulatory outpatient clinics in university hospitals), located in 20 cities in four brazilian geographic regions (north / northeast, midwest, southeast and south). briefly, all patients received health care from the national brazilian health care system (nbhcs). each clinic provided data from a minimum of 50 consecutive outpatients with an initial diagnosis of t1d who regularly attended the clinic. the inclusion criteria consisted of a diagnosis of t1d by a physician that was based on the typical clinical presentation, including variable degrees of weight loss, polyuria, polydipsia, and polyphagia, and the need for continuous insulin use since t1d diagnosis. the following variables were assessed in each interview during the clinical visit : current age, age at diagnosis, duration of diabetes (y), height (m), weight (kg), mean blood pressure (systolic and diastolic in mmhg from three consecutive measurements in one day using a standard clinical sphygmomanometer), modality of diabetic treatment, comorbidities, frequency of sbgm, and smoking status. the levels of glycated hemoglobin (hba1c), fasting plasma glucose (fpg), total cholesterol, ldl cholesterol, hdl cholesterol, and triglycerides on the last clinical visit were obtained from medical records. the screenings for retinopathy, using fundoscopy ; nephropathy, according to microalbuminuria ; and foot examinations in patients with diabetes duration equal or greater than five years were noted when these procedures were performed within one year of the study assessment. patients with diabetes for less than five years were not included in the analysis of diabetic chronic microvascular complications (n = 1, 160, 32.3%). the brazilian diabetes society (bds) monitored and reviewed all study - related documents and approved all amendments and publications. the following ada goals for adequate metabolic and clinical control were adopted by the brazdiab1sg : hba1c 7.5% and 40 mg / dl for men (1.1 mmol / l) and > 50 mg / dl (1.3 mmol / l) for women ; ldl cholesterol 18 years were considered adults. a detailed description of the study sample calculation has been given previously. briefly, the study sample represented the distribution of t1d cases across four geographic regions in brazil. the proportion of cases from each region was estimated using the overall population distribution reported in the 2000 brazilian institute of geography and statistics population census (ibge) ; 38.8%, 31.7%, 23.0%, and 6.6% of the population was distributed in the southeast, north / northeast, south, and midwest regions, respectively. these data were combined with the national estimates of the prevalence of diabetes, which were derived from a 1988 survey, to determine the minimum number of patients to be studied in each region. recruitment in each region of the country enrolled > 95% of the estimated number of t1d patients for the region. this classification also takes into account education level, which is categorized as illiterate / incomplete primary education, complete primary education / incomplete secondary education, complete secondary education / incomplete high school, complete high school / some college, or complete college education. the following classes of economic status were considered for this analysis : high, middle, low, and very low. data are presented as the means (sd) for continuous variables and as counts (relative frequencies) for discrete variables. for analyzing blood pressure data, the mean from three consecutive measurements in a single day comparisons between numeric variables were performed using independent two - sided t - tests and two - sided z - tests for discrete variables with a normal approximation to the binomial distribution. an unadjusted pearson 's correlation coefficient was calculated when indicated. a multiple logistic regression (forward - wald) the following independent variables were included : race (caucasian or non - caucasian based on self - reporting), age, bmi, geographic region, gender, urine albumin excretion rate, and economic status. all of the analyses were performed using spss version 16.0 (statistical package of social sciences, chicago, il, usa). odds ratios with 95% confidence intervals (ci) were performed when indicated. a two - sided p value less than 0.05 was considered significant. the majority of the patients evaluated were less than 30 years old (n = 1, 077, 30%). due to missing data, in the total population of 3,591 patients, 268 (7.5%) could not be classified as either hypertensive or normotensive. among the 3,323 (92.5%) t1d patients evaluated, a total of 689 (19.2%) of the studied patients were considered hypertensive, 236 (6.6%) were based on actual blood pressure measurements, and 453 (12.6%) were based on a history of or treatment for hypertension (self - reported). hypertension was more frequent in adults than in children or adolescents (n = 562 (31.3%) versus n = 127 (8.3%), respectively, p < 0.001). patients with hypertension were also older, exhibited longer duration of diabetes and had higher bmi, total cholesterol, triglycerides, ldl - c, and hdl - c values than patients without hypertension (p < 0.001 for all comparisons). these data are listed in table 1. a greater number of children and adolescents had missing blood pressure data than did adults (258 (96.3%) versus 10 (3.7%), respectively, p < 0.001), and these data are indicated in table 2. the mean age at the time of hypertension diagnosis was 20 10.3 years, and the self - reported duration of hypertension was 3 years (range < 1 to 44 years). patients who were aware of their hypertension were older (p < 0.001) and exhibited higher sbp (p = 0.001) and fewer borderline sbp of 140 mmhg (p = 0.01) and borderline dbp of 90 mmhg (p = 0.02) compared to patients who were unaware. more patients aware of their hypertensive status received treatment than did patients who were unaware of their condition (p < 0.001). higher sbp and dbp values were also observed in treated patients compared to untreated patients (sbp : 132.99 19.4 versus 123.1 21.2 mmhg, respectively, p < 0.001, and dbp : 83.00 12.32 versus 79.12 14.20 mmhg, respectively, p < 0.001). in total, 207 (55.9%) of the 370 treated patients were administered only one antihypertensive agent ; of these, 161 (43.5%) patients used ace inhibitors and 46 (12.4%) patients received monotherapy with calcium channel blockers (n = 5, 1.3%), beta blockers (n = 10, 2.7%), angiotensin receptor blockers (arbs) (n = 15, 4.1%), or diuretics (n = 16, 4.3%). a total of 122 (33%) patients received two drugs in the following combinations : ace inhibitors plus diuretics, arbs, beta blockers or calcium channel blockers, and arbs plus diuretics or calcium channel blockers. forty - one (11.1%) patients received triple therapy with ace inhibitors and diuretics plus arbs, beta blockers or calcium channel blockers. a total of 76 (22.9%) treated hypertensive patients achieved the targeted blood pressure range. the patients ' sbp values correlated with age (r = 0.47, p = 0.001), diabetes duration (r = 0.41, p < 0.001), total insulin dose (r = 0.17, p < 0.001), aer (r = 0.16, p < 0.001), bmi (r = 0.44, p < 0.001), total cholesterol (r = 0.11, p = 0.001), triglycerides (r = 0.10, p = 0.001), hdl cholesterol (r = 0.05, p = 0.01) and ldl cholesterol (r = 0.07, p = 0.001). the dbp values correlated with age (r = 0.40, p = 0.001), diabetes duration (r = 0.32, p < 0.001), total insulin dose (r = 0.12, p < 0.001), aer (r = 0.16, p < 0.001), bmi (r = 0.38, p < 0.001), total cholesterol (r = 0.15, p = 0.001), triglycerides (r = 0.14, p < 0.001), hdl cholesterol (r = 0.05, p = 0.01), and ldl cholesterol (r = 0.07, p = 0.001). patients with proliferative retinopathy or nonproliferative retinopathy had higher sbp and dbp values than patients without retinopathy (sbp : 124.5 20.6 versus 121.2 19.2 versus 113.1 15.6 mmhg, respectively, p < 0.001, and dbp : 78.5 11.9 versus 76.9 11.5 versus 72.5 10.8 mmhg, respectively, p < 0.001). additionally, patients with clinical nephropathy or microalbuminuria had higher sbp and dbp values than patients without nephropathy (sbp : 123.3 21.1 versus 120.9 17.9 versus 113.3 15.8 mmhg, respectively, p < 0.001, and dbp : 78.7 12.6 versus 76.6 10.7 versus 72.5 10.7 mmhg, respectively, p < 0.001). multivariate logistic analysis revealed that hypertension was directly associated with age (or = 1.06 ; 95% ci (1.051.076 ; p < 0.001)), bmi (or = 1.13 ; 95% ci (or = 1.091.17 ; p < 0.001)), aer level [or = 1.02 ; 95% ci (1.011.03 ; p < 0.001) ] and male gender [or = 1.35 ; 95% ci (1.021.80 ; p < 0.001) ]. caucasian race was also associated with a lower odds ratio of hypertension (or = 0.68 ; 95% ci (0.510.91 ; p = 0.01)). this study demonstrated that, while nearly 20% of the patients examined exhibited hypertension, only 53.7% of these patients received treatment. moreover, only 22.3% of the treated hypertensive patients achieved the targeted sbp and dbp values. hypertension was more common in non - caucasian adults and was associated with microvascular complications and other cardiovascular risk factors, such as being overweight or obese and exhibiting dyslipidemia. the ada provides recommended blood pressure levels for all diabetic patients, but approximately 7.5% of the patients participating in the current study received no such evaluation in the year prior to the study. this was commonly observed primarily in children and adolescents, as well as individuals from the north / northeast and midwest regions of brazil. some diabetes clinical care centers in brazil may not include blood pressure evaluations in their routine care of children and adolescents. although hypertension was more frequent among adults (31.3%), in our diabetic study population, 8.3% of diabetic children and adolescents also were hypertensive. few studies of hypertension in t1d patients have been conducted ; the majority of these studies analyzed hypertension in adult diabetic patients and reported a prevalence of 24 to 43% [1214, 2529 ], which is similar to those observed in the current study. in addition, an observational study in rio de janeiro, brazil, identified a hypertension prevalence of 6.8% in nondiabetic children and adolescents. our data on children and adolescents are similar to those published by the search study (5.9%), although our prevalence figures were higher than those published (4%) in a recent norwegian study. studies of elevated sbp or dbp in children and adolescents (greater than the 90th percentile for age, gender, and height) have reported a prevalence of hypertension between 6% and 23%, depending on the presence of other cardiovascular risk factors [2629 ]. additionally, the prevalence of hypertension has been shown to increase fourfold in overweight or obese children and adolescents. age, diabetes duration, the presence of chronic complications, race, and the number of medical visits with available blood pressure evaluations may account for the differences between our study and those conducted previously. more than one - third of our patients who were unaware of their hypertensive condition were children and adolescents. importantly, all of these patients were treated by an endocrinologist in secondary and tertiary care settings. diabetes treatment in public clinics is financed by the nbhcs, and our data reveal that factors other than medical recommendations might likely interfere with diabetes care in brazil [18, 31 ]. the guidelines recommend aggressive hypertension treatment in t1d patients, but only 53.7% of our patients received such treatment ; similar results were described in the eurodiab study. in the current study, the majority of treated patients (55.9%) received only one antihypertensive drug, whereas 44.1% received two or more drugs. these results are in contrast with the results of previous studies reporting that up to 19% of t1d patients received two antihypertensive agents [12, 13 ]. in addition, only 11.1% of the patients in the current study received triple therapy, which is higher than the percentage described in the cact1 (7%) and eurodiab (1.9%) studies. the abovementioned studies were conducted 5 to 10 years before our study, suggesting that an increase in the intensity of hypertension treatment has occurred in recent years, as previously observed in a temporal analysis of eurodiab. however, less than one - third of our patients and t1d patients in the eurodiab study exhibited controlled sbp and dbp levels, which suggests that factors beyond pharmacological treatment might influence blood pressure control. additionally, compared to our study, a larger percentage of the cact1 patients (up to 64%) exhibited controlled blood pressure levels. this difference may be attributed to study design, as the patients in the eurodiab and our corresponding studies were not volunteers. the pittsburgh epidemiology of diabetes complications study utilized different targets for blood pressure and demonstrated small improvements in hypertension control, primarily in younger - aged groups of t1d patients, over a 10-year follow - up period. one study that was performed at academic medical centers observed a low rate of medication management when t1d patients remained above their blood pressure goal. as factors such as hypertension, obesity, and being overweight are indicators of cvd risk, we concluded that the young patients that were evaluated represent a high - risk group for the development of microvascular and macrovascular complications associated with diabetes, as described previously [4, 5, 2628 ]. furthermore, our study demonstrated a clear association between the different stages of retinopathy and nephropathy and increasing levels of blood pressure. the brazdiab1sg is the only national registry on the prevalence, awareness, and treatment of hypertension in t1d ; the principal strength of our study is our large sample size, which included a representative sample of t1d distribution in the young brazilian population. importantly, our study included patients from a wide range of racial backgrounds from all geographic regions of the country, and it maintained a uniform, standard recruitment protocol at all of the participating centers. we used a clinical definition of t1d that was assigned by physicians and was applicable to all patients, which is similar to previous studies [15, 16 ]. nevertheless, it is important to emphasize that 93.1% of our patients were diagnosed before the age of 30, which supports the high probability that these patients had t1d. also, as all of the patients in this study lived in large cities and were seen in a public center by a specialist, patients who relied on primary care facilities and lived in rural areas may have been overlooked. although 14% of the brazilian population lives in rural areas, the prevalence of t1d in this group is very low, and consequently rural t1d patients represent the minority of patients who receive treatment in brazil. additionally, patients recruitment within each center may have produced a selection bias for age because the majority of our patients were younger than 30 years of age. moreover, there were missing data for blood pressure measurements, which were primarily observed in the youngest patients. additionally, the prevalence of hypertension may have been overestimated because diagnosis was based on the measurement of a blood pressure in one day rather than two separate measurements on two separate days. although we used a standard clinical sphygmomanometer, the possibility for misclassification remains, especially, at borderline diagnosis levels for sbp and dbp. misclassification was noted in our sample in the analysis of the lack of hypertension awareness, which was more frequent in the borderline group of patients. the use of self - reported hypertension as a criterion for awareness may have also produced a bias in the diagnosis of this condition. therefore, to our knowledge, this research constitutes the first national report on the prevalence of hypertension in t1d in brazil, a disease with increasing incidence in our country. our results demonstrate that many t1d patients with hypertension do not receive antihypertensive treatment ; moreover, few treated t1d patients receive combined therapy, and few of these patients achieve their targeted sbp and dbp values. the evaluation of blood pressure in children and adolescents is likely not included in all routine diabetic clinical care centers. thus, greater attention should be paid to blood pressure evaluation and hypertension diagnosis and treatment for t1d patients in brazil.	objective. this study evaluated the prevalence, awareness, and type of treatment for hypertension in brazil in patients with type 1 diabetes (t1d). methods. this was a cross - sectional, multicenter study that was conducted from december 2008 to december 2010 in 28 public clinics located in 20 brazilian cities. results. a total of 3,591 patients were studied, 56% female, average age 21.2 11.7 years, with a median duration of diabetes 9.6 8.1 years. blood pressure levels were available for a total of 3,323 patients and 689 (19.2%) patients were hypertensive. hypertensive patients were older, exhibited longer duration of diabetes, and had higher body mass index (bmi), total cholesterol, triglycerides, and ldl - c values (p < 0.001, for all comparisons), but only 370 (53.7%) received treatment. patient awareness of hypertension was documented in 453 (65.5%) patients. however, only 76 (22.9%) of the treated patients attained the target systolic (sbp) and diastolic blood pressures (dbp). conclusions. our results demonstrate that a large number of t1d patients with hypertension do not receive appropriate treatment ; few of the treated t1d patients achieved the target sbp and dbp values. greater attention should be paid to blood pressure evaluation, hypertension diagnosis, and treatment of t1d patients in brazil.
cells achieve polarity in part by localization of mrna, which allows protein synthesis to be confined to a subcellular compartment (meignin and davis, 2010). in the studies of mrna localization in somatic cells, visualization has played a crucial role since the first observation almost 30 years ago (jeffery., 1983 ; lawrence and singer, 1986). although in situ hybridization is still widely considered as the standard tool, a variety of techniques in imaging and labeling have enabled detection of single rna molecules not only in fixed cells (femino., 1998) but also in live cells in real time (bertrand.,1998). although imaging techniques are highly sophisticated, analysis of mrna localization has been mostly limited by the qualitative interpretation. for instance, transcripts expressed during drosophila embryogenesis are classified into ~35 localization categories (lcuyer., 2007). a conventional method to quantify rna localization is based on manual counting of the cells by two independent observers who are blind to the experimental conditions. although it may eliminate potential bias in data selection and processing, ambiguities still remain due to individual variation. in addition to the need for an objective analysis, the importance of quantitative measurement has been recognized for several reasons. first, localization can be described as a continuous process rather than an all - or- nothing occurrence (zimyanin., 2008). second, a metric for localization could identify distinct populations that may be missed by a binary analysis. moreover, quantification could be automated to facilitate high - throughput image analysis where differential response of cells could be examined under manifold conditions. a few quantification methods have been suggested in the literature to analyze subcellular localization of mrna. in one approach, the most dense and least dense regions of the cell were selected, and the ratio of the highest rna density to the lowest density was calculated (lawrence and singer,1986). 1994) counted a cell as localizing if 80% signal was concentrated in leading lamella area comprising a quarter of the cell area. 2009) selected regions of interest (roi), and calculated the ratio of the mrna concentration at the leading edge and in the perinuclear region. for the transcripts that localize in the vicinity of a certain cellular compartment, the distance between the mrna and the cellular objects may be used to quantify localization (jourdren., 2010). these varying analysis methods could lead to significantly different conclusions, and demonstrate the need for an objective quantitative analysis of rna localization. here we demonstrate an analytical method applied to three different cell types : budding yeast, chicken embryonic fibroblasts (cef), and mouse embryonic fibroblasts (mef). when mrna distribution is asymmetric in a cell, the centroid of the mrna should deviate from the centroid of the cell. we define the displacement vector point- ing from the center of the cell to the center of mrna as the rna polarization vector (figure 1a). the polarization index (pi) is determined by dividing the size of the polarization vector by the radius of gyration of the cell (rgcell) pi=(xrnaxcell)2+(yrnaycell)+(zrnazcell)2rgcell where xrna, yrna, zrna are the coordinates of the centroid for the rna, and, xcell, ycell, zcell are those for the cell in a general threedimensional case. the radius of gyration rgcell is calculated by the root gyration rg cell is calculated by the root - mean - square distance of all pixels within the cell from the centroid of the cell. the displacement between the two centroids is divided by rgcell in order to assess the polarization normalized to the size and the elongation of the cell. the second quantity, the dispersion of mrna, is measured by calculating the second moment 2 of rna positions : 2=1ni1n{(xixrna)2+(yyrna)2+(zizrna)2=x2+y2+z2 where n is the total number of mrna molecules, xi, yi, zi are the coordinates of the ith mrna, and x2, y2, z2 are the variances of the positions. the second moment is dependent on the shape and size of the cell as well as the rna distribution. to normalize the effect from the cell morphology, we divide the second moment of mrna 2 by the second moment of the hypothetical uniform distribution 2. a binary mask image of each cell is generated and 2 is calculated as the second moment of each pixel s coordinates within the mask 2=1mi1m{(xixrna)2+(yyrna)2+(zizrna)2 } where m is the total number of pixels within the mask, and xi, yi, zi are the coordinates of the ith pixel. then we define the dispersion index (di) as di=22 di has by definition a value of 1 if the cell has an absolutely uniform distribution of mrna (figure s1a). when mrna is concentrated in a certain region, di is less than 1 (figures s1b s1d). if mrna is spread around the rim of the cell, di becomes larger than 1 (figure s1e). to test the two - quantity approach (pi and di), we analyzed localization of the budding yeast ash1 mrna. ash1 expression is cell - cycle regulated and reaches a peak during mitosis. in wild - type (wt) cells, the majority of ash1 transcripts are actively localized to the bud (long., 1997), she2p is the primary rna - binding protein (niessing., 2004). in the absence of she2p, ash1 mrna becomes homogeneously distributed between the mother cell and the bud tip (long., 1997) we performed single - molecule fluorescence in situ hybridization (fish) (femino., 1998) on wt and she2 cells as described previously (trcek., 2012 ; zenklusen., multiple fields were imaged and maximum intensity projections of z stacks were generated for two - dimensional analysis. by using a least - squares gaussian fitting routine, we obtained the intensity and spatial information of each fluorescent particle and processed them to quantify polarization and dispersion of rna distribution. in order to analyze the posttranscriptional localization of mrna, the multiple copies of mrna at the transcription sites were excluded from the analysis. in wild - type strain, pi was 0.71 0.04 (sem) and di was 0.50 0.04. in she2 strain, pi was 0.16 0.02 and di was 0.82 0.02. consistent with the human perception of the representative images (figures 1b and 1d), the objective metrics indicate that the highly polarized localization of ash1 mrna in wild - type cells was disrupted by the deletion of she2 gene. in order to assess the relationship between the polarization and dispersion of mrna in each strain, we computed the correlation coefficient of the two metrics. the pearson correlation coefficient was 0.72 in wild - type and 0.13 in the she2 strain. in wild - type cells, higher polarization of ash1 mrna was strongly associated with tighter confinement of the mrna. this negative correlation between the polarization and the dispersion disappeared in she2 strain (figures 1c and 1e). we next applied the analysis method for the distribution of mrna in chicken embryonic fibroblasts. by comparing the distribution of -actin mrna and gapdh mrna in the same cell, we investigated if localization is a general effect for any mrna or a specific effect for -actin mrna. figure 2a shows a representative image of primary cef cells in which -actin mrna is localized to the leading edge (indicated with arrowheads), whereas gapdh mrna is more uniformly distributed. because the densities of these mrna species are too high to distinguish individual molecules, the distribution of pixel intensity values were analyzed instead of the distribution of discrete mrna particles. after background subtraction, the second moment m2 was calculated by 2=i, jrij2iiji, jiij where rij is the distance from the centroid of the cell to the pixel (i, j) within the cell boundary, and iij is the intensity value of the pixel in a two - dimensional image. a representative cell shown in the left side of figure 2a exhibits polarized localization of -actin mrna (pi = 1.14, di = 0.35) and more uniform distribution of gapdh mrna (pi = 0.33, di = 1.17). in a randomly - chosen population of cef cells (n = 99), the mean pi was 0.47 0.03 for -actin mrna and 0.29 0.02 for gapdh mrna. in 82% of the cells (in the upper triangle above the red dashed line in figure 2b), the distribution of -actin mrna was more polarized than gapdh mrna. however, the difference in the mean di was not as significant : 0.98 0.05 for -actin mrna and 0.82 0.04 for gapdh mrna. unlike yeast ash1 mrna, the mean values of pi and di for -actin mrna indicate a moderate polarization and a loose dispersion on average because of the intrinsic cell - to - cell variation in primary cef culture. (1994) reported that only 30%35% of primary cef localized -actin mrna to the cell periphery using a binary counting method. these cells showing -actin mrna localization may represent a subpopulation of motile cells (kislauskis., 1997). although the cell population was heterogeneous, the correlation coefficient between pi and di for -actin mrna was highly negative (r = 0.61) in contrast to the low value for gapdh mrna (r = 0.27) (figures s2c and s2d). this result shows that -actin mrna tends to exhibit polarized localization whereas gapdh mrna does not. the correlation coefficient between pi and di describes the distinct localization property of each mrna species in a population of cells. finally, we examined the performance of this method to quantify the dynamics of -actin mrna localization in living mammalian cells. because the correlation between pi and di for -actin mrna was high, the polarization of mrna distribution was used as a single metric in live cell analysis. we visualized the endogenous -actin mrna using the cells from the actb - mbs mouse that contains 24 repeats of ms2 binding site (mbs) cassette in the 3 untranslated region (utr) of the -actin gene (lionnet., primary mouse embryonic fibroblasts (mef) were isolated from the mouse, infected with lentivirus that expresses ms2 capsid protein fused with gfp (mcp - gfp) to allow fluorescent labeling of the -actin transcript, and stained with membrane - permeable cytoplasmic dye. a nuclear localization sequence (nls) was added to the n - terminus of mcp - gfp so that free nls - mcp - gfps preferentially localize in the nucleus lowering the background in the cytoplasm (bertrand., 1998). as a negative control, we infected wild - type mef with nls - mcp - gfp expressing lentivirus and observed little fluorescence in the cytoplasm (figure s3a). in the actb - mbs mef cells, nls - mcp - gfp binds to mbs - tagged -actin mrna and the complex is exported out to the cytoplasm (figure s3b). using time - lapse imaging, we monitored the localization pattern of -actin mrna as the cell migrated on fibronectin - coated glass surface (figure 3a and movie s1). the velocity of the cell centroid was measured at 1-min interval, and defined as the protrusion vector. we found that the mrna polarization vector (yellow arrows, figure 3a) and the protrusion vector (red arrows, figure 3a) were highly correlated in space and in time. in all of the migrating cells that we monitored (n = 11), the autocorrelation curve of protrusion vector decays faster than the one for mrna polarization vector, indicating that mrna localization is a slower process than random protrusions. from the mean auto - correlation curves of polarization vector (black curve, figure 3b) and protrusion vector (red curve, figure 3b), the half - lives are estimated to be ~16 min for mrna localization and ~4 min for random protrusions. to quantify the relationship between rna localization and cell protrusion, we calculated the correlation coefficients of two vectors with varying time lags. within our time resolution, however, the skewed correlation in the negative time lag suggests that cell protrusion precedes mrna localization in overall cell movement. it has been shown that mrna localization is not necessary for random protrusions, but required for directed migration (shestakova., 2001). we found that the net migration distance in 2 hr is highly correlated with the mean polarization index of the cell (r = 0.75) (figure 3d). this result supports that localization of -actin mrna has a physiological role in directed cell migration. in summary, the combination of polarization and dispersion of mrna distribution enables an effective assessment of mrna localization. this approach will allow us to quantify subtle changes in rna distribution by gene deletion or mutation, and to compare the localization characteristics of different transcripts. moreover, a quantitative analysis of a single cell in time - lapse images will facilitate studies on the kinetics and the physiological role of mrna localization. we have demonstrated the utility of the new method in budding yeast and primary chicken and mouse embryonic fibroblasts. there is a requirement for this analysis that the entire cell area is imaged while maintaining sufficient sensitivity for detecting rna signals. in order to apply this method for larger cells such as neurons (bassell., 1998 ; lyles., 2006), drosophila embryos (l cuyer., 2007), and syncytial muscle cells (kislauskis., 1993 ; sigrist., 2000), it may be necessary to image multiple fields in three dimensions and stitch them together to reconstruct the whole specimen (preibisch., 2009). in case of further application to the cells in vivo in the tissue environment, it will be useful to outline the cells by cellsurface markers or plasma membrane stains because the surrounding tissues may obscure the regions of the cell for analysis. the simple unbiased analysis of intracellular localization presented in this work may considerably enrich studies on the local regulation and function of many mrna species. yeast cells were grown in rich media until they reach early log phase with od600 ~0.5 (table s1). cells were fixed by adding 8 ml of 32% paraformaldehyde to 42 ml of culture for 45 min at room temperature. the rest of the spheroplasting and in situ hybridization followed the procedure described previously (trcek. briefly, we synthesized four different single stranded dna probes each of which was 50-nucleotide - long and labeled with four cy3 dyes (table s2). the labeling efficiency of each probe was above 90%, indicating efficient coupling of fluorescent dye with each probe. cells in g2 and mitosis were selected using morphological markers and analyzed for ash1 mrna localization (trcek., 2011). primary chicken embryonic fibroblast (cef) cells were obtained from charles river laboratories (wilmington, ma). cells were plated on 10 cm culture dishes and grown at 37c in mem supplemented with 10% fcs in an atmosphere of 95% air/5% co2 for 12 days. cells were trypsinized and seeded on coverslips at a density of 1 10 cells / ml. after overnight incubation, cells were washed with dpbs and fixed in 4% paraformaldehyde in pbs for 20 min. the coverslips were stored in 70% ethanol at 4 c for a few days and processed for fluorescence in situ hybridization as described in singer lab protocols (http://singerlab.org/protocols/). slides were imaged using an olympus bx-61 microscope equipped with an x - cite 120 pc lamp (exfo), a uplanapo 100 1.35 na oil immersion objective (olympus) and a coolsnap hq ccd camera (photometrics). we used chroma filter set 31000 (dapi), 41007a (cy3), and 41008 (cy5). cells were imaged with 0.2 mz steps in each channel using metamorph software (molecular devices). primary mefs were cultured from 14-day - old embryos isolated from a pregnant female of actb - mbs mouse (lionnet., 2011). the head and dark cardiac tissue was removed from the embryo and the rest of the body was digested with trypsin edta for 20 min. after adding media (dmem, 10% fbs, 1% pen / strep), we selected fibroblasts by plating the cells on 10 cm culture dishes for 1 hr and washing off the unattached cells with fresh media. the next day, cells were plated on fibronectin - coated mattek dishes (mattek), infected with lentivirus, and incubated for 48 hr to express nls - mcp - gfp. we stained the cells with 5 m celltracker orange cmra (invitrogen) and replaced the culture media with l-15 media containing 10% fbs, 1% pen / strep, and 1% oxyrase (oxyrase) prior to the experiment. time - lapse images were taken on an olympus ix-71 inverted microscope equipped with a uapo/340 40 1.35 na oil immersion objective (olympus), an ms-2000 xyz automated stage (asi) and an ixon electron - multiplying charge - coupled device (emccd) camera (andor). the temperature was kept at 37c with 60% humidity in an environmental chamber (precision plastics). the excitation sources were 488 nm line from an argon ion laser (melles griot) and a 561 nm diode - pumped solid state laser (cobolt). multiple fields of images were acquired every 1 min using metamorph (molecular devices). if the cell was larger than the field of view, the 2d/3d stitching plugin available through fiji was used to create a tiled image (preibisch., 2009). to analyze the fixed - cell data, cells were segmented using the dic or auto - fluorescence image. a binary mask was generated and the centroid of the cell was calculated by the mean value of x- and y - coordinate of the pixels within the mask region. for single - molecule fish images, a maximum intensity projection of z stacks was used to quantify mrna distribution. in budding yeast images, each fluorescent spot was fit with a two - dimensional gaussian function, yielding the amplitude and the position of the fluorescent particle. from the histogram of fluorescence intensity the average intensity of single probes was used to calculate the number of probes bound in each fluorescent spot inside a cell. only the bright spots binding more than four probes were selected to analyze the number and the position of the target mrna. the mean and the variance of the x- and y - coordinates of the rna molecules except for the ones at the transcription sites were used to calculate the polarization and dispersion indices. for fish images of cefs, after background subtraction, the intensity - weighted centroid and the second moment were calculated. to analyze the live - cell data, masks were generated that define the cell and the nucleus from the celltracker dye image and the nls - mcp - gfp image, respectively. the center of the cell was identified by the intensity - weighted centroid of the cytoplasmic dye image. after background subtraction, the nls - mcp - gfp image was divided by the celltracker image to obtain rna distribution image normalized by the cytoplasmic volume. the center of rna was determined by the weighted centroid of the resulting image excluding the nuclear region. for each time point, the rna polarization vector was calculated as the vector pointing from the center of the cell to the center of rna. the protrusion vector was defined as the instantaneous velocity of the cell calculated every 1 min. the net migration distance was determined to be the distance between the beginning and ending points of the cell trajectory. auto - correlation and cross - correlation of any two sets of vectors were computed by the dot product of the vectors with varied time lags using a similar method demonstrated by weiger. the half - lives of mrna polarization and cell protrusion were determined at the correlation coefficient of 0.5 from the autocorrelation curves. yeast cells were grown in rich media until they reach early log phase with od600 ~0.5 (table s1). cells were fixed by adding 8 ml of 32% paraformaldehyde to 42 ml of culture for 45 min at room temperature. the rest of the spheroplasting and in situ hybridization followed the procedure described previously (trcek. briefly, we synthesized four different single stranded dna probes each of which was 50-nucleotide - long and labeled with four cy3 dyes (table s2). the labeling efficiency of each probe was above 90%, indicating efficient coupling of fluorescent dye with each probe. cells in g2 and mitosis were selected using morphological markers and analyzed for ash1 mrna localization (trcek., 2011). primary chicken embryonic fibroblast (cef) cells were obtained from charles river laboratories (wilmington, ma). cells were plated on 10 cm culture dishes and grown at 37c in mem supplemented with 10% fcs in an atmosphere of 95% air/5% co2 for 12 days. cells were trypsinized and seeded on coverslips at a density of 1 10 cells / ml. after overnight incubation, cells were washed with dpbs and fixed in 4% paraformaldehyde in pbs for 20 min. the coverslips were stored in 70% ethanol at 4 c for a few days and processed for fluorescence in situ hybridization as described in singer lab protocols (http://singerlab.org/protocols/). slides were imaged using an olympus bx-61 microscope equipped with an x - cite 120 pc lamp (exfo), a uplanapo 100 1.35 na oil immersion objective (olympus) and a coolsnap hq ccd camera (photometrics). we used chroma filter set 31000 (dapi), 41007a (cy3), and 41008 (cy5). cells were imaged with 0.2 mz steps in each channel using metamorph software (molecular devices). primary mefs were cultured from 14-day - old embryos isolated from a pregnant female of actb - mbs mouse (lionnet., 2011). the head and dark cardiac tissue was removed from the embryo and the rest of the body was digested with trypsin edta for 20 min. after adding media (dmem, 10% fbs, 1% pen / strep), we selected fibroblasts by plating the cells on 10 cm culture dishes for 1 hr and washing off the unattached cells with fresh media. the next day, cells were plated on fibronectin - coated mattek dishes (mattek), infected with lentivirus, and incubated for 48 hr to express nls - mcp - gfp. we stained the cells with 5 m celltracker orange cmra (invitrogen) and replaced the culture media with l-15 media containing 10% fbs, 1% pen / strep, and 1% oxyrase (oxyrase) prior to the experiment. time - lapse images were taken on an olympus ix-71 inverted microscope equipped with a uapo/340 40 1.35 na oil immersion objective (olympus), an ms-2000 xyz automated stage (asi) and an ixon electron - multiplying charge - coupled device (emccd) camera (andor). the temperature was kept at 37c with 60% humidity in an environmental chamber (precision plastics). the excitation sources were 488 nm line from an argon ion laser (melles griot) and a 561 nm diode - pumped solid state laser (cobolt). multiple fields of images were acquired every 1 min using metamorph (molecular devices). if the cell was larger than the field of view, the 2d/3d stitching plugin available through fiji was used to create a tiled image (preibisch., 2009). to analyze the fixed - cell data, cells were segmented using the dic or auto - fluorescence image. a binary mask was generated and the centroid of the cell was calculated by the mean value of x- and y - coordinate of the pixels within the mask region. for single - molecule fish images, a maximum intensity projection of z stacks was used to quantify mrna distribution. in budding yeast images, each fluorescent spot was fit with a two - dimensional gaussian function, yielding the amplitude and the position of the fluorescent particle. from the histogram of fluorescence intensity the average intensity of single probes was used to calculate the number of probes bound in each fluorescent spot inside a cell. only the bright spots binding more than four probes the mean and the variance of the x- and y - coordinates of the rna molecules except for the ones at the transcription sites were used to calculate the polarization and dispersion indices. for fish images of cefs, after background subtraction, the intensity - weighted centroid and the second moment were calculated. to analyze the live - cell data, masks were generated that define the cell and the nucleus from the celltracker dye image and the nls - mcp - gfp image, respectively. the center of the cell was identified by the intensity - weighted centroid of the cytoplasmic dye image. after background subtraction, the nls - mcp - gfp image was divided by the celltracker image to obtain rna distribution image normalized by the cytoplasmic volume. the center of rna was determined by the weighted centroid of the resulting image excluding the nuclear region. for each time point, the rna polarization vector was calculated as the vector pointing from the center of the cell to the center of rna. the protrusion vector was defined as the instantaneous velocity of the cell calculated every 1 min. the net migration distance was determined to be the distance between the beginning and ending points of the cell trajectory. auto - correlation and cross - correlation of any two sets of vectors were computed by the dot product of the vectors with varied time lags using a similar method demonstrated by weiger. (2010). the half - lives of mrna polarization and cell protrusion were determined at the correlation coefficient of 0.5 from the autocorrelation curves. figure s1. monte carlo simulation of localization patterns, related to figure 1 (a - e) simulated localization patterns of 1,000 particles in a bounded circle. (f) each data point indicates the polarization index and the dispersion index of the corresponding localization pattern shown in (a)-(e). specificity of single - molecule fish and quantification of mrna distribution, related to figure 2 (a and b) fish with ms2_lk51 probe to wild - type mef (a) and actb - mbs mef (b). the intensity range is the same for the two images. because each -actin mrna in the actb - mbs mef contains 12 repeats of the ms2 linker, the bright spots in (b) indicate the presence of mbs - tagged -actin mrna with high specificity. low fluorescence from nonspecific binding of single probes as shown in (a) is excluded from the analysis by thresholding the intensity value. the specificity of single - molecule fish method has been also demonstrated previously (femino., 1998 and zenklusen., 2008). (c and d) quantification of -actin mrna and gapdh mrna distributions in chicken embryonic fibroblasts. scatter plot of polarization index in x axis and dispersion index in y axis for -actin mrna (a) and for gapdh mrna (b). comparison of nls - mcp - gfp fluorescence in wild - type mef and actb - mbs mef, related to figure 3 (a and b) fluorescence images of nls - mcp - gfp expressed in wild - type mef (a) and actb - mbs mef (b). the intensity range is the same for the two images. in the absence of mbs, nls - mcp - gfp localizes to the nucleus as shown in (a). in actb - mbs mef, nls - mcp - gfp bound to mbs - tagged -actin mrna appears in the cytoplasm. the color map shows the fluorescence intensity of nls - mcp - gfp divided by the intensity of cytoplasmic dye, representing the concentration of -actin mrna. the localization pattern of -actin mrna is highly dynamic and correlated with the cell protrusion and migration.	summarylocalization of mrna is a critical mechanism used by a large fraction of transcripts to restrict its translation to specific cellular regions. although current high- resolution imaging techniques provide ample information, the analysis methods for localization have either been qualitative or employed quantification in non - randomly selected regions of interest. here, we describe an analytical method for objective quantification of mrna localization using a combination of two characteristics of its molecular distribution, polarization and dispersion. the validity of the method is demonstrated using single - molecule fish images of budding yeast and fibroblasts. live - cell analysis of endogenous -actin mrna in mouse fibroblasts reveals that mrna polarization has a half- life of ~16 min and is cross - correlated with directed cell migration. this novel approach provides insights into the dynamic regulation of mrna localization and its physiological roles.
there exists a distinct tension between the transformationist and the emergentist paradigm : the recognition of genuine (de novo : sereno, 2007, p. 575 ; see also wagner, 2014, p. 132) evolutionary novelties with no homologous correspondent in the ancestral lineage as a problem agenda of evolutionary developmental research, coupled with the recognition that such evolutionary novelties develop from embryonic rudiments (anlagen) shared with the ancestral lineage (see also wagner, 2014, p. 126). we propose to articulate and analyze this tension from a perspective which one of us called the semaphorontic view of homology, we will first introduce willi hennig 's concept of the semaphoront and discuss its relation to homology. we use the gnathostome jaw as an example to examine the understanding of evolutionary novelties under the semaphorontic view of homology. subsequently, we address the issues of the identity of homologues and the consequent transitivity of homology relations that have traditionally been invoked by biologists and philosophers alike. our main finding is that ontogenetic identity relations maintained throughout developmental trajectories are not transitive with phylogenetic identity relations maintained throughout evolutionary trajectories. however, our discussion also shows how and why the understanding and judgment as to whether a structure is or is not an evolutionary innovation depends on the adoption of one of two alternative viewpoints on homology : that of the emergentist or the transformationist paradigm. in other words, assessments of evolutionary novelty are preconditioned by theoretical commitments rather than decided empirically on a casebycase basis. the concept of the semaphoront was first introduced by hennig (1947, p. 276) as the characterbearer [merkmalstrger] the individual organisms within a short timespan of their life, during which they do not themselves change and hence also do not change their relations to others. the technical term semaphoront hennig introduced in his grundzge of 1950, defined as an individual during a very small temporal duration (not a point in time) of its life another, refined definition appeared in the english version of his textbook, published in 1966 : [] we should not regard the organism or the individual as the ultimate element of the biological system. rather it should be the organism or the individual at a particular point of time, or even better, during a certain, theoretically infinitely small, period of its life (hennig, 1966, p. 6) these definitions reveal that for hennig, the concept of the semaphoront had a metaphysical as well as an epistemological dimension. hennig (1950, p. 5) followed the philosophers nicolai hartmann and ludwig von bertalanffy when he interpreted an organism as a dynamic, processual system, i.e., a system of causal interactions he used the semaphoront to slice through this processual system to obtain a timeslice of theoretically minimal, but heuristically suitable thickness, i.e., a characterbearer that represents the organism at different stages of its life cycle (rieppel, 2007). the epistemological dimension is rooted in hennig 's critique of the practice of describing separate species for larvae and imagoes of holometabolous insects. the leading german limnologist of the time, august thienemann, had argued that separate species descriptions, and corresponding identification keys, should be developed for larvae, pupae and imagoes of aquatic insects, so that nonspecialists such as water hygienists would be enabled to readily identify any potential water pests (e.g., thienemann and krger, 1937 ; thienemann was the first to distinguish oligotrophic [healthy ] from eutrophic [undesirable ] waters : blackbourne, 2006, p. 232). hennig (1943) retorted that the natural system of greatest theoretical relevance to comparative biology is the phylogenetic system, one that represents the successive species lineage splitting events through time. but since there could be only one phylogenetic process, there could also be only one phylogenetic system, a system that had to provide a unifying perspective on biodiversity. the semaphoront thus serves as a tool to capture organisms at different stages of their life cycle, providing characterbearers suitable for phylogeny reconstruction. the fact that hennig (1950, p. 5) conceptualized the organism as a system of causal interactions has important theoretical, and metaphysical, consequences. the theoretical consequence is that although the semaphoront slices through the organism that is a processual system, the series of semaphoronts representing that organism at different stages of its life cycle is causally integrated and interconnected. this in turn has the metaphysical consequence, which hennig (1950, p. 114, 66, p. 81) drew from the work of the philosopher theodor ziehen (1934) that the (self) identity of the organism is preserved through time and change (metamorphosis). following zimmerman 's (37, 43) concept of character phylogeny (see also donoghue and kadereit, 1992), hennig ordered the characters gleaned from semaphoronts of the organisms under comparison into transformation series : [d]ifferent characters that are to be regarded as transformation stages of the same original character are generally called homologous. [] naturally, in determining homologies we are limited to erecting hypotheses such as that particular characters a, a, a the phylogenetic continuity of causal integration and interdependence underlying phylogenetic transformation series individuates homologues just as the ontogenetic causal integration and interdependence of a series of semaphoronts individuates the organism that they represent. some authors, including hennig (1966), platnick (1979) and patterson (1982) did not see a difference between character and character states, in contrast to others who did, such as wiley (1981). in a recent analysis of the relation of homology in terms of evolutionary transformation series, wagner (2014, p. 54) emphasizes the distinction of character and character states in relation to the issue of character identity. the transformation series as a whole represents the character ; the various conditions of form comprised by the transformation series are the different (transformational) states of that character. an evolutionary transformation series thus represents a homologue (homologous character) ; the states comprised by a transformation series represent this homologue in two or more organisms under a different condition of form, and possibly also of different function (homologous character states) : the same but different again. it is therefore not similarity, but the continuity of causal integration and interdependence underlying a transformation series that individuates the homologue. a homologue, on wagner 's (2014) account, is subject not to a similarity relation, but to the identity relation : the identity of a morphological character is not tied to similarity ; rather, it is tied to the historical continuity of descent (wagner, 2014, p. 53). on that account, homologues form evolutionary lineages. wagner (2014) invokes character identity as individuating homologues, the latter maintained through transformational stages by the genealogical continuity of the underlying genetic character identity networks (chin : wagner, 2014, p. 97 ; see also wagner, 2007). but just as the concept of the semaphoront can be applied to an individual organism and its successive stages in a complex life cycle, so can the concept of the semaphoront also be applied to parts of an individual organism (organs and organ systems) and their successive developmental stages. and in that context again, it is the causal integration of the developmental process that maintains the (self or character) identity of organs and organ systems through developmental change. it is the application of the concept of the semaphoront to parts of an organism that provides the platform for the development of a semaphorontic view of homology, which one of us paraphrasing hennig 's definition of the semaphoront (66, p. 6 ; see above)formulated as follows (assis, in press) : [] we should not regard the [part ] or the [organ ] as the ultimate element of [homology ]. rather it should be the [part ] or the [organ ] at a particular point of time, or even better, during a certain, theoretically infinitely small, period of its [ontogeny ]. [] we should not regard the [part ] or the [organ ] as the ultimate element of [homology ]. rather it should be the [part ] or the [organ ] at a particular point of time, or even better, during a certain, theoretically infinitely small, period of its [ontogeny ]. parts of organisms (organs or organ systems) at different developmental stages can thus be understood as different semaphoronts of those parts. homology relations may then obtain between semaphorontic parts of two or more organisms throughout different developmental stages of those parts in these same organisms. on the semaphorontic view of homology, two different dimensions of identity relations obtain for parts (organs and organ systems) and their corresponding homologues. the first is the relation that ties together different developmental stages of a homologue within an organism, e.g., from precursor anlagen through to the fully differentiated organ, an identity that is anchored in the continuous causal integration of the developmental (ontogenetic) process. the second dimension is the relation that ties together homologues at successive developmental states as character states of a character transformation series across species, an identity that is anchored in the continuous causal integration of the evolutionary (phylogenetic) process. the first dimension is an intraorganismal character identity maintained throughout developmental change ; the second dimension is an interspecies character (state) identity maintained throughout evolutionary change. in line with this, two questions might be raised : (1) why is the semaphorontic definition productive for evolutionary biology ? and (2) is it substantially different than the characterstate / character equation ? with respect to the first question, the semaphorontic view of homology reinforces the importance of ontogenetic evidence in support of homology hypotheses and evolutionary relationships (assis, in press). the view also develops novel resources for solving the transitivity problem for identitybased theories of homology, as we demonstrate in the penultimate section of this paper. with respect to the second question, the semaphorontic view is not in disagreement with wagner 's distinction between character states and characters. rather, it identifies another crucial distinction residing within wagner 's and others views on homology : a distinction between the ontogenetic and phylogenetic states of a homologous character. again, recognizing this distinction provides the resources for solving the problem of transitivity that arises with a strong conception of homology built on identity relations (wiley, 2008, following ghiselin, 2005), with important consequences for the understanding of evolutionary novelties, consequences we will discuss immediately after the presentation of an illustrative empirical case. we have chosen the origin of the gnathostome jaw as an empirical example to illustrate the application of the semaphorontic view to homology assessment, and its theoretical consequences. the example is based on the work of shigeru kuratani and his collaborators (e.g., shigetani., 2012, 2005, ; kuratani, 2004, 2005, 2012 ; kuratani., 2012), who compared the pharyngula and postpharyngula larval (ammocoete) stages of the lamprey lethenteron japonicum with gnathostome development (e.g., kuratani., 2004). the ammocoete oral apparatus comprises the upper and lower lips, and a pair of muscular flaps, the velum. this oral apparatus is compared to that of gnathostomes, in particular the primary upper (palatoquadrate) and lower (meckel 's cartilage) jaws, their associated musculature and innervation patterns. at the pharyngula stage, both lamprey and gnathostome show the same fundamental tripartite configuration of the rostralmost [neural ] crestderived ectomesenchyme the anteriormost compartment of the rostral ectomesenchyme lies in a preocular position. behind the optic vesicle lies the trigeminal ectomesenchyme, which is subdivided into a postocular (premandibular) and a mandibular compartment, in accordance with the partitioning of the trigeminal nerve into an ophthalmic and a maxillarymandibular division. the mandibular compartment of the ectomesenchyme corresponds to the mandibular arch domain, as it is from this compartment of the ectomesenchyme that the gnathostome jaw (palatoquadrate and meckel 's cartilage) develops. rathke 's classic account of the development of the gnathostome jaw distinguished two anterior visceral arches (the mandibular and hyoid arch) from the succeeding branchial arches. the serial patterning of the visceral / branchial arches is controlled by a hierarchically structured the ectomesenchyme located in the premandibular (postoptic) domain of gnathostomes is dlxnegative, and develops into the trabeculae cranii that floor the prechordal neurocranium on either side of the head (kuratani. while the premandibular (postoptic) and mandibular arch domains respectively of the trigeminal ectomesenchyme can be considered to be homologous morphological structures (homologous tissues : compagnucci., 2013, p. 444) at the pharyngula stage of the lamprey and gnathostomes, the entire trigeminal ectomesenchyme is dlxpositive in the lamprey, whereas the premandibular (postoptic) compartment is dlxnegative in gnathostomes. the premandibular (postoptic) ectomesenchyme, which in gnathostomes gives rise to the trabeculae cranii, in the ammocoete larva gives rise to mucocartilaginous structures in the upper lip (see de beer 37, p. 44). the ectomesenchyme of the mandibular arch domain, which in gnathostomes gives rise to the jaws, in the ammocoete larva gives rise to mucocartilaginous structures in the lower lip and velum. and unlike the gnathostome trabeculae, which derive from prechordal ectomesenchyme, the trabeculae of the lamprey develop from anterior parachordal mesoderm (see kuratani, 2004, fig. 6 ; kuratani., 2012). on a semaphorontic account, and given a causal integration of developmental processes, there obtains ontogenetic character identity (a form of semaphorontic or intraorganismal identity maintained throughout developmental trajectories) of the premandibular (postoptic) ectomesenchyme and the upper lip skeleton in the lamprey, as well as ontogenetic character identity of the premandibular (postoptic) ectomesenchyme and the trabeculae cranii in gnathostomes. similarly for the mandibular arch domain ectomesenchyme : it is ontogenetically identical with the skeletal structures of the lower lip and velum in the ammocoete larva, and with the jaws in gnathostomes respectively. there also obtains, at the pharyngula stage, phylogenetic character identity (a form of ancestral or interspecies identity maintained throughout evolutionary trajectories) of the premandibular (postoptic) and mandibular arch domains respectively of the trigeminal ectomesenchyme in the lamprey and gnathostomes. at the genetic level, however, the premandibular (postoptic) domain of the ectomesenchyme is dlxpositive in the lamprey, dlxnegative in gnathostomes. problems of homology, i.e., of phylogenetic identity at the morphological level come into focus in postpharyngula stages. what does it mean to say that the mucocartilaginous structures in the upper lip of the ammocoete larva are homologous (i.e., phylogenetically identical) with the trabeculae cranii in gnathostomes, as would have to be the case if derivation from common embryonic rudiments (anlagen) is the key to homology ? on the latter criterion, the jaws of gnathostomes would have to be homologous in the phylogenetic dimension with mucocartilaginous structures in the lower lip and velum of the ammocoete larva.. given heterotopic effects in dlxfamily gene regulation, they speak of a loss (kuratani 2004 : 339, 342), an obliteration (kuratani, 2005, p. 439), or a disruption (shigetani., 2005, the consequence is that the gnathostome jaw qualifies as an evolutionary novelty sensu mller and wagner (1991) : homologous throughout gnathostomes, it has no homologue in the ancestral lineage (kuratani, 2004, p. 342). a transformationist approach to the study of the origin of the gnathostome jaw is further rejected on the grounds that there obtains no primitive condition of the anteriormost branchial arch in any adult agnathan, extant or fossil, from which the gnathostome jaw could seamlessly be derived. the reason is that the branchial arch skeleton in all adult agnathans is always specialized, i.e., adapted to specific modes of life (kuratani, 2005, p. 489). the conclusion to a loss, obliteration, or disruption of relations of homology through heterotopic effects in development is predicated on the way homologues are conceptualized. owen 's definition (see above) renders homologues the same but different in two or more organisms. on that account, mucocartilaginous structures in the lower lip and velum of the ammocoete larva and the jaws of gnathostomes could well qualify as the same but different. elsewhere, owen characterized homologues as namesakes : a homologue is a part or organ in one organism so answering to that in another as to require the same name the question then arises as to whether the names of homologues are proper names or general names (natural kind terms) ? alternative conceptualizations of homologues as individuals or natural kinds have been discussed by brigandt (2007, 2009) and assis and brigandt (2009). if the names of homologues are proper names, then homologues are individuals (for a characterization of homologues as individuals see brigandt, 2009, p. 87). according to wagner (2014, p. 70), a homologue [] behaves like an entity that forms lineages of descent ; homologues consequently are individuals, not sets or classes (see also ghiselin, 2005). the relation of homology (in which homologues take part) is then governed by the identity relation ; for instance, wagner (2007, p. 473 ; see also wagner, 2014) characterized homology as character identity. identity in the strong sense is transitive (wiley, 2008) : if a is identical to b, and b is identical to c, then a is also identical to c. brigandt (2002, p. 391) on good grounds rejected the equation of the sameness of homologues (as in the same but different) with numerical or tokenidentity, but instead characterized the relation of homology as one of correspondence. but correspondence, just as identity, has been claimed to be transitive as well (ghiselin, 2005, p. 96). either way, the transitivity of the relation of homology qua character identity has important consequences, if the relation of homology is also symmetrical as it indeed is (if a is homologous with b, then b is also homologous with a). call the mandibular arch domain of the ectomesenchyme in the lamprey a1, the mucocartilaginous structures in the ammocoete lower lip and velum a2 : on the semaphorontic view, a1 is (ontogenetically, through development) identical with a2. call the mandibular arch domain of the ectomesenchyme in gnathostomes b1, the gnathostome jaw b2 : on the semaphorontic view, b1 is (ontogenetically, through development) identical with b2. on morphological grounds, if the homology relation is an identity relation that is transitive and symmetrical, a2 must also be homologous with b2. ghiselin (2005, p. 98) attributes any dissatisfaction with this counterintuitive conclusion to a propensity to conceptualize taxa as classes, rather than individuals of which homologues are parts. in line with this, wagner (2014, p. 91) highlights the important caveat that character identity arises at a certain stage of development and not earlier. this could imply that the character identity of the gnathostome jaw (b2) is not yet established by the mandibular domain ectomesenchyme at the pharyngula stage, rendering its nonhomology at both ontogenetic and phylogenetic levels with skeletal structures in the lamprey lower lip and velum (a2) unproblematic. conversely, if the identity of the gnathostome jaw had been established at the pharyngula stage, its nonhomology with the lamprey lower lip and velum would require the disruption of character identity during development, as was concluded by shigetani. an alternative is to ask (see also brigandt, 2002) whether numerical (or token) identity is simply too strong to be applied to what winther (2006, p. 471) has identified as compositional biology, one that is based on the notion of parts and wholes, as well as their respective functions and capacities, such as the capacity of the mandibular arch domain ectomesenchyme to develop into jaws in gnathostomes (see also winther, 2011). correspondingly, we introduce the notion of compositional identity : identity that is grounded in relations prevailing between parts of a whole, rather than in numerical selfidentity. for compositional biology so concerned with parts as well as wholes and especially for evolutionary studies in compositional biology, it is important to understand partpart relations as well as the more traditional partwhole relations. for example, in the semaphorontic view of homology, it is not just the organism at different developmental stages between which one can pick out identity relations (these would be hennig 's temporalparttowhole relations) ; it is also the different developmental stages of parts (organs or organ systems) of an organism between which one can pick out identity relations (these would be temporalparttopart relations). likewise, as wagner 's distinction between homologous characters and character states shows, studies in comparative morphology are not limited to comparisons among whole organisms of different species at different points in the evolutionary trajectory. it is also possible to pick out just a part of these organisms of different species a homologous character and then to compare instances of this part a character state at different points in the evolutionary trajectory. to put the point in other words, to do this kind of comparison is to treat a part of an organism (a character state) like its own hennigean temporalpart of an evolutionaryratherthandevelopmental whole (a homologous character). just as identity holds between hennigean temporalparts and the whole organism throughout development, so too does identity hold between character states and the homologous character throughout evolution. adopting the compositional view entails that, with ontogenetic identity in general, the relevant wholes are whole organisms, while the relevant parts are the timeslices or temporal parts of those wholes i.e., hennig 's original semaphoronts. the ontogenetic identity of homologous parts of an organism is thus an identity relation that holds not between the whole organism and its temporal parts, but rather that homologous part and its temporal partparts i.e., the various states of just a fly 's wing throughout the development of the fly. with phylogenetic identity in general, the relevant wholes are higher taxa (like clades), while the relevant parts are the smaller taxa contained within the higher taxa (like species). the phylogenetic identity of homologous parts is thus an identity relation that holds not between whole clades and the species they contain, but rather it is a relation that holds between one part (in wagner 's terms : character) possessed (or not) by organisms throughout the clade and that part 's particular instantiations (again, in wagner 's terms : character states) in different species within the relevant clade. so, a particular human arm has a parttopart ontogenetic identity from embryonic stages to adulthood and through senescence ; human arms also share a certain parttopart phylogenetic identity with seal flippers, bat wings and other tetrapod forelimbs. ontogenetic identity comes from shared developmental history whereas phylogenetic identity comes from shared evolutionary history ; what the semaphorontic view helps to illustrate is that both shared and divergent developmental tracks are nested within evolutionary history, which is how we end up with confusing situations like the one detailed in this paper, where a1 and b1 share a certain, limited phylogenetic identity but then diverge developmentally, producing nonhomologous ontogenetic states (a2 and b2) from their homologous precursors. it is crucial to realize that the identity of one temporal part of an organism with the whole organism or the identity of a character state with the homologous character does not imply the identity of that part with others within the whole. to illustrate this point consider identical twins. these are organisms which share a genetic identity ; and they each have a personal identity that persists throughout organismal changes in development. the fact that they share a genetic identity does not mean that they are semaphoronts of the same organism, sharing personal identity as well. hence, we need a conception of identity that allows us to make clear that we are picking out identity relations among parts across wholes, rather than that conception of numerical identity which holds between all of a thing and only itself. our notion of compositional identity ought not to be confused with the composition as identity thesis (as formulated in baxter, 1988a, 1988b ; lewis 1991) from analytic philosophy. whereas the composition as identity thesis deals with partwhole relations (defining numerical identity of a whole in terms of composition by its parts), our notion of compositional identity deals with partpart relations, i.e., defining identity relations between parts of an organism through different developmental stages, or parts of different organisms throughout different evolutionary trajectories. in the notation defined above, there prevails an ontogenetic form of compositional identity between a1 and a2, since these represent two semaphoronts (qua parts of an organism) of the same developmental (ontogenetic) transformation series. the same holds of b1 and b2, which are ontogenetic semaphoronts of the same developmental sequence. the compositional identity of a part of an organism that is maintained in two (or more) different developmental stages of this part is thus rooted in ontogenetic relations. turning to the putative homology of a1 with b1, it can be stated that a1 and b1 are ontogenetically similar phylogenetically homologous semaphoronts, i.e., semaphoronts whose character identity is grounded in phylogenetic relations, i.e., in their common evolutionary origin. in other words, there is a phylogenetic form of compositional identity between a1 and b1. in contrast, a2 and b2 are ontogenetically dissimilar and phylogenetically nonhomologous semaphoronts, i.e., semaphoronts that have differentiated along distinct and different developmental (ontogenetic) trajectories. the fact that character identity between developmental stages of the same part of an organism is grounded in ontogenetic relations, whereas character identity between a homologous part of two or more organisms is grounded in different, i.e., phylogenetic relations, breaks the transitivity of homology relations relative to ontogenetic relations. an ontogenetic form of compositional identity holds between a1 and a2 as well as between b1 and b2, but this form of compositional identity is not transitive with the phylogenetic form of compositional identity that holds between a1 and b1. such transitivity would necessitate the homology of the gnathostome jaw with the mucocartilaginous structures in the lower lip and velum of the ammocoete oral apparatus. to deny the homology of a2 and b2 means to acknowledge the fact that although a2 and b2 both derive from similar, indeed homologous embryonic precursors (a1 and b1), they subsequently undergo radically divergent development that leads to the emergence of novel structures in both lampreys and gnathostomes. divergence is the fundamental developmental trajectory articulated in von baer 's (1828, p. 224) laws of individual development. the essence of von baer 's conception of embryonic development that he equated with a process of individuation of an organism has been aptly captured by richards (92, p. 59) : embryological evolution, in von baer 's view, is a process of differentiation a movement from the more homogeneous and universal to the more heterogeneous and individual. a group of related organisms will show similar early embryonic stages, but progressively deviate from one another during subsequent development as the more particular, or individual characteristics become differentiated. viewed from the semaphorontic perspective, it is possible not only to talk about the individuation of the whole organism, but also about the individuation of parts of organisms through development. on that account, von baerian differentiation results in the individuation of parts of organisms (organs and organ systems) through development which, as was stressed by wagner (2014, p. 91), establishes character identity at a certain time in development, and not before. on the classic, transformationist account, the vertebrate jaws (palatoquadrate and meckel 's cartilage respectively) evolved from the first visceral, i.e., mandibular arch (epi and ceratobranchial respectively) such homology is rejected by kuratani (2005, p. 489) not only because of divergent developmental trajectories in the lamprey and gnathostomes, but also because the first gill arch is always specialized in its own way not just in extant, but also in extinct agnathans. according to this argument, there does not exist, in any fully differentiated agnathan, a primitive condition of the first gill arch that could easily have transformed into the gnathostome jaw. kuratani 's (2005) argument in this respect recalls remane 's (48, p. 258) rejection of the possibility of a (nullwertahne) on the grounds that a completely unspecialized, hence nonadapted organism would not be viable. enough reason, then, to turn away from consideration of the transformation of fully differentiated ancestral into descendent structures, and to compare embryonic conditions of form instead. within the emergentist paradigm, and in accordance with von baer 's conception of development, the lamprey and gnathostomes both being vertebrates share structural similarities at early developmental stages, i.e., at the phylotypic pharyngula stage (phylotypic period sensu richardson, 95, 98). they all share a central nervous system, neural crest, a series of branchial arch primordia etc., at the pharyngula stage. at that early developmental stage, it is possible to identify a mandibular arch domain of ectomesenchyme, associated with the maxillary and mandibular divisions of the trigeminal nerve (v2/3), that is ontogenetically similar and phylogenetically homologous in the lamprey and in gnathostomes. the subsequent development of the mandibular arch domain is radically divergent, however, in the lamprey and gnathostome, resulting in the individuation of ontogenetically dissimilar and phylogenetically nonhomologous structures (i.e., different character identities) in both lineages : mucocartilaginous skeletal elements of the lower lip and velum in the ammocoete larva, jaws in gnathostomes. these then represent alternative characters, not character states forming a transformation series. in sum : from the transformationist perspective, the gnathostome jaw must be a homologue of the first gill arch of agnathans, but this assumption is not borne out by the empirical situation described by kuratani (2005). on an emergentist account such as wagner 's, both the mucocartilaginous skeletal elements in the ammocoete larva and jaws in gnathostomes must be evolutionary novelties, but this assessment downplays the ontogenetically similar and phylogenetically homologous developmental precursor states in both lineages. the semaphorontic view of homology, however, captures both the element of sameness (between a1 and b1) and that of difference (between a2 and b2), without falling afoul of the logical problem of transitivity. with respect to the phylogenetic meaning of the characters, the mandibular arch domain of the ectomesenchyme is a synapomorphy of vertebrates (present in a1 and b1). the jaw (palatoquadrate and meckel 's cartilage) is a synapomorphy of gnathostomes (b2), whereas the mucocartilaginous structures in the lower lip and velum of the lamprey ammocoete larva are a synapomorphy of lampreys (a2). under von baerian differentiation, which results in the individuation of alternate nonhomologous characters (different character identities) through deviation in development, homology this is not the case in under the transformationist paradigm, where homology reigns over an entire transformation series (either linear or ramifying), thus encompassing both the plesiomorphic as well as the apomorphic character states (for further discussion see brower and de pinna, 2012 ; nixon and carpenter, 2012 ; assis, 2013). furthermore, if evolutionary novelties are defined as characters that have no homologue in the ancestral condition, then evolutionary novelties can only obtain under the emergentist paradigm of deviating developmental trajectories. under the transformationist paradigm,	abstractthe relation of homology is generally characterized as an identity relation, or alternatively as a correspondence relation, both of which are transitive. we use the example of the ontogenetic development and evolutionary origin of the gnathostome jaw to discuss identity and transitivity of the homology relation under the transformationist and emergentist paradigms respectively. token identity and consequent transitivity of homology relations are shown to be requirements that are too strong to allow the origin of genuine evolutionary novelties. we consequently introduce the concept of compositional identity that is grounded in relations prevailing between parts (organs and organ systems) of a whole (organism). we recognize an ontogenetic identity of parts within a whole throughout the sequence of successive developmental stages of those parts : this is an intraorganismal character identity maintained throughout developmental trajectory. correspondingly, we recognize a phylogenetic identity of homologous parts within two or more organisms of different species : this is an interspecies character identity maintained throughout evolutionary trajectory. these different dimensions of character identity ontogenetic (through development) and phylogenetic (via shared evolutionary history)break the transitivity of homology relations. under the transformationist paradigm, the relation of homology reigns over the entire character (state) transformation series, and thus encompasses the plesiomorphic as well as the apomorphic condition of form. in contrast, genuine evolutionary novelties originate not through transformation of ancestral characters (states), but instead through deviating developmental trajectories that result in alternate characters. under the emergentist paradigm, homology is thus synonymous with synapomorphy. j. exp. zool. (mol. dev. evol.) 324b : 578587, 2015. 2015 the authors. journal of experimental zoology part b : molecular and developmental evolution published by wiley periodicals, inc.

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