APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death

p53 is a transcriptional activator which has been implicated as a key regulator of neuronal cell death after acute injury. We have shown previously that p53-mediated neuronal cell death involves a Bax-dependent activation of caspase 3; however, the transcriptional targets involved in the regulation of this process have not been identified. In the present study, we demonstrate that p53 directly upregulates Apaf1 transcription as a critical step in the induction of neuronal cell death. Using DNA microarray analysis of total RNA isolated from neurons undergoing p53-induced apoptosis a 5–6-fold upregulation of Apaf1 mRNA was detected. Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons. In both in vitro and in vivo neuronal cell death processes of p53-induced cell death, Apaf1 protein levels were increased. We addressed whether p53 directly regulates Apaf1 transcription via the two p53 consensus binding sites in the Apaf1 promoter. Electrophoretic mobility shift assays demonstrated p53–DNA binding activity at both p53 consensus binding sequences in extracts obtained from neurons undergoing p53-induced cell death, but not in healthy control cultures or when p53 or the p53 binding sites were inactivated by mutation. In transient transfections in a neuronal cell line with p53 and Apaf1 promoter–luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites. The importance of Apaf1 as a p53 target gene in neuronal cell death was evaluated by examining p53-induced apoptotic pathways in primary cultures of Apaf1-deficient neurons. Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates. Together, these results demonstrate that Apaf1 is a key transcriptional target for p53 that plays a pivotal role in the regulation of apoptosis after neuronal injury

Direct molecular mimicry enables off-target cardiovascular toxicity by an enhanced affinity TCR designed for cancer immunotherapy

Natural T-cell responses generally lack the potency to eradicate cancer. Enhanced affinity T-cell receptors (TCRs) provide an ideal approach to target cancer cells, with emerging clinical data showing significant promise. Nevertheless, the risk of off target reactivity remains a key concern, as exemplified in a recent clinical report describing fatal cardiac toxicity, following administration of MAGE-A3 specific TCR-engineered T-cells, mediated through cross-reactivity with an unrelated epitope from the Titin protein presented on cardiac tissue. Here, we investigated the structural mechanism enabling TCR cross-recognition of MAGE-A3 and Titin, and applied the resulting data to rationally design mutants with improved antigen discrimination, providing a proof-of-concept strategy for altering the fine specificity of a TCR towards an intended target antigen. This study represents the first example of direct molecular mimicry leading to clinically relevant fatal toxicity, mediated by a modified enhanced affinity TCR designed for cancer immunotherapy. Furthermore, these data demonstrate that self-antigens that are expressed at high levels on healthy tissue should be treated with extreme caution when designing immuno-therapeutics

Capturing complex tumour biology in vitro: Histological and molecular characterisation of precision cut slices

Precision-cut slices of in vivo tumours permit interrogation in vitro of heterogeneous cells from solid tumours together with their native microenvironment. They offer a low throughput but high content in vitro experimental platform. Using mouse models as surrogates for three common human solid tumours, we describe a standardised workflow for systematic comparison of tumour slice cultivation methods and a tissue microarray-based method to archive them. Cultivated slices were compared to their in vivo source tissue using immunohistochemical and transcriptional biomarkers, particularly of cellular stress. Mechanical slicing induced minimal stress. Cultivation of tumour slices required organotypic support materials and atmospheric oxygen for maintenance of integrity and was associated with significant temporal and loco-regional changes in protein expression, for example HIF-1α. We recommend adherence to the robust workflow described, with recognition of temporal-spatial changes in protein expression before interrogation of tumour slices by pharmacological or other means

Dynamic Imaging of CD8+ T Cells and Dendritic Cells during Infection with Toxoplasma gondii

To better understand the initiation of CD8+ T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8+ T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8+ T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8+ T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8+ T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis

The SITLESS project: Exercise referral schemes enhanced by self-management strategies to battle sedentary behaviour in older adults: Study protocol for a randomised controlled trial

Abstract Background Older adults are the fastest growing segment of the world‘s population. Recent evidence indicates that excessive sitting time is harmful to health, independent of meeting the recommended moderate to vigorous physical activity (PA) guidelines. The SITLESS project aims to determine whether exercise referral schemes (ERS) can be enhanced by self-management strategies (SMSs) to reduce sedentary behaviour (SB), increase PA and improve health, quality of life and function in the long term, as well as psychosocial outcomes in community-dwelling older European citizens from four countries, within a three-armed pragmatic randomised controlled trial, compared with ERS alone and also with general recommendations about PA. Methods A total of 1338 older adults will be included in this study, recruited from four European countries through different existing primary prevention pathways. Participants will be randomly allocated into an ERS of 16 weeks (32 sessions, 45–60 min per session), ERS enhanced by seven sessions of SMSs and four telephone prompts, or a control group. Outcomes will be assessed at baseline, month 4 (end of ERS intervention), month 16 (12 months post intervention) and month 22 (18 months post intervention). Primary outcomes will include measures of SB (time spent sedentary) and PA (counts per minute). Secondary outcomes will include muscle and physical function, health economics’ related outcomes, anthropometry, quality of life, social networks, anxiety and depressive symptoms, disability, fear of falling, executive function and fatigue. A process evaluation will be conducted throughout the trial. The full analysis set will follow an intention-to-treat principle and will include all randomised participants for whom a baseline assessment is conducted. The study hypothesis will be tested with mixed linear models with repeated measures, to assess changes in the main outcomes (SB and PA) over time (baseline to month 22) and between study arms. Discussion The findings of this study may help inform the design and implementation of more effective interventions to reduce SB and increase PA levels, and hence improve long-term health outcomes in the older adult population. SITLESS aims to support policy-makers in deciding how or whether ERS should be further implemented or restructured in order to increase its adherence, impact and cost-effectiveness. Trial registration ClinicalTrials.gov, NCT02629666 . Registered 19 November 2015

Cardiac troponin may be released by ischemia alone, without necrosis

Whilst it is formally stated that cardiac troponin is only released when cardiac myocytes undergo necrosis, there are a number of clinical situations where troponin is present in the circulation, without any apparent cardiac injury. In these cases, troponin half-life in the circulation is usually substantially shorter than that seen when troponin is released following myocardial infarction with frank necrosis. A mechanism has been described in liver, where large cytoplasmic molecules can pass from the intra- to extra-cellular space without cellular necrosis occurring. This occurs by the formation of membranous blebs which bud off from the plasma membrane of the cell. Blebs develop during cellular ischemia. If the ischemia is limited and re-oxygenation occurs, the blebs may be released into the circulation without rupture of the plasma membrane, resulting in a one-off release of cytoplasmic contents including macromolecules. Evidence from cardiac studies is presented supporting the presence of membranous blebs in cardiac myocytes, enabling troponin to be released from cardiac cells due to ischemia alone, without necrosis. Copyright © 2009 Elsevier B.V. All rights reserved

MyD88-dependent influx of monocytes and neutrophils impairs lymph node B cell responses to chikungunya virus infection via Irf5, Nos2 and Nox2

Author summary Elucidating mechanisms by which viruses subvert B cell immunity and establish persistent infection is essential for the development of new therapeutic strategies against chronic viral infections. The humoral immune response initiates in the lymph node draining the site of viral infection. However, how persistent viruses evade B cell responses is poorly understood. In this study, we find that infection with pathogenic, persistent chikungunya virus triggers rapid recruitment of neutrophils and monocytes to the draining lymph node, which impair structural organization, lymphocyte accumulation, and downstream virus-specific B cell responses that are important for control of infection. This work enhances our understanding of the pathogenesis of acute and chronic CHIKV disease and highlights how local innate immune responses in draining lymphoid tissue dictate the effectiveness of downstream adaptive immunity. Humoral immune responses initiate in the lymph node draining the site of viral infection (dLN). Some viruses subvert LN B cell activation; however, our knowledge of viral hindrance of B cell responses of important human pathogens is lacking. Here, we define mechanisms whereby chikungunya virus (CHIKV), a mosquito-transmitted RNA virus that causes outbreaks of acute and chronic arthritis in humans, hinders dLN antiviral B cell responses. Infection of WT mice with pathogenic, but not acutely cleared CHIKV, induced MyD88-dependent recruitment of monocytes and neutrophils to the dLN. Blocking this influx improved lymphocyte accumulation, dLN organization, and CHIKV-specific B cell responses. Both inducible nitric oxide synthase (iNOS) and the phagocyte NADPH oxidase (Nox2) contributed to impaired dLN organization and function. Infiltrating monocytes expressed iNOS through a local IRF5- and IFNAR1-dependent pathway that was partially TLR7-dependent. Together, our data suggest that pathogenic CHIKV triggers the influx and activation of monocytes and neutrophils in the dLN that impairs virus-specific B cell responses