Methamphetamine mediates immune dysregulation in a murine model of chronic viral infection

Methamphetamine (METH) is a highly addictive psychostimulant that not only affects the brain and cognitive functions but also greatly impacts the host immune system, rendering the body susceptible to infections and exacerbating the severity of disease. Although there is gathering evidence about METH abuse and increased incidence of HIV and other viral infections, not much is known about the effects on the immune system in a chronic viral infection setting. We have used the lymphocytic choriomeningitis virus (LCMV) chronic mouse model of viral infection in a chronic METH environment and demonstrate that METH significantly increases CD3 marker on splenocytes and programmed death -1 (PD-1) expression on T cells, a cell surface signaling molecule known to inhibit T cell function and cause exhaustion in a lymphoid organ. Many of these METH effects were more pronounced during early stage of infection, which are gradually attenuated during later stages of infection. An essential cytokine for T-lymphocyte homeostasis, Interleukin-2 (IL-2) in serum was prominently reduced in METH-exposed infected mice. In addition, the serum pro-inflammatory (TNF, IL12 p70, IL1β, IL-6 and KC-GRO) and Th2 (IL-2, IL-10 and IL-4) cytokine profiles were also altered in the presence of METH. Interestingly CXCR3, an inflammatory chemokine receptor, showed significant increase in the METH treated LCMV infected mice. Similarly, compared to only infected mice, epidermal growth factor receptor (EGFR) in METH exposed LCMV infected mice were up regulated. Collectively, our data suggest that METH alters systemic, peripheral immune responses and modulates key markers on T cells involved in pathogenesis of chronic viral infection

Inhibition of glycogen synthase kinase 3β promotes tight junction stability in brain endothelial cells by half-life extension of occludin and claudin-5.

Neuroinflammatory conditions often involve dysfunction of the Blood-Brain Barrier (BBB). Therefore, identifying molecular targets that can maintain barrier fidelity is of clinical importance. We have previously reported on the anti-inflammatory effects that glycogen synthase kinase 3β (GSK3β) inhibition has on primary human brain endothelial cells. Here we show that GSK3β inhibitors also promote barrier tightness by affecting tight junction (TJ) protein stability. Transendothelial electrical resistance (TEER) was used to evaluate barrier integrity with both pharmacological inhibitors and mutants of GSK3β. Inhibition of GSK3β produced a gradual and sustained increase in TEER (as much as 22% over baseline). Analysis of subcellular membrane fractions revealed an increase in the amount of essential tight junction proteins, occludin and claudin-5, but not claudin-3. This phenomenon was attributed to a decrease in TJ protein turnover and not transcriptional regulation. Using a novel cell-based assay, inactivation of GSK3β significantly increased the half-life of occludin and claudin-5 by 32% and 43%, respectively. A correlation was also established between the enhanced association of β-catenin with ZO-1 as a function of GSK3β inhibition. Collectively, our findings suggest the possibility of using GSK3β inhibitors as a means to extend the half-life of key tight junction proteins to promote re-sealing of the BBB during neuroinflammation

Death and afterlife in religious notions and praxis of inhabitans of ancient Etruria

Cílem práce je na základě analýzy pramenů písemného i ikonografického charakteru popsat eschatologické představy Etrusků a s nimi související kultickou praxi. Práce postupně popisuje rituály v pořadí, ve kterém následovaly bezprostředně po úmrtí člověka. S tím souvisí také možné představení posvátného dramatu na počest zesnulého. Dále je zde věnována pozornost rituálům, které byly údajně zaznamenány v posvátných knihách, a které umožňovaly zbožštění duší zesnulých. V této souvislosti se u Etrusků potvrzuje konání lidské oběti. V druhé části se pak práce věnuje etruským představám o cestě do zásvětí a zásvětí jako takovému. Probrána je zde také etruská démonologie popisující celou řadu postav, včetně podsvětních vládců

Impaired Subset Progression and Polyfunctionality of T Cells in Mice Exposed to Methamphetamine during Chronic LCMV Infection

<div><p>Methamphetamine (METH) is a widely used psychostimulant that severely impacts the host’s innate and adaptive immune systems and has profound immunological implications. T cells play a critical role in orchestrating immune responses. We have shown recently how chronic exposure to METH affects T cell activation using a murine model of lymphocytic choriomeningitis virus (LCMV) infection. Using the TriCOM (trinary state combinations) feature of GemStone^™ to study the polyfunctionality of T cells, we have analyzed how METH affected the cytokine production pattern over the course of chronic LCMV infection. Furthermore, we have studied in detail the effects of METH on splenic T cell functions, such as cytokine production and degranulation, and how they regulate each other. We used the Probability State Modeling (PSM) program to visualize the differentiation of effector/memory T cell subsets during LCMV infection and analyze the effects of METH on T cell subset progression. We recently demonstrated that METH increased PD-1 expression on T cells during viral infection. In this study, we further analyzed the impact of PD-1 expression on T cell functional markers as well as its expression in the effector/memory subsets. Overall, our study indicates that analyzing polyfunctionality of T cells can provide additional insight into T cell effector functions. Analysis of T cell heterogeneity is important to highlight changes in the evolution of memory/effector functions during chronic viral infections. Our study also highlights the impact of METH on PD-1 expression and its consequences on T cell responses.</p></div

PD-1 expression in T cell subsets in the spleen.

<p>Percentages of PD-1 expression in CD4 (<b>A</b>) and CD8 (<b>B</b>) subsets from the PSM analysis, Data are presented as mean ± SEM, *<i>P</i><0.05.</p

Correlation analysis between PD-1 and cytokine expression in CD4 and CD8 T cells in chronic LCMV infection.

<p>Plots represent correlation analysis of PD-1 expression with IL-2, IFN-γ or TNF-α cytokine expression during chronic LCMV infection. Day 14, 28 and 56 time points are noted by different symbols. Pearson correlation value (r) and <i>P</i> value (if any) are indicated in each plot.</p

Probability State Modeling of T cell progression modulated by METH in chronic LCMV infection.

<p>Average Probability State Model (PSM) progression plots of CD4+ (top panel) and CD8+ (bottom panel) T cells from spleens from LCMV (n = 6) and LCMV-METH (n = 3) treated mice (14 days post-infection). The subsets are defined by the correlated measurements CD3, SSC (side-scatter), CD8, CD4, CD62L and CD44. The x-axis represents CD4 or CD8 T-cell antigen-dependent progression with units of cumulative percentage of events. The y-axis is the relative dynamic range of the measurement intensities between 0 and 100. The end of the naïve stage (red) is defined as the beginning of the up-regulation of CD44 (first black diamond). The end of the CM (green) stage is defined by the down-regulation of CD62L (black diamond), while the end of the EM stage (blue) and the beginning of the EF stage (brown) are the end of the down-regulation of CD44 (second black diamond). Each controlled definition point (CDP) in an expression profile (EP) is shown as a white or black diamond. Each CDP in the progression plot has a vertical box whisker for examining the variability of measurement intensities and a horizontal box whisker for examining the variability of associated cumulative percentages. Table shows the mean ± SEM of the percentage of the T cell subsets in the CD4 and CD8 cell types at day 14.</p

Analysis of co-expression of cytokines modulated by METH in CD4 and CD8 splenocytes in chronic LCMV infection.

<p>Pie charts show the changing patterns of cytokines of two (<b>A</b>) and three (<b>B</b>) co-expressed cytokines respectively, in CD4 and CD8 T cells in LCMV and LCMV-METH groups during chronic viral infection (days 14, 28 and 56). Numbers in the pie charts are averages of the percentages from TriCOM analysis. Bar graphs show the actual percentages of two (<b>C</b>) or three (<b>D</b>) co-expressed cytokines during LCMV infection.</p