Antibodies against insulin measured by electrochemiluminescence predicts insulitis severity and disease onset in non-obese diabetic mice and can distinguish human type 1 diabetes status

Abstract Background The detection of insulin autoantibodies (IAA) aids in the prediction of autoimmune diabetes development. However, the long-standing, gold standard 125I-insulin radiobinding assay (RBA) has low reproducibility between laboratories, long sample processing times and requires the use of newly synthesized radiolabeled insulin for each set of assays. Therefore, a rapid, non-radioactive, and reproducible assay is highly desirable. Methods We have developed electrochemiluminescence (ECL)-based assays that fulfill these criteria in the measurement of IAA and anti-insulin antibodies (IA) in non-obese diabetic (NOD) mice and in type 1 diabetic individuals, respectively. Using the murine IAA ECL assay, we examined the correlation between IAA, histopathological insulitis, and blood glucose in a cohort of female NOD mice from 4 up to 36 weeks of age. We developed a human IA ECL assay that we compared to conventional RBA and validated using samples from 34 diabetic and 59 non-diabetic individuals in three independent laboratories. Results Our ECL assays were rapid and sensitive with a broad dynamic range and low background. In the NOD mouse model, IAA levels measured by ECL were positively correlated with insulitis severity, and the values measured at 8-10 weeks of age were predictive of diabetes onset. Using human serum and plasma samples, our IA ECL assay yielded reproducible and accurate results with an average sensitivity of 84% at 95% specificity with no statistically significant difference between laboratories. Conclusions These novel, non-radioactive ECL-based assays should facilitate reliable and fast detection of antibodies to insulin and its precursors sera and plasma in a standardized manner between laboratories in both research and clinical settings. Our next step is to evaluate the human IA assay in the detection of IAA in prediabetic subjects or those at risk of type 1 diabetes and to develop similar assays for other autoantibodies that together are predictive for the diagnosis of this common disorder, in order to improve prediction and facilitate future therapeutic trials.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Mtss1 promotes cell-cell junction assembly and stability through the small GTPase Rac1

Cell-cell junctions are an integral part of epithelia and are often disrupted in cancer cells during epithelial-to-mesenchymal transition (EMT), which is a main driver of metastatic spread. We show here that Metastasis suppressor-1 (Mtss1; Missing in Metastasis, MIM), a member of the IMD-family of proteins, inhibits cell-cell junction disassembly in wound healing or HGF-induced scatter assays by enhancing cell-cell junction strength. Mtss1 not only makes cells more resistant to cell-cell junction disassembly, but also accelerates the kinetics of adherens junction assembly. Mtss1 drives enhanced junction formation specifically by elevating Rac-GTP. Lastly, we show that Mtss1 depletion reduces recruitment of F-actin at cell-cell junctions. We thus propose that Mtss1 promotes Rac1 activation and actin recruitment driving junction maintenance. We suggest that the observed loss of Mtss1 in cancers may compromise junction stability and thus promote EMT and metastasis

Deletion of Nlrp3 protects from inflammation-induced skeletal muscle atrophy

BACKGROUND: Critically ill patients develop atrophic muscle failure, which increases morbidity and mortality. Interleukin-1β (IL-1β) is activated early in sepsis. Whether IL-1β acts directly on muscle cells and whether its inhibition prevents atrophy is unknown. We aimed to investigate if IL-1β activation via the Nlrp3 inflammasome is involved in inflammation-induced atrophy. METHODS: We performed an experimental study and prospective animal trial. The effect of IL-1β on differentiated C2C12 muscle cells was investigated by analyzing gene-and-protein expression, and atrophy response. Polymicrobial sepsis was induced by cecum ligation and puncture surgery in Nlrp3 knockout and wild type mice. Skeletal muscle morphology, gene and protein expression, and atrophy markers were used to analyze the atrophy response. Immunostaining and reporter-gene assays showed that IL-1β signaling is contained and active in myocytes. RESULTS: Immunostaining and reporter gene assays showed that IL-1β signaling is contained and active in myocytes. IL-1β increased Il6 and atrogene gene expression resulting in myocyte atrophy. Nlrp3 knockout mice showed reduced IL-1β serum levels in sepsis. As determined by muscle morphology, organ weights, gene expression, and protein content, muscle atrophy was attenuated in septic Nlrp3 knockout mice, compared to septic wild-type mice 96 h after surgery. CONCLUSIONS: IL-1β directly acts on myocytes to cause atrophy in sepsis. Inhibition of IL-1β activation by targeting Nlrp3 could be useful to prevent inflammation-induced muscle failure in critically ill patients

The academic backbone: longitudinal continuities in educational achievement from secondary school and medical school to MRCP(UK) and the specialist register in UK medical students and doctors

Background: Selection of medical students in the UK is still largely based on prior academic achievement, although doubts have been expressed as to whether performance in earlier life is predictive of outcomes later in medical school or post-graduate education. This study analyses data from five longitudinal studies of UK medical students and doctors from the early 1970s until the early 2000s. Two of the studies used the AH5, a group test of general intelligence (that is, intellectual aptitude). Sex and ethnic differences were also analyzed in light of the changing demographics of medical students over the past decades. Methods: Data from five cohort studies were available: the Westminster Study (began clinical studies from 1975 to 1982), the 1980, 1985, and 1990 cohort studies (entered medical school in 1981, 1986, and 1991), and the University College London Medical School (UCLMS) Cohort Study (entered clinical studies in 2005 and 2006). Different studies had different outcome measures, but most had performance on basic medical sciences and clinical examinations at medical school, performance in Membership of the Royal Colleges of Physicians (MRCP(UK)) examinations, and being on the General Medical Council Specialist Register. Results: Correlation matrices and path analyses are presented. There were robust correlations across different years at medical school, and medical school performance also predicted MRCP(UK) performance and being on the GMC Specialist Register. A-levels correlated somewhat less with undergraduate and post-graduate performance, but there was restriction of range in entrants. General Certificate of Secondary Education (GCSE)/O-level results also predicted undergraduate and post-graduate outcomes, but less so than did A-level results, but there may be incremental validity for clinical and post-graduate performance. The AH5 had some significant correlations with outcome, but they were inconsistent. Sex and ethnicity also had predictive effects on measures of educational attainment, undergraduate, and post-graduate performance. Women performed better in assessments but were less likely to be on the Specialist Register. Non-white participants generally underperformed in undergraduate and post-graduate assessments, but were equally likely to be on the Specialist Register. There was a suggestion of smaller ethnicity effects in earlier studies. Conclusions: The existence of the Academic Backbone concept is strongly supported, with attainment at secondary school predicting performance in undergraduate and post-graduate medical assessments, and the effects spanning many years. The Academic Backbone is conceptualized in terms of the development of more sophisticated underlying structures of knowledge ('cognitive capital’ and 'medical capital’). The Academic Backbone provides strong support for using measures of educational attainment, particularly A-levels, in student selection

“It’s hard to tell”. The challenges of scoring patients on standardised outcome measures by multidisciplinary teams: a case study of Neurorehabilitation

Background Interest is increasing in the application of standardised outcome measures in clinical practice. Measures designed for use in research may not be sufficiently precise to be used in monitoring individual patients. However, little is known about how clinicians and in particular, multidisciplinary teams, score patients using these measures. This paper explores the challenges faced by multidisciplinary teams in allocating scores on standardised outcome measures in clinical practice. Methods Qualitative case study of an inpatient neurorehabilitation team who routinely collected standardised outcome measures on their patients. Data were collected using non participant observation, fieldnotes and tape recordings of 16 multidisciplinary team meetings during which the measures were recited and scored. Eleven clinicians from a range of different professions were also interviewed. Data were analysed used grounded theory techniques. Results We identified a number of instances where scoring the patient was 'problematic'. In 'problematic' scoring, the scores were uncertain and subject to revision and adjustment. They sometimes required negotiation to agree on a shared understanding of concepts to be measured and the guidelines for scoring. Several factors gave rise to this problematic scoring. Team members' knowledge about patients' problems changed over time so that initial scores had to be revised or dismissed, creating an impression of deterioration when none had occurred. Patients had complex problems which could not easily be distinguished from each other and patients themselves varied in their ability to perform tasks over time and across different settings. Team members from different professions worked with patients in different ways and had different perspectives on patients' problems. This was particularly an issue in the scoring of concepts such as anxiety, depression, orientation, social integration and cognitive problems. Conclusion From a psychometric perspective these problems would raise questions about the validity, reliability and responsiveness of the scores. However, from a clinical perspective, such characteristics are an inherent part of clinical judgement and reasoning. It is important to highlight the challenges faced by multidisciplinary teams in scoring patients on standardised outcome measures but it would be unwarranted to conclude that such challenges imply that these measures should not be used in clinical practice for decision making about individual patients. However, our findings do raise some concerns about the use of such measures for performance management

Metastases: the glycan connection

An association between protein glycosylation and tumorigenesis has been recognized for over 10 years. Associations linking the importance of glycosylation events to tumor biology, especially the progression to metastatic disease, have been noted over many years, Recently, a mouse model in which β1,6-N-acetylglucosaminyltransferase V (a rate-limiting enzyme in the N-glycan pathway) has been knocked out, was used to demonstrate the importance of glycosylation in tumor progression. By crossing mice lacking this enzyme with a transgenic mouse model of metastatic breast cancer, metastatic progression of the disease was dramatically reduced. These experiments provide in vivo evidence for the role of N-linked glycosylation in metastatic breast cancer and have significant implications for the development of new treatment strategies

Construct-level predictive validity of educational attainment and intellectual aptitude tests in medical student selection: meta-regression of six UK longitudinal studies

Background: Measures used for medical student selection should predict future performance during training. A problem for any selection study is that predictor-outcome correlations are known only in those who have been selected, whereas selectors need to know how measures would predict in the entire pool of applicants. That problem of interpretation can be solved by calculating construct-level predictive validity, an estimate of true predictor-outcome correlation across the range of applicant abilities. Methods: Construct-level predictive validities were calculated in six cohort studies of medical student selection and training (student entry, 1972 to 2009) for a range of predictors, including A-levels, General Certificates of Secondary Education (GCSEs)/O-levels, and aptitude tests (AH5 and UK Clinical Aptitude Test (UKCAT)). Outcomes included undergraduate basic medical science and finals assessments, as well as postgraduate measures of Membership of the Royal Colleges of Physicians of the United Kingdom (MRCP(UK)) performance and entry in the Specialist Register. Construct-level predictive validity was calculated with the method of Hunter, Schmidt and Le (2006), adapted to correct for right-censorship of examination results due to grade inflation. Results: Meta-regression analyzed 57 separate predictor-outcome correlations (POCs) and construct-level predictive validities (CLPVs). Mean CLPVs are substantially higher (.450) than mean POCs (.171). Mean CLPVs for first-year examinations, were high for A-levels (.809; CI: .501 to .935), and lower for GCSEs/O-levels (.332; CI: .024 to .583) and UKCAT (mean = .245; CI: .207 to .276). A-levels had higher CLPVs for all undergraduate and postgraduate assessments than did GCSEs/O-levels and intellectual aptitude tests. CLPVs of educational attainment measures decline somewhat during training, but continue to predict postgraduate performance. Intellectual aptitude tests have lower CLPVs than A-levels or GCSEs/O-levels. Conclusions: Educational attainment has strong CLPVs for undergraduate and postgraduate performance, accounting for perhaps 65% of true variance in first year performance. Such CLPVs justify the use of educational attainment measure in selection, but also raise a key theoretical question concerning the remaining 35% of variance (and measurement error, range restriction and right-censorship have been taken into account). Just as in astrophysics, ‘dark matter’ and ‘dark energy’ are posited to balance various theoretical equations, so medical student selection must also have its ‘dark variance’, whose nature is not yet properly characterized, but explains a third of the variation in performance during training. Some variance probably relates to factors which are unpredictable at selection, such as illness or other life events, but some is probably also associated with factors such as personality, motivation or study skills

Can Score Databanks Help Teaching?

Basic courses in most medical schools assess students' performance by conferring scores. The objective of this work is to use a large score databank for the early identification of students with low performance and to identify course trends based on the mean of students' grades. METHODOLOGY/PRINCIPAL FINDINGS: We studied scores from 2,398 medical students registered in courses over a period of 10 years. Students in the first semester were grouped into those whose ratings remained in the lower quartile in two or more courses (low-performance) and students who had up to one course in the lower quartile (high-performance). ROC curves were built, aimed at the identification of a cut-off average score in the first semesters that would be able to predict low performances in future semesters. Moreover, to follow the long-term pattern of each course, the mean of all scores conferred in a semester was compared to the overall course mean obtained by averaging 10 years of data. Individuals in the low-performance group had a higher risk of being in the lower quartile of at least one course in the second semester (relative risk 3.907; 95% CI: 3.378-4.519) and in the eighth semester (relative risk 2.873; 95% CI: 2.495-3.308). The prediction analysis revealed that an average score of 7.188 in the first semester could identify students that presented scores below the lower quartiles in both the second and eighth semesters (p<0.0001 for both AUC). When scores conferred by single courses were compared over time, three time-trend patterns emerged: low variation, upward trend and erratic pattern. CONCLUSION/SIGNIFICANCE: An early identification of students with low performance may be useful in promoting pedagogical strategies for these individuals. Evaluation of the time trend of scores conferred by courses may help departments monitoring changes in personnel and methodology that may affect a student's performance

Downregulation of metastasis suppressor 1(MTSS1) is associated with nodal metastasis and poor outcome in Chinese patients with gastric cancer

<p>Abstract</p> <p>Background</p> <p>The putative tumor metastasis suppressor 1(MTSS1) is an actin-binding scaffold protein that has been implicated to play an important role in carcinogenesis and cancer metastasis, yet its role in the development of gastric cancer has not been well illustrated. In this study, we detected MTSS1 expression and explored its clinical significance in gastric cancer.</p> <p>Methods</p> <p>Immunohistochemistry was performed using tissue microarrays containing gastric adenocarcinoma specimens from 1,072 Chinese patients with normal adjacent mucosa, primary gastric cancer and lymph node (LN) metastasis and specific antibody against MTSS1. MTSS1 mRNA and protein expression were detected by reverse transcription-polymerase chain reaction and Western blotting. The clinical follow-up was done in the 669 patients living in Shanghai that was chose from the 1072 cases.</p> <p>Results</p> <p>Complete loss of MTSS1 expression was observed in 751 cases (70.1%) of the 1,072 primary tumors and 103 (88%) of 117 nodal metastases; and loss of MTSS1 expression was significantly associated with poorly differentiated tumors, large tumor size, deep invasion level, the presence of nodal metastases and advanced disease stage. Moreover, multivariate analysis demonstrated that loss of MTSS1 expression correlated significantly with poor survival rates (RR = 0.194, 95% CI = 0.144-0.261, P < 0.001).</p> <p>Conclusions</p> <p>MTSS1 expression decreased significantly as gastric cancer progressed and metastasized, suggesting MTSS1 may serve as a useful biomarker for the prediction of outcome of gastric cancer.</p

Immortalized, premalignant epithelial cell populations contain long-lived, label-retaining cells that asymmetrically divide and retain their template DNA

Abstract Introduction During selective segregation of DNA, a cell asymmetrically divides and retains its template DNA. Asymmetric division yields daughter cells whose genome reflects that of the parents, simultaneously protecting the parental cell from genetic errors that may occur during DNA replication. We hypothesized that long-lived epithelial cells are present in immortal, premalignant cell populations, undergo asymmetric division, retain their template DNA strands, and cycle both during allometric growth and during pregnancy. Methods The glands of 3-week-old immune-competent Balb/C female mice were used intact or cleared of host epithelium and implanted with ductal-limited, lobule-limited, or alveolar-ductal progenitor cells derived from COMMA-D1 pre-malignant epithelial cells. 5-Bromo-2-deoxyuridine (5-BrdU) was administered to identify those cells that retain their template DNA. Nulliparous mice were then either injected with [3H]-thymidine (3H-TdR) to distinguish 5-BrdU label-retaining cells that enter the cell cycle and euthanized, or mated, injected with 3H-TdR, and euthanized at various days after coitus. Sections were stained for estrogen receptor-&#945; (ER-&#945;) or progesterone receptor (PR) with immunohistochemistry. Cells labeled with both 5-BrdU and 3H-TdR were indicative of label-retaining epithelial cells (LRECs). Results Cells that retained a 5-BrdU label and cells labeled with [3H]-thymidine were found in all mice and were typically detected along the branching epithelium of mature mouse mammary glands. Cells containing double-labeled nuclei (LRECs) were found in the intact mammary glands of both pregnant and nulliparous mice, and in mammary glands implanted with premalignant cells. Double-labeled cells (3H-TdR/5-BrdU) represent a small portion of cells in the mammary gland that cycle and retain their template DNA (5-BrdU). Some label-retaining cells were also ER-&#945; or PR positive. LRECs distributed their second label (3H-TdR) to daughter cells, and this effect persisted during pregnancy. LRECs, and small focal hyperplasia, were found in all immortalized premalignant mammary-implant groups. Conclusions The results indicate that a subpopulation of long-lived, label-retaining epithelial cells (LRECs) is present in immortal premalignant cell populations. These LRECs persist during pregnancy, retain their original DNA, and a small percentage express ER-&#945; and PR. We speculate that LRECs in premalignant hyperplasia represent the long-lived (memory) cells that maintain these populations indefinitely.Peer Reviewe