In periodontitis, gingival fibroblasts (GF) appear to produce a multitude of paracrine factors. However, the influence of GF-derived soluble factors on osteoclastogenesis remains unclear. In this case study, production of paracrine factors by GF was assessed under inflammatory and non-inflammatory conditions, as well as their effect on osteoclastogenesis. Human primary GF were cultured in a transwell system and primed with a cocktail of IL-1β, IL-6 and TNF-α to mimic inflammation. GF were co-cultured directly and indirectly with human peripheral blood mononuclear cells (PBMC). Cytokines and chemokines in supernatants (flow cytometry based multiplex assay), osteoclastogenesis (TRAcP staining) and gene expression (qPCR) were quantified on days 7 and 21. Results from this case study showed that GF communicated via soluble factors with PBMC resulting in a two-fold induction of osteoclasts. Reversely, PBMC induced gene expression of IL-6, OPG and MCP-1 by GF. Remarkably, after priming of GF with cytokines, this communication was impaired and resulted in fewer osteoclasts. This could be partly explained by an increase in IL-10 expression and a decrease in MCP-1 expression. Intriguingly, the short priming of GF resulted in significantly higher expression of inflammatory cytokines that was sustained at both 7 and 21 days. GF appear to produce paracrine factors capable of stimulating osteoclastogenesis in the absence of physical cell-cell interactions. GF cultured in the presence of PBMC or osteoclasts had a remarkably inflammatory phenotype. Given profound expression of both pro- and anti-inflammatory cytokines after the inflammatory stimulus, it is probably the effector hierarchy that leads to fewer osteoclasts.

Extracellular vesicles (EVs), exhibiting their functional activity after internalization by recipient cells, are involved in the pathogenesis of drug-induced polyneuropathy (DIPN), a common complication of antitumor therapy. In this work, the internalization of EVs obtained from colorectal cancer patients undergoing polychemotherapy and its relationship with neurotoxicity were assessed using a model system of mononuclear leukocytes. Circulating EVs were isolated from 8 colorectal cancer patients who received antitumor therapy according to the FOLFOX or XELOX regimens before the start of chemotherapy (point 1) and after 3-4 courses (point 2). Mononuclear leukocytes of a healthy donor served as a cellular model system for EV internalization in vitro. EV internalization was assessed using fluorescence microscopy. It was shown that internalization of EVs obtained from colorectal cancer patients with high neurotoxicity was higher than in the group with low neurotoxicity. The ability of CD11b-positive (CD11b⁺) and CD11b-negative (CD11b⁻) mononuclear leukocytes of a healthy donor to internalize EVs obtained from patients before and after chemotherapy did not reveal significant differences. A direct relationship was found between the relative number of CD11b⁻ cells with internalized EVs and the integral index of neurotoxicity according to the NRS scale at the peak of its manifestation (point 2) (r=0.675, p.

Immune-related drug delivery systems (DDSs) in humanized mouse models are at the forefront of cancer research and serve as bridges between preclinical studies and clinical applications. These systems offer unique platforms for exploring new therapies and understanding their interactions with human cells and the immune system. Here, we focus on a DDS and a peripheral blood mononuclear cell (PBMC)-engrafted humanized mouse model that we recently developed, and consider some of the key components, challenges, and applications to advance these systems towards better cancer treatment on the basis of a better understanding of the immune response. Our DDS is unique and has a dual function, an anticancer effect and a capacity to fine-tune the immune reaction. The PBL-NOG-hIL-4-Tg mouse system is superior to other available humanized mouse systems for the development of such multifunctional DDSs because it supports the rapid reconstruction of an individual donor\'s immunity and avoids the onset of graft-versus-host disease.

BACKGROUND: The immunological background responsible for the severe course of COVID-19 and the immune factors that protect against SARS-CoV-2 infection are still unclear. The aim of this study was to investigate immune system status in persons with high exposure to SARS-CoV-2 infection.
METHODS: Seventy-one persons employed in the observation and infectious diseases unit were qualified for the study between November 2020 and October 2021. Symptomatic COVID-19 was diagnosed in 35 persons. Anti-SARS-CoV-2 antibodies were also found in 8 persons. Peripheral blood mononuclear cells subpopulations were analyzed by flow cytometry, and the concentrations of cytokines and anti-SARS-CoV-2 antibodies were determined by ELISA.
RESULTS: The percentages of cytotoxic T lymphocytes (CTLs), CD28+ and T helper (Th) cells with invariant T-cell receptors were significantly higher in persons with symptomatic COVID-19 than in those who did not develop COVID-19\' symptoms. Conversely, symptomatic COVID-19 persons had significantly lower percentages of: a) CTLs in the late stage of activation (CD8+/CD95+), b) NK cells, c) regulatory-like Th cells (CD4+/CTLA-4+), and d) Th17-like cells (CD4+/CD161+) compared to asymptomatic COVID-19\' persons. Additionally, persons with anti-SARS-CoV-2 antibodies had a significantly higher lymphocyte count and IL-6 concentration than persons without these antibodies.
CONCLUSIONS: Numerous lymphocyte populations are permanently altered by SARS-CoV-2 infection. High percentages of both populations: NK cells-as a part of the non-specific response, and T helper cells\' as those regulating the immune response, could protect against the acute COVID-19 symptoms development. Understanding the immune background of COVID-19 may improve the prevention of this disease by identifying people at risk of a severe course of infection.
BACKGROUND: This is a retrospective observational study without a trial registration number.

By analyzing two large atlases of almost 4 million cells, we show that immune-senescence involves a gradual loss of cellular identity, reflecting increased cellular heterogeneity, for effector, and cytotoxic immune cells. The effects are largely similar in both males and females and were robustly reproduced in two atlases, one assembled from 35 diverse studies including 678 adults, the other the OneK1K study of 982 adults. Since the mean transcriptional differences among cell-types remain constant across age deciles, there is little evidence for the alternative mechanism of convergence of cell-type identity. Key pathways promoting activation and stemness are down-regulated in aged T cells, while CD8 TEM and CD4 CTLs exhibited elevated inflammatory, and cytotoxicity in older individuals. Elevated inflammatory signaling pathways, such as MAPK and TNF-alpha signaling via NF-kB, also occur across all aged immune cells, particularly amongst effector immune cells. This finding of lost transcriptional identity with age carries several implications, spanning from a fundamental biological understanding of aging mechanisms to clinical perspectives on the efficacy of immunomodulation in elderly people.

BACKGROUND: Mycobacterium cell wall fraction (MCWF) is derived from nonpathogenic Mycobacterium phlei and is used as an immunomodulatory compound in clinical practice, yet its mode-of-action requires further research.
OBJECTIVE: To evaluate the host response to MCWF in canine peripheral blood mononuclear cells (PBMCs) by using enzyme-linked immunosorbent assays (ELISA) and quantitative reverse transcription (qRT)-PCR for assessment of cytokines.
METHODS: Eight healthy Labrador retrievers.
METHODS: PBMCs were isolated from whole blood using density centrifugation. The cells were cultured with different concentrations of MCWF or a potent stimulator of cytokine production, phorbol 12-myristate 13-acetate/ionomycin, or left in cell culture medium for 24, 48 and 72 h. Cytokines were measured by ELISA for interleukin (IL)-4, IL-10 and interferon-gamma (IFN-γ), and by qRT-PCR for IL-4, IL-10, IL-13, IFN-γ, tumour necrosis factor alpha (TNF-α) and transforming growth factor-beta.
RESULTS: A significant increase of IL-10 messenger ribonucleic acid (mRNA) was detected at all time points for all concentrations of MCWF (p < 0.05). Protein analysis reflected this finding, with a maximum IL-10 concentration of 300.6 ± 38.3 μg/mL. Compared to the negative control, post-stimulation elevation of IFN-γ mRNA was noted at 24 h with all concentrations of MCWF (p < 0.01), and TNF-α mRNA was increased for 0.5 μg/dL MCWF only at 72 h (p < 0.05).
CONCLUSIONS: MCWF stimulation of PBMCs results in the elevation of both proinflammatory and regulatory cytokine mRNA. Further research into the role of MCWF as a systemically administered regulatory immunomodulator or adjuvant to allergen-specific immunotherapy should be considered.

BACKGROUND: The apolipoprotein E (APOE) ε4 allele exerts a significant influence on peripheral inflammation and neuroinflammation, yet the underlying mechanisms remain elusive.
METHODS: The present study enrolled 54 patients diagnosed with late-onset Alzheimer\'s disease (AD; including 28 APOE ε4 carriers and 26 non-carriers). Plasma inflammatory cytokine concentration was assessed, alongside bulk RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) analysis of peripheral blood mononuclear cells (PBMCs).
RESULTS: Plasma tumor necrosis factor α, interferon γ, and interleukin (IL)-33 levels increased in the APOE ε4 carriers but IL-7 expression notably decreased. A negative correlation was observed between plasma IL-7 level and the hippocampal atrophy degree. Additionally, the expression of IL-7R and CD28 also decreased in PBMCs of APOE ε4 carriers. ScRNA-seq data results indicated that the changes were mainly related to the CD4+ Tem (effector memory) and CD8+ Tem T cells.
CONCLUSIONS: These findings shed light on the role of the downregulated IL-7/IL-7R pathway associated with the APOE ε4 allele in modulating neuroinflammation and hippocampal atrophy.
CONCLUSIONS: The apolipoprotein E (APOE) ε4 allele decreases plasma interleukin (IL)-7 and aggravates hippocampal atrophy in Alzheimer\'s disease. Plasma IL-7 level is negatively associated with the degree of hippocampal atrophy. The expression of IL-7R signaling decreased in peripheral blood mononuclear cells of APOE ε4 carriers Dysregulation of the IL-7/IL-7R signal pathways enriches T cells.

Immunosuppressive drugs (ISDs) are given to avoid the allograft rejection after transplantation. The concentrations of ISDs should be closely monitored owing to their wide inter-individual variability in its pharmacokinetics and narrow therapeutic window. Currently, the whole blood concentration measurement is the major approach of therapeutic drug monitoring of clinical ISDs in organ transplantation. Its correlation with the efficacy of ISDs remains elusive. While the acute rejection after transplantation may occur even when whole-blood ISDs concentrations are within the target range. Since the site of action of ISDs are within the lymphocyte, direct measurement of drug exposure in target cells may more accurately reflect the clinical efficacy of ISDs. Although several methods have been developed for the peripheral blood mononuclear cells (PBMCs) extraction and drug concentration measurement, the complex pre-processing has limited the study of the relationship between intracellular ISDs concentrations and the occurrence of rejection. In this study, the extraction of ISDs in PBMCs was carried out by the liquid-liquid extraction with low temperature purification, without centrifugation. The lower limit of quantitation were 0.2 ng/mL for cyclosporine A, tacrolimus and sirolimus, 1.0 ng/mL for mycophenolic acid, and the within-run and between-run coefficient of variations were both less than 12.4 %. The calibration curves of mycophenolic acid had a linear range (ng/mL): 1.0-128.0 (r2 = 0.9992). The calibration curves of other three ISDs had a linear range (ng/mL): 0.2-20.48 (r2 > 0.9956). A total of 157 clinical samples were analyzed by the UPLC-MS/MS for ISDs concentration in blood or plasma ([ISD]blood or plasma) and the concentration within PBMCs ([ISD]PBMC). Although there was strong association between [ISD]PBMC and [ISD]blood or plasma, the large discrepancies between concentration within [ISD]blood or plasma and [ISD]PBMC were observed in a small proportion of clinical samples. The developed method with short analysis time and little amounts of blood sample can be successfully applied to therapeutic drug monitoring of ISDs in PBMCs for analysis of large numbers of clinical samples and is helpful to explore the clinical value of ISDs concentration in PBMCs.

Human papillomavirus (HPV) 11/16 E6/E7 proteins have been recognized to be pivotal in viral pathogenesis. This study sought to uncover the potential mechanisms of how HPV11/16 E6/E7-transfected keratinocytes inhibit cytokine secretion in peripheral blood mononuclear cells (PBMC). Upon co-culturing HPV11/16 E6/E7-transfected keratinocytes with PBMC in a non-contact manner, we observed a marked decrease in various cytokines secreted by PBMC. To determine if this suppression was mediated by specific common secreted factors, we conducted transcriptomic sequencing on these transfected cells. This analysis identified 53 common differentially secreted genes in all four HPV-transfected cells. Bioinformatics analysis demonstrated these genes were predominantly involved in immune regulation. Results from quantitative PCR (qPCR) and an extensive literature review suggested the downregulation of 12 genes (ACE2, BMP3, BPIFB1, CLU, CST6, CTF1, HMGB2, MMP12, PDGFA, RNASE7, SULF2, TGM2), and upregulation of 7 genes (CCL17, CCL22, FBLN1, PLAU, S100A7, S100A8, S100A9), may be crucial in modulating tumor immunity and combating pathogenic infections, with genes S100A8 and S100A9, and IL-17 signaling pathway being particularly noteworthy. Thus, HPV11/16 E6/E7 proteins may inhibit cytokine secretion of immune cells by altering the expression of host-secreted genes. Further exploration of these genes may yield new insights into the complex dynamics of HPV infection.

Exposure to 2.45 GHz electromagnetic radiation (EMR) emitted from commonly used devices has been reported to induce oxidative stress in several experimental models. Our study aims to evaluate the efficacy of sulforaphane, a well-known natural product, in preventing radiation-induced toxic effects caused by a 24 h exposure of SH-SY5Y neuronal-like cells and peripheral blood mononuclear cells (PBMCs) to 2.45 GHz EMR. Cells were exposed to radiation for 24 h in the presence or absence of sulforaphane at different concentrations (5-10-25 µg/mL). Cell viability, mitochondrial activity alterations, the transcription and protein levels of redox markers, and apoptosis-related genes were investigated. Our data showed a reduction in cell viability of both neuronal-like cells and PBMCs caused by EMR exposure and a protective effect of 5 µg/mL sulforaphane. The lowest sulforaphane concentration decreased ROS production and increased the Mitochondrial Transmembrane Potential (Δψm) and the NAD+/NADH ratio, which were altered by radiation exposure. Sulforaphane at higher concentrations displayed harmful effects. The hormetic behavior of sulforaphane was also evident after evaluating the expression of genes coding for Nrf2, SOD2, and changes in apoptosis markers. Our study underlined the vulnerability of neuronal-like cells to mitochondrial dysfunction and oxidative stress and the possibility of mitigating these effects by supplementation with sulforaphane. To our knowledge, there are no previous studies about the effects of SFN on these cells when exposed to 2.45 GHz electromagnetic radiation.