A Population of CD4+CD8+ Double-Positive T Cells Associated with Risk of Plasma Leakage in Dengue Viral Infection

According to the WHO 2009 classification, dengue with warning signs is at the risk of developing severe form of dengue disease. One of the most important warning signs is plasma leakage, which can be a serious complication associated with higher morbidity and mortality. We report that the frequency of CD4+CD8+ double-positive (DP) T cells is significantly increased in patients at risk of developing plasma leakage. Transcriptomic analysis demonstrated that CD4+CD8+ DP cells were distinct from CD4+ Single Positive (SP) T cells but co-clustered with CD8+ SP cells, indicating a largely similar transcriptional profile. Twenty significant differentially expressed (DE) genes were identified between CD4+CD8+ DP and CD8+ SP cells. These genes encode OX40 and CCR4 proteins as well as other molecules associated with cell signaling on the cell surface (NT5E, MXRA8, and PTPRK). While comparing the profile of gene expression in CD4+CD8+ DP cells from patients with and without warning signs of plasma leakage, similar expression profile was observed, implying a role of CD4+CD8+ DP cells in plasma leakage through a quantitative increase rather than functional alteration. This study provided novel insight into the host immune response during the acute febrile phase of DENV infection and the role of CD4+CD8+ DP T cells in the pathogenesis of plasma leakage.

The Clinicopathological Characteristics, Prognosis and Immune Microenvironment Mapping in MSI-H/MMR-D Endometrial Carcinomas

Endometrial cancer had a relatively high prevalence of MMR deficiency. MMR-D/MSI-H endometrial cancer patients are suggested to be potential beneficiaries of PD-1/PD-L1 inhibitor therapy. Here, we explored the prognostic value of MSI subtype in endometrial cancer and its correlation with immune environment. Based on expression and clinical data of 78 POLE, 123 MSI and 299 Other EC samples from the TCGA-UCEC project, we found that the MSI tumors were identified more often in early stage, had a lower age, better patient survival, enriched CD8+ T cells, and regulatory T cells and less M2 macrophages and activated dendritic cells than the Other subgroup, and shared a relatively similar expression profile with POLE subgroup by differential analysis. In addition, we established the immune landscape of an MMR-D endometrial cancer tissue using unbiased single-cell RNA-seq analysis of 4518 cells. By immunohistochemistry analysis, we found that the MMR-D tumors showed a higher trend of CD20+ B cells infiltration. Our study might expand our understanding of the role of immune subsets in MSI endometrial carcinomas and provide guidance of immunotherapy for endometrial cancer.

Ligand-Regulated Expression of TNF Receptors 1 and 2 Determines Receptor-Mediated Functional Responses

<b><i>Introduction:</i></b> Modulating specific biological effects through the changes in cytokine receptors’ expression level remains poorly understood. This study aimed to investigate the influence of the dose-dependent effect of TNF on the balance between proapoptotic and proliferation response depending on the parameters of TNFR1/2 expression density. <b><i>Methods:</i></b> Tumor cell lines (HEp-2, K-562, MCF-7, ZR-75/1, MOLT-4, IM-9, and Raji) were characterized for TNFR1/2 co-expression using flow cytometry and were studied to reveal the dose-dependent effect of rhTNF on cell cycle and apoptosis parameters. The associations among the studied parameters were estimated by correlation and regression analysis. <b><i>Results:</i></b> It was found for ZR-75/1 cells (the cell line characterized by high expression of both types) that a dose-dependent increase in expression of both types of TNF-α receptors on cells reduces the proliferative activity of cells. For MOLT-4 cells (which are characterized by lower expression), an increase in proliferative response of cells was positively associated with the percentage of both TNFR1⁺ and TNFR2⁺ cells. However, opposite effects on the cells were shown for the K-562 and MCF-7 lines having a similar expression profile. A similarity (a large percentage of double-positive cells) was revealed for the lines having similar effects (K-562 and ZR-75/1). <b><i>Conclusions:</i></b> High expression of TNF receptor type 1 is not always associated with predominant activation of proapoptotic pathways. However, in the case of simultaneous high expression of both types of receptors, the proportion of double-positive cells is crucial for the activation of either the proapoptotic or proliferation pathways.

Trem2 promotes anti-inflammatory responses in microglia and is suppressed under pro-inflammatory conditions

Genome-wide association studies have reported that, amongst other microglial genes, variants in TREM2 can profoundly increase the incidence of developing Alzheimer’s disease (AD). We have investigated the role of TREM2 in primary microglial cultures from wild type mice by using siRNA to decrease Trem2 expression, and in parallel from knock-in mice heterozygous or homozygous for the Trem2 R47H AD risk variant. The prevailing phenotype of Trem2 R47H knock-in mice was decreased expression levels of Trem2 in microglia, which resulted in decreased density of microglia in the hippocampus. Overall, primary microglia with reduced Trem2 expression, either by siRNA or from the R47H knock-in mice, displayed a similar phenotype. Comparison of the effects of decreased Trem2 expression under conditions of lipopolysaccharide (LPS) pro-inflammatory or IL-4 anti-inflammatory stimulation revealed the importance of Trem2 in driving a number of the genes up-regulated in the anti-inflammatory phenotype. RNA-seq analysis showed that IL-4 induced the expression of a program of genes including Arg1 and Ap1b1 in microglia, which showed an attenuated response to IL-4 when Trem2 expression was decreased. Genes showing a similar expression profile to Arg1 were enriched for STAT6 transcription factor recognition elements in their promoter, and Trem2 knockdown decreased levels of STAT6. LPS-induced pro-inflammatory stimulation suppressed Trem2 expression, thus preventing TREM2’s anti-inflammatory drive. Given that anti-inflammatory signaling is associated with tissue repair, understanding the signaling mechanisms downstream of Trem2 in coordinating the pro- and anti-inflammatory balance of microglia, particularly mediating effects of the IL-4-regulated anti-inflammatory pathway, has important implications for fighting neurodegenerative disease.

Shared transcriptomic responses of Aedes aegypti to arboviral infections : example of dengue and Rift Valley fever viruses

BackgroundArthropod borne virus infections cause several emerging and resurgent infectious diseases. Among the diseases caused by arboviruses, dengue and Rift Valley fever are responsible for a high rate of severe human diseases worldwide. Understanding the effects of viral infection on gene expression in the mosquito is crucial to the development of early diagnostic tools and may enable researchers and policy makers to better anticipate outbreaks in the next future. Methods Here we investigate the alterations in gene expression across the entire Aedes aegypti genome during infection with DENV and RVF over time.ResultsWe describe several up-regulated genes that share a similar expression profile during infection with both viruses at early and late phases of infection. Family B and D clip-domain serine proteases (CLIP) are clearly overrepresented as well as C-type lectins and transferrin. ConclusionsOur results provide an extensive amount of data highlighting viral gene targets in the mosquito during infection. This data may also be used to develop broad-spectrum anti-viral diagnostic tools based on mosquitoes rather than the mammalian hosts and would help to predict and manage the emergence of outbreaks.

Abstract TP281: Transcriptome Analysis of the Human Brain Microvascular Endothelial Cells During and After Oxygen-Glucose Deprivation Stress as a Tool to Identify Novel Targets

Background: The blood-brain barrier (BBB) constitutes an important component of the neurovascular unit (NVU) by providing a physical and a chemical barrier critical for the brain homeostasis. The disruption of the BBB during cerebral ischemia constitutes a key event of the disease, resulting in its opening and ultimately the formation of cerebral edema. Therefore, targeting such BBB disruption by restoring the barrier function could mitigate such insult. In this study, we investigated change in gene expression profile of the human BBB in vitro using an induced pluripotent stem cell (iPSC) based model. Methods: BMECs derived from two human iPSC lines (CTR90F and CTR65M) were exposed to acute OGD stress (1% O2, no glucose) for 6h, followed by reoxygenation (21% O2, 1g/L glucose) for 18h and compared to control (21% O2, 1g/L glucose, 24h) Total RNA was extracted for cDNA microarray analysis to the University of Texas Southwestern Genomic Core Facility using a ClariomD® chip. Fold-change >2 was considered statistically significant (P<0.05). Results: OGD/reoxygenation resulted in almost 3700 genes differentially expressed, including over 600 coding genes. Most of such differential expression occurred during the OGD phase. Reoxygenation yielded to quasi-similar expression profile than controls. Several original pathways have been identified and include nuclear receptors, PI3K/Akt signaling pathways, microRNAs, and adhesion signaling pathways. Conclusions: Our preliminary findings suggest that our iPSC-derived model of the BBB based on BMECs showed an overlap of several genes commonly identified as hypoxic/ischemic responsive genes, but also highlighted novel signaling pathways that have little or no literature on their function at the BBB. We are currently refining our list of genes and focusing our attention on genes that have shown the highest enrichment or depletion.

PSXIII-18 Conceptus stimuli in peripheral blood mono and polymorphonuclear cells at the beginning of pregnancy

The objective of this experiment was to compare the expression of Interferon-tau Stimulated Genes (ISGs) in peripheral blood mono and polymorphonuclear cells (PBMCs and PMNs) in heifers following insemination. Twenty-nine Nelore heifers had estrous cycle synchronized, and FTAI occurred on D0. Pregnancy diagnosis was performed by ultrasonography on D28 post FTAI. On D0, 10, 14, 16, 18 and 20, blood (25mL) from the jugular vein was collected in heparinized tubes for isolation of PMNs and PBMCs. The isolation was performed using Ficoll®Paque Plus (GE Healthcare). PMNs and PBMCs samples from 8 pregnant and 9 non-pregnant heifers were subjected to RNA extraction using the DirectZol-RNA kit (Zymo-Research) and Trizol (Invitrogen), respectively. The expression of the target genes (ISG15, OAS-1, MX1 and MX2) was normalized in relation to the two reference genes (GAPDH/ACTB for PMNs and GAPDH/PPIA for PBMCs). The abundance of transcripts was evaluated by analysis of variance considering fixed effects of group, day and group by day interaction using the PROC MIXED procedure in SAS. PMNs and PBMCs had a similar expression profile of ISG15 and OAS-1, showing a relative increase (P &lt; 0.05) from D18, and a significant increase in (P &lt; 0.05) expression in pregnant compared to non-pregnant females on D18 and D20. These results were also observed for MX1 in PBMCs. In PMNs, no significant effects for MX1 were found. For MX2, in both cells types, only a group effect (P &lt; 0.05) was observed, indicating a higher expression in pregnant heifers (0.57±0.11 vs. 0.21±0.03) on the days evaluated. When comparing the relative expression of the target genes to D0, no significant (P &gt; 0.1) differences were found between PMNs and PBMCs. In summary, ISG expression is similar in PMNs and PBMCs, specifically for ISG15 and OAS-1, which both seem to be suitable biomarkers for potential early pregnancy determination in heifers.

IMMUNOHISTOCHEMICAL IDENTIFICATION AND DISTRIBUTION OF GLUTAMATERGIC NMDA AND mGlu1 RECEPTORS IN THE PONTINE INTERTRIGEMINAL REGION IN RATS

Local glutamate simulation of intertrigeminal region (ITR) in the lateral pons evoked immediate cardiovascular and respiratory effects proposing its role in central cardiorespiratory control. Since pharmacological studies provided only functional evidence for the existence of glutamate receptors in the ITR and thereby specifying putative neurochemical substrate involved in this control, here we employed immunohistochemistry to examine expression and distribution of NMDA and mGlu1 receptors in this structure. Thirty adult male Sprague-Dawley rats were perfuse-fixed, their brains frozen and cut into sequential series of 20 µm thick sections through the ITR. Immunohistochemistry was performed using polyclonal antibodies against NMDA-NR1, NMDA-NR2A and mGlu1 receptors. Labeled neurons in the ITR were analyzed using light microscope and computerized image analysis system for quantification of relative immunoreactivity as the mean of integrated optical density (IOD), and counting the immunopositive cells. Light microscopic analyses demonstrated NMDA-NR1-immunoreactivity mainly localized in the neuronal cell bodies with sparse distribution on primary dendrites, while NMDA-NR2A-immunoreactivity was basically somatically distributed. The mGlu1-immunoreactivity was moderate and observed both in neuronal bodies and primary dendrites or extracellular matrix suggesting somatodendritic localization. Quantitative analyses of IOD showed very strong expression of NMDA-NR1, weak of NMDA-NR2A and strong-to-moderate expression of mGluR1, with differences in immunostaining signal distribution over rostro-caudal span of the ITR. Counting of immunopositive cells followed similar expression profile. Our data directly confirm the presence of glutamatergic NMDA and mGlu1 receptors in the ITR apparently involved in signaling pathways by which this region modulates cardiorespiratory functions such as blood pressure, heart rate and breathing.

Heightened condition-dependence of the sexual transcriptome as a function of genetic quality in Drosophila melanogaster head tissue

Theory suggests sexual traits should show heightened condition-dependent expression. This prediction has been tested extensively in experiments where condition has been manipulated through environmental quality. Condition-dependence as a function of genetic quality has, however, only rarely been addressed, despite its central importance in evolutionary theory. To address the effect of genetic quality on expression of sexual and non-sexual traits, we here compare gene expression in Drosophila melanogaster head tissue between flies with intact genomes (high condition) and flies carrying a major deleterious mutation (low condition). We find that sex-biased genes show heightened condition-dependent expression in both sexes, and that expression in low condition males and females regresses towards a more similar expression profile. As predicted, sex-biased expression was more sensitive to condition in males compared to females, but surprisingly female-biased, rather than male-biased, genes show higher sensitivity to condition in both sexes. Our results thus support the fundamental predictions of the theory of condition-dependence when condition is a function of genetic quality.

Knockdown of PTOV1 and PIN1 Exhibit Common Phenotypic Anti-Cancer Effects in MDA-MB-231 Cells

BackgroundEarlier, we have identified PTOV1 as a novel interactome of PIN1 in PC-3 cells. This study aims to explore the functional similarity and the common role of both genes in breast cancer cell proliferation.MethodsCTG, crystal violet assay, clonogenic assay, wound healing assay, cell cycle analysis, Hoechst staining and ROS measurement were performed to assess cell viability were performed after knocking down of PTOV1 and PIN1 by siRNAs in MDA-MB-231 and MCF-7 cells. CO-IP, qPCR and western blot were performed for interaction, transcriptional and translational regulation of both genes.ResultsKnockdown of PTOV1 and PIN1 inhibited the cell proliferation, colony formation, migration cell cycle, and induces nuclear condensation as well as ROS production. Interaction of PTOV1 and PIN1 was validated by Co-IP in MDA-MB-231 cells. Genes involved in cell proliferation, migration, cell cycle, and apoptosis were regulated by PIN1 and PTOV1. PTOV1 knockdown inhibited Bcl-2, Bcl-xL and induces BAX, LC3 and Beclin-1. Overexpression of PIN1 increased the expression of PTOV1. Knockdown of both genes inhibited the expression of cyclin D1, c-Myc, and β-catenin.ConclusionsPTOV1 and PIN1 interacts and exert oncogenic role in MDA-MB-231 cells by sharing the similar expression profile at transcriptional and translational level which can be a promising hub for therapeutic target.