PD-L1 Co-Stimulation Contributes to Ligand-Induced T Cell Receptor Down-Modulation on CD8+CD8^+ T Cells

T cell receptor (TCR) down-modulation after antigen presentation is a fundamental process that regulates TCR signal transduction. Current understanding of this process is that intrinsic TCR/CD28 signal transduction leads to TCR down-modulation. Here, we show that the interaction between programmed cell death 1 ligand 1 (PD-L1) on dendritic cells (DCs) and programmed death 1 (PD-1) on CD8 T cells contributes to ligand-induced TCR down-modulation. We provide evidence that this occurs via Casitas B-lymphoma (Cbl)-b E3 ubiquitin ligase up-regulation in CD8 T cells. Interference with PD-L1/PD-1 signalling markedly inhibits TCR down-modulation leading to hyper-activated, proliferative CD8 T cells as assessed in vitro and in vivo in an arthritis model. PD-L1 silencing accelerates anti-tumour immune responses and strongly potentiates DC anti-tumour capacities, when combined with mitogen-activated kinase (MAPK) modulators that promote DC activation

Towards integrated flood inundation modelling in groundwater-dominated catchments

Traditionally in flood inundation modelling the contribution of groundwater is either neglected or highly simplified. Long-duration groundwater-induced events, such as those that occur across Chalk catchments of northern Europe, can, however, incur significant economic and social cost. We present a new methodology for integrated flood inundation modelling by coupling the 2D hydrodynamic model LISFLOOD-FP with the 3D finite-difference groundwater model ZOOMQ3D. We apply the model to two adjacent Chalk catchments in southern England, the Lambourn and Pang, over two flooding events, during the winters of 2000/01 and 2013/14. A dense network of monitoring boreholes reveals local-scale heterogeneities in the aquifer not captured by the model. However, we demonstrate through inundation extent and streamflow comparisons that, on a regional scale, groundwater levels are simulated sufficiently well to capture groundwater inundation extent. The role of the unsaturated zone is discussed and contrasted between the two events. Currently, predictive tools to simulate groundwater flood events are limited, and this new, computationally efficient methodology will help to fill this gap

Atp2c2 Is Transcribed From a Unique Transcriptional Start Site in Mouse Pancreatic Acinar Cells

Proper regulation of cytosolic Ca2+ is critical for pancreatic acinar cell function. Disruptions in normal Ca2+ concentrations affect numerous cellular functions and are associated with pancreatitis. Membrane pumps and channels regulate cytosolic Ca2+ homeostasis by promoting rapid Ca2+ movement. Determining how expression of Ca2+ modulators is regulated and the cellular alterations that occur upon changes in expression can provide insight into initiating events of pancreatitis. The goal of this study was to delineate the gene structure and regulation of a novel pancreas-specific isoform for Secretory Pathway Ca2+ ATPase 2 (termed SPCA2C), which is encoded from the Atp2c2 gene. Using Next Generation Sequencing of RNA (RNA-seq), chromatin immunoprecipitation for epigenetic modifications and promoter-reporter assays, a novel transcriptional start site was identified that promotes expression of a transcript containing the last four exons of the Atp2c2 gene (Atp2c2c). This region was enriched for epigenetic marks and pancreatic transcription factors that promote gene activation. Promoter activity for regions upstream of the ATG codon in Atp2c2’s 24th exon was observed in vitro but not in in vivo. Translation from this ATG encodes a protein aligned with the carboxy terminal of SPCA2. Functional analysis in HEK 293A cells indicates a unique role for SPCA2C in increasing cytosolic Ca2+. RNA analysis indicates that the decreased Atp2c2c expression observed early in experimental pancreatitis reflects a global molecular response of acinar cells to reduce cytosolic Ca2+ levels. Combined, these results suggest SPCA2C affects Ca2+ homeostasis in pancreatic acinar cells in a unique fashion relative to other Ca2+ ATPases. J. Cell. Physiol. 231: 2768–2778, 2016. © 2016 Wiley Periodicals, Inc

Oxidized phospholipids are proinflammatory and proatherogenic in hypercholesterolaemic mice.

Oxidized phospholipids (OxPL) are ubiquitous, are formed in many inflammatory tissues, including atherosclerotic lesions, and frequently mediate proinflammatory changes 1 . Because OxPL are mostly the products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them are unavailable and their roles in vivo are largely unknown. We previously cloned the IgM natural antibody E06, which binds to the phosphocholine headgroup of OxPL, and blocks the uptake of oxidized low-density lipoprotein (OxLDL) by macrophages and inhibits the proinflammatory properties of OxPL2-4. Here, to determine the role of OxPL in vivo in the context of atherogenesis, we generated transgenic mice in the Ldlr-/- background that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter. E06-scFv was secreted into the plasma from the liver and macrophages, and achieved sufficient plasma levels to inhibit in vivo macrophage uptake of OxLDL and to prevent OxPL-induced inflammatory signalling. Compared to Ldlr-/- mice, Ldlr -/- E06-scFv mice had 57-28% less atherosclerosis after 4, 7 and even 12 months of 1% high-cholesterol diet. Echocardiographic and histologic evaluation of the aortic valves demonstrated that E06-scFv ameliorated the development of aortic valve gradients and decreased aortic valve calcification. Both cholesterol accumulation and in vivo uptake of OxLDL were decreased in peritoneal macrophages, and both peritoneal and aortic macrophages had a decreased inflammatory phenotype. Serum amyloid A was decreased by 32%, indicating decreased systemic inflammation, and hepatic steatosis and inflammation were also decreased. Finally, the E06-scFv prolonged life as measured over 15 months. Because the E06-scFv lacks the functional effects of an intact antibody other than the ability to bind OxPL and inhibit OxLDL uptake in macrophages, these data support a major proatherogenic role of OxLDL and demonstrate that OxPL are proinflammatory and proatherogenic, which E06 counteracts in vivo. These studies suggest that therapies inactivating OxPL may be beneficial for reducing generalized inflammation, including the progression of atherosclerosis, aortic stenosis and hepatic steatosis

Metabolic, health and lifestyle profiling of breast cancer radiotherapy patients and the risk of developing fatigue

Background: Fatigue is commonly reported by cancer patients. In some instances it can persist after treatment is com-pleted. In order to develop effective treatment strategies it is important to understand the mechanisms underlying the development of fatigue and to be able to predict those that may be at greatest risk of experiencing fatigue during and following treatment. The current paper examines predisposing factors for fatigue including altered fatty acid homeosta-sis in a cohort of breast cancer radiotherapy patients. Methodology: Patients had undergone breast-conserving surgery and adjuvant breast irradiation. Prior to radiotherapy the patients were free from significant fatigue. Levels of fatigue were determined prior to and following radiotherapy using the Functional Assessment of Cancer Therapy fatigue sub-scale. Plasma fatty acid levels, urinary and plasma amino acid levels, blood biochemistry factors and general health and lifestyle characteristics were assessed. Results: Following radiotherapy, significant fatigue affected approximately one third of the 26 patients and these subjects were then assigned to the fatigued cohort. Univariate analysis revealed that higher levels of the fatty acids myristic acid and eicosadienoic acid were present for the fatigued cohort prior to radio-therapy. Multivariate analysis also revealed that fatty acid homeostasis was altered between the fatigued and non-fatigued groups at baseline. Orthogonal partial least squares discriminant analysis of the general health, lifestyle and metabolic data revealed that the fatigued and non-fatigued patients could be clustered into two clearly separate groups. Conclusions: The results supported the proposition that the fatigued patients had an underlying metabolic ho-meostasis which may predispose them to the development of fatigue. Biochemical and general health profiling of breast cancer patients has the potential to identify those at most risk of developing significant fatigue following radiotherapy

Amyloid β-peptide directly induces spontaneous calcium transients, delayed intercellular calcium waves and gliosis in rat cortical astrocytes

The contribution of astrocytes to the pathophysiology of AD (Alzheimer's disease) and the molecular and signalling mechanisms that potentially underlie them are still very poorly understood. However, there is mounting evidence that calcium dysregulation in astrocytes may be playing a key role. Intercellular calcium waves in astrocyte networks in vitro can be mechanically induced after Aβ (amyloid β-peptide) treatment, and spontaneously forming intercellular calcium waves have recently been shown in vivo in an APP (amyloid precursor protein)/PS1 (presenilin 1) Alzheimer's transgenic mouse model. However, spontaneous intercellular calcium transients and waves have not been observed in vitro in isolated astrocyte cultures in response to direct Aβ stimulation in the absence of potentially confounding signalling from other cell types. Here, we show that Aβ alone at relatively low concentrations is directly able to induce intracellular calcium transients and spontaneous intercellular calcium waves in isolated astrocytes in purified cultures, raising the possibility of a potential direct effect of Aβ exposure on astrocytes in vivo in the Alzheimer's brain. Waves did not occur immediately after Aβ treatment, but were delayed by many minutes before spontaneously forming, suggesting that intracellular signalling mechanisms required sufficient time to activate before intercellular effects at the network level become evident. Furthermore, the dynamics of intercellular calcium waves were heterogeneous, with distinct radial or longitudinal propagation orientations. Lastly, we also show that changes in the expression levels of the intermediate filament proteins GFAP (glial fibrillary acidic protein) and S100B are affected by Aβ-induced calcium changes differently, with GFAP being more dependent on calcium levels than S100B

Do young children get the message? The effects of repeated video viewing on explicit and implicit information

The aim of this study was to explore the effects of repeat viewing on comprehension of explicitly and implicitly presented information in an animated movie. Seventy-three pre-school children watched an animated film and were tested for comprehension after either their single or fifth viewing. Only children&rsquo;s comprehension of explicitly presented information was facilitated by repeat viewing. However, post hoc analyses revealed that children&rsquo;s explicit and implicit comprehension of a central character Thunderbolt significantly increased across viewing conditions, whereas, repeat viewing only facilitated children&rsquo;s explicit comprehension of the central character Patch. The theoretical and practical implications of these findings are discussed.<br /

STAT3 activation by E6 is essential for the differentiation-dependent HPV18 life cycle.

Human papillomaviruses (HPV) activate a number of host factors to control their differentiation-dependent life cycles. The transcription factor signal transducer and activator of transcription (STAT)-3 is important for cell cycle progression and cell survival in response to cytokines and growth factors. STAT3 requires phosphorylation on Ser727, in addition to phosphorylation on Tyr705 to be transcriptionally active. In this study, we show that STAT3 is essential for the HPV life cycle in undifferentiated and differentiated keratinocytes. Primary human keratinocytes containing high-risk HPV18 genomes display enhanced STAT3 phosphorylation compared to normal keratinocytes. Expression of the E6 oncoprotein is sufficient to induce the dual phosphorylation of STAT3 at Ser727 and Tyr705 by a mechanism requiring Janus kinases and members of the MAPK family. E6-mediated activation of STAT3 induces the transcription of STAT3 responsive genes including cyclin D1 and Bcl-xL. Silencing of STAT3 protein expression by siRNA or inhibition of STAT3 activation by small molecule inhibitors, or by expression of dominant negative STAT3 phosphorylation site mutants, results in blockade of cell cycle progression. Loss of active STAT3 impairs HPV gene expression and prevents episome maintenance in undifferentiated keratinocytes and upon differentiation, lack of active STAT3 abolishes virus genome amplification and late gene expression. Organotypic raft cultures of HPV18 containing keratinocytes expressing a phosphorylation site STAT3 mutant display a profound reduction in suprabasal hyperplasia, which correlates with a loss of cyclin B1 expression and increased differentiation. Finally, increased STAT3 expression and phosphorylation is observed in HPV positive cervical disease biopsies compared to control samples, highlighting a role for STAT3 activation in cervical carcinogenesis. In summary, our data provides evidence of a critical role for STAT3 in the HPV18 life cycle