Muscle injury induces an increase in total and non-rapid eye movement sleep time.

STUDY OBJECTIVES: This study describes macro- and micro-sleep responses to a myotoxic skeletal muscle injury and investigates possible mechanisms. METHODS: We recorded the electroencephalogram (EEG)/electromyogram (EMG) of 24 Wistar rats before and after induction of tibialis anterior muscle injury (n = 8 per group: control, control + buprenorphine and injured). A top-down analysis of sleep characteristics was processed from total sleep time (TST), sleep stages, sleep stability, spectral analysis, and spindles. To further investigate the mechanisms involved, we analyzed the protein level of sleep regulatory molecules including tumor necrosis factor- α (TNF-α), interleukin-1ß (IL-1ß), insulin-like growth factor-1 (IGF-1), and brain and muscle ARNT-like 1 (BMAL1) in plasma, frontal cortex, hippocampus, and tibialis anterior, collected at day +2 after injury from non-EEG/EMG implanted rats. RESULTS: Muscle injury induces a significant increase in TST at 48 and 72 h post-injury, specific to non-rapid eye movement (NREM) sleep. These increases occur during the dark period and are associated with the higher stability of sleep over 24 h, without change in the different power/frequency spectral bands of NREM/REM sleep. There was no corresponding sleep increase in slow-wave activity or spindle density, nor were there changes in brain levels of the sleep-regulating proinflammatory cytokine IL-1ß, which is otherwise involved in the local response to injury. Conversely, decreased protein levels of brain IGF-1 and muscle BMAL1, a core circadian clock gene, after injury may play a role in increased sleep time. CONCLUSION: Muscle injury induces an increase in total sleep time at 48- and 72-h post-injury, specific to NREM sleep during the dark period in rats and is associated with higher sleep stability over 24 h.

TREatment of ATopic eczema (TREAT) Registry Taskforce: protocol for a European safety study of dupilumab and other systemic therapies in patients with atopic eczema.

BACKGROUND: A long-term prospective observational safety study is essential to characterize fully the safety profile of systemic immunomodulating therapies for patients with atopic eczema. The TREatment of ATopic eczema (TREAT) Registry Taskforce offers a large platform to conduct such research using national registries that collect the same data using a predefined core dataset. OBJECTIVES: To present a protocol for a safety study comparing dupilumab with other systemic immunomodulating therapies in children and adults with moderate-to-severe atopic eczema, to assess the long-term safety risk of these therapies in a routine clinical care setting. METHODS: We describe a registry-embedded international observational prospective cohort study. Adult and paediatric patients who start treatment with dupilumab or another systemic immunomodulating agent for their atopic eczema will be included. The primary end point is the incidence of malignancies (excluding nonmelanoma skin cancer) compared between the treatment groups. Secondary end points include other serious adverse events and adverse events of special interest, such as eye disorders and eosinophilia. CONCLUSIONS: This protocol delineates a safety study for dupilumab in adult and paediatric patients with atopic eczema, using a standardized methodological approach across several national registries. The protocol could also be used for other novel systemic immunomodulating therapies, and could provide licensing and reimbursement authorities, pharmaceutical companies and clinicians with safety evidence from a routine clinical care setting. What's already known about this topic? There is a need for long-term data on the safety of systemic immunomodulating therapies in patients with atopic eczema. Regulatory bodies, such as the European Medicines Agency, increasingly stipulate the collection of such data as part of the licensing agreement for new treatments, to assess the new agent's long-term safety profile against established therapies. Large numbers of patients with a long duration of follow-up are necessary in order to detect rare events like malignancies. What does this study add? The TREAT Registry Taskforce offers a platform to conduct such research with a network of multiple national atopic eczema research registries. We present a protocol for an investigator-initiated multicentre safety study comparing dupilumab with other systemic immunomodulating therapies in adults and subsequently adolescents and children with moderate-to-severe atopic eczema. This protocol can be used as a framework for similar studies for other novel systemic immunomodulating therapies across both adult and paediatric populations.

Liver Stiffness at the Time of Sustained Virological Response Predicts the Clinical Outcome in People Living With Human Immunodeficiency Virus and Hepatitis C Virus With Advanced Fibrosis Treated With Direct-acting Antivirals.

BACKGROUND: Some people living with hepatitis C virus (HCV) with sustained virological response (SVR) develop hepatic complications. Liver stiffness (LS) predicts clinical outcome in people living with human immunodeficiency virus (HIV) with active HCV coinfection, but information after SVR is lacking. We aimed to analyze the predictive ability of LS at SVR for liver complications in people living with HIV/HCV with advanced fibrosis treated with direct-acting antivirals (DAA). METHODS: In sum, 640 people living with HIV/HCV fulfilling the following criteria were included: (i) Achieved SVR with DAA-including regimen; (ii) LS ≥ 9.5 kPa before therapy; and (iii) LS measurement available at SVR. The primary endpoint was the occurrence of a liver complication-hepatic decompensation or hepatocellular carcinoma (HCC)-or requiring liver transplant after SVR. RESULTS: During a median (Q1-Q3) follow-up of 31.6 (22.7-36.6) months, 19 (3%) patients reached the primary endpoint. In the multivariate analysis, variables (subhazard ratio [SHR] [95% confidence interval]) associated with developing clinical outcomes were: prior hepatic decompensations (3.42 [1.28-9.12]), pretreatment CPT class B or C (62.5 [3.08-1246.42]) and MELD scores (1.37 [1.03-1.82]), CPT class B or C at SVR (10.71 [1.32-87.01]), CD4 cell counts <200/µL at SVR time-point (4.42 [1.49-13.15]), FIB-4 index at SVR (1.39 [1.13-1.70]), and LS at SVR (1.05 [1.02-1.08] for 1 kPa increase). None of the 374 patients with LS <14kPa at SVR time-point developed a liver complication or required hepatic transplant. CONCLUSIONS: LS at the time of SVR after DAA therapy predicts the clinical outcome of people living with HIV/HCV with advanced fibrosis. These results suggest that LS measurement may be helpful to select candidates to be withdrawn from surveillance programs.

Publisher Correction: Barcoding reveals complex clonal behavior in patient-derived xenografts of metastatic triple negative breast cancer.

The original version of this Article contained an error in Fig. 4. In the left histogram of the right panel of Fig. 4d, several data points were inadvertently deleted from the histogram during the production process. This error has been corrected in both the PDF and HTML versions of the Article. The original, incorrect version of Fig. 4 is presented in the accompanying Publisher Correction.

Barcoding reveals complex clonal behavior in patient-derived xenografts of metastatic triple negative breast cancer.

Primary triple negative breast cancers (TNBC) are prone to dissemination but sub-clonal relationships between tumors and resulting metastases are poorly understood. Here we use cellular barcoding of two treatment-naïve TNBC patient-derived xenografts (PDXs) to track the spatio-temporal fate of thousands of barcoded clones in primary tumors, and their metastases. Tumor resection had a major impact on reducing clonal diversity in secondary sites, indicating that most disseminated tumor cells lacked the capacity to 'seed', hence originated from 'shedders' that did not persist. The few clones that continued to grow after resection i.e. 'seeders', did not correlate in frequency with their parental clones in primary tumors. Cisplatin treatment of one BRCA1-mutated PDX model to non-palpable levels had a surprisingly minor impact on clonal diversity in the relapsed tumor yet purged 50% of distal clones. Therefore, clonal features of shedding, seeding and drug resistance are important factors to consider for the design of therapeutic strategies.

Impact of interferon-free regimens on the glomerular filtration rate during treatment of chronic hepatitis C in a real-life cohort.

Little data are available on renal toxicity exerted by direct-acting antivirals (DAAs) in real life. The aim of this study was to assess the impact of direct-acting antivirals against hepatitis C virus infection currently used in Spain and Portugal on the estimated glomerular filtration rate (eGFR) in clinical practise. From an international, prospective multicohort study, patients treated with DAAs for at least 12 weeks and with eGFR ≥30 mL/min per 1.73 m2 at baseline were selected. eGFR was determined using the CKD-EPI formula. A total of 1131 patients were included; 658 (58%) were HIV/HCV-coinfected patients. Among the 901 patients treated for 12 weeks, median (interquartile range) eGFR was 100 (87-107) at baseline vs 97 (85-105) mL/min per 1.73 m2 at week 12 of follow-up (FU12) post-treatment (P < .001). For HIV-coinfected subjects who received tenofovir plus a ritonavir-boosted HIV protease inhibitor (PI/r), baseline vs FU12 eGFR were 104 (86-109) vs 104 (91-110) mL/min per 1.73 m2 (P = .913). Among subjects receiving ombitasvir/paritaprevir with or without dasabuvir, eGFR did not show any significant change. Of 1100 subjects with eGFR >60 mL/min per 1.73 m2 at baseline, 22 (2%) had eGFR <60 mL/min per 1.73 m2 at FU12, but none presented with eGFR <30 mL/min per 1.73 m2 . In conclusion, eGFR slightly declines during therapy with all-oral DAAs and this effect persists up to 12 weeks after stopping treatment in subjects with normal to moderately impaired renal function, regardless of HIV status. Concomitant use of tenofovir plus PI/r does not seem to have an impact on eGFR.

Self-Assembly and Anti-Amyloid Cytotoxicity Activity of Amyloid beta Peptide Derivatives.

The self-assembly of two derivatives of KLVFF, a fragment Aß(16-20) of the amyloid beta (Aß) peptide, is investigated and recovery of viability of neuroblastoma cells exposed to Aß (1-42) is observed at sub-stoichiometric peptide concentrations. Fluorescence assays show that NH2-KLVFF-CONH2 undergoes hydrophobic collapse and amyloid formation at the same critical aggregation concentration (cac). In contrast, NH2-K(Boc)LVFF-CONH2 undergoes hydrophobic collapse at a low concentration, followed by amyloid formation at a higher cac. These findings are supported by the ß-sheet features observed by FTIR. Electrospray ionization mass spectrometry indicates that NH2-K(Boc)LVFF-CONH2 forms a significant population of oligomeric species above the cac. Cryo-TEM, used together with SAXS to determine fibril dimensions, shows that the length and degree of twisting of peptide fibrils seem to be influenced by the net peptide charge. Grazing incidence X-ray scattering from thin peptide films shows features of ß-sheet ordering for both peptides, along with evidence for lamellar ordering of NH2-KLVFF-CONH2. This work provides a comprehensive picture of the aggregation properties of these two KLVFF derivatives and shows their utility, in unaggregated form, in restoring the viability of neuroblastoma cells against Aß-induced toxicity.

B-Cell-Activating Factor Levels Are Associated With Antibody-Mediated Histological Damage in Kidney Transplantation.

INTRODUCTION: Along with death engraftment, in recent years, antibody-mediated damage has been identified as the leading cause of loss of kidney transplants. Despite the recognition of the role of the B-lymphocyte subpopulation in the development of both tolerance and rejection, little is known about the trigger mechanisms and effectors of this humoral response. BACKGROUND: We analyzed the relationship between B lymphocyte subpopulations and levels of B-cell-activating factor (BAFF) with the histological findings in biopsies of renal transplantation. MATERIAL AND METHODS: We selected 35 patients whose kidney transplant biopsy was performed between January and November 2015. The biopsy specimens were classified according to Banff criteria. At the moment of the biopsy BAFF levels and B-lymphocyte subpopulations in blood were measured using enzyme-linked immunosorbent assay (ELISA) and using flow cytometry, respectively. RESULTS: Mean BAFF levels were 493 ± 245 pg/mL. The median performance of biopsy post-transplantation was 12.9 (11.7-23.9) months. BAFF levels correlated with pretransplantation antibodies (r = 0.523; P = .002) but not with kidney function. In biopsies performed more than 1 year after transplantation BAFF levels correlated with the severity of chronic glomerular (cg) involvement (r = 0.625; P = .003). Histological variables related to antibody-mediated injury selected by principal component analysis (glomerulitis, peritubular capillary, and chronic glomerulopathy) related to BAFF levels (B factor, 116; 95% confidence interval [CI], 12-220; P = .029). Biopsy specimens with transplant glomerulopathy (TG) showed lower levels of circulating naive CD19 + subpopulation, IgD+, and CD27- (32.7 ± 28.1 vs 87.9 ± 79.1; P = .017) compared with biopsy specimens without TG. CONCLUSIONS: Elevated levels of BAFF are associated with increased presence and severity of TG and a set of variables related to antibody-mediated histological damage. TG is associated with changes in circulating B-lymphocyte subpopulations that could contribute to its pathogenesis.

Targeting BCL-2 to enhance vulnerability to therapy in estrogen receptor-positive breast cancer.

The last three decades have seen significant progress in our understanding of the role of the pro-survival protein BCL-2 and its family members in apoptosis and cancer. BCL-2 and other pro-survival family members including Mcl-1 and BCL-XL have been shown to have a key role in keeping pro-apoptotic 'effector' proteins BAK and BAX in check. They also neutralize a group of 'sensor' proteins (such as BIM), which are triggered by cytotoxic stimuli such as chemotherapy. BCL-2 proteins therefore have a central role as guardians against apoptosis, helping cancer cells to evade cell death. More recently, an increasing number of BH3 mimetics, which bind and neutralize BCL-2 and/or its pro-survival relatives, have been developed. The utility of targeting BCL-2 in hematological malignancies has become evident in early-phase studies, with remarkable clinical responses seen in heavily pretreated patients. As BCL-2 is overexpressed in ~75% of breast cancer, there has been growing interest in determining whether this new class of drug could show similar promise in breast cancer. This review summarizes our current understanding of the role of BCL-2 and its family members in mammary gland development and breast cancer, recent progress in the development of new BH3 mimetics as well as their potential for targeting estrogen receptor-positive breast cancer.

Pro-apoptotic Bim suppresses breast tumor cell metastasis and is a target gene of SNAI2.

Evasion of cell death is fundamental to the development of cancer and its metastasis. The role of the BCL-2-mediated (intrinsic) apoptotic program in these processes remains poorly understood. Here we have investigated the relevance of the pro-apoptotic protein BIM to breast cancer progression using the MMTV-Polyoma middle-T (PyMT) transgenic model. BIM deficiency in PyMT females did not affect primary tumor growth, but substantially increased the survival of metastatic cells within the lung. These data reveal a role for BIM in the suppression of breast cancer metastasis. Intriguingly, we observed a striking correlation between the expression of BIM and the epithelial to mesenchymal transition transcription factor SNAI2 at the proliferative edge of the tumors. Overexpression and knockdown studies confirmed that these two genes were coordinately expressed, and chromatin immunoprecipitation analysis further revealed that Bim is a target of SNAI2. Taken together, our findings suggest that SNAI2-driven BIM-induced apoptosis may temper metastasis by governing the survival of disseminating breast tumor cells.

Impact of conditional deletion of the pro-apoptotic BCL-2 family member BIM in mice.

The pro-apoptotic BH3-only BCL-2 family member BIM is a critical determinant of hematopoietic cell development and homeostasis. It has been argued that the striking hematopoietic abnormalities of BIM-deficient mice (accumulation of lymphocytes and granulocytes) may be the result of the loss of the protein throughout the whole animal rather than a consequence intrinsic to the loss of BIM in hematopoietic cells. To address this issue and allow the deletion of BIM in specific cell types in future studies, we have developed a mouse strain with a conditional Bim allele as well as a new Cre transgenic strain, Vav-CreER, in which the tamoxifen-inducible CreER recombinase (fusion protein) is predominantly expressed in the hematopoietic system. We show that acute loss of BIM in the adult mouse rapidly results in the hematopoietic phenotypes previously observed in mice lacking BIM in all tissues. This includes changes in thymocyte subpopulations, increased white blood cell counts and resistance of lymphocytes to BIM-dependent apoptotic stimuli, such as cytokine deprivation. We have validated this novel conditional Bim knockout mouse model using established and newly developed CreER strains (Rosa26-CreER and Vav-CreER) and will make these exciting new tools for studies on cell death and cancer available.

Both leukaemic and normal peripheral B lymphoid cells are highly sensitive to the selective pharmacological inhibition of prosurvival Bcl-2 with ABT-199.

Overexpression of the prosurvival protein Bcl-2 marks many B-lymphoid malignancies and contributes to resistance to many commonly used chemotherapeutic agents. The first effective BH3 mimetic inhibitors of Bcl-2, ABT-737 and navitoclax, also target Bcl-xL, causing dose-limiting thrombocytopenia. This prompted the development of the Bcl-2-selective antagonist, ABT-199. Here we show that in lymphoid cells, ABT-199 specifically causes Bax/Bak-mediated apoptosis that is triggered principally by the initiator BH3-only protein Bim. As expected, malignant cells isolated from patients with chronic lymphocytic leukaemia are highly sensitive to ABT-199. However, we found that normal, untransformed mature B cells are also highly sensitive to ABT-199, both in vitro and in vivo. By contrast, the B-cell precursors are largely spared, as are cells of myeloid origin. These results pinpoint the probable impact of the pharmacological inhibition of Bcl-2 by ABT-199 on the normal mature haemopoietic cell lineages in patients, and have implications for monitoring during ABT-199 therapy as well as for the clinical utility of this very promising targeted agent.

Systematic review, meta-analysis and meta-regression of the effect of tranexamic acid on surgical blood loss.

BACKGROUND: Tranexamic acid (TXA) reduces blood transfusion in surgery but the extent of the reduction in blood loss and how it relates to the dose of TXA is unclear. METHODS: A systematic review of randomized trials was performed. Data were extracted on blood loss from trials comparing intravenous TXA with no TXA or placebo in surgical patients. A Bayesian linear regression was used to describe the relationship between the reduction in blood loss with TXA and the extent of bleeding as measured by the mean blood loss in the control group. A meta-analysis of the log-transformed data was conducted to quantify the effect of TXA on blood loss, stratified by type of surgery, timing of TXA administration and trial quality. Meta-regression was used to explore the effect of TXA dosage. RESULTS: Data from 104 trials were examined. Although the absolute reduction in blood loss with TXA increased as surgical bleeding increased, the percentage reduction was similar. TXA reduced blood loss by 34 per cent (pooled ratio 0·66, 95 per cent confidence interval 0·65 to 0·67; P < 0·001). The percentage reduction in blood loss with TXA differed by type of surgery, timing of TXA administration and trial quality, but the differences were small. The effect of TXA on blood loss did not vary over the range of doses assessed (5·5-300 mg/kg). CONCLUSION: TXA reduces blood loss in surgical patients by about one-third. A total dose of 1 g appears to be sufficient for most adults. There is no evidence to support the use of high doses.

Development and validation of a prognostic model to predict death in patients with traumatic bleeding, and evaluation of the effect of tranexamic acid on mortality according to baseline risk: a secondary analysis of a randomised controlled trial.

BACKGROUND: Severe bleeding accounts for about one-third of in-hospital trauma deaths. Patients with a high baseline risk of death have the most to gain from the use of life-saving treatments. An accurate and user-friendly prognostic model to predict mortality in bleeding trauma patients could assist doctors and paramedics in pre-hospital triage and could shorten the time to diagnostic and life-saving procedures such as surgery and tranexamic acid (TXA). OBJECTIVES: The aim of the study was to develop and validate a prognostic model for early mortality in patients with traumatic bleeding and to examine whether or not the effect of TXA on the risk of death and thrombotic events in bleeding adult trauma patients varies according to baseline risk. DESIGN: Multivariable logistic regression and risk-stratified analysis of a large international cohort of trauma patients. SETTING: Two hundred and seventy-four hospitals in 40 high-, medium- and low-income countries. PARTICIPANTS: We derived prognostic models in a large placebo-controlled trial of the effects of early administration of a short course of TXA [Clinical Randomisation of an Antifibrinolytic in Significant Haemorrhage (CRASH-2) trial]. The trial included 20,127 trauma patients with, or at risk of, significant bleeding, within 8 hours of injury. We externally validated the model on 14,220 selected trauma patients from the Trauma Audit and Research Network (TARN), which included mainly patients from the UK. We examined the effect of TXA on all-cause mortality, death due to bleeding and thrombotic events (fatal and non-fatal myocardial infarction, stroke, deep-vein thrombosis and pulmonary embolism) within risk strata in the CRASH-2 trial data set and we estimated the proportion of premature deaths averted by applying the odds ratio (OR) from the CRASH-2 trial to each of the risk strata in TARN. INTERVENTIONS: For the stratified analysis according baseline risk we considered the intervention TXA (1 g over 10 minutes followed by 1 g over 8 hours) or matching placebo. MAIN OUTCOME MEASURES: For the prognostic models we included predictors for death in hospital within 4 weeks of injury. For the stratified analysis we reported ORs for all causes of death, death due to bleeding, and fatal and non-fatal thrombotic events associated with the use of TXA according to baseline risk. RESULTS: A total of 3076 (15%) patients died in the CRASH-2 trial and 1705 (12%) in the TARN data set. Glasgow Coma Scale score, age and systolic blood pressure were the strongest predictors of mortality. Discrimination and calibration were satisfactory, with C-statistics > 0.80 in both CRASH-2 trial and TARN data sets. A simple chart was constructed to readily provide the probability of death at the point of care, while a web-based calculator is available for a more detailed risk assessment. TXA reduced all-cause mortality and death due to bleeding in each stratum of baseline risk. There was no evidence of heterogeneity in the effect of TXA on all-cause mortality (p-value for interaction = 0.96) or death due to bleeding (p= 0.98). There was a significant reduction in the odds of fatal and non-fatal thrombotic events with TXA (OR = 0.69, 95% confidence interval 0.53 to 0.89; p= 0.005). There was no evidence of heterogeneity in the effect of TXA on the risk of thrombotic events (p= 0.74). CONCLUSIONS: This prognostic model can be used to obtain valid predictions of mortality in patients with traumatic bleeding. TXA can be administered safely to a wide spectrum of bleeding trauma patients and should not be restricted to the most severely injured. Future research should evaluate whether or not the use of this prognostic model in clinical practice has an impact on the management and outcomes of trauma patients.

The CRASH-2 trial: a randomised controlled trial and economic evaluation of the effects of tranexamic acid on death, vascular occlusive events and transfusion requirement in bleeding trauma patients.

BACKGROUND: Among trauma patients who survive to reach hospital, exsanguination is a common cause of death. A widely practicable treatment that reduces blood loss after trauma could prevent thousands of premature deaths each year. The CRASH-2 trial aimed to determine the effect of the early administration of tranexamic acid on death and transfusion requirement in bleeding trauma patients. In addition, the effort of tranexamic acid on the risk of vascular occlusive events was assessed. OBJECTIVE: Tranexamic acid (TXA) reduces bleeding in patients undergoing elective surgery. We assessed the effects and cost-effectiveness of the early administration of a short course of TXA on death, vascular occlusive events and the receipt of blood transfusion in trauma patients. DESIGN: Randomised placebo-controlled trial and economic evaluation. Randomisation was balanced by centre, with an allocation sequence based on a block size of eight, generated with a computer random number generator. Both participants and study staff (site investigators and trial co-ordinating centre staff) were masked to treatment allocation. All analyses were by intention to treat. A Markov model was used to assess cost-effectiveness. The health outcome was the number of life-years (LYs) gained. Cost data were obtained from hospitals, the World Health Organization database and UK reference costs. Cost-effectiveness was measured in international dollars ($) per LY. Deterministic and probabilistic sensitivity analyses were performed to test the robustness of the results to model assumptions. SETTING: Two hundred and seventy-four hospitals in 40 countries. PARTICIPANTS: Adult trauma patients (n = 20,211) with, or at risk of, significant bleeding who were within 8 hours of injury. INTERVENTIONS: Tranexamic acid (loading dose 1 g over 10 minutes then infusion of 1 g over 8 hours) or matching placebo. MAIN OUTCOME MEASURES: The primary outcome was death in hospital within 4 weeks of injury, and was described with the following categories: bleeding, vascular occlusion (myocardial infarction, stroke and pulmonary embolism), multiorgan failure, head injury and other. RESULTS: Patients were allocated to TXA (n = 10,096) and to placebo (n = 10,115), of whom 10,060 and 10,067 patients, respectively, were analysed. All-cause mortality at 28 days was significantly reduced by TXA [1463 patients (14.5%) in the TXA group vs 1613 patients (16.0%) in the placebo group; relative risk (RR) 0.91; 95% confidence interval (CI) 0.85 to 0.97; p = 0.0035]. The risk of death due to bleeding was significantly reduced [489 patients (4.9%) died in the TXA group vs 574 patients (5.7%) in the placebo group; RR 0.85; 95% CI 0.76 to 0.96; p = 0.0077]. We recorded strong evidence that the effect of TXA on death due to bleeding varied according to the time from injury to treatment (test for interaction p < 0.0001). Early treatment (≤ 1 hour from injury) significantly reduced the risk of death due to bleeding [198 out of 3747 patients (5.3%) died in the TXA group vs 286 out of 3704 patients (7.7%) in the placebo group; RR 0.68; 95% CI 0.57 to 0.82; p < 0.0001]. Treatment given between 1 and 3 hours also reduced the risk of death due to bleeding [147 out of 3037 patients (4.8%) died in the TXA group vs 184 out of 2996 patients (6.1%) in the placebo group; RR 0.79; 95% CI 0.64 to 0.97; p = 0.03]. Treatment given after 3 hours seemed to increase the risk of death due to bleeding [144 out of 3272 patients (4.4%) died in the TXA group vs 103 out of 3362 patients (3.1%) in the placebo group; RR 1.44; 95% CI1.12 to 1.84; p = 0.004]. We recorded no evidence that the effect of TXA on death due to bleeding varied by systolic blood pressure, Glasgow Coma Scale score or type of injury. Administering TXA to bleeding trauma patients within 3 hours of injury saved an estimated 755 LYs per 1000 trauma patients in the UK. The cost of giving TXA to 1000 patients was estimated at $30,830. The incremental cost of giving TXA compared with not giving TXA was $48,002. The incremental cost per LY gained of administering TXA was $64. CONCLUSIONS: Early administration of TXA safely reduced the risk of death in bleeding trauma patients and is highly cost-effective. Treatment beyond 3 hours of injury is unlikely to be effective. Future work [the Clinical Randomisation of an Antifibrinolytic in Significant Head injury-3 (CRASH-3) trial] will evaluate the effectiveness and safety of TXA in the treatments of isolated traumatic brain injury (http://crash3.lshtm.ac.uk/). TRIAL REGISTRATION: Current Controlled Trials ISRCTN86750102, ClinicalTrials.gov NCT00375258 and South African Clinical Trial Register DOH-27-0607-1919. FUNDING: The project was funded by the Bupa Foundation, the J P Moulton Charitable Foundation and the NIHR Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 17, No. 10. See HTA programme website for further project information.

ASCORE: an up-to-date cardiovascular risk score for hypertensive patients reflecting contemporary clinical practice developed using the (ASCOT-BPLA) trial data.

A number of risk scores already exist to predict cardiovascular (CV) events. However, scores developed with data collected some time ago might not accurately predict the CV risk of contemporary hypertensive patients that benefit from more modern treatments and management. Using data from the randomised clinical trial Anglo-Scandinavian Cardiac Outcomes Trial-BPLA, with 15 955 hypertensive patients without previous CV disease receiving contemporary preventive CV management, we developed a new risk score predicting the 5-year risk of a first CV event (CV death, myocardial infarction or stroke). Cox proportional hazard models were used to develop a risk equation from baseline predictors. The final risk model (ASCORE) included age, sex, smoking, diabetes, previous blood pressure (BP) treatment, systolic BP, total cholesterol, high-density lipoprotein-cholesterol, fasting glucose and creatinine baseline variables. A simplified model (ASCORE-S) excluding laboratory variables was also derived. Both models showed very good internal validity. User-friendly integer score tables are reported for both models. Applying the latest Framingham risk score to our data significantly overpredicted the observed 5-year risk of the composite CV outcome. We conclude that risk scores derived using older databases (such as Framingham) may overestimate the CV risk of patients receiving current BP treatments; therefore, 'updated' risk scores are needed for current patients.

Trypanosoma cruzi discrete typing units in Chagas disease patients from endemic and non-endemic regions of Argentina.

Genetic diversity of Trypanosoma cruzi may play a role in pathogenesis of Chagas disease forms. Natural populations are classified into 6 Discrete Typing Units (DTUs) Tc I-VI with taxonomical status. This study aimed to identify T. cruzi DTUs in bloodstream and tissue samples of Argentinean patients with Chagas disease. PCR-based strategies allowed DTU identification in 256 clinical samples from 239 Argentinean patients. Tc V prevailed in blood from both asymptomatic and symptomatic cases and Tc I was more frequent in bloodstream, cardiac tissues and chagoma samples from immunosuppressed patients. Tc II and VI were identified in a minority of cases, while Tc III and Tc IV were not detected in the studied population. Interestingly, Tc I and Tc II/VI sequences were amplified from the same skin biopsy slice from a kidney transplant patient suffering Chagas disease reactivation. Further data also revealed the occurrence of mixed DTU populations in the human chronic infection. In conclusion, our findings provide evidence of the complexity of the dynamics of T. cruzi diversity in the natural history of human Chagas disease and allege the pathogenic role of DTUs I, II, V and VI in the studied population.

Alternative splicing of Bim and Erk-mediated Bim(EL) phosphorylation are dispensable for hematopoietic homeostasis in vivo.

The pro-apoptotic BH3-only protein Bim has a major role in hematopoietic homeostasis, particularly in the lymphocyte compartment, where it strongly affects immune function. The three major Bim isoforms (Bim(EL), Bim(L) and Bim(S)) are generated by alternative splicing. Bim(EL), the most abundant isoform, contains a unique sequence that has been reported to be the target of phosphorylation by several MAP kinases. In particular, Erk1/2 has been shown to interact with Bim(EL) through the DEF2 domain of Bim(EL) and specifically phosphorylate this isoform, thereby targeting it for ubiquitination and proteasomal degradation. To examine the physiological importance of this mechanism of regulation and of the alternative splicing of Bim, we have generated several Bim knock-in mouse strains and analyzed their hematopoietic system. Although mutation in the DEF2 domain reduces Bim(EL) degradation in some circumstances, this mutation did not significantly increase Bim's pro-apoptotic activity in vivo nor impact on the homeostasis of the hematopoietic system. We also show that Bim(EL) and Bim(L) are interchangeable, and that Bim(S) is dispensable for the function of Bim. Hence, we conclude that physiological regulation of Bim relies on mechanisms independent of its alternative splicing or the Erk-dependent phosphorylation of Bim(EL).

Bim must be able to engage all pro-survival Bcl-2 family members for efficient tumor suppression.

Overexpression of the transcriptional regulator Myc is thought to be the cause or a contributing factor in the development of a large number of human lymphomas and certain other cancers. Apoptotic cell death constitutes a tumor suppressive mechanism, particularly in the context of Myc overexpression. Accordingly, lymphoma development in Eµ-Myc transgenic mice, which mimic the Myc/IgH chromosomal translocation that causes Burkitt lymphoma, is accelerated by concomitant overexpression of anti-apoptotic Bcl-2 family members or loss of pro-apoptotic BH3-only proteins, such as Bim. Bim binds with high affinity to all pro-survival Bcl-2-like proteins and can also interact with Bax/Bak, but it remains unclear which of these interactions are critical for its tumor suppressive function. We have previously generated knock-in mutant mice in which the BH3 region of Bim has been exchanged with that for Bad, Noxa or Puma so that it can only bind to select pro-survival Bcl-2-like proteins: Bim(Bad) binding to Bcl-2, Bcl-x(L) and Bcl-w, but not Mcl-1 or A1; Bim(Noxa) binding only to Mcl-1 and A1 and as a control, Bim(Puma), which can still bind all pro-survival Bcl-2-like proteins. We have now inter-crossed these Bim mutant mice with Eµ-Myc transgenic mice, and found that both the Bim(Bad) and the Bim(Noxa) mutations but not the Bim(Puma) mutation greatly accelerate Myc-induced lymphoma development and increase leukemic burden. These results demonstrate that for optimal tumor suppressive activity, Bim must be able to interact with all and not just select pro-survival Bcl-2 family members.

Effect of one night of sleep loss on changes in tumor necrosis factor alpha (TNF-α) levels in healthy men.

Total sleep deprivation in humans is associated with increased daytime sleepiness, decreased performance, elevations in inflammatory cytokines, and hormonal/metabolic disturbances. To assess the effects of 40 h of total sleep deprivation (TSD) under constant and well controlled conditions, on plasma levels of TNF-α and its receptor (TNFR1), interleukin-6 (IL-6), cortisol and C-reactive protein (CRP), sleepiness and performance, 12 healthy men (29±3 years) participated in a 5-days sleep deprivation experiment (two control nights followed by a night of sleep loss and one recovery night). Between 0800 and 2300 (i.e. between 25 and 40 h of sleep deprivation), a serial of blood sampling, multiple sleep latency, subjective levels of sleepiness and reaction time tests were completed before (day 2: D2) and after (day 4: D4) one night of sleep loss. We showed that an acute sleep deprivation (i.e. after 34 and 37 h of sleep deprivation) induced a significant increase in TNF-α (P<0.01), but there were no significant changes in TNFR1, IL-6, cortisol and CRP. In conclusion, our study in which constant and controlled experimental conditions were realized with healthy subjects and in absence of psychological or physical stressors, an acute total sleep deprivation (from 34 h) was sufficient to induce secretion of pro-inflammatory cytokine such as TNF-α, a marker more described in chronic sleep restriction or deprivation and as mediators of excessive sleepiness in humans in pathological conditions.