Response Of Peripheral Blood Mononuclear Cells Research Articles

Activation of Peripheral Blood Mononuclear Cells and Leptin Secretion: New Potential Role of Interleukin-2 and High Mobility Group Box (HMGB)1.

Activation of innate immunity and low-grade inflammation contributes to hyperglycemia and an onset of Type 2 Diabetes Mellitus (T2DM). Interleukin-2 (IL-2), leptin, High Mobility Group Box-1 (HMGB-1), and increased glucose concentrations are mediators of these processes also by modulating peripheral blood mononuclear cells (PBMCs) response. The aim of this study was to investigate if HMGB-1 and IL-2 turn on PBMCs and their leptin secretion. In isolated human PBMCs and their subpopulations from healthy individuals and naïve T2DM patients, leptin release, pro-inflammatory response and Toll-like Receptors (TLRs) activation was measured. After treatment with IL-2 and HMGB1, NK (Natural Killer) have the highest amount of leptin secretion, whilst NK-T have the maximal release in basal conditions. TLR4 (TAK242) and/or TLR2 (TLR2-IgA) inhibitors decreased leptin secretion after IL-2 and HMGB1 treatment. A further non-significant increase in leptin secretion was reported in PBMCs of naive T2DM patients in response to IL-2 and HMGB-1 stimulation. Finally, hyperglycemia or hyperinsulinemia might stimulate leptin secretion from PBMCs. The amount of leptin released from PBMCs after the different treatments was enough to stimulate the secretion of IL-1β from monocytes. Targeting leptin sera levels and secretion from PBMCs could represent a new therapeutic strategy to counteract metabolic diseases such as T2DM.

Noble Metals for Modern Implant Materials: MOCVD of Film Structures and Cytotoxical, Antibacterial, and Histological Studies.

This work is aimed at developing the modification of the surface of medical implants with film materials based on noble metals in order to improve their biological characteristics. Gas-phase transportation methods were proposed to obtain such materials. To determine the effect of the material of the bottom layer of heterometallic structures, Ir, Pt, and PtIr coatings with a thickness of 1.4–1.5 μm were deposited by metal–organic chemical vapor deposition (MOCVD) on Ti6Al4V alloy discs. Two types of antibacterial components, namely, gold nanoparticles (AuNPs) and discontinuous Ag coatings, were deposited on the surface of these coatings. AuNPs (11–14 nm) were deposited by a pulsed MOCVD method, while Ag films (35–40 nm in thickness) were obtained by physical vapor deposition (PVD). The cytotoxic (24 h and 48 h, toward peripheral blood mononuclear cells (PBMCs)) and antibacterial (24 h) properties of monophase (Ag, Ir, Pt, and PtIr) and heterophase (Ag/Pt, Ag/Ir, Ag/PtIr, Au/Pt, Au/Ir, and Au/PtIr) film materials deposited on Ti-alloy samples were studied in vitro and compared with those of uncoated Ti-alloy samples. Studies of the cytokine production by PBMCs in response to incubation of the samples for 24 and 48 h and histological studies at 1 and 3 months after subcutaneous implantation in rats were also performed. Despite the comparable thickness of the fibrous capsule after 3 months, a faster completion of the active phase of encapsulation was observed for the coated implants compared to the Ti alloy analogs. For the Ag-containing samples, growth inhibition of S. epidermidis, S. aureus, Str. pyogenes, P. aeruginosa, and Ent. faecium was observed.

Peripheral blood mononuclear cell response to YKL-40 and Galectin-3 in cystic fibrosis

BackgroundElevated circulating levels of YKL-40 correlate with disease severity in Cystic Fibrosis (CF), but the role of YKL-40 in the inflammatory response in CF is still under investigation. Our main goal was to evaluate if YKL-40 can modulate the expression of major cytokines (IL-6, IL-10, IL-13) implicated in the inflammatory response in CF. A secondary goal was to explore the interactions between YKL-40 and other circulating proteins to determine the impacts on cytokine modulation. MethodPeripheral blood mononuclear cells (PBMCs) were isolated from the blood of 83 adult CF patients in stable clinical condition. PBMCs were treated with human YKL-40 followed by the measure of IL-6, IL-10 and IL-13 gene expression. Protein arrays were used to explore the interactions between YKL-40 and circulating proteins. Interaction with Galectin-3 (GAL3) was identified, and confirmed by binding assay. Cytokine gene expressions were again monitored by RT-qPCR after PBMC treatment with GAL3, with or without YKL-40 co-stimulation. ResultsFollowing YKL-40 stimulation, PBMC gene expression of IL-6, IL-10 and IL-13 varies across patients. IL-6 and IL-13 are coexpressed, but this response was different in male and female patients. GAL3 protein was detected in the blood of CF patients, and a molecular interaction with YKL-40 was identified. GAL3 did not interfere with the YKL-40 stimulation of IL-6, IL-10 and IL-13 but may modulate the coexpression. ConclusionWe observed that YKL-40 stimulation had a variable impact on IL-6, IL-10, and IL-13 gene expression in CF PBMCs and uncovered an interaction between GAL3 and YKL-40 in the serum of CF patients. Our findings suggest that YKL-40 is not only a biomarker of disease severity in CF, but it might play an active role in the inflammatory pathophysiology of the disease.

Abstract LB049: Preliminary analysis of immunogenicity of HB-201 and HB-202, an arenavirus-based cancer immunotherapy, in patients with advanced HPV16-positive cancers

Abstract Background: Human Papillomavirus 16 (HPV16) is linked to most HPV-associated cancers such as cervical, head and neck, vaginal and anal cancers. Treatment options are limited for patients with HPV16+ recurrent or metastatic cancers. The generation and maintenance of the HPV16+ malignant state requires the stable expression of HPV16-specific E7 and E6 oncogenes, which therefore constitute attractive targets for immunotherapy. HB-201 and HB-202 are both replicating live-attenuated vectors based on arenaviruses LCMV and PICV, respectively, expressing the same non-oncogenic HPV16 E7E6 fusion protein for induction of tumor specific T-cell responses. In preclinical models, administration of HB-201 alone and sequential administration of HB-202 followed by HB-201 was safe and demonstrated potent immunogenicity by induction of E7 and E6 -specific CD8+ T cell responses and efficient tumor control of HPV+ TC-1 tumors. Methods: A first-in-human, Phase I/II open-labelled clinical trial of HB-201 single vector therapy and HB-201 & HB-202 two-vector therapy in patients with treatment-refractory HPV16+ cancers is currently ongoing (NCT04180215). Here, we present first immunogenicity results from the dose escalation phase I of this study. The phase I of the trial is designed to evaluate different dose levels and dosing schedules of HB-201 as a single-vector therapy or as an alternating two-vector therapy together with HB-202. Peripheral blood mononuclear cells (PBMCs) were collected before and after treatment from all patients. PBMCs from a subset of patients were examined for HPV16-specific T cell responses measured by IFN-γ enzyme-linked immunospot. Paired tissue biopsy and serum samples were also collected and being currently evaluated for histology and pharmacokinetics. Results: We demonstrated induction of a directly ex vivo (i.e. no expansion) detectable HPV16-specific T-cell response in PBMCs from patients receiving a single dose of HB-201 or HB-202. Additional exploratory analysis will be available at the time of the meeting. Conclusion: These preliminary data demonstrate for the first time with arenavirus vectors, the induction of HPV16-specific T cells in cancer patients following a single injection of HB-201 or HB-202. Arenavirus vectors expressing E7E6 may constitute a new potential therapy for patients with treatment refractory HPV16+ cancers. Clinical data will be presented in an upcoming scientific meeting. Additional schedules, alternating two-vector therapy with HB-201/HB-202, and combination with anti-PD-1 mAbs are being explored in additional cohorts. Citation Format: Kia Katchar, Michael Schwendinger, Diane DaSilva, Henning Lauterbach, Klaus Orlinger, Xiaoping Qing, Daniel Pinschewer, Igor Matushansky. Preliminary analysis of immunogenicity of HB-201 and HB-202, an arenavirus-based cancer immunotherapy, in patients with advanced HPV16-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB049.

Label-free quantitative lymphocyte activation profiling using microfluidic impedance cytometry

Circulating lymphocytes are integral components of our adaptive immunity with emerging clinical applications in immune status monitoring in infectious diseases and cell-mediated cancer immunotherapies. Herein we present a novel impedance-based microfluidic assay for label-free lymphocyte activation profiling based on native or antigen-specific T-lymphocyte biophysical responses. Single cell impedance profiling of T-lymphocytes first revealed distinct biophysical differences in cell size and membrane electrical impedance of healthy, activated (CD3/CD28) and dead lymphocyte populations. Impedance characterization of peripheral blood mononuclear cells (PBMCs) stimulated with mitogen phytohemagglutinin (PHA) or Tuberculin Purified Protein Derivative antigen (PPD) after 24 h also showed an increase in lymphocyte cell size (∼8 to 10 μm) which corresponded to activated lymphocytes (CD69+CD137+). We next developed a spiral inertial microfluidics cell sorter integrated with coplanar electrodes for direct impedance quantification of activated lymphocytes. By removing non-activated smaller lymphocytes (< 8 μm) and employing hydrodynamic-based single stream particle focusing, we demonstrated significant enrichment of activated lymphocytes (∼11.7-fold) to electrically detect low levels of lymphocyte activation (< 5%). Finally, the developed biochip is coupled with magnetic activated cell sorting (MACS) to quantify CD4+ T-lymphocytes response in PBMCs stimulated with PPD. A differential impedance cell count ratio (stimulated/unstimulated) was defined to distinguish activated T-lymphocytes, which showed better sensitivity as compared to immunophenotyping by flow cytometry. Taken together, the integrated impedance biosensor can be further developed as a rapid multiplexed screening assay to detect antigen-specific T-lymphocyte responses to characterize host immunity and diagnosis of infectious diseases (e.g tuberculosis, dengue and COVID-19).

Dietary Sodium Nitrate Activates Antioxidant and Mitochondrial Dynamics Genes after Moderate Intensity Acute Exercise in Metabolic Syndrome Patients.

Exercise can induce a pro-inflammatory response in aged subjects with metabolic disorders and nitrate supplementation has shown anti-inflammatory effects. We evaluated the influence of dietary nitrate on the response of the antioxidant and mitochondrial dynamics genes to acute exercise in peripheral blood mononuclear cells (PBMCs), as well as the antioxidant and the inflammatory response of PBMCs against immune stimulation. Metabolic syndrome patients participated in a crossover study in which they consumed a beverage containing 16 mM sodium nitrate or a placebo with the same composition without nitrate before performing a submaximal test at 60–70% of their maximal heart rate for 30 min. The intake of nitrate increased the nitrate plus nitrite plasma levels about 8-fold and induced the upregulation of catalase, superoxide dismutase, glutathione peroxidase, mitofusin 2 and PGC1α in PBMCs after exercise. The gene expression of catalase and TNFα was enhanced by phorbol myristate acetate (PMA) only in the placebo group, while the glutathione peroxidase expression was enhanced by PMA only after nitrate intake. The intake of nitrate by metabolic syndrome patients induces an antioxidant and mitochondrial response to exercise at the same time that it attenuates the pro-inflammatory response to immune stimulation.

The role of sirtuin 1 on the induction of trained immunity

Sirtuin 1 (SIRT1) has been described to modify immune responses by modulation of gene transcription. As transcriptional reprogramming is the molecular substrate of trained immunity, a de facto innate immune memory, we investigated the role of SIRT1 in the induction of trained immunity. We identified various SIRT1 genetic single nucleotide polymorphisms affecting innate and adaptive cytokine production of human peripheral blood mononuclear cells (PBMCs) in response to various stimuli on the one hand, and in vitro induction of trained immunity on the other hand. Furthermore, inhibition of SIRT1 upregulated pro-inflammatory innate cytokine production upon stimulation of PBMCs. However, inhibition of SIRT1 in vitro had no effect on cytokine responses upon induction of trained immunity, while activation of SIRT1 mildly modified trained immunity responses. In conclusion, SIRT1 modifies innate cytokine production by PBMCs in response to various microbes, but has only a secondary role for BCG and β-glucan-induced trained immunity responses.

Pathogenesis, MicroRNA-122 Gene-Regulation, and Protective Immune Responses After Acute Equine Hepacivirus Infection.

Background and AimsEquine hepacivirus (EqHV) is phylogenetically the closest relative of HCV and shares genome organization, hepatotropism, transient or persistent infection outcome, and the ability to cause hepatitis. Thus, EqHV studies are important to understand equine liver disease and further as an outbred surrogate animal model for HCV pathogenesis and protective immune responses. Here, we aimed to characterize the course of EqHV infection and associated protective immune responses.Approach and ResultsSeven horses were experimentally inoculated with EqHV, monitored for 6 months, and rechallenged with the same and, subsequently, a heterologous EqHV. Clearance was the primary outcome (6 of 7) and was associated with subclinical hepatitis characterized by lymphocytic infiltrate and individual hepatocyte necrosis. Seroconversion was delayed and antibody titers waned slowly. Clearance of primary infection conferred nonsterilizing immunity, resulting in shortened duration of viremia after rechallenge. Peripheral blood mononuclear cell responses in horses were minimal, although EqHV‐specific T cells were identified. Additionally, an interferon‐stimulated gene signature was detected in the liver during EqHV infection, similar to acute HCV in humans. EqHV, as HCV, is stimulated by direct binding of the liver‐specific microRNA (miR), miR‐122. Interestingly, we found that EqHV infection sequesters enough miR‐122 to functionally affect gene regulation in the liver. This RNA‐based mechanism thus could have consequences for pathology.ConclusionsEqHV infection in horses typically has an acute resolving course, and the protective immune response lasts for at least a year and broadly attenuates subsequent infections. This could have important implications to achieve the primary goal of an HCV vaccine; to prevent chronicity while accepting acute resolving infection after virus exposure.

Lipopolysaccharide stimulation test on cultured PBMCs assists the discrimination of cryopyrin-associated periodic syndrome from systemic juvenile idiopathic arthritis

Systemic juvenile idiopathic arthritis (sJIA) and cryopyrin-associated periodic syndrome (CAPS) share many common manifestations. We aim to identify an applicable method to assist disease discrimination. Inflammatory cytokines were measured in the plasma of patients with CAPS, sJIA with persistent disease course and healthy controls. Supernatants collected from non-stimulated peripheral blood mononuclear cells (PBMCs) and those undergone inflammasome stimulation tests utilizing lipopolysaccharide (LPS) with and without adenosine triphosphate (ATP) were investigated. Inflammatory cytokines in patient plasma fail to differentiate sJIA from CAPS. PBMCs from sJIA secrets higher amount of IL-1β and IL-18 while CAPS PBMCs produces more caspase-1 without stimulation. IL-1β, IL-18, and caspase-1 were significantly elevated among CAPS PBMCs (all p < 0.05) upon LPS stimulation, but not when additional ATPs were provided. Levels of cytokines and PBMC responses to the stimulation assays were similar among all sJIA patients regardless of their history of macrophage activation syndrome. Unstimulated PBMC activities and the LPS inflammasome stimulation assay without exogenic ATPs can assist the differentiation of CAPS from sJIA with persistent disease course.

Proteomic Analysis of Peripheral Blood Mononuclear Cells Following a Single High Fat High Carbohydrate Meal Challenge

ObjectivesAn individual’s ability to adapt to a dynamic environment is termed ‘phenotypic flexibility’ and can be weakened by chronic stress, leading to dysregulation of normal homeostatic processes. A long-term high energy and high fat high carbohydrate (HFHC) diet increases risk of metabolic diseases, however, the acute effect of a single HFHC meal on disturbances in metabolic homeostasis is less understood. In this study, we aimed to characterise the effect of a single HFHC meal on the postprandial proteome of peripheral blood mononuclear cells (PBMCs). MethodsTwelve healthy men (22.7 ± 3.4 years, BMI of 22.2 ± 1.5 kg/m2) were recruited. Participants consumed a HFHC milkshake after fasting overnight. The meal contained 16 g of carbohydrate and 15 g of fat per 100 g and had a total energy content of 64 kJ/kg body weight. PBMCs were collected at fasting, 3 and 6 hours following the meal. The proteome was measured via LC-MS/MS using untargeted label-free quantification. Differentially expressed proteins were identified with a modified paired t test (limma, R v.4.0.3). Gene ontology analysis was performed with ClueGO v2.5.7 in Cytoscape v3.8.2. ResultsAfter filtering to include only high confident proteins consistently detected in each condition, 6007 proteins were identified and included in analysis. Changes in protein expression relative to fasting were observed at 3 hours (143 proteins) and 6 hours (210 proteins) post meal (fold change > ±1.2, p < 0.05). The most highly altered biological processes observed at each timepoint were ‘zymogen activation’ (GO:0,031,638, 31.25% of groups at 3 hours) and ‘ribosome biogenesis’ (GO:0,042,254, 19.15% of groups at 6 hours). ConclusionsThese results reveal adaptations that occur in the PBMC proteome in response to a single HFHC meal. Changes to proenzyme activation shows a general response in PBMCs to meal-induced changes in the cellular environment, whilst regulation of ribosome synthesis pathways may reflect changes in energy regulation pathways caused by increased nutrient availability. This study characterises the PBMC response in healthy men, and provides a useful reference for future studies that investigate how PBMCs lose the ability to maintain homeostasis following a HFHC meal. Funding SourcesMonash University, Melbourne, Australia

Direct and indirect immune effects of CMP-001, a virus-like particle containing a TLR9 agonist

BackgroundCMP-001, also known as vidutolimod, is a virus-like particle containing a TLR9 agonist that is showing promise in early clinical trials. Our group previously demonstrated that the immunostimulatory effects of...

Dynamic insulin-stimulated mTOR/GSK3 signaling in peripheral immune cells: Preliminary evidence for an association with lithium response in bipolar disorder.

A key mechanism of lithium is the inhibition of glycogen synthase kinase-3β (GSK3β) and activation of mammalian target of rapamycin (mTOR), two contributors to insulin signaling. We explored the relationship between these markers and clinical response to lithium in bipolar disorder (BD). Thirty-four subjects with BD who had been taking lithium for ≥2years and had a maintenance lithium Alda score defined as either high (≥7; n=20) or low (≤2; n=14) were included in the study. Baseline protein expression of GSK3β and mTOR (total and phosphorylated (p)) was obtained from a buffy coat. Peripheral blood mononuclear cells (PBMCs) from a subset of each group (n=11) were stimulated with insulin (10µg) and change in protein expression was determined using Western blot. In buffy coat samples, significantly higher levels of pmTOR were present in subjects with an Alda score ≤2 (lithium non-responsive), relative to those with scores ≥7 (lithium-responsive). No differences were observed for pGSK3β. In contrast, functional PBMC responses to 5min of insulin stimulation demonstrated robust increases in pGSK3β (87.05±43.41%) and pmTOR (105.7±66.48%) in the lithium responsive group only. This contrasted observed decreases in pGSK3β (34.08±16.12%) and pmTOR (37.84±14.39%) 5mins post-insulin in non-responders. Dynamic increases in pmTOR and pGSK3β post-insulin stimulation may reflect an immunometabolic state that facilitates lithium response. Further prospective analyses are needed to replicate and extend these preliminary findings and further investigate the role of insulin signaling in lithium response in BD.

Inhibition of Epigenetic Reader BET Proteins by Apabetalone Counters Inflammation in Activated Innate Immune Cells from Fabry Disease Patients Receiving ERT

Abstract Fabry disease (FD) is a rare genetic disorder with a deficit in the degradation pathway of a glycolipid, globotriaocylceramide (Gb3). Gb3 deposits in various tissues evoke immune-mediated systemic inflammation, driving the progression of FD complications. In particular, cardiac and renal problems, leading causes of mortality in FD patients, are insufficiently managed by enzyme replacement therapy (ERT) in the long term. Here, we examined immune profiles in unstimulated peripheral blood mononuclear cells (PBMCs) from FD patients on ERT relative to untreated patients. The results showed an upregulation of the CCR2/MCP-1 axis, particularly in the presence of renal dysfunction. We also observed an increase of IL12B transcripts in unstimulated PBMCs when tracking the immune status over two years of ERT in patients with early-stage chronic kidney disease (CKD). Interestingly, pro-inflammatory responses in LPS-stimulated PBMCs were countered by apabetalone, a clinical-stage candidate that inhibits Bromodomain and Extra Terminal (BET) proteins, epigenetic readers. Apabetalone treatment dose dependently inhibited MCP-1 and IL-12 production by up to 90%. Apabetalone downregulated the transcription of other pro-inflammatory cytokines including TNF-α, IL-6 and IL-8 by 70%, 48% and 54%, respectively. Reactive oxygen species (ROS) are an indicator of oxidative damage caused by intracellular Gb3 deposits in FD patients. Apabetalone suppressed ROS levels by up to ~70% in LPS-stimulated neutrophils. Hence, apabetalone treatment may reduce pathological inflammation and oxidative stress in FD patients and thus complement ERT to optimize patient outcomes, warranting further investigation of apabetalone as a therapeutic for FD.

The Impact of Advanced Age on Vaccine-Induced Tissue Immune Recall Responses

Abstract Infectious diseases account for one-third of deaths in individuals over the age of 65. Vaccines are a primary tool to combat infection, yet they are less effective in the aged population. Many have focused on vaccine-induced peripheral blood responses in old age; however, work from our lab demonstrates that immune responses to vaccination and infection in the periphery and local infection site (tissue) differ. To improve health outcomes in our aged population, we must study both systemic and tissue vaccine responses. We do so using a model of Mycobacterium bovis BCG vaccination and subsequent tuberculin skin test (TST) eight weeks after vaccination in adult and aged baboons. Vaccination generates BCG-specific immune cells that are recruited to the skin upon TST challenge, which we can study in skin biopsies reflecting tissue-specific responses. Three days or one week after TST we examine BCG-specific responses in skin biopsies. We also determine BCG-induced responses in the blood to compare tissue and systemic responses. We find increased oxidation and decreased production of immune proteins in aged baboon skin at the site of TST challenge, despite no alterations in antigen-specific peripheral blood mononuclear cell (PBMC) responses between age groups. Interestingly, cell migration assays show increased migration of PBMCs from aged BCG-vaccinated baboons, reinforcing the notion that systemic responses are not altered and that study of the tissue environment is important to understand responses to antigenic challenge with older age.

Impact of heat stress and a feed supplement on hormonal and inflammatory responses of dairy cows

The aim of this trial was to evaluate the effects of an immunomodulatory supplement (OmniGen AF, OG; Phibro Animal Health Corp.) and heat stress on hormonal, inflammatory, and immunological responses of lactating dairy cows. Sixty multiparous Holstein cows were randomly assigned to 4 treatments in a 2 × 2 factorial arrangement using 2 environments: cooled using fans and misters, or noncooled, and 2 top-dressed feed supplements (56 g/d): OG or a placebo (CTL). Temperature-humidity index averaged 78 during the 8-wk trial. Blood was drawn to analyze cortisol, prolactin, and circulating tumor necrosis factor (TNF)-α and IL-10. Peripheral blood mononuclear cells (PBMC) were isolated and stimulated with hydrocortisone, prolactin, or lipopolysaccharide (LPS), individually or in several combinations, to assess induced proliferation and cytokine production. At d 52, 6 cows per treatment were injected i.v. with an LPS bolus (ivLPS) to assess hormone and cytokine responses. For cooled cows, feeding OG increased plasma cortisol concentration relative to CTL. Noncooled cows fed CTL had lower circulating TNF-α concentrations than noncooled-OG and cooled-CTL cows, with cooled-OG intermediate. Hydrocortisone+LPS-stimulated PBMC from OG cows tended to proliferate more than CTL. Relative to cooled cows, PBMC from noncooled cows produced more TNF-α and IL-10 when stimulated with LPS. Following ivLPS, cooled-OG cows had a greater cortisol response than the other treatments. In conclusion, OG supplementation enhanced cortisol release under basal condition and induced inflammation with cooling compared with CTL. This suggests that heat stress inhibits OG-mediated cortisol release. Heat stress seemed to enhance the inflammatory responses of PBMC from lactating cows. However, OG supplementation promoted PBMC proliferation under stress, or in the presence of hydrocortisone.

Inflammatory cytokines dysfunction in autism spectrum disorder

IntroductionAutism spectrum disorder (ASD) is a common neurodevelopmental disorder. Its underlying causes and pathophysiologies remain unclear. Recent data support the potential involvement of neuroinflammation in the onset of this disorder.ObjectivesThe aim of our study was to investigate the potential link between ASD and inflammatory mediators.MethodsThis descriptive study was conducted among ASD outpatients followed-up at the child and adolescent psychiatry department in the Military Hospital of Instruction of Tunis. Blood samples were collected for inflammatory cytokines dosage, notably the interleukin 1β (IL-1β), interleukin 6 (IL-6) and the Tumor Necrosis Factor α (TNF-α) immunodosage.ResultsTwenty-four patients were included in this study, aged between four and ten years old (mean age= 6,55 years; minimum=4; maximum=10 years). Our sample was mainly represented by male patients (95,6%). TNF-α plasmatic levels were high (&gt;5pg/mL) among all of our sample with a mean of 11,6 pg/mL (minimum= 6,87; maximum=17,7 pg/mL; standard deviation= 3,52 pg/mL), suggesting abnormal peripheral blood mononuclear cells response. However, IL-1β and IL-6 plasmatic levels were relatively normal.ConclusionsAn immune response dysregulation was detected in our sample. Multiple clinical and experimental studies investigated the implication of inflammatory cytokines in neurodevelopmental disruption. Their results, however, remain controversial and limited by small samples. Further studies need to be done in order to investigate the neuroimmunological factors linked with ASD.

Mesenchymal Stem Cells Regulate Type 2 Innate Lymphoid Cells via Regulatory T Cells through ICOS-ICOSL Interaction

Group 2 innate lymphoid cells (ILC2s) are recognized as key controllers and effectors of type 2 inflammation. Mesenchymal stem cells (MSCs) have been shown to alleviate type 2 inflammation by modulating T lymphocyte subsets and decreasing TH2 cytokine levels. However, the effects of MSCs on ILC2s have not been investigated. In this study, we investigated the potential immunomodulatory effects of MSCs on ILC2s in peripheral blood mononuclear cells (PBMCs) from allergic rhinitis patients and healthy subjects. We further investigated the mechanisms involved in the MSC modulation using isolated lineage negative (Lin−) cells. PBMCs and Lin− cells were cocultured with induced pluripotent stem cell‐derived MSCs (iPSC‐MSCs) under the stimulation of epithelial cytokines IL‐25 and IL‐33. And the ILC2 levels and functions were examined and the possible mechanisms were investigated based on regulatory T (Treg) cells and ICOS‐ICOSL pathway. iPSC‐MSCs successfully decreased the high levels of IL‐13, IL‐9, and IL‐5 in PBMCs in response to IL‐25, IL‐33, and the high percentages of IL‐13+ILC2s and IL‐9+ILC2s in response to epithelial cytokines were significantly reversed after the treatment of iPSC‐MSCs. However, iPSC‐MSCs were found directly to enhance ILC2 levels and functions via ICOS‐ICOSL interaction in Lin− cells and pure ILC2s. iPSC‐MSCs exerted their inhibitory effects on ILC2s via activating Treg cells through ICOS‐ICOSL interaction. The MSC‐induced Treg cells then suppressed ILC2s by secreting IL‐10 in the coculture system. This study revealed that human MSCs suppressed ILC2s via Treg cells through ICOS‐ICOSL interaction, which provides further insight to regulate ILC2s in inflammatory disorders.

MiR‑101‑3p negatively regulates inflammation insystemic lupus erythematosus via MAPK1targeting and inhibition of the NF‑κB pathway.

Systemic lupus erythematosus (SLE) is an autoimmune disease often used as a model in genomics research. The downregulation of microRNA‑101‑3p (miR‑101‑3p) participates in the progression of SLE, although the underlying mechanisms remain to be elucidated. The present study aimed to evaluate the specific roles of miR‑101‑3p in the SLE inflammatory response and its potential mechanisms. Reverse transcription‑quantitative (RT‑q) PCR was used to profile miR‑101‑3p expression in the peripheral blood mononuclear cells (PBMCs) from 40 female patients with SLE and 20 female healthy volunteers. The interactions between miR‑101‑3p and MAPK1 were identified and evaluated using dual‑luciferase reporter and RNA pull‑down assays. The levels of IL‑10 and IFN‑γ were evaluated by enzyme‑linked immunosorbent assay. The expression of NF‑κB p65 and phosphorylated IκBα were evaluated using western blotting. miR‑101‑3p expression was demonstrated to be downregulated in SLE PBMCs. miR‑101‑3p negatively regulated IL‑10 and IFN‑γ expression in SLE samples and was demonstrated to target MAPK1. Increases in MAPK1 expression eliminated miR‑101‑3p inhibition of IL‑10 and IFN‑γ. MAPK1 activated the NF‑κB pathway in SLE PBMCs and this activation was inhibited when miR‑101‑3p was overexpressed. In addition, treatment with BAY11‑7085 (NF‑κB activator) was demonstrated to reverse the inhibitory effects of miR‑101‑3p expression on both IL‑10 and IFN‑γ in SLE PBMCs. BAY11‑7082 also markedly reduced MAPK1‑induced increases in IL‑10 and IFN‑γ in SLE PBMCs. miR‑101‑3p overexpression attenuated the inflammatory response in SLE PBMCs by inhibiting the expression of MAPK1 and blocking the NF‑κB pathway. The results revealed a novel regulatory mechanism in SLE inflammation and offer a new direction for the development of SLE treatments.

Echinacea purpurea extract (cichoric acid) exerts an anti-inflammatory effect on yak PBMCs and regulates the TLR4 signalling pathway

IntroductionInflammation is one of the main causes of impaired health in livestock and some of its processes weaken animal productivity and impact human health. The present study was conducted to evaluate the effect of echinacea extract (cichoric acid – CA) on yak peripheral blood mononuclear cells (PBMCs), inflammatory-related factors, and the toll-like receptor (TLR)4 signalling pathway induced by lipopolysaccharide (LPS) in these PBMCs.Material and MethodsYak PBMCs were co-cultured with LPS and CA in vitro. The proliferative activity of cells was detected using the cell-counting kit-8 method, the optimal stimulation concentration of LPS was selected, the effect of CA on the content of inflammation-related factors was evaluated using an ELISA kit, and the mRNA expression of these factors was detected by RT-PCR.ResultsCA inhibited the inflammatory response of yak PBMCs induced by LPS. CA inhibited gene and protein expression of key nodes of the TLR4 signalling pathway in yak PBMCs.ConclusionIt is suggested that CA has anti-inflammatory and immunomodulatory effects on yak PBMCs via the TLR4 pathway.

Effects of relocation on immunological and physiological measures in female squirrel monkeys (Saimiri boliviensis boliviensis).

In the present study, we have quantified the effects of transport, relocation and acclimate/adapt to their new surroundings on female squirrel monkey. These responses are measured in blood samples obtained from squirrel monkeys, at different time points relative to their relocation from their old home to their new home. A group of squirrel monkeys we transported, by truck, for approximately 10 hours. Peripheral blood mononuclear cells (PBMCs) were assayed in order to evaluate the phenotype of lymphocyte subsets by flow, mitogen-specific immune responses of PBMCs in vitro, and levels of cytokines at various time points including immediately before transport, immediately upon arrival, and after approximately 150 days of acclimation. We observed significant changes in T cells and subsets, NK and B cells (CD4+, CD8+, CD4+/CD8+, CD16+, and CD20+). Mitogen specific (e.g. PHA, PWM and LPS) proliferation responses, IFN-γ by ELISPOT assay, and cytokines (IL-2, IL-4 and VEGF) significant changes were observed. Changes seen in the serum chemistry measurements mostly complement those seen in the hematology data. The specific goal was to empirically assess the effects of relocation stress in squirrel monkeys in terms of changes in the numbers and functions of various leukocyte subsets in the blood and the amount of time required for acclimating to their new environment. Such data will help to determine when newly arrived animals become available for use in research studies.