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Innate immune toll-like receptors (TLRs) are emerging as nutrient sensors. Oxidative stress in the adipose tissue in obesity acts as a critical early trigger of altered pathophysiology. TLR2/TLR4 adipose upregulation has been associated with insulin resistance in humans; however, it remains unclear whether oxidative stress can modulate expression of TLR2/4 and related immune-metabolic regulators (IRF3/5) in immune cells. We, therefore, assessed their expression along with proinflammatory cytokines in the human PBMC following induction of oxidative stress. PBMC were isolated from blood of healthy donors using Ficoll-Paque method and cells were treated with H O to induce oxidative stress. ROS was measured by DCFH-DA assay. Target gene and protein expression was determined using real-time RT-PCR and flow cytometry/confocal microscopy, respectively. TLR2/4 expression by H O in presence of ROS-inhibitors or leptin/LPS/fatty acids was also assessed. Expression of phosphorylated/total ERK1/2, c-Jun, p38, and NF-κB was determined by western blotting. The data (mean±SEM) were compared using unpaired student's t-test or ANOVA; all P-values <0.05 were considered significant. TLR2/4 mRNA/protein expression was elevated by oxidative stress in PBMC compared to controls (P<0.001). This induction was abrogated by apocynin/N-acetyl cysteine treatments (P<0.01). H O -induced TLR2/4 gene expression was further enhanced by leptin, LPS, oleate, or palmitate (P<0.05). Oxidative stress also promoted expression of IRF3/5 and proinflammatory cytokines including IFN-γ, IL-1β, IL-6, TNF-α, and MCP-1/CCL2. This oxidative stress in PBMC involved MAPK/NF-κB dependent signaling. Taken together, oxidative stress upregulates expression of TLR2/4, IRF3/5 and signature proinflammatory cytokines in PBMC, involving MAPK/NF-κB dependent signaling, all of which may have implications for metabolic inflammation.

Background: The recent SARS-CoV-2 coronavirus pandemic has spread worldwide, with the first cases recorded in December 2019 in China. Symptoms include fever, cough, dyspnea, and gastrointestinal issues. Patients with SARS-CoV-2 are classified as mild, severe, or critical, highlighting the necessity for earlier treatment methods and reliable biomarkers. MicroRNAs have consistently been considered essential biological biomarkers. Objectives: In this case-control study, we investigated the levels of miR-26a and miR-125b in PBMCs of SARS-CoV-2 patients in northwestern Iran. Methods: Blood samples were collected from 100 COVID-19-infected patients, divided into two groups: 50 patients with no serious lung damage and 50 patients with COVID-19 infection and a lung abscess. RNA extraction techniques were used to examine PBMC specimens from COVID-19 patients. mRNA was extracted from WBCs, and the expression levels of miR-26a and miR-125b were evaluated using qRT-PCR in the two study groups. Results: The expression levels of miR-26a and miR-125b in PBMCs of patients with a poor prognosis were significantly lower compared to those with a good prognosis, as shown by qRT-PCR. The receiver operating characteristic (ROC) analysis indicated significant diagnostic potential for both miRNAs (miR-26a [area under the ROC curve (AUC)] = 0.8458, P < 0.0001; miR-125b AUC = 0.7944, P < 0.0001). Conclusions: The relative expression of miR-26a and miR-125b in SARS-CoV-2 infection was significantly different between the two study groups. These findings suggest their potential as disease severity indicators and prognostic markers.

Aging leads to a progressive functional decline of the immune system, rendering the elderly increasingly susceptible to disease and infection. The degree to which immune cell senescence contributes to this decline remains unclear, however, since markers that label immune cells with classical features of cellular senescence accurately and comprehensively have not been identified. Using a second‐generation fluorogenic substrate for β‐galactosidase and multi‐parameter flow cytometry, we demonstrate here that peripheral blood mononuclear cells (PBMCs) isolated from healthy humans increasingly display cells with high senescence‐associated β‐galactosidase (SA‐βGal) activity with advancing donor age. The greatest age‐associated increases were observed in CD8+ T‐cell populations, in which the fraction of cells with high SA‐βGal activity reached average levels of 64% in donors in their 60s. CD8+ T cells with high SA‐βGal activity, but not those with low SA‐βGal activity, were found to exhibit features of telomere dysfunction‐induced senescence and p16‐mediated senescence, were impaired in their ability to proliferate, developed in various T‐cell differentiation states, and had a gene expression signature consistent with the senescence state previously observed in human fibroblasts. Based on these results, we propose that senescent CD8+ T cells with classical features of cellular senescence accumulate to levels that are significantly higher than previously reported and additionally provide a simple yet robust method for the isolation and characterization of senescent CD8+ T cells with predictive potential for biological age. Senescent CD8+ T cells in peripheral blood can be detected, quantified, and isolated using a fluorogenic and self‐immobilizing substrate of senescence‐associated β‐galactosidase. Characterization of CD8+ T cells with high SA‐βGal activity isolated from healthy donors in their 20s and 60s revealed a significantly greater abundance of SA‐βGal expressing CD8+ T cells with a unique transcriptional signature and features telomere dysfunction‐induced senescence and p16‐mediated senescence in older humans.

In recent years, functional genomics approaches combining genetic information with bulk RNA-sequencing data have identified the downstream expression effects of disease-associated genetic risk factors through so-called expression quantitative trait locus (eQTL) analysis. Single-cell RNA-sequencing creates enormous opportunities for mapping eQTLs across different cell types and in dynamic processes, many of which are obscured when using bulk methods. Rapid increase in throughput and reduction in cost per cell now allow this technology to be applied to large-scale population genetics studies. To fully leverage these emerging data resources, we have founded the single-cell eQTLGen consortium (sc-eQTLGen), aimed at pinpointing the cellular contexts in which disease-causing genetic variants affect gene expression. Here, we outline the goals, approach and potential utility of the sc-eQTLGen consortium. We also provide a set of study design considerations for future single-cell eQTL studies.

Teucrium polium has been used in traditional medicine around the world for centuries in treatment of various conditions and diseases. Many studies have confirmed pharmacological effects of its extracts, although the immunomodulatory effect has not been investigated. Therefore, the aim of our study was to examine the immunomodulatory effect of methanolic extract of T. polium (TPE) on peripheral blood mononuclear cells (PBMCs) derived from healthy donors and patients with hepatitis C virus HCV infection. We analyzed the effect of the extract on PBMCs viability using the MTT test. The cell death type was determined using Annexin V-FITC/7-AAD staining. Immunophenotyping using anti-CD8 FITC, anti-CD4 PE, anti-CD3 ECD, anti-CD20 PC5, anti-CD14 FITC and anti-CD25 PC7 was performed by flow cytometry. Results of the MTT test indicate that TPE stimulates proliferation of healthy PBMCs, while the HCV PBMCs viability was slightly reduced. The percentage of apoptotic HCV PBMCs was higher after TPE treatment compared to the control. The proportion of CD25-expressing cells was higher among the untreated HCV PBMCs than in the untreated healthy PBMCs. TPE treatment significantly and gradually increased CD25 expression in healthy PBMCs, whereas CD25 expression on HCV PBMCs increased only at the highest TPE concentration. The upregulation of double-positive CD3+CD25+, CD20+CD25+ and CD14+CD25+ cells was significant in TPE treated healthy PBMCs, while only the highest concentration was effective on HCV PBMCs. In summary, TPE exerts a strong immunomodulatory effect on healthy PBMCs and, only at the highest concentration, on HCV PBMNCs.

Background: Endometriosis is believed to be associated with dysfunction of the lymphocyte population and cytotoxicity of natural killer (NK) cells, induced by the production of interleukin-2 (IL-2). Objective: This study aimed to investigate T lymphocytes and NK cell activity in the peripheral blood mononuclear cells (PBMCs) of women with endometriosis. Materials and Methods: PBMCs were obtained from the peripheral venous blood samples of 14 women with and without endometriosis (n = 7 for each group). Then, the PBMCs were co-cultured for 4 days and were treated with recombinant IL-2 for cytotoxic activity toward target cells (Daudi and K562 cells). The cytotoxicity activity was determined using the 51 chromium release assay before and after stimulation. Flow cytometry measurement was used to examine the expression of T lymphocytes and NK cells before and after being treated with IL-2. Results: The concentration of CD3+CD28+ (co-stimulatory) was significantly lower in the endometriosis group (65.62 ± 5.38) compared to in its counterpart (50.24 ± 4.22) (p = 0.04) before stimulation. However, no significant differences were observed in any other T lymphocytes and NK cells. It was also found that there was a significant increase of CD3-CD28+ after treatment with IL-2 only in the healthy control but not in women with endometriosis. Conclusion: Increased expression of CD160 and decreased CD28 play a role in inhibiting NK cell activation and T cell response in women with endometriosis. Key words: CD28, CD160, Cytotoxic, Endometriosis, PBMC.

Background Interest in single-cell transcriptomic analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. In almost all reported works investigators have used live cells, which introduces cell stress during preparation and hinders complex study designs. Recent studies have indicated that cells fixed by denaturing fixative can be used in single-cell sequencing, however they did not usually work with most types of primary cells including immune cells. Methods The methanol-fixation and new processing method was introduced to preserve human peripheral blood mononuclear cells (PBMCs) for single-cell RNA sequencing (scRNA-Seq) analysis on 10× Chromium platform. Results When methanol fixation protocol was broken up into three steps: fixation, storage and rehydration, we found that PBMC RNA was degraded during rehydration with PBS, not at cell fixation and up to 3-month storage steps. Resuspension but not rehydration in 3× saline sodium citrate (SSC) buffer instead of PBS preserved PBMC RNA integrity and prevented RNA leakage. Diluted SSC buffer did not interfere with full-length cDNA synthesis. The methanol-fixed PBMCs resuspended in 3× SSC were successfully implemented into 10× Chromium standard scRNA-seq workflows with no elevated low quality cells and cell doublets. The fixation process did not alter the single-cell transcriptional profiles and gene expression levels. Major subpopulations classified by marker genes could be identified in fixed PBMCs at a similar proportion as in live PBMCs. This new fixation processing protocol also worked in several other fixed primary cell types and cell lines as in live ones. Conclusions We expect that the methanol-based cell fixation procedure presented here will allow better and more effective batching schemes for a complex single cell experimental design with primary cells or tissues.

In early pregnancy, approximately 70% of women experience nausea and vomiting, with hyperemesis gravidarum (HG) can potentially lead to severe fluid and nutritional imbalances that require hospitalization. Although HG often resolves on its own in the early stages of pregnancy, its severity is linked to ketosis and elevated serum urea levels, as well as an increased risk of neurodevelopmental disorders in children. To further investigate the immune status of HG patients, we plan to conduct single-cell transcriptomic sequencing and plasma proteomic analysis of peripheral blood samples. This approach aims to elucidate the interactions and mechanisms of PBMCs and provide new insights into potential therapeutic interventions. Our findings indicate an increased proportion of neutrophils in HG patients, along with the upregulation of interferon genes and associated pathways. Notably, the activity of interferon-related TFs, such as STAT1, IRF7, and IRF9, was significantly elevated. Additionally, we observed a decrease in T cell activity in HG patients, while the functionality of NK cells and CD14 monocytes was enhanced. The elevated plasma levels of NDEL1 may also have implications for fetal development. We have constructed a single-cell atlas of PBMCs from pregnant women with HG, which is expected to enhance our understanding of the immune response in HG and identify potential therapeutic targets for this condition.

Background Increasing evidence suggests a double-faceted role of alpha-synuclein (α-syn) following infection by a variety of viruses, including SARS-CoV-2. Although α-syn accumulation is known to contribute to cell toxicity and the development and/or exacerbation of neuropathological manifestations, it is also a key to sustaining anti-viral innate immunity. Consistently with α-syn aggregation as a hallmark of Parkinson's disease, most studies investigating the biological function of α-syn focused on neural cells, while reports on the role of α-syn in periphery are limited, especially in SARS-CoV-2 infection. Results Results herein obtained by real time qPCR, immunofluorescence and western blot indicate that α-syn upregulation in peripheral cells occurs as a Type-I Interferon (IFN)-related response against SARS-CoV-2 infection. Noteworthy, this effect mostly involves α-syn multimers, and the dynamic α-syn multimer:monomer ratio. Administration of excess α-syn monomers promoted SARS-CoV-2 replication along with downregulation of IFN-Stimulated Genes (ISGs) in epithelial lung cells, which was associated with reduced α-syn multimers and α-syn multimer:monomer ratio. These effects were prevented by combined administration of IFN-β, which hindered virus replication and upregulated ISGs, meanwhile increasing both α-syn multimers and α-syn multimer:monomer ratio in the absence of cell toxicity. Finally, in endothelial cells displaying abortive SARS-CoV-2 replication, α-syn multimers, and multimer:monomer ratio were not reduced following exposure to the virus and exogenous α-syn, suggesting that only productive viral infection impairs α-syn multimerization and multimer:monomer equilibrium. Conclusions Our study provides novel insights into the biology of α-syn, showing that its dynamic conformations are implicated in the innate immune response against SARS-CoV-2 infection in peripheral cells. In particular, our results suggest that promotion of non-toxic α-syn multimers likely occurs as a Type-I IFN-related biological response which partakes in the suppression of viral replication. Further studies are needed to replicate our findings in neuronal cells as well as animal models, and to ascertain the nature of such α-syn conformations.

Parasitic diseases, caused by intestinal helminths, remain a very serious problem in both human and veterinary medicine. While searching for new nematicides we examined a series of 1,2,4-triazole derivatives 9–22, obtained during reactions of N3-substituted amidrazones with itaconic anhydride. Two groups of compounds, 9–16 and 17–22, differed in the position of the double bond on the methacrylic acid moiety. The toxicity of derivatives 9–22 and the anti-inflammatory activity of 12 and 19–22 were studied on peripheral blood mononuclear cells (PBMC). Antiproliferative activity of compounds 12 and 19–22 was tested cytometrically in PBMC cultures stimulated by phytohemagglutinin. The influence of derivatives 12 and 19–22 on the TNF-α, IL-6, IL-10 and IFN-γ production was determined by ELISA in lipopolysaccharide-stimulated PBMC cultures. Anthelmintic activity of compounds 10–22 was studied in the Rhabditis sp. nematodes model. Most compounds (11–22) proved to be non-toxic to human PBMC. Derivatives 19–22 showed anti-inflammatory activity by inhibiting the proliferation of lymphocytes. Moreover, compounds 12 and 19–22 significantly reduced the production of TNF-α and derivatives 19–21 decreased the level of INF-γ. The strongest anti-inflammatory activity was observed for compound 21. Compounds 12 and 14 demonstrated anthelmintic activity higher than albendazole and may become promising candidates for anthelmintic drugs.