The present study aimed at assessing the psychometric properties of psychosocial determinants of physical activity-related measures in Iranian adolescent girls.Several measures of psychosocial determinants of physical activity were translated from English into Persian using the back-translation technique. These translated measures were administered to 512 ninth and tenth-grade Iranian high school students.The results of a series of factor analysis showed that the self-efficacy scale contained a single factor, the social support scale contained two factors: family support and friend support, the physical activity 'pros & cons' scale contained two factors: physical activity pros scale and physical activity cons scale, the change strategies scale contained a single factor, the environment scale also contained a single factor. Chronbach's alphas, mean inter-item correlations and test-retest coefficients showed that these solutions were reliable. These preliminary results provide support for using the mentioned scales to measure psychosocial determinants of physical activity in Iranian adolescent girls. Promotion of physical activity level is one of the most important and effective strategies for reducing the risk of several chronic diseases including cardiovascular diseases, non-insulin-dependent diabetes mellitus, osteoporosis, obesity and some types of cancer .Physical activity habits fostered and developed during the early stages of life may be expected to persist into adulthood, reducing the incidence of chronic diseases associated with a sedentary lifestyle in later life . Given tPromotion of physical activity level among adolescents can be desired by behavioral interventions. More effective interventions are needed because half of individuals who initiate a physical activity program drop out within six months .Data from three national surveys among Iranian adults have shown that more than 80% of the Iranian population is physically inactive . A few lA major issue in physical activity programs and research among adolescents is the accurate measurement psychosocial determinants of physical activity which may contribute to physical activity in this population. This has led healthcare professionals and researchers to develop exercise interventions based on theoretical models of behavior change in an attempt to increase physical activity levels .To understand the levels of physical activity among individuals, various researchers have identified a number of promising variables that may influence levels of physical activity. These variables include demographics, cognition, behaviour, social environment, and physical environment. In intervention programmes, cognitive variables are particularly targeted, because they may be more amenable to change than the less mutable variables such as age and income . AlthougIn this study, the instruments were used from PACE-Adolescent Physical Activity Survey and translated from English into Persian using the back-translation technique. Social Cognitive Theory (SCT) and the Transtheorical Model (TTM) guided instrument development ,10.SCT iThe Transtheoretical Model (TTM) of behavior change can also provide a useful framework for examining the issue of adoption and maintenance of physical activity with adolescents . The TTMThere are no theoretically based instruments in the literature that measure physical activity related psychosocial determinates among Iranian adolescents. Thus, the present study is the first research for the development of physical activity related psychosocial determinant measures.It also examines the psychometric characteristics of several physical activity-related psychosocial determinant measures in Iranian adolescent girls. Acknowledging the low physical activity during adolescence, standardized, reliable and valid measures of influence of physical activity for this population is essential. In this study, some 512 high school students were administered the questionnaires of physical activity along with other measures to evaluate their reliability and validity for this population. We conducted tests of internal consistency, test-retest reliability and factor analysis in constructs of physical activity self-efficacy, physical activity social support, physical activity pros and cons, physical activity change strategies, and physical activity environmental factors. Consistent with the initial test development, we predicted a good internal consistency among the scales, and high test-retest reliability.th or 10th grades) and being able to attend two survey sessions). The eligible subjects were recruited from 12 high schools in Tehran. The age of participants ranged from 15 to 17 years with average age 16.15 years (SD = 0.77). A total of 545 students were recruited into the study, 33 subjects were omitted from the analysis due to missing data on one or more of the determinants physical activity items of interest. Popular textbooks on factor analysis give specific advice on sample size for factor analysis, the required variable to subject ratio lies between 1:5 and 1:10 [Are the questionnaires a valid and reliable measure for Iranian adolescents?"Participants were female students who meet the inclusion criteria of the study  believes in six aspects of construct validation including content, substantive, structural, generalizability, external, and consequential as they apply to performance assessment. Also, Samuel Messick argues that 'it is not sufficient merely to select tasks that are relevant to the construct domain. In addition, the assessment should assemble tasks that are representative of the domain... The intent is to insure that all important parts of the construct domain are covered [Samuel Messick  covered .'In the research literature of nursing and other health care professions, factor analysis is most often used as a part of the instrument development process. Factor analysis may be a vital step in creating a new measurement tool; it is a method for organizing the items into factors. A factor is a group of items that could be said to be related to each other .After comprehensive literature review on the existing instruments of measuring physical activity determinants in adolescents, we used measures of physical activity related psychosocial determinants that have previously been adapted and developed among the American adolescents by Norman & Sallis . First, A panel of eight Iranian experts in the areas of health education and clinical psychology were asked to quantify the clarity linguistic appropriateness of the translated questionnaires . The panel members were asked to evaluate the pilot instrument for the appropriateness and relevance of the items. Furthermore, the expert panel was asked to evaluate item wording, response format, and instrument length.A pilot study was conducted to test whether the physical activity questionnaires were easy to read and to comprehend by the students. A convenience sample of 12- students completed the physical activity questionnaires and gave comments on their understanding of the items. The changes made to the original version include adoption of age-appropriate words and the development of a format more appealing to adolescence girls.I'm sure I can't" to five "I'm sure I can".This variable asked the individuals about their confidence in being able to carry out a regular schedule of exercise as well as the barriers they perceived in exercising. A six-item physical activity self-efficacy scale was used based on the previous scales ,21.The p"not important" to five "extremely important".Decisional balance consisted of two constructs labeled the 'Pros' and 'Cons' of change that address cognitive and motivational aspects of human decision-making. Marcus, Rakowski and Rossi  modifiedFour items of family support on physical activity assessed family influences on physical activity . The ite(1) Watched you participate in physical activity or play sports? (2) Encouraged you to do sports or physical activity? (3) Provided transportation to a place where you can do physical activity or play sports? (4) Done a physical activity or played sports with you?Never" to five "Every Day".Items were asked in reference to a typical week and participants responded using a 5-point scale from one ""Never" to five "Every Day".Items similar to the family support items assessed friend support related to physical activity. The five items assessed the frequency that friends provided encouragement and support for participating in physical activity. The items were: (1) Do your friends encourage you to do sports or physical activities?, (2) Do your friends do physical activity or play sports with you?, (3) Do your friends or classmates tease you about not being good at physical activities or sports?, (4) Do your friends ask you to walk or bike to school or to a friend's house? And (5) Do your friends tell you that you are doing well in physical activities or sports? The items were asked in reference to a typical week and the participants responded using a 5-point scale from one Never" to five "Many Times".The change strategies were similar to the constructs described as processes of change in the Transtheoretical Model  and were"Disagree a lot" and five "Agree a lot". The items were: (1) There are enough supplies and pieces of sports equipment  At home to use for physical activity; (2) There are playgrounds, parks or gyms close to my home or that I can get to easily; (3) It is safe to walk or jog alone in my neighborhood during the day; and (4) It is difficult to walk or jog in my neighborhood because of things like traffic, no sidewalks, dogs and gangs. The item number four was reverse-scored before all analyses.The measure of perceived environment that assessed the neighborhood environment in terms of facilitating physical activity included four items rated on a 5-point scale with anchors of one These items were originally from the Amherst Health and Activity Study .Each scale's reliability was estimated by calculating its internal consistency and test-retest stability. Internal consistency measured by coefficient alpha is the proportion of a scale's total variance that is attributable to a common source, the true score of a latent variable underlying the items . A minimAnother estimate of a scale's reliability is its temporal stability assessed by a test-retest design. The following standards were used to evaluate the reliability coefficients: (1) less than 0.00, poor; (2) 0.00–0.20, slight; (3) 0.21–0.40, fair; (4) 0.41–0.60, moderate; (5) 0.61–0.80, substantial; and (6) 0.81–1.00, excellent .In this study, exploratory factor analysis (EFA) was used to summarize the data by grouping the intercorrelated variables together. Most often, this occurs in the early stages of research. The direct purpose of exploratory factor analysis (EFA) is to reduce a set of data so that it may be described and used easily. Other purposes include instrument development and theory construction .Principa2. Household income was unfairly distributed across the four income categories. Some %62.8 of the participating girls' family had < $320 household income, %48.5 (n = 248) of fathers had completed high school, with %16.8 (n = 86) completing a college or graduate degree, and %54 (n = 276) of mothers had completed high school, with %09.0 (n = 46) completing a college or graduate degree.Demographic characteristics of the participating girls are shown in Table The sample size of 512 was sufficient to produce reliable correlation coefficients so that popular textbooks on factor analysis give specific advice on sample size for factor analysis. The required variable to subject ratio lies between 1:5 and 1:10 .Prior to performing Principal Components Analysis (PCA), the suitability of data for factor analysis was assessed. An inspection of the correlation matrix in each subscale revealed that most of the correlations were greater than 0.30, therefore, some clustering of items was expected and exploratory factor analysis was deemed appropriate in the early stage of research . The KaiFor physical activity self-efficacy Table , one facTwo sub-scales were identified for the physical activity social support Table , family For the physical activity decisional balance Table , two subPrincipal Component Analysis (PCA) with oblique was performed on the students' responses to the 15 change strategies items. Oblique rotation, which allows the factors to be statistically related , was useThe eigenvalues for the first 2 consecutive components were 5.70 and 1.06. Examination of the eigenvalues greater than 1 indicated that a 2-factor solution may be appropriate. The examination of the scree plot also suggested that 2 dimensions underlie change strategies scale. Although these two methods are the most popular heuristic, they are potentially unreliable ,31, and , and 31,Using the procedure recommended by Thompson and Daniel , 50 randFor the physical activity environmental factors, two sub-scales were also identified. The eigenvalues for the first 2 consecutive components were 2.03 and 1.009. Examination of the eigenvalues greater than 1 indicated that a 2-factor solution may be appropriate. The examination of the scree plot also suggested that 2 dimensions underlie environmental factors scale. In this study parallel analysis (PA)  was alsoThe findings showed intercorrelations among the physical activity-related psychosocial measures. Physical activity self-efficacy was significantly and positively correlated with the physical activity pros scale (perceived benefits) and change strategies, while it was negatively correlated with the physical activity cons scale (perceived barrier).Those girls with higher scores on physical activity self-efficacy reported higher scores on physical activity pros, change strategies and lower scores on the physical activity cons.Reliability was determined by examining both the internal consistency and test-retest stability of the physical activity-related psychosocial measures. The physical activity-related psychosocial measures showed adequate internal consistency   with theIn addition, table Almost 20% (93 subjects) of the original sample (512) were randomly selected to complete the physical activity-related psychosocial measures again 15 days after the initial assessment. Pearson Product Moment Correlations were calculated between the Time 1 and Time 2 assessments for the five scales. Results showed that the relationships were in the large effect size range for scales of physical activity self-efficacy, physical activity social support, physical activity pros and cons, physical activity change strategies and physical activity environmental factors, respectively .The purpose of this study was to identify and evaluate the psychometric characteristics of physical activity-related psychosocial scales. This preliminary testing provides evidence for the reliability and validity of the physical activity- related psychosocial determinants questionnaires in Iranian high school girls.An instrument containing the five scales was developed through a focus group with Iranian adolescent girls; the items were selected based on the consideration of contextually cultural relevance and language issues. Content validity of the instruments was established by having a panel of experts evaluating the instruments to obtain the most appropriate item content. The scales items were drawn from Norman studies  and confThe obtained results from this study demonstrated acceptable internal consistency, good test-retest reliability and validity of the instruments in a large sample of Iranian adolescent girls. Of these five scales, four showed adequate internal consistency, using Chronbach alpha  , while tA comparison of psychometric properties of scores from the translated measures with those from the original measures shows that both are similar, therefore, researches can use physical activity-related psychosocial scales to help promote physical activity levels among adolescents.The exploratory factor analysis also identified subscales within the two of the five scales including: physical activity social support scale: family support and friend support, and physical activity decisional balance scale: pros and cons. The self-efficacy scale contained a single factor, the change strategies scale contained a single factor, and the environment scale also contained a single factor. Self-efficacy for physical activity had been used in previous studies and was kept as single dimensional scale. Also, social support and pros and cons for physical activity had been used in previous studies and thus were kept as multidimensional scales.The most closely related previous study reported evidence in support of a one-dimensional scale of physical activity change strategies and scale of environmental factors ,41, whilThese findings extend previous research by supporting single dimensional scale of physical activity self-efficacy ,41, and , and 41,Intercorrelations between the physical activity-related psychosocial scales were fair to moderate suggesting that psychosocial sub-scales are generally independent.The analysis reported here provides further empirical support for the relevance of Bandura's social cognitive theory to the studies of the psychosocial determinants of physical activity.In spite of the suitable design and use of exploratory factor analysis in this study, several limitations were noted. First, there are no theoretically based questionnaires of physical activity related psychosocial determinants for adolescents in Iran. Second, since our sample consisted of adolescents from a specific education area in Tehran, our results could not be generalized to adolescents who live in other geographic locations in Iran. Therefore, future research should replicate this study with a sample of adolescents living in others education areas.In summary, development of questionnaires to measure physical activity- related psychosocial determinants in Iranian adolescents is still in its developmental stage. These measures warrant further study to strengthen their measurement properties, but may be useful in future studies for examining the factors that contribute to physical activity in Iranian adolescent girls. We believe the behavior change construct measures that demonstrated strong psychometric properties will be useful instruments for measuring adolescents in observational and experimental studies of physical activity. Further work is needed to refine the measures that need to be improved and to assess the construct validity of these measures.In conclusion, the results of this study provide evidence for the soundness of factor structure and acceptable reliability of the scales of physical activity- related psychosocial determinants in the Iranian population.The authors declare that they have no competing interests.AP, AH, SF, FG and AA contributed equally to the design and conduct of the survey, analysis of the results, drafting and critical revision of the manuscript. AP, AH, AA, SF and FG read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:Self – efficacy of physical activity. There are many barriers in the way of physical activity. Mark the extent of your capability for doing physical activity in the following situations. Answer all the questions. 1. Can you do exercise or physical activity even when you are sad or under stress? 2. Can you dedicate a specific time for doing exercise or physical activity on most days of the week?. 3. Can you do exercise or physical activity even when your family or friends ask you to do something else?. 4. Can you get up early to do your exercise or physical activity even on the weekends?. 5. Can you do your exercise or physical activity even when you have a lot of homework to do?. 6. Can you do your exercise or physical activity even when the weather is hot or it is rainy?. Family support of physical activity. 1. How many days a week do your family members watch your exercise or physical activity?. 2. How many days a week do your family members encourage you to do exercise or physical activity?. 3. How many days a week do your family members provide you transportation go to a place for doing exercise or physical activity?. 4. How many days a week do your family members do their exercise or physical activity with you?. Friend support of physical activity. 1. How many days a week do your friends encourage you to do exercise or physical activity?. 2. How many days a week do your friends do their exercise or physical activity with you?. 3. How many days a week do your friends tease you for doing well your exercise or physical activity?. 4. How many days a week do your friends ask you to walk or bike from your house to the school or their houses?. 5. How many days a week do your friends tell you that you are doing well your exercise or physical activity?. 6. How many of your 5 closest friends do their physical activity regularly?. Positive and negative of physical activity. The following sentences are different beliefs about physical activity. Please choose the degree which indicates the importance of each sentence to you whenever deciding whether or not to do your physical activity. 1. I would feel embarrassed if people saw me doing physical activity. 2. Physical activity helps me to keep fit. 3. My parents would become happy if I do physical activity. 4. I have to learn a lot of things to be able to do physical activity. 5. If I do physical activity I would feel better about myself. 6. I would need too much help from my parents to be able to do physical activity. 7. Physical activity and exercise makes me unpleasant feeling. 8. I would have fun doing physical activity or playing sports with my friends. 9. If I do physical activity I would have more energy. 10. Physical activity reduces the time I spend for being with my friends. The strategies to change physical activity. The followings are activities/thoughts, and feelings people use to help them change their physical activity. Think about yourself similar cases that you experience or have experienced during the past month then indicate HOW OFTEN you do each of the followings:. 1. I look for information about physical activity or sports. 2. I keep the account of my physical activity. 3. I find out the ways to overcome the obstacles of doing physical activity. 4. I think about the effect of facilities (such as having sports equipments at home or in the near by park) on the amount of my physical activity. 5. To be more physically active I keep things at home to remind me that. 6. I encourage myself for doing physical activity or exercise. 7. I do things to make physical activity more enjoyable. 8. I think about the benefits I get from the physical activity or exercise. 9. I think about the benefits of physical activity than its troubles. 10. I say to myself about the positive sides of physical activity. 11. When my physical activity plans are stopped for a while,. I tell myself I can start again and become active. 12. I have a friend or family member who encourages me to do physical activity. 13. I try different kinds of physical activity to have more options to choose from. 14. I set goals for doing physical activity. 15. I consider alternative plans to assure myself of having physical activity. Environmental factors of physical activity:. Define your attitude about your life environment and indicate how much do you agree with the following sentences?. 1. There is enough sports equipment at home to use for physical activity. 2. Walking or jogging is difficult around my house, because of traffic, lack of side walks, dogs, gangs, and so on. 3. It is possible to access playgrounds, parks or gyms near to my home for doing physical activity or exercise. 4. It is safe to walk or jog during the day around my home. Physical activity enjoyment. 1. I enjoy doing physical activity or exercise. Recreation choices of physical activity. 2. Which of the following activities do you usually choose to spend on your leisure time?. (1) I almost always choose the activities like watching T.V, studying, listening to music, or working with computer. (2)I usually most always choose the activities like watching T.V, studying, listening to music, or working with computer. (3) It is possible to choose active entertainments as much as inactive ones. (4) I usually choose the activities like riding, bike, skating, and games played, outside or active sports. (5) I almost always choose the activities like riding, bike, skating, and games played, outside or active sports.Final translation of psychosocial determinants of physical activity questionnaires. Translation and back translation procedure was used to develop culturally equivalent questionnaires. Click here for file


The purpose of this study was to use magnetic resonance (MR) imaging to track in vivo the transition from in situ neoplasia to invasive cancer in a transgenic mouse model of human cancer.Because of the small size of in situ (DCIS) and is considered a precursor of invasive tumors. Images were serially obtained from 10-21 weeks of age at 2-3 week intervals. MIN lesions were identified based on their morphology on MR images. Lesions were followed over time and several lesion features were measured including volume, growth rate and morphology. For those MIN lesions that progressed to invasive cancer the progression time was measured.MR images of 12 female C3(1) SV40 Tag mice that develop mammary intraepithelial neoplasia (MIN) were obtained. MIN is believed to be similar to human ductal carcinoma 3 with an average growth rate of -0.15 ± 0.66 week-1. Even though all mice were inbred to express the SV40 Tag transgene in the mammary epithelium and expected to develop invasive carcinoma, the individual MIN lesions took vastly different progression paths: (i) 9 lesions progressed to invasive tumors with an average progression time of 4.6 ± 1.9 weeks; (ii) 2 lesions regressed, i.e., were not detected on future images; and (iii) 5 were stable for over 8 weeks, and were demonstrated by a statistical model to represent indolent disease.Overall, 21 MIN lesions were initially detected at an average initial volume of 0.3 ± 0.2 mmin situ neoplasia to invasive carcinoma and provide image-based evidence that DCIS may be a non-obligate precursor lesion with highly variable outcomes. In addition, this study represents a first step towards developing methods of image acquisition for identifying radiological characteristics that might predict which in situ neoplasias will become invasive cancers and which are unlikely to progress.To our knowledge, the results reported here are the first measurements of the timescale and characteristics of progression from  DCIS is a heterogeneous disease in which neoplastic cells are confined by the basement membrane of lobuloductal or ductal lumen. Progression to invasive ductal carcinoma (IDC) is thought to occur by first degradation of the basement membrane, microinvasion of cancer cells into the surrounding stroma and growth of a solid tumor. The use of screening mammography has increased rates of detection of DCIS . C3. C310]. type (as defined above). Lesions within each region were grouped together (if necessary) and the following features were then evaluated: age at initial lesion detection (weeks), volume (mm3), further morphologic classification  and distance from the intramammary lymph node (mm).All image analysis was performed using software written in Interactive Data Language (IDL) . The images of mouse mammary glands were analyzed in a manner analogous to methods used when evaluating human breast images. In women, cancers are often assigned a location by dividing the breast in quadrants relative to the nipple: upper-outer, upper-inner, lower-outer and lower-inner. Cancers within a quadrant are usually grouped as one, and the worst pathology determines the overall diagnosis; in other words, an invasive cancer with nearby extensive DCIS is considered an invasive tumor. For the mice, we proceeded with a similar analysis Figure . The ingUsing the agar grid and lesion location relative to the intramammary lymph node, lesion development was followed over time. This could only be assessed in cases where the lesion had been imaged at least twice. Growth rate was calculated according to the following equation:V is lesion volume (mm3), t is the time in weeks, and α is the growth rate (per week). This was calculated separately for MIN (αMIN) and invasive tumors (αtumor). Changes in morphology as lesions developed were also examined separately for in situ and invasive tumors.- where Reproducibility of MR assessments of lesions type (as 'nonmass' or 'mass'), morphology descriptors and size was evaluated for three mice with imaging repeated under different conditions.prog (in weeks) was calculated: Tprog = age at initial detection of invasive tumor - age at initial detection of MIN. The average progression time μprog and the standard deviation of progression times σprog were then calculated. If an invasive tumor was not found in subsequent images, the latency time Tlat (in weeks) was determined: Tlat = age at final imaging session - age at initial detection of MIN. To determine whether some latent lesions truly represented non-progressing or 'indolent' disease, a threshold T0 was found so that MIN lesions with Tlat > T0 could be considered a biologically distinct class of 'indolent' lesion. Details of the statistical method used are in the following section. In addition, the prior images of all invasive tumors were evaluated to determine how many were preceded by MIN.Each MIN was followed over time to determine whether invasive cancer developed in that region in the future. Specifically, if any invasive tumor developed in the same region on subsequent images, the lesion was classified as having progressed and the progression time TMIN) were predictors of whether that MIN lesion would progress in the future to an invasive tumor. In addition, to test whether cancer progression is related to lesion location in the gland, the distance from the lymph node was also evaluated as a predictor. The average value of each of these parameters was calculated separately in both progressing and indolent MIN, and compared using the Student's t-test. Receiver operating curve (ROC) analysis  was performed to determine the diagnostic accuracy for each parameter in the task of distinguishing progression from indolent MIN. For each parameter, ROC analysis yielded area under the curve (Az) values that quantified diagnostic accuracy.We tested if a larger volume, earlier age at initial detection and a larger growth rate  lesions in which progression to invasive cancer was not observed, with a range of latency times Tlat. Our goal is to determine which lesions in the second group, if any, truly represent non-progressing or indolent disease. We begin first with the population of MIN lesions in which progression to invasive cancer was observed. We assume the progression time Tprog for each lesion is normally distributed N, and denote Nprog as the number of lesions in the progressing group. We next consider a subset of the latent MIN lesions with latency times Tlat longer than a threshold T0, and denote N0 the number of lesions with Tlat > T0. The probability that after randomly drawing n = Nprog+ N0 lesions from N, we have selected at most Nprog lesions with Tprog < T0 can be found using cumulative form of the binomial distribution, given by the following density function:We consider two groups of MIN: (i) lesions in which progression to invasive cancer was observed, with average time to progression Tp is the probability that one progressing lesion can have Tprog < T0  and k = Nprog. Thus, for each T0 we obtain a probability that lesions with Tlat > T0 are part of the progressing group.- where 3. MIN typically presented in a ductal or segmental shape and a homogenous pattern. Invasive tumors also developed predominantly in the mid (8/14) or lower (5/14) gland region. Tumors appeared at an average age of 16.3 ± 3.2 weeks at an initial volume at the time of detection of 17.2 ± 41.6 mm3. The latter value was skewed due to two tumors that presented initially at a very large volume (>120 mm3); excluding these two very large tumors reduced the average initial tumor volume to 1.96 ± 2.01 mm3. Both of the very rapidly growing large tumors were observed to have developed only three weeks after the in situ phase in the same animal. The typical invasive tumor morphology at initial detection was a round shape with smooth margins and a homogeneous pattern.Overall, 2 of 12 mice did not develop any cancerous lesion in the imaged inguinal glands. In the remaining 10 mice, a total of 21 MIN lesions developed. In eight of these mice a total of 14 invasive tumors developed . The lesion features at initial detection are summarized in Table MIN = -0.15 ± 0.66 weeks. Several lesions exhibited close to zero growth . Although the growth rates of invasive tumors varied considerably  are highly predictive but not conclusive proof of true disease stage, we proceeded to use image-based features to analyze in situ neoplastic progression. Based on image-based evidence alone, nine of 21 MIN lesions progressed into invasive cancers with an average progression time of Tprog = 4.6 ± 1.9 weeks . One of 21 MIN developed at the last imaging time point (21 weeks of age) and thus subsequent development was unknown. Nine of 14 invasive tumors were preceded by MIN that was detected by MR imaging; MIN was not detected in the prior images of four tumors, and one tumor was detected at the first imaging session (age 12 weeks) and thus prior history was unknown.With the caveat that image-based assessments of lesion histology  the invasive tumors that developed in the same region had an average invasive growth rate of αtumor = 0.36 ± 0.38 per week. In comparison, for the three MIN lesions with the highest growth rate (average αMIN = 0.31 ± 0.25 per week) the invasive tumors that developed in the same region had a higher average growth rate of αtumor = 0.83 ± 0.37 per week, although this difference was not significant (P = 0.19).We compared the growth rates of those MIN lesions that progressed to invasive cancer to the growth rates of the invasive tumors that subsequently developed in the same region. This was possible in six of the nine MIN lesions that progressed to invasive cancer. There was a tendency for increased MIN growth rate to be correlated with an increased growth rate of the subsequent invasive cancer; for the three MIN lesions with the lowest growth rates  SV40 Tag mice that can only be derived from repeated non-invasive imaging were measured. Significantly, we found that even in these mice that are genetically predisposed to develop invasive carcinoma, a substantial proportion of in situ cancers did not progress to invasive tumors within 21 weeks of monitoring. To our knowledge, these results provide the first detailed, high-resolution measurements of early mammary cancer natural history in mice. Our work complements work by Abbey and colleagues that investigated malignant transformation of in situ mammary cancer in a transplantable tissue model, using positron emission tomography (PET) to provide metabolic characterization at lower spatial resolution (5 mm3 voxel size compared with 0.0068 mm3 in our study) [We have used serial MR imaging to study the natural history of r study) ,16.In the present study, we measured changes in image-based features that are highly predictive of disease stage. As this was a serial imaging study, we could not perform histologic confirmation of the image-based findings. This underscores a significant challenge with noninvasive imaging of disease progression: it is very difficult to determine lesion histology without sacrificing the animal, thereby losing longitudinal information. With larger and more detailed sensitivity/specificity studies that correlate image-based features with a wide variety of histologic presentations, this limitation can be mitigated.in situ vs. invasive disease (via determination of lesion type) is reproducible, as are measurements related to size and morphology of early invasive tumors. However, in situ lesion morphology  may not be adequately reproducible. Further studies are needed to quantify reproducibility of MR measurements of early murine mammary cancer.If image-based measurements are to be robust predictors of disease stage, their reproducibility must be established. Our preliminary work suggests that assessment of in situ cancer regression, which if validated in larger numbers with detailed pathology correlation, would be direct demonstration of spontaneous breast cancer regression [in situ cancers, and none decreased in size or regressed. There was a trend for increased MIN growth rate to be a predictor of both the eventual development of invasive carcinoma, and a higher invasive tumor growth rate, in the same region. Unfortunately, in this pilot study the number of cases was too small to draw conclusions with statistical significance.The C3(1) SV40 Tag mouse model is being used for a wide variety of studies, ranging from evaluating effects of interventional and preventive therapies -30, to ugression -39. The It is important to note that our results pertain to this specific mouse model; it will be important to establish imaging techniques and assess similar characteristics in other mouse models of human breast cancer. If some features can be found that persist across mouse models, they may ultimately demonstrate applicability to human disease.in situ stage [in situ lesions in fact originate in lobules, and that a separate more aggressive disease representing a duct-forming invasive carcinoma is being incorrectly included with other in situ cancers [in situ cancers, such theories may be difficult to evaluate. Our work and extensions thereof, for example the use of dynamic contrast-enhanced MR imaging to probe changes in vasculature, can provide detailed and direct measurements of tumorigenesis on which these mathematical and physiologic models of disease initiation and progression can be evaluated.The natural history of breast cancer is still an open question, and there are many theories of the mechanisms governing the growth and progression of early breast cancers in women. The 'angiogenic switch' is thought to be a crucial step during breast tumorigenesis, and has been hypothesized to occur at or before the tu stage . Franks tu stage -43. Taba cancers ,45. Due There are several limitations to this study. First, lesion morphology was assessed using 2D axial slices rather than a 3D rendering of the inguinal glands. This could compromise the assessment of lesion morphology, particularly of MIN located in the lower gland area.Second, there may have been some errors in lesion identification. Although the descriptors of 'nonmass' and 'mass' are highly specific to MIN and invasive tumors, respectively, the 'mass' descriptor is not perfectly correlated  implyingThird, the numbers of lesions studied was rather small, limiting the statistical significance of our findings. To address this, both an increased number of mice should be imaged as well as an increased number of mammary glands in each mouse . In this way, cancer development can be assessed in the whole mouse, and data can be analyzed to determine whether lesions in the same animal can be considered as independent.Fourth, in this study we did not consistently perform end-point histologic evaluation of imaged mammary glands.in situ cancer progression.Fifth, mice were followed to 20 or 21 weeks of age rather than until natural death, so that studies of cancer progression could not be performed past this age. Although the range of cancer development in this model spans 10 to 24 weeks of age, and most female mice must be sacrificed at this time due to increased tumor burden in the mammary glands, additional time points should be acquired to definitively assess Sixth, changes in the parenchyma that preceded the development of MIN could not be easily observed because of the poor signal-to-noise ratio of the normal tissue. Recent improvements in imaging methods have provided greatly enhanced images of normal parenchyma, opening up the possibility of studying changes in the normal mammary glandular tissue that are precursors to cancer development.Finally, the new framework we have presented for analyzing early carcinogenesis in mice may need improvement. For example, the number of MIN lesions that progressed to invasive tumors may have been over-estimated. Our criterion was only that an invasive tumor appeared in the same region on subsequent imaging; however, this tumor may have been independent of the original MIN detected previously. The transition from MIN to invasive tumors was rarely observed directly. In addition, cancer growth rates could only be calculated for lesions imaged at least twice, that is, 15 of 21 MIN and 8 of 14 invasive tumors. The remaining lesions were excluded from any analysis of growth rates, which may have introduced a bias. These two limitations could be addressed by conducting serial imaging at higher frequency  so that each MIN lesion can be definitively linked with its subsequent invasive phase, and so that growth rates can be measured for all. Lastly, the statistical model we used to identify indolent lesions could most likely be improved or modified.In prior work, we introduced MR imaging methods for imaging early murine mammary neoplasias and invasive cancers, and subsequently reported on how those techniques could be used to better interpret clinical MR imaging of the breast . Here, win situ mammary cancers did not progress to invasive cancers, and investigated potential predictive markers of progression. This pilot study represents a first step towards detailed studies of functional and morphologic characteristics of mammary tumorigenesis, and developing methods for image acquisition and analysis that can predict which in situ cancers will become invasive and which would not. Such investigations would have an important impact on clinical management of patients with DCIS.We have used longitudinal noninvasive imaging to gain new insights into the natural history of early mammary cancer in the C3(1) SV40 Tag mouse model of human breast cancer. We found image-based evidence that some in situ; GRE: gradient recalled echo; IDC: invasive ductal carcinoma; IDL: Interactive Data Language; MIN: mammary intraepithelial neoplasia; MR: magnetic resonance; PET: positron emission tomography; ROC: receiver operating curve; SV40 Tag: simian virus 40 large T antigen.Az: area under the curve; BI-RADS: Breast Imaging Reporting and Data System; DCIS: Ductal carcinoma The authors declare that they have no competing interests.SJ conceived of the study and experiment design, conducted the imaging experiments, performed the data analysis and drafted the manuscript. SC participated in conception and design of the study and provided transgenic mice for imaging. XF helped to draft the manuscript and perform data analysis. EJ is the veterinary technician that participated in all imaging experiments. GM helped conceive and design the study. GK conceived of the study and experiment design, and participated in its coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


To assess whether an expanded (five level) risk stratification system can be used to identify the sub-group of intermediate risk patients with prostate cancer who benefit from combining androgen deprivation therapy (ADT) with external beam radiotherapy (EBRT).Using a previously validated 5-risk group schema, a prospective non-randomized data set of 1423 men treated at the British Columbia Cancer Agency was assessed for the primary end point of biochemical control (bNED) with the RTOG-ASTRO "Phoenix" definition (lowest PSA to date + 2 ng/mL), both with and without adjuvant ADT. The median follow-up was 5 years.There was no bNED benefit for ADT in the low or low intermediate groups but there was a statistically significant bNED benefit in the high intermediate, high and extreme risk groups. The 5-year bNED rates with and without ADT were 70% and 73% respectively for the low intermediate group (p = non-significant) and 72% and 58% respectively for the high intermediate group (p = 0.002).There appears to be no advantage to ADT where the Gleason score is 6 or less and PSA is 15 or less. ADT is beneficial in patients treated to standard dose radiation with Gleason 6 disease and a PSA greater than 15 or where the Gleason score is 7 or higher. Androgen deprivation therapy (ADT) has a proven role in the treatment of metastatic prostate cancer. Some groups of patients undergoing external beam radiotherapy (EBRT) for localized prostate cancer also benefit from adjuvant ADT. An EORTC trial randomized patients with T1–2 high grade or T3–4 N0–1 prostate cancer to either radiotherapy alone or with 3 years of ADT and showed an improved overall survival at 5 years[Sub-division of prostate cancer patients into risk groups can be used to guide management decisions based on their risk of relapse. The National Comprehensive Cancer Network (NCCN) classifies three risk groups: Low risk , Intermediate risk (T2b-T2c or Gleason score 7 or PSA 10.1–20 ng/ml) and High risk (≥ T3a or Gleason 8–10 or PSA > 20 ng/ml) . HoweverA prospective non-randomized patient data set was analyzed, comprising 1583 men treated with EBRT between 1994 and 2001 identified from the Prostate Cohort Outcomes Initiative Database of the British Columbia Cancer Agency (BCCA). After exclusions descried below, 1423 men were available for analysis. All patients received radical EBRT with photon irradiation and CT planning. Those treated with hypofractionated radiation  were excluded, as were those who had neoadjuvant ADT of duration <2 months or >12 months, n = 24. Three-dimensional conformal radiotherapy was used from 1998. The median dose administered was 66 Gy (range 66 – 72 Gy) in 2 Gy fractions. Most patients were treated with small volumes to the prostate alone but 177 patients also had a first phase with whole pelvic radiotherapy. Three patients enrolled in a study of ADT versus ADT and EBRT  were excluded.Patients with higher risk cancers were often selected for combined therapy with neoadjuvant ADT and radiation, but there was no formal policy governing this until 1997. Additionally, because of waiting lists in the early 1990's men with lower risk cancers were also given ADT. Prior to 1997, ADT was delivered by a combination of low-dose stilboestrol (0.1 mg) and cyproterone acetate (50 mg), which has been shown to provide castrate levels of testosterone , the BCCGenerally, patients were seen every 6 months for 3 years, then annually for 3 years, then every 2 years. At each visit clinical examination, PSA and testosterone assays, and toxicity were scored. Follow-up is timed from the completion of radiation therapy. Data for all patients was entered prospectively into the database. Additional PSA results from other sources, such as general practice requests, were also incorporated into the database. Institutional ethics review boards approval was obtained for this study.The standard risk stratification schema was that of the National Comprehensive Cancer Network (NCCN) with three levels: high risk – those with either PSA > 20 ng/mL or T stage T3 or more, or Gleason score 8–10; low risk – those with a PSA <10 ng/mL and T2a or less stage, and GS 6 or less; intermediate risk – all those not high or low risk. Our five level investigational risk schema is shown in Table The primary endpoint for the study is the absence of biochemical evidence of disease (bNED), which has been shown to be an independent predictor of overall survival when radiotherapy alone has been used to treat prostate cancer  and is fComparison of patient characteristics was made using chi-squared tests for categorical data and Kruskal-Wallis tests for non-parametric tests. Time to end-point events was derived using the Kaplan-Meier technique with corresponding log-rank tests of significance. Patients were censored at the time of last clinical follow-up. P values of <0.05 were considered significant.The median follow-up was 5 years for biochemical status, (range 1 month – 11 years) and 7.7 years for survival, range 3 months – 12 years). Patient characteristics by expanded and NCCN risk groups are shown in table The Kaplan-Meier survival curves for biochemical control by NCCN risk groups are presented in figure With the expanded 5-risk grouping significant differences in bNED was seen in the high-intermediate, high and extreme risk groups but not the low and low-intermediate risk groups figure . For theThe use of androgen deprivation therapy in combination with EBRT has increased substantially over recent years, with, for example, data from the Cancer of the Prostate Strategic Urologic Research Endeavor (CAPSURE) in the United States suggesting that the rate of use has increased from 9.8% to 74.6% between 1989–1990 and 2000–2001. This isTo date, accurately identifying which patients will benefit from the addition of ADT to EBRT has been difficult, due to inter-trial differences in factors such as the stage and grade of the patients, dose and volume of radiation and the duration and timing of ADT. In terms of the tumour characteristics, most early studies have focused on patients with locally advanced disease. The Radiation Therapy Oncology Group (RTOG) 86–10 study  randomizPossibly more relevant to contemporary cohorts, the trial by D'Amico et al  randomizLogically, the duration of ADT has since become the focus in a number of studies. A Canadian study which included T1c-T4 tumors, compared 3 and 8 months neo-adjuvant ADT and showed improvements in cause-specific survival in a high risk sub-set with longer ADT durations, according to a recent oral update of a prior publication . The RTONo significant effect on cancer specific or overall survival was able to be demonstrated. This may be because the follow-up is too short and/or it may reflect the relatively high short-term efficacy of salvage ADT. Another possible explanation could be that the ADT duration for the high and extreme risk groups was insufficient. Prolonging the duration of ADT appears to benefit patients with a higher chance of relapse following EBRT ,19. A prThe study presented here used prospectively collected data but a valid criticism of the comparison made is that it is not a randomized trial. There was likely case selection between those patients who received ADT and those who did not. A further issue is that the doses of radiation that were used, while typical for the era, are below contemporary levels. Whether or not dose-escalation beyond the doses used in the era of this study (66–70 Gy) would obviate any benefit of ADT in higher risk cancers is currently unknown, although subject to ongoing randomized trials. Improved bNED rates seen with escalated doses of radiation would suggest that the benefit might be less than that achieved with ADT. For example the absolute improvement in Phoenix bNED with 78 Gy versus 68 Gy in the Dutch randomized trial was 6% , which iADT often causes significant side effects. During therapy, patients can suffer hot flashes, weight gain, gynaecomastia, impotence, loss of libido, fatigue and depression ,23. LongOverall, trials looking at the combination of ADT and EBRT have shown the greatest benefits for patients with locally advanced or high-grade tumors. This is reflected by our practice, in that higher risk patients were more likely to receive ADT. This was also the case in another retrospective review published recently . The benThis analysis divides the intermediate risk patients with localized prostate cancer and identifies the sub-groups who do, and do not obtain a bNED benefit from ADT. There appears to be no advantage to ADT where the Gleason score is 6 or less and PSA is 15 or less. ADT provides a bNED benefit in patients treated to standard dose radiation with Gleason 6 disease with a PSA greater than 15 or where the Gleason score is higher than 6.The authors declare that they have no competing interests.MB participated in the design of the study, checked data integrity, carried out the analyses and drafted the manuscript. SGW developed the new risk group system, participated in the design of the study and drafts of the manuscript. TP conceived of the study, co-developed the prospective data sources, participated in the design of the study and manuscript drafts. All authors have read and approved the final manuscript.


Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in calpain 3 (CAPN3). Calpain 3 plays different roles in muscular cells, but little is known about its functions or in vivo substrates. The aim of this study was to identify the genes showing an altered expression in LGMD2A patients and the possible pathways they are implicated in. Ten muscle samples from LGMD2A patients with in which molecular diagnosis was ascertained were investigated using array technology to analyze gene expression profiling as compared to ten normal muscle samples. Upregulated genes were mostly those related to extracellular matrix (different collagens), cell adhesion (fibronectin), muscle development (myosins and melusin) and signal transduction. It is therefore suggested that different proteins located or participating in the costameric region are implicated in processes regulated by calpain 3 during skeletal muscle development. Genes participating in the ubiquitin proteasome degradation pathway were found to be deregulated in LGMD2A patients, suggesting that regulation of this pathway may be under the control of calpain 3 activity. As frizzled-related protein (FRZB) is upregulated in LGMD2A muscle samples, it could be hypothesized that β-catenin regulation is also altered at the Wnt signaling pathway, leading to an incorrect myogenesis. Conversely, expression of most transcription factor genes was downregulated . Finally, the upregulation of IL-32 and immunoglobulin genes may induce the eosinophil chemoattraction explaining the inflammatory findings observed in presymptomatic stages. The obtained results try to shed some light on identification of novel therapeutic targets for limb-girdle muscular dystrophies. It has been reported to play different roles in the cell. Calpain 3 has a certain role in direct and indirect regulation of conventional calpains by proteolytic degradation of calpains and calpastatin respectively Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in calpain 3 (CAPN3), a muscle-specific, calcium-dependent cystein protease. Calpain 3 structure is similar to that of the ubiquitous calpains 1 and 2, but calpain 3 has specific regions  that confer it special characteristics such as autocatalytic and nuclear translocation capacity. Although calpain 3 was identified in 1989 Calpain 3 was shown to be in complex with dysferlin, suggesting a membrane homeostasis role of calpain 3 in vitro and suggested that FLNC may be an in vivo substrate for calpain 3, functioning to regulate protein-protein interactions with sarcoglycans. Thus, calpain-mediated remodeling of cytoskeletal-membrane interactions, such as those occurring during myoblast fusion and muscle repair, may involve regulation of FLNC-sarcoglycan interactions On the other hand, it has been confirmed that calpain 3 can cleave the C-terminal portion of FLNC Its presence in the nucleus has led to suggest that calpain 3 plays an important role in regulation of transcription factors indirectly controlling apoptotic processes Based on the observation of the C3 knockout (C3KO) mice, it has been suggested that calpain 3 is necessary for ubiquitination and acts upstream of the ubiquitination machinery Inflammatory cells have been detected in muscle tissue from patients with mutations in the CAPN3 gene in early stages In this study, the RNA expression profiling in muscle from biopsies of LGMD2A patients and control subjects were compared in order to determine the potential functions and the pathways in which calpain 3 is implicated.Muscle biopsies were taken from 10 LGMD2A patients  and 10 controls . Two out of the 10 LGMD2A patients showed an inflammatory pattern with eosinophilic infiltrates in their biopsies.For diagnostic purposes deltoid, quadriceps, and biceps muscle specimens were collected using institutionally approved protocols and after obtaining informed consent . Muscle The quality of all RNAs obtained from muscle biopsies  was verified using spectrophotometry and the Bioanalyzer system (Agilent). All of them showed acceptable quality and integrity (RIN above 7) to be eligible for the experiment.in vitro transcription . Fragmented cRNA of each sample was hybridized individually to human HG-U133A (22.283 probe sets) and HG-U133B (22.645 probe sets) GeneChips  in order to analyze the expression of 44.928 probes, comprising more than 33.000 genes.All RNAs were reverse-transcribed, and biotinylated cRNA probes were generated by In-depth quality controls were performed to analyze the validity of the hybridization processes in accordance with four criteria. First, the correct presence of the signal corresponding to the spike control BioB. Second, the expression ratio between the 3′ and 5′ ends of the housekeeping GAPDH should not exceed a value of three. Third, the full percentage of presences detected by the Affymetrix Detection algorithm for each array must be in the range 40–60. And finally, the percentage of outlier probe sets detected within each microarray should be less than 5%. All hybridized arrays on the study met all four quality criteria, demonstrating the reliability of data generated.The hybridized arrays were scanned, and raw data were extracted using the Microarray Analysis Suite 5.0 . The raw data were normalized using RMA (Robust Multichip Average) expression summary in Bioconductor The sensitivity of microarray-generated data to noise from experimental variables is well documented First, in order to identify significantly different genes between LGMD2A patients and normal controls, a geometric fold-change analysis was used t-test was applied to the unaffected control data set vs. the LGMD2A data set. The use of p-values implies hypothesis testing. It is assumed in the null hypothesis that there is no fold change and then evidence was looked for to reject it using a type-1 error. The threshold was set at p 0.001.Second, Class Comparison Difference Analyses were performed using BRB-ArrayTools developed by Dr. Richard Simon and BRB-ArrayTools Development Team. In order to identify probe sets with significant intensity differences between disease classes, a two-sample univariate To minimize false positives, only the probe sets commonly yielded by both methodologies were included into the final list of genes differentially expressed in LGMD2A.Moreover, as an additional supporting process, two machine learning feature selection techniques were run. Symmetrical uncertainty ranking The same procedure was used to compare samples from patients who were asymptomatic but had eosinophilic infiltrates in their muscle biopsies (2 cases) and samples from healthy controls (10 cases).Microarray data have been submitted to the GEO (Gene Expression Omnibus) public database (accession GSE11681).To investigate the validity of array data, expression levels of the differentially expressed genes were measured using the TaqMan quantitative RT-PCR assay. Relative expression levels initially determined with the cDNA microarrays were correlated to the expression levels assessed using quantitative RT-PCR for each patient sample.Whereas microarrays identify target genes of interest among thousands of genes, truly quantitative information relies on quantitative RT-PCR. Some of the significantly regulated changes found on the microarray could be replicated by quantitative RT-PCR. Quantitative RT-PCR was performed using the 7900 HT Fast Real-Time PCR System (Applied Biosystems). Because of the limiting RNA amount isolated from muscle biopsies used for microarray analysis, only a few samples (6 cases) were used for confirmation with quantitative RT-PCR experiments.The TaqMan Low Density Arrays (TLDA) were purchased from Applied Biosystems, and the protocol recommended by the manufacturer was used. Customer-designed TLDAs were used in order to test a series of 63 genes. In order to select these genes, genes with unknown function, hypothetical proteins, and open reading frame regions were excluded. Gene families were represented including only some of the members, such as collagens, etc. Moreover, genes showing differential expression profiling in the comparison between patients with eosinophilic infiltrates and healthy controls were included in the TLDA, as well as genes with expression variation in other studies.Expression levels for all transcripts were determined relative to the internal housekeeping control gene GAPDH in the TLDAs which, as expected, did not demonstrate altered expression according to microarray analysis.In order to identify probe sets with significant intensity differences, the method applied to the unaffected control data set vs. the LGMD2A data set was Benjamini-Hochberg method using Stat Miner program (Integromics).After having adjusted the background, normalized and summarized the data, the fold change obtained generates a list by magnitude of response. As a result of this method, the fold change analysis identified 156 differentially expressed probe sets in LGMD2A skeletal muscle compared to control skeletal muscle. Of these, 92 were significantly overexpressed and 64 showed a reduced expression in LGMD2A patients compared to the unaffected controls.PCA grouped together on the one hand patient samples and on the other hand control samples and a greater variability was seen among controls due to the heterogeneity of this group .t-test to the unaffected control data set vs. the LGMD2A data set. This method identified 627 probe sets with a p value higher than 0.001.On the other hand, the additional statistical method used to analyze the data, the Class Comparison Differences method, applied a two-sample univariate www.ncbi.nlm.nih.gov/LocusLink/): extracellular matrix proteins/phosphate transport, cell adhesion, muscle development, transcription factors, signaling pathways, metabolic process, transport, ubiquitin cycle, and other functions.However, the final list of genes comprised 86 probe sets (74 genes) commonly yielded by the two methodologies which were differentially expressed in LGMD2A compared to unaffected muscle biopsies. Of these 74 genes, 53 were overexpressed and 21 had a reduced expression in the LGMD2A patients and all the genes were clustered into functional groups . TranscrThe additional supporting process, the Correlation-based Feature Subset selection (CFS) Some transcript classes were of particular interest in this analysis . Most geAs a whole, in most of the biologically classified processes, the same trend to up- or downregulation was seen for all genes involved in the same process. Genes associated with extracellular matrix , cell adhesion, muscle development , signaling pathways, and ubiquitin cycle predominated among upregulated genes. However, all genes involved in metabolic processes and transcription factors (except for the E2F8 gene) were downregulated .On the other hand, upregulation of IGF1, which is a regulator of somatic growth and cell proliferation, was seen in this study. IGFa is an inducer of different pathways such as the phosphatidylinositol 3-kinase survival , the calcineurin.mediated signaling pathways, and of GATA2 activation.HERC1 and ANAPC1 are genes implicated in the ubiquitin cycle and showed upregulation in LGMD2A muscle samples. HERC1, ubiquitously expressed, is located in the cytosol in the Golgi apparatus, stimulating guanine nucleotide, forming a cytosolic ternary complex with clathrin and Hsp70, and is involved in protein trafficking. ANAPC1 is a component of the anaphase promoting complex/cyclosome (APC/C), a cell cycle-regulated E3 ubiquitin ligase that controls progression through mitosis and the G1 phase of the cell cycle.There are two deregulated genes according to our results whose cell location is the mitochondrion matrix, one of which is involved in the metabolic process, ALDH2  (downregulated), while the other, the PPM2C gene  (upregulated) is implicated in protein amino acid dephosphorylation.Twenty four out of the 74 deregulated genes with altered expression in LGMD2A were also deregulated in other muscular dystrophies  A comparison was made between biopsies of control muscles (10 cases) and biopsies from two cases showing eosinophil infiltrates. Results of this comparison are summarized in Genes involved in immune response such as IL-32, IGHG1, and IGKC were upregulated, as well as CAPN6, while those involved in chemotaxis or regulation of the protein kinase B signaling cascade were downregulated in asymptomatic cases comparing with controls.The main correlation between the two assays is showed in The main correlation between the two assays, microarrays and quantitative RT-PCR, was high for all genes, indicating a good agreement between both assays for identification of deregulated genes.On the other hand, by means of the additional supporting process, a high correlation degree among results was shown and it provided more reliability to the final list of genes. Therefore, results of the two machine learning approaches support the degree of relevance of the 74 genes identified.Calpain 3 was not abnormally regulated in the microarray study (data not shown). While protein analysis usually shows an absence of protein in patients, the microarray data did not reveal a reduction of calpain 3 mRNA indicating that this primary genetic defect cannot be identified by expression profiling. It is worthwhile mentioning that the presence of different missense mutations in most of the patients may explain this observation as well as the variability found in the Western Blot including normal patterns In LGMD2A muscles, genes associated with ECM/membrane-related, cell adhesion genes, muscle development genes, signaling pathway genes, and ubiquitin cycle genes were upregulated .The general trend for structural genes to be expressed at higher levels in patients could reflect a general upregulation of structural genes in the mutant muscle, as previously reported for other types of muscular dystrophies It is interesting to note that a large proportion of genes associated to the extracellular matrix were probably upregulated as a result of fibrotic infiltration. These genes include extracellular matrix proteins such as collagen types I and III (the two major collagens in the ECM), cell adhesion proteins such as CD9, CD44, and fibronectin.SPARC, overexpressed in the fibroblasts of skin biopsy specimens obtained from patients with systemic sclerosis Our results showed that MYH3  is highly upregulated in samples from LGMD2A patients. Expression of embryonic myosin heavy chain is a hallmark of muscle regeneration after birth and a characteristic marker of human muscular dystrophies. During normal human development, expression is restricted to the embryonic period of development in vivo substrates for CAPN3. Identification of MLC1 as a potential substrate for CAPN3 was of interest because, in a previous study, Kramerova et al Another upregulated gene is myosin light chain 1 slow A (MLC1SA), a transcriptional regulator promoting muscle cell proliferation expressed in both slow-twitch skeletal muscle and non-muscle tissue. This gene showed a high individual correlation with class phenotype (0.442), and was one of the seven genes included in the CFS output. This fact flawlessly demonstrates its importance not only from an individual point of view, but also because of its potential interactions. MLC1SA is one of the two phosphorylable regulatory light chains forming the myosin complex. Cohen et al LGMD2A patients showed upregulation of the IGF-1 gene as previously observed in other muscular dystrophies. Normal skeletal muscle is able to efficiently repair itself in response to injury. IGF-1 has been implicated as a factor that may affect many steps in gene expression control, including cell proliferation, differentiation, and degradation processes. IGF-1 is a peptide that has been shown to have anabolic effects on muscle cells. This action can be explained based on the molecular signaling events initiated by its receptor, a tyrosine kinase activated on IGF-1 binding, and transmitted through a cascade of intracellular events, leading to a general increase in protein synthesis in vitro differentiation of the C2C12 murine myogenic cell line, and is regulated during in vivo skeletal muscle development Integrin β1 binding protein (ITGB1BP2 = melusin), also upregulated in LGMD2A muscles, is present in a costamere-like pattern consisting of two rows flanking a-actinin at Z line. Melusin expression is upregulated during It is still unclear whether CAPN3 directly cleaves proteins to make them available for ubiquitination or whether the effect of CAPN3 is indirect (i.e. through regulation of other proteins involved in ubiquitination) Moreover, Ono et al As ubiquitination tags proteins for degradation, decreased ubiquitination may lead to excessive accumulation of the proteins that should otherwise be degraded. This in turn could trigger a cell stress response, one manifestation of which is upregulation of heat shock proteins The protein coded by HERC1, upregulated in LGMD2A patients, has a C-terminal HECT (homologous to E6-AP C-terminus) domain, which suggests that it has an ubiquitin ligase activity.β-catenin plays a critical role in many cellular and morphogenic processes by performing two distinct functions: in the nucleus, it acts as a mandatory coactivator of TCF/LEF transcription factors in response to Wnt signaling during both embryonic development and adult muscle regeneration, while at the cell membrane, β-catenin associates with the cadherin complex that links adhesion molecules to the cytoskeleton. In both cases, the concentration of β-catenin has been shown to be tightly regulated through ubiquitin-mediated degradation Two distinct ubiquitin ligase complexes control β-catenin levels in cytoplasm and at the membrane On the other hand, frizzled-related protein (FRZB) is upregulated in LGMD2A muscle samples. It could therefore be hypothesized that β-catenin regulation is also altered at the Wnt signaling pathway, leading to an abnormal myotube fusion or incorrect myogenesis.in vivo substrate for calpain 3 In our results, the mitochondrial genes found to be deregulated were ALDH2 and PPM2C. ALDH2 was downregulated in patient samples and is implicated in the glycolysis/gluconeogenesis pathway. On the contrary, expression of the PPM2C mitochondrial gene was upregulated in our study. Protein phosphatase 1J  was found to be an In later stages of the disease, the muscle pathology is characterized mainly by the presence of lobulated fibers (LF), which are composed of misaligned myofibrils that form a lobular pattern, in addition to fiber size variation and interstitial fibrosis. Lobulated muscle fibers reflect an abnormal spatial distribution of the intermyofibrillar mitochondria network mRNA expression profiles were specifically altered in LGMD2A muscles with lobulated fibers Keira et al According to these results, identification of these mitochondrial proteins suggests that CAPN3 may be involved in mitochondrial protein turnover.According to In muscles from patients with Duchenne muscular dystrophy, upregulated genes were mostly those related to immune response, sarcomeric, ECM, and cell growth, whereas downregulated genes were associated to energy metabolism, transcription/translation, signaling, and proteasomes c-fos and c-jun proteins have been described as showing strong cytoplasmic expression related to the degeneration process occurring in Duchenne and Becker muscular dystrophies in vivo. Since genes coding for proteins in this pathway are dysregulated in LGMD2A patients, it may be suggested that the downregulation of c-jun and other transcription factors observed in LGMD2A patients are regulated in an indirect way by calpain 3.Recently Gan et al TFRC (transferrin receptor) and VLDLR (very low density lipoprotein receptor) are upregulated in LGMD2A patients as occurred in FSHD muscles According to the available information, it could be suggested that FSHD shared the greatest quantity of differentially expressed genes and the deregulation tendencies (up/downregulation) are the same and in a similar range of variation. However it would be difficult to establish any correlation given that in the FSHD, even in patients with the same deletion fragment, high variability of impairment and of muscle affectation grade is observed. Therefore, the data depend enormously on the place and on the moment that biopsy has been taken.S100A6  and S100A8 , dysregulated in LGMD2A muscles, are involved in various intracellular and extracellular regulatory activities Dysferlin was not abnormally regulated in LGMD2A patients in the microarray study. The value obtained for the expression of the DYSF gene did not fulfil the established criteria to be considered as differently expressed (data not shown).In this study mRNA levels are analysed, not protein quantities. Even if the protein is reduced in the Western Blot, this reduction may not be regulated at a transcriptional level, it may happen at a post-translation level. Since calpain 3 was shown to be in complex with dysferlin and it has been demonstrated that AHNAK, a novel component of the dysferlin protein complex, serves as a direct substrate of calpain 3 in cell culture, the lack of one of these proteins may justify the reduction of the other.Dysf mice, the microarray data did not reveal a reduction of dysferlin mRNA indicating that this primary genetic defect cannot be identified by expression profiling As described in previous works The vast majority of upregulated genes in Fukuyama-type congenital muscular dystrophy (FCMD) and laminin-a2 deficient congenital muscular dystrophy (MDC1A) encode extracellular matrix components, presumably related to fibrotic change. However, mature muscle components were extremely downregulated in congenital muscular dystrophies Muscle regeneration is also a process that depends on the skeletal muscle basement membrane. Basement membrane is thought to not only maintain cell integrity but also to mediate signal transmission in cell differentiation, growth, attachment, survival, polarity, proliferation, and apoptosis Costameric proteins can interact with many components of both the sarcolemma and cytoskeleton. Different publications support a role for the costamere/Z-disk axis in mechanotransduction, the dynamic process through which mechanical stimuli are sensed by muscle cells and converted into biochemical responses Based on our results and since collagens, melusin and fibronectin, were deregulated, we may hypothesize that upregulation of ECM genes found in LGMD2A patients may result from signal transduction defects due to basement membrane dysfunction. Calpain 3 recognizes a wide range of substrates, including cytoskeletal proteins and myofibrillar proteins Presence of eosinophilic cells has recently been detected in muscle tissue from patients with mutations in the CAPN3 gene In our study, IL-32 was upregulated in LGMD2A patients with eosinophilic infiltrates . AlthougEosinophilia has also been reported as a prominent feature of the necrotic phase in dystrophin-deficient mdx mice. This study suggested that eosinophilia was promoted by at least perforin-dependent cytotoxicity of effector T cells and T-cell production of interleukin-5 (IL-5) Inflammatory features may be seen in some muscular dystrophies, such as facioscapulohumeral muscular dystrophy Although the comparison between gene expression profiles between LGMD2A with and without eosinophilia would be interesting, it was not possible to perform. The methods used needed a higher quantity of samples to obtain significant results. Moreover, when a PCA plot was performed for LGMD2A patients only (including cases with and without eosinophilia), no different groups were created and this may be due to the low sample number too.It seems that the comparison between asymptomatic patients with eosinophilia with control samples is more indicated to shed some light onto the initial mechanism that triggers the eosinophilic cell attraction to muscle. The comparison between asymptomatic patients and controls allows a clearer view due to a lower interfering expression variation.In a first approach asymptomatic cases were considered as affected and were included into the patient group in the general analysis. These cases were included in the affected group due to their abnormal muscle biopsy pattern. Additionally reinforcing this decision, the PCA plots clustered together the LGMD2A case with or without eosinophilia.Finally in an additional analysis, however, it was decided to consider them as a different group compared to the controls in order to obtain information about eosinofilic attraction in the early stage of the disease.In conclusion, upregulated genes were mostly those related to extracellular matrix, cell adhesion, sarcomeric proteins, and signal transduction. It is therefore suggested that different proteins located at or participating in the costameric region are involved in processes regulated by calpain 3 during skeletal muscle development. Upregulation of these proteins may indicate a compensatory attempt of the muscle, and since most of these genes are also upregulated in other dystrophic processes, upregulation might be relatively nonspecific.It was also found that genes participating in the ubiquitin proteasome degradation pathway are deregulated in LGMD2A patients, which suggests that regulation of this pathway may be under the control of calpain 3 activity.Finally, the upregulation of IL-32 and immunoglobulin genes may cause the eosinophil chemoattraction observed in the inflammatory findings in presymptomatic stages. This upregulation seems to disappear when the disease progresses. However, they might be quite specific markers for the disease.Though samples taken from different muscles could add variability to the results of the expression array analysis, correlation of the results with the quantitative RT-PCR results gave strength to the findings. Gene expression profiling is presented as a useful approach to mine new data and hopefully open new perspectives for muscular disorders, shedding some light on identification of novel therapeutic targets for limb-girdle muscular dystrophies.Looking ahead, each of these methods should be individually analyzed in the animal model and in cell models.This analysis gives a total of 24 genes that may be considered as potential diagnostic or evolutionary biomarkers of the disease. However, this question will not be solved until the predictive value of these markers is proved in a series of patients with different evolutive status and secondly until the consistency of the results in different muscles and different laboratories is proved.


Cerebral oedema is associated with significant neurological damage in patients with traumatic brain injury. Bradykinin is an inflammatory mediator that may contribute to cerebral oedema by increasing the permeability of the blood-brain barrier. We evaluated the safety and effectiveness of the non-peptide bradykinin B2 receptor antagonist Anatibant in the treatment of patients with traumatic brain injury. During the course of the trial, funding was withdrawn by the sponsor.Adults with traumatic brain injury and a Glasgow Coma Scale score of 12 or less, who had a CT scan showing an intracranial abnormality consistent with trauma, and were within eight hours of their injury were randomly allocated to low, medium or high dose Anatibant or to placebo. Outcomes were Serious Adverse Events (SAE), mortality 15 days following injury and in-hospital morbidity assessed by the Glasgow Coma Scale (GCS), the Disability Rating Scale (DRS) and a modified version of the Oxford Handicap Scale (HIREOS).228 patients out of a planned sample size of 400 patients were randomised. The risk of experiencing one or more SAEs was 26.4% (43/163) in the combined Anatibant treated group, compared to 19.3% (11/57) in the placebo group . All cause mortality in the Anatibant treated group was 19% and in the placebo group 15.8% . The mean GCS at discharge was 12.48 in the Anatibant treated group and 13.0 in the placebo group. Mean DRS was 11.18 Anatibant versus 9.73 placebo, and mean HIREOS was 3.94 Anatibant versus 3.54 placebo. The differences between the mean levels for GCS, DRS and HIREOS in the Anatibant and placebo groups, when adjusted for baseline GCS, showed a non-significant trend for worse outcomes in all three measures.This trial did not reach the planned sample size of 400 patients and consequently, the study power to detect an increase in the risk of serious adverse events was reduced. This trial provides no reliable evidence of benefit or harm and a larger trial would be needed to establish safety and effectiveness.This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN23625128. Cerebral oedema is associated with significant mortality and morbidity after traumatic brain injury (TBI). It develops soon after injury, reaching a maximum between 3 and 5 days post injury . An incrBradykinin B2 receptor antagonists (BB2) block the activity of the kinin-kallikrein system, preventing the release of BK, and may reduce neuronal damage after TBI . A systeAnatibant, a non-peptide, selective BB2 antagonist, reduces brain oedema in animal TBI models -11. No sAnatibant inhibits the binding of BK to the B2 receptor. After subcutaneous injection Anatibant is bio-available and crosses the BBB. Pharmacokinetic (PK) studies show brain interstitial fluid levels in excess of the concentration required to produce half maximum inhibition at the B2 receptor. Anatibant has a plasma half-life after subcutaneous administration of 30 and 70 hours in healthy volunteers and TBI patients respectively and can be given as a once daily subcutaneous injection. Metabolism and excretion is mainly through hepatic and biliary routes. PK parameters are increased 1.5-2 fold in TBI patients compared to healthy volunteers, possibly due to decreased hepatic clearance ,12.Because plausible treatment effects in TBI are likely to be modest, we planned to conduct a large phase III trial with at least five thousand patients. As a prelude to this, we set out to conduct a phase II trial of 400 patients to assess safety and to inform the dose selection for the phase III trial.All collaborating investigators were required to secure the relevant ethics and regulatory approvals before recruitment could begin. Consent was obtained in accordance with the requirements of each ethics committee.All non-pregnant adults (ages 16 to 65 years inclusive) with a traumatic brain injury, who had a score on the Glasgow Coma Scale (GCS) of 12 or less (out of a maximum score of 15) and who had a CT scan showing an intracranial abnormality consistent with trauma, and were within eight hours of their injury, were eligible for trial entry if the responsible doctor considered there was the potential for them to benefit from the trial treatment. Patients known to have been treated with another investigational drug therapy within 30 days of injury were excluded.Patients were randomly allocated to receive a low (10 mg loading dose and 5 mg/day), medium (20 mg loading dose and 10 mg/day) or high (30 mg loading dose and 15 mg/day) dose of Anatibant or matching placebo. The loading dose was given as soon as possible and within 8 hours of injury and administered as two simultaneous subcutaneous injections of 5 mg, 10 mg and 15 mg respectively. The maintenance dose was given 24 hours after the loading dose and continued daily for 4 days.The primary study objective was to compare the proportion of patients with at least one SAE in those receiving Anatibant and those receiving placebo. The secondary study objectives were to assess the effect of Anatibant on early mortality and morbidity and to establish the dose to be used in the phase III trial. Samples for pharmacokinetic analyses were collected but these data are not reported here.A Serious Adverse Event (SAE) was defined as any untoward medical occurrence that was (1) fatal; (2) life threatening; (3) required or prolonged hospitalisation; (4) resulted in persistent or significant disability or incapacity; (5) medically significant in that it may jeopardise the patient and may require medical or surgical intervention to prevent one of the outcomes listed above; or (6) congenital anomalies. Medical occurrences that were expected in the course of traumatic brain injury, or which were known to occur during routine diagnostic or therapeutic procedures, were excluded. Any SAE occurring after the administration of the first dose of study medication and up to 15 days following injury was included. Mortality was assessed up to 15 days following the injury. In-hospital morbidity was assessed 15 days after injury or at withdrawal using the Glasgow Coma Scale (GCS), the Disability Rating Scale (DRS) and a modified version of the Oxford Handicap Scale which is referred to here as the Head Injury Related Early Outcome Score (HIREOS).We planned to randomise a total of 400 patients, 100 to each of the four groups. Assuming that the proportion of patients with at least one SAE would be 20% in the placebo group, the trial would have had 90% statistical power to detect a doubling of this proportion (to 40%) in the combined Anatibant groups using a 1-sided significance level of 5%. A 1-sided test was considered appropriate since a decision to not continue to a Phase III trial would only have been taken if the proportion of patients with at least one SAE had been higher in the combined Anatibant groups.Randomisation was done centrally by Sealed Envelope Ltd, UK, using an Interactive Voice Response System (IVRS). Initial patient information confirming eligibility was first collected via the IVRS. The central study computer then randomly assigned a treatment pack number corresponding to one of a number of blinded treatment packs available in the emergency department of the participating hospital. The information collected was used to achieve balance with respect to key prognostic factors using minimisation on the basis of the following variables: sex, age , time since injury: ; GCS: 3 to 5; 6 to 8; 9 to 12; and pupil reactivity .Active drug and placebo were visually matched and packed by an independent clinical trials supply company, Bilcare, UK. All study personnel and participants were to be blinded to treatment assignment for the duration of the study. Only the independent Data Safety Monitoring Board (DSMB) and their independent statistician were to see un-blinded data but none had any contact with study participants.All data analyses were carried out according to a pre-defined statistical analysis plan. Both intention-to-treat and per-protocol analyses were undertaken. As per the analysis plan the primary analyses reported here compare results in the combined Anatibant groups with those in the placebo group. Because the trial stopped early, formal statistical comparisons between dose groups, although specified in the analysis plan, are not reported here. In addition, for the updated meta-analysis of effect of Bradykinin B2 receptor antagonists on mortality after TBI, the relative risks (RR) and 95% confidence intervals (CIs) for mortality were calculated for each trial and pooled using the fixed-effect model.The proportion of patients with at least one SAE in the combined Anatibant groups was compared with that in the placebo group using a Fisher's exact test. The relative risk and 95% confidence interval were also calculated. The result was declared significant if the p-value was less than 0.05 (1-sided test).All cause mortality in the period up to and including 15 days post randomisation was compared using Fisher's exact test. GCS, DRS and HIREOS scores at 15 days post randomisation were compared using an analysis of covariance model to adjust for baseline GCS scores. Since these variables are not normally distributed, non-parametric bias-corrected and accelerated 95% bootstrap confidence intervals  were computed and used to infer statistical significance at the 5% level . No subgDuring the trial, interim analyses of all data including SAEs, mortality and morbidity were carried out by an independent DSMB. The DSMB had the responsibility for deciding whether, while recruitment was in progress, the un-blinded results (or the un-blinded results for a particular subgroup), should be revealed to the Trial Steering Committee (TSC). Because the trial was collecting data on a range of potential safety end points and it would have been impossible to specify in advance the potential adverse effects, there were no formal stopping rules. Instead, the DSMB was required to reveal the un-blinded results to the TSC if, taking into account both statistical and clinical issues and exercising their best clinical and statistical judgement, the un-blinded results provided sufficient evidence that the trial treatment was on balance harmful for all, or for a particular category of patients.Xytis Pharmaceutical Sarl (Switzerland) was legal sponsor and funder and was involved in the development of the protocol. Xytis had one non-voting representative on the TSC but did not contribute to this data analysis. The TSC provided overall supervision of the trial. In particular, the TSC concentrated on the progress of the trial, adherence to the protocol, patient safety and consideration of new information. The trial was coordinated and managed by the London School of Hygiene & Tropical Medicine (LSHTM).The first patient was enrolled in March 2007. On 1 November 2007, after reviewing un-blinded data on 140 patients, the DSMB disclosed limited un-blinded results to the TSC because of patient safety concerns. By that time a total of 228 patients had been randomised. The TSC suspended recruitment until a review of the data on all 228 patients could be undertaken by the DSMB. Presentation of this data to the DSMB was delayed due to a legal dispute between LSHTM and Xytis which was heard in the High Court of London in March 2008. The DSMB finally reviewed the data on 6 June 2008 and recommended that recruitment could recommence. After funding was withdrawn the trial was terminated.http://www.trialscoordinatingcentre.Lshtm.ac.uk/.In the course of the trial a number of protocol deviations occurred, including LSHTM not receiving all trial data and information on SAEs. LSHTM withdrew Clinical Research Associates' (CRA) access to the clinical trial database for a period of time when this occurred. In addition, the High Court of Justice in London granted an injunction preventing the TSC or the independent DSMB from making any determination or recommendation to terminate the trial. The Sponsor obtained the randomisation code from the company responsible for drug packing and conducted three un-blinded analyses on different data sets prior to the termination of the trial. In March 2008, by order of the High Court of Justice, all claims against the LSHTM were dismissed and the injunctions were discharged. All public domain documentation is available on the trial coordinating centre web-site The 228 patients randomised to Anatibant or placebo group Figure  up to 1 Both per protocol (PP) and intention to treat analyses (ITT) were conducted. PP analysis is presented only for the primary analysis (number and percentage of patients with at least one SAE). ITT analysis is presented for all other analyses since there were no material differences between them.p= 0.19). There were no SAEs suspected to be related to the study drug as judged by investigators.There were 43 26.4%) patients with at least one SAE within two weeks of randomisation among the 163 patients allocated Anatibant , compared with 11 (19.3%) among the 57 allocated placebo (Table 6.4% patip = 0.38).Vital status at two weeks following injury was known for 220 (96%) randomised patients. There were 31 (19.0%) deaths in those allocated Anatibant , compared with 9 (15.8%) among those allocated placebo (RR = 1.20 (0.61 to 2.36), 1 sided p > 0.05).The mean GCS in patients allocated Anatibant  was 12.48 compared with 9.73 among those allocated placebo (a lower GCS signifies worse outcome). Adjusting for baseline GCS score the difference in the means (δ) was -0.55 [95% bootstrap CI (-1.42 to 0.59)], a difference that was not statistically significant (p > 0.05) difference in the means (δ) was 1.61 [95% bootstrap CI (-1.16 to 4.23)].The mean DRS among patients allocated Anatibant  was 11.18 compared with 9.73 among those allocated placebo (a higher DRS signifies a worse outcome). Adjusting for baseline GCS score the  difference in the means (δ) was 0.42 [95% bootstrap CI (-0.08 to 0.86)].The mean HIREOS among patients allocated Anatibant  was 3.94 compared with 3.54 among those allocated placebo (a higher HIREOS signifies a worse outcome). Adjusting for baseline GCS score the , 11.7% developed erythema at an injection site that was classified as moderate, intense or very intense. There were no such reactions in the placebo group (1 sided P = 0.003).There was no emergency un-blinding of randomised patients.Our intention was to randomise 400 patients to this phase II trial of Anatibant in patients with traumatic brain injury. Had we done so, we would have had more than 90% power to detect a doubling in the number of patients with one or more SAEs among patients on active treatment. In fact, we were only able to randomise 228 patients and as a result the trial is underpowered. The statistically non-significant increase in the proportion of patients with at least one SAE that we observed is consistent with a sizeable increase or a modest reduction. We also observed statistically non-significant increases in mortality and morbidity.We conducted an international multi-centre randomised controlled trial of an emergency treatment for traumatic brain injury. Despite the well documented challenges of recruiting patients in emergency situations, patient recruitment was rapid and had it not been prematurely terminated the trial is likely to have reached its planned sample size of 400 patients. Allocation was well concealed through the use of central computerised telephone randomisation. Although the use of matching placebo helped to ensure that outcome assessment was blind to treatment allocation, the subcutaneous administration of Anatibant was associated with an increase in local skin reactions at the injection site which could have un-blinded treatment allocation in some cases. Skin reactions are unlikely to have biased assessment of all cause mortality, but the extent to which they might have biased the assessment of more subjective outcomes is open to question. There was minimal loss to follow up.Although the TSC temporarily stopped recruitment in response to patient safety concerns from the DSMB, when the data on all randomised patients were reviewed, it was recommended that the trial could continue. Stopping recruitment until the data on all randomised patients could be represented to the DSMB, ensured that patient safety was the prime consideration. It would be inappropriate to conclude that this temporary stop was unjustified, since even extreme differences in patient outcome can occur by chance alone.The primary outcome measure was the proportion of patients with one or more SAEs. In the context of critical illness however, the usual International Conference on Harmonisation Good Clinical Practice (ICH GCP) definition of an SAE  is problAlthough this is the largest trial of a Bradykinin B2 receptor antagonist in TBI conducted so far, it was small, and even in aggregate the total number of participants in clinical trials of B2 Receptor antagonists is less than 400 patients. The pooled relative risk for death from all trials combined is 0.84 (95% CI = 0.55 to 1.29) which is consistent with a modest increase or a modest decrease in the risk of death  were reported.The litigation delayed the conduct of the trial and incurred a considerable opportunity cost to the trial investigators. At a time when universities are encouraged to collaborate with industry in drug development the risk of litigation must be considered carefully.BBB: blood-brain barrier; BB2: Bradykinin B2; BK: Bradykinin; CT scan: Computerised Tomography of the head; CI: confidence interval; DSMB: Data Safety Monitoring Board; DRS: Disability Rating Scale; GCS: Glasgow Coma Scale; HIREOS: Head Injury Related Early Outcome Score; ITT: intention to treat; IVRS: interactive voice response system; PK: Pharmacokinetic; PP: per protocol; RR: relative risks; SAE: serious adverse event; TBI: traumatic brain injury; TSC: Trial Steering Committee.This trial was developed and coordinated by LSHTM and was commercially funded by Xytis. The Independent TSC and DSMB were reimbursed costs incurred for meetings. The funder (Xytis) was involved through the Protocol Committee in the design of the trial. The collection of data was done by the collaborators listed and payments made were based on a per patient schedule. Monitoring of the trial data at site was done by monitors employed by the funder. The funder had view of the manuscript prior to submission and their comments were reviewed and accepted or rejected by the TSC.The factual contents of this paper and the details provided about the clinical trial reported therein have not been verified by BioMed Central Limited ('the publisher') its agents or employees. This article has been published in accordance with this journal's obligation to report all clinical trials, in the public interest. No liability is accepted by the publisher, its agents or employees in connection with the contents of this paper.The trial was designed by HS and IR and the protocol was developed by the Protocol Committee and reviewed and accepted by the Trial Steering Committee. OF and CF were responsible for the data analysis. All authors were involved in the interpretation of the data and revised the manuscript critically for important intellectual content. All authors read and approved the submitted manuscript.