Non-stimulated Peripheral Blood Mononuclear Cells Research Articles

Anti-inflammatory effects of Mentha pulegium L. extract on human peripheral blood mononuclear cells are mediated by TLR-4 and NF-κB suppression

There is great interest in evaluating the anti-inflammatory properties of new herbal products. Thus, the effects of Mentha pulegium L. extract on gene and protein expressions of pro-inflammatory mediators and transcription factors were determined.The hydro-ethanolic extract of Mentha pulegium L. was obtained and optimal non-cytotoxic concentrations of the extract were determined by MTT assay. Then, three different concentrations of Mentha pulegium L. (10, 30, and 90 μg/mL) were used to pre-treat the lipopolysaccharide (LPS)-stimulated and non-stimulated peripheral blood mononuclear cells (PBMCs) of 10 healthy individuals. Finally, the tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, Toll-like receptor-4 (TLR-4), nuclear factor-kappa B (NF-κB) p65, activator protein-1 (AP-1), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) gene expressions and TNF-α, IL-1β, IL-6, TLR-4, prostaglandin E2 (PGE2), and COX-2 protein levels were measured.MTT results showed that there is no significant difference in cell viability among 10, 20, 40, and 80 μg/mL concentrations of Mentha pulegium L. extract at 24, 48, and 72 h (P > 0.05). The IC50 values were 236.1, 147.0, and 118.0 μg/mL after 24, 48, and 72 h respectively. TNF-α, IL-1β, IL-6, TLR-4, iNOS, and NF-κB p65 mRNA levels in the pre-treated LPS-stimulated PBMCs were concentration-dependently reduced (P < 0.01 for TNF-α, TLR-4, and NF-κB p65; P < 0.05 for IL-1β, IL-6, and iNOS). Also, the protein levels of pro-inflammatory mediators decreased and these differences were significant for TNF-α, IL-1β, and TLR-4 (P < 0.001, P < 0.01, and P < 0.001, respectively).Mentha pulegium L. extract decreased the expression and biosynthesis of pro-inflammatory mediators. These effects are mainly mediated by TLR-4 and NF-κB suppression. Thus, Mentha pulegium L. could be useful in treating or ameliorating chronic inflammatory diseases.

Peripheral Blood Mononuclear Cells from Patients with Type 1 Diabetes and Diabetic Retinopathy Produce Higher Levels of IL-17A, IL-10 and IL-6 and Lower Levels of IFN-γ-A Pilot Study.

Inflammation is key to the pathogenesis of diabetic retinopathy (DR). This prospective study investigated alterations in inflammatory cytokines in peripheral blood mononuclear cells (PBMCs) in 41 people with type 1 diabetes (T1D), sub-grouped into mild non-proliferative DR (mNPDR; n = 13) and active and inactive (each n = 14) PDR. Age/gender-matched healthy controls (n = 13) were included. PBMCs were isolated from blood samples. Intracellular cytokine expression by PBMCs after 16-h stimulation (either E. coli lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate plus ionomycin, D-glucose or D-mannitol) were assessed by flow cytometry. Cytokine production in plasma, non-stimulated and LPS-stimulated PBMC supernatant was also assessed. Increased BMC IL-10 secretion and reduced expression of IL-6 and IFN-γ in CD3+ cells were observed in mNPDR. Reduced IL-6 and IL-10 secretion, and higher levels of intracellular IL-6 expression, especially in CD11b+ PBMCs, was detected in aPDR; levels were positively correlated with DR duration. Patients with T1D demonstrated increased intracellular expression of IL-17A in myeloid cells and reduced IFN-γ expression in CD3+ cells. Plasma levels of IL-1R1 were increased in mNPDR compared with controls. Results suggest that elevated PBMC-released IL-10, IL-6, in particular myeloid-produced IL-17A, may be involved in early stages of DR. IL-6-producing myeloid cells may play a role in PDR development.

Serum Levels and in vitro CX3CL1 (Fractalkine), CXCL8, and IL-10 Synthesis in Phytohemaglutinin-Stimulated and Non-stimulated Peripheral Blood Mononuclear Cells in Subjects With Schizophrenia.

IntroductionAlthough schizophrenia is a severe mental illness, whose etiology is still largely unknown, its pathogenesis may be associated with dysregulation of the immune mechanisms. The present study compares the levels of interleukin (IL)-10, interleukin-8 (CXCL8), and fractalkine (CX3CL1) between schizophrenia patients and healthy controls. It also assesses the ability of peripheral peripheral blood mononuclear cells (PBMCs) to produce these cytokines spontaneously and following mitogen-stimulation.Materials and MethodsA prospective study was performed of 60 adult schizophrenia patients and 32 controls. CXCL8, IL-10, and fractalkine concentrations were measured in serum and supernatants from cultured PBMCs. Anthropometric (BMI, WHR) and body composition measurements were taken using bioimpedance analysis (BIA) and dual-energy X-ray absorptiometry (DXA).Results and ConclusionThe schizophrenia patients demonstrated significantly higher levels of serum CXCL8 (schizophrenia: 13.4 ± 15.7 pg/mL, control: 6.9 ± 4.2 pg/mL, p = 0.001) and lower level of serum fractalkine (schizophrenia: 22.8 ± 9.9 pg/mL, control: 45.4 ± 84.5 pg/mL, p = 0.041). Serum IL-10 levels did not significantly differ. No in vitro synthesis of fractalkine was observed. Neither unstimulated or PHA-stimulated CXCL8 secretion differed between the two groups (p >0.05). The patients not taking mood stabilizers (MS–) demonstrated significantly higher CXCL8 levels than those on mood stabilizers (MS+) (p = 0.03) and control (p < 0.001). In addition, the MS- sub-group demonstrated significantly lower serum fraktalkine than controls (p = 0.009). These effects could be described as pseudo-normalization of CXCL8 and fractalkine in schizophrenia patients taking mood stabilizers.

T-lymphocyte activation markers in patients with HIV-1-associated neurocognitive disorder.

Despite immune-reconstitution, after TARc HIV-positive patients, neurocognitive disorders related to HIV-1 (HAND) have been observed in these patients. The diagnosis occurs, in most cases, in the advanced stage. The objective of this study is to quantify activation markers (CD25, CD38, CD69, and HLA-DR) in the blood of patients with chronic HIV-1 infection and relate to HAND and premature senescence. The level of activation markers was quantified in the blood of 10 HIV-positive patients with HAND, 10 cases without HAND undergoing regular follow-up at the Secondary Immunodeficiency Clinic (ADEE3002) at the Hospital of Clinics of the Medical School of São Paulo, and 10 healthy seronegative volunteers using the flow cytometry method. Subsequently, the analysis was performed using the FlowJo™ v10.6.1 program and GraphPad Prism 8.3.0. In addition to T CD4+ cells, T CD4+ bright/CD8+ dim and T CD4+/CD8+/CD45RA-/CD27 cells, a specific HIV-1 phenotype, also proved to be relevant to differentiate patients with HAND. Between the activation marker, CD38 in T CD4+ and T CD4+/CD8+/CD45RA-/CD27+ cells and the activation marker HLA-DR in T CD8+/CD45RA-/CD27+ managed to differentiate our HAND group. Importantly, only non-stimulated peripheral blood mononuclear cells (PBMCs) were used in this study. A combination of activation and senescence markers CD38 and HLA-DR and subgroups of T lymphocytes can be used to indicate seropositive patients who are progressing to a HAND condition. Thus, it can contribute to an early diagnosis and opportunity for possible reversal of dementia with alternative treatments, with high penetration in the blood-brain barrier.

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.

Ex vivo Nitrite Exposure and Response of Peripheral Blood Mononuclear Cells from Osteoarthritis Cases and Controls: Proliferation and Nitric Oxide Generation

This ex vivo pilot study examined the effect of low dose nitrite exposure on peripheral blood mononuclear cells (PBMC) proliferation and nitric oxide (NO) generation among people with osteoarthritis (cases) and without osteoarthritis (controls). The hypothesis was that nitrite exposure produced different effects on proliferation and NO generation between the cases and controls. Human PBMC, composed of lymphocytes and monocytes, were isolated from the whole blood of 31 participants (15 cases and 16 controls) using density gradient centrifugation, then exposed to five different nitrite concentrations: 0 (negative controls), 1 mg/l (0.02 mM), 5 mg/l (0.10 mM), 10 mg/l (0.21 mM), and 15 mg/l (0.32 mM). PBMC proliferation and NO generation were determined after 96-hours and 48-hours, respectively, using a Muse Unit (Cell Analyzer/Flow Cytometer). Increasing nitrite exposure significantly suppressed the non-stimulated PBMC proliferation (F = 7.07, p<.0001). The study also showed PBMC proliferation was significantly suppressed among all participants when exposed to 5 mg/l nitrites, but that the degree of suppression was higher among participants with osteoarthritis (F=4.23, p<0.05). Increasing nitrite exposure together with stressful life events, use of multiple drugs (prescribed and supplements), and chronic disease also significantly impacted the NO generating cells and PBMC proliferation.

Pharmacodynamic assessment of mycophenolic acid in resting and activated target cell population during the first year after renal transplantation.

AimsTo explore the pharmacodynamics of mycophenolic acid (MPA) through inosine monophosphate dehydrogenase (IMPDH) capacity measurement and purine levels in peripheral blood mononuclear cells (PBMC) longitudinally during the first year after renal transplantation (TX).MethodsPBMC were isolated from renal recipients 0–4 days prior to and 6–9 days, 5–7 weeks and 1 year after TX (before and 1.5 hours after dose). IMPDH capacity and purine (guanine and adenine) levels were measured in stimulated and nonstimulated PBMC.ResultsTwenty‐nine patients completed the follow‐up period, of whom 24 received MPA. In stimulated PBMC, the IMPDH capacity (pmol 10−6 cells min−1) was median (interquartile range) 127 (95.8–147) before TX and thereafter 44.9 (19.2–93.2) predose and 12.1 (4.64–23.6) 1.5 hours postdose across study days after TX. The corresponding IMPDH capacity in nonstimulated PBMC was 5.71 (3.79–6.93), 3.35 (2.31–5.62) and 2.71 (1.38–4.08), respectively. Predose IMPDH capacity in nonstimulated PBMC increased with time, reaching pre‐TX values at 1 year. In stimulated PBMC, both purines were reduced before (median 39% reduction across days after TX) and after (69% reduction) dose compared to before TX. No alteration in the purine levels was observed in nonstimulated PBMC. Patients needing dose reductions during the first year had lower pre‐dose IMPDH capacity in nonstimulated PBMC (1.87 vs 3.00 pmol 10−6 cells min−1, P = .049) at 6–9 days.ConclusionThe inhibitory effect of MPA was stronger in stimulated PBMC. Nonstimulated PBMC became less sensitive to MPA during the first year after TX. Early IMPDH capacity appeared to be predictive of dose reductions.

P840 Immunomodulatory effects of two different postbiotics on primary human cell culture

Abstract Background Inflammatory bowel disease (IBD) is characterised by chronic, relapsing intestinal inflammation with extensive damage of the colonic mucosa and a remarkable dysbiosis. Since the current treatments show limitations in the response of some patients and many of them are associated with important side effects, new and safer strategies are needed. Postbiotics are any soluble factor resulting from the metabolic activity of a live probiotic bacteria or any released molecule capable of providing health benefits through a direct or indirect mechanism. Nevertheless, the effects of certain postbiotics in the context of intestinal inflammation have not been extensively addressed. Our aim is to study the immunomodulatory properties of two different postbiotics on primary human cell culture and their protective effects on human intestinal ex vivo 2D culture. Methods Postbiotics from Streptococcus salivarius subsp. thermophilus and Lactobacillus paracasei Lpc-37 were obtained after the microbial culture of each bacterial strain. Supernatants were collected at OD600 = 0.6, filtered and frozen. Immunostimulatory, immunosuppressor or immunomodulatory effects of each postbiotic were tested in peripheral blood mononuclear cells (PBMCs) isolated from healthy donors (n = 4). PBMCs are a heterogenous cell population that includes myeloid as well as lymphoid immune cells. Postbiotics were used to assess whether bacterial metabolites could modulate the cytokine release in particular IL-12p40 and IL-10 by LPS-stimulated PBMC, mimicking innate immune activation. Dendritic cells (DCs) differentiated from isolated human monocytes were also stimulated with all postbiotics (n = 2) to study the innate-adaptive immune crosstalk by analyzing IL-10 and IL-12p70 secretion. Postbiotics stimulation was also performed in differentiated human intestinal epithelial monolayers derived from EpOCs (Epithelial Intestinal Organoids Cells) to analyse the outcome in an ex vivo organ culture. Results Postbiotics derived from S.thermophilus and Lpc-37 were able to increase the secretion of IL10 in both PBMCs and monocyte-derived DCs, while no change was shown in IL-12p40/IL-12p70 production. Interestingly, S. thermophilus also showed a ‘per se’ anti-inflammatory effect due to an increase of IL-10 in non-stimulated PBMCs. On the other hand, postbiotics were able to up-regulate MUC2 expression in the 2D organ culture while down-regulating LGR5. Conclusion Overall we conclude that the tested postbiotics show an immunomodulatory effect as well as important properties on intestinal epithelial-cells stemness and differentiation, being S. thermophilus the best candidate due to its remarkable anti-inflammatory effect in both stimulated and non-stimulated cells.

Adipose-derived stromal cell therapy combined with a short course nonmyeloablative conditioning promotes long-term graft tolerance in vascularized composite allotransplantation.

The risks of chronic immunosuppression limit the utility of vascularized composite allotransplantation (VCA) as a reconstructive option in complex tissue defects. We evaluated a novel, clinically translatable, radiation-free conditioning protocol that combines anti-lymphocyte serum (ALS), tacrolimus, and cytotoxic T-lymphocyte-associated protein 4 immunoglobulin (CTLA4-Ig) with adipose-derived stromal cells (ASCs) to allow VCA survival without long-term systemic immunosuppression. Full-mismatched rat hind-limb-transplant recipients received tacrolimus (0.5mg/kg) for 14days and were assigned to 4 groups: controls (CTRL) received no conditioning; ASC-group received CTLA4-Ig (10mg/kg body weight i.p. postoperative day [POD] 2, 4, 7) and donor ASCs (1×106 iv, POD 2, 4, 7, 15, 28); the ASC-cyclophosphamide (CYP)-group received CTLA4-Ig, ASC plus cyclophosphamide (50mg/kg ip, POD 3); the ASC-ALS-group received CTLA4-Ig, ASCs plus ALS (500µL ip, POD 1, 5). Banff grade III or 120days were endpoints. ASCs suppressed alloresponse in vitro. Median rejection-free VCA survival was 28days in CTRL (n=7), 34 in ASC (n=6), and 27.5 in ASC-CYP (n=4). In contrast, ASC-ALS achieved significantly longer, rejection-free VCA survival in 6/7 animals (86%), with persistent mixed donor-cell chimerism, and elevated systemic and allograft skin Tregs , with no signs of acute cellular rejection. Taken together, a regimen comprised of short-course tacrolimus, repeated CTLA4-Ig and ASC administration, combined with ALS, promotes long-term VCA survival without chronic immunosuppression.

Beyond the Biological Effect of a Chemically Characterized Poplar Propolis: Antibacterial and Antiviral Activity and Comparison with Flurbiprofen in Cytokines Release by LPS-Stimulated Human Mononuclear Cells.

Bee propolis, especially Euro-Asian poplar propolis, is among the most well-known natural products traditionally used to treat pharyngitis and minor wounds. The aim of this research was to investigate the pharmacological properties responsible for poplar propolis effectiveness using, for the first time, different in vitro approaches applied to a chemically characterized sample. The anti-inflammatory activity was compared with flurbiprofen by determining pro-inflammatory cytokines released by lipopolysaccharide-stimulated human peripheral blood mononuclear cells (PBMC). The antibacterial activity against Gram+ and Gram- bacteria was assessed, as well as antiviral effects on H1N1 influenza a virus. Poplar propolis (5 and 25 µg/mL) exerted a concentration-dependent anti-inflammatory activity. In this range of concentrations, propolis effect was not inferior to flurbiprofen on cytokines released by lipopolysaccharide (LPS)-stimulated human PBMC. Poplar propolis was found to upregulate IL-6 and IL-1β in non-stimulated PBMC. S. aureus, S. pyogenes, and S. pneumoniae were the most susceptible bacterial strains with inhibitory concentrations ranging from 156 to 625 µg/mL. A direct anti-influenza activity was not clearly seen. Effective anti-inflammatory concentrations of propolis were significantly lower than the antibacterial and antiviral ones and results suggested that the anti-inflammatory activity was the most important feature of poplar propolis linked to its rationale use in medicine.

The Effects of Dental Pulp Stem Cell Conditioned Media on the Proliferation of Peripheral Blood Mononuclear Cells

Background: Dental Pulp Stem Cells (DPSCs) are multipotent mesenchymal stem cells. DPSCs can renew themselves and differentiate into various cell types such as adipocytes, osteocytes, neurons, etc. DPSCs possess immunomodulatory properties and can inhibit peripheral blood mononuclear cell (PBMC) proliferation. Recent studies showed that conditioned-medium mesenchymal stem cells also had immunosuppressive activity. The ability of DPSC conditioned medium to suppress proliferation of allogeneic PBMC determined using BrdU (5-bromo-2’-deoxyuridine) proliferation assay. Materials and Methods: Dental pulp stem cells were extracted from a wisdom tooth. These cells are characterized for differentiation potential to adipogenic and osteogenic lineage and expression of mesenchymal stem cells markers, including CD105, CD73, CD90, CD14, CD-34, and CD45. The characterized DPSCs were cultured, and the conditioned medium (CM) got isolated. Stimulated and non-stimulated PBMCs from the allogeneic donor were cultured with DPSC-CM for 24, 48, and 72 hours. The proliferation of PBMCs was measured with BrdU assay. Results: The BrdU test results showed that DPSC-CM reduced allogeneic PBMC proliferation at different time points. DPSC-CM could inhibit stimulated and non-stimulated PBMC in 48 and 72 hours after incubation.Conclusion: This study demonstrated that DPSC-CM had an immunomodulatory effect on the proliferation of allogeneic cells.

Low-dose endosulfan inhibits proliferation and induces senescence and pro-inflammatory cytokine production in human lymphocytes, preferentially impacting cytotoxic cells

Endosulfan is a DDT-era organochlorine pesticide. Due to past and current environmental contamination, investigation of endosulfan exposure is of current importance. Acute high dose exposure precipitates neural/endocrine system damage, but the effects on the immune system and of lower doses are not well-characterized. Two relatively low concentrations of endosulfan (i.e. 0.1 and 17 µM ENDO) were investigated in an in vitro study using human peripheral blood mononuclear cells (PBMC) to understand effects of relatively low doses (0.1–25.0 µM [≈0.04–10 ppm/40–10,000 ppb]) of ENDO upon normal human T- and B-lymphocytes and NK cells. The study here found that 17 µM ENDO inhibited phytohemagglutinin-M (PHA)-induced human PBMC proliferation. It was also seen that senescence and apoptosis among non-stimulated cells was increased, specifically within CD8 and NK populations, and that CD4:CD8 ratios also were increased. Treatment of non-stimulated PBMC with ENDO led to overall increases in production of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-2, -4, and -6, and decreased production of anti-inflammatory IL-10, suggesting an immunosenescence secretory phenotype. Interestingly, when the cells were pre-stimulated with mitogen (PHA), ENDO became inhibitory against the mitogen-induced proliferation and cytokine formation – with the exception of that of TNFα and IL-6, suggesting differential effects of ENDO on activated cells. Thus, at the organismal level, ENDO might also display differential effects during states of autoimmune disease or chronic viral infection in the exposed host.

Can the Toll-like receptors 4 expression in peripheral blood mononuclear cells help assess the effectiveness of immunosuppression and the chance of a future good renal transplant function?

BackgroundA small percentage of peripheral blood mononuclear cells (PBMCs) circulating during the kidney transplantation (KT) period remain in the blood long after transplantation. A part of the PBMCs penetrates the graft. AimTo examine if the choice of immunosuppression may change TLR4ex and how TLR4ex affects the transplant function in the future. MaterialThe study population-143 transplanted patients (pts) (55 females, 88 males), mean age on recruitment day 50.33 ± 12.8 years old, mean BMI 25.04 ± 4.18. 41 pts. experienced delayed graft function (DGF+). 55 pts. were treated with cyclosporine A (CsA) and 88 with tacrolimus (Tac). All were treated with mofetil mycophenolate (MMF). The PBMCs acquisition and starting point of the follow-up (TLR-day) was at least one month after KT. MethodWe investigated averaged mRNA expression of Toll-like receptors 4 (TLR4ex) in non-stimulated peripheral blood mononuclear cells with the use of real-time polymerase chain reaction. The KT pts. (All, Tac, CsA, DGF+) were divided by the respective median of their TLR4ex (lower: L-TLR4ex, higher: H-TLR4ex). Main clinical parameters and transplant biopsy files (if available) were assessed on TLR-day and post follow-up. ResultsWe found that TLR4ex was reduced for a long time in patients who experienced delayed graft function. L-TLR4ex had a higher proportion of DGF+ patients, and patients treated with CsA but lower of those treated with Tac than in H-TLR4ex. The amplitude of changes in renal function parameters (ΔEGFR%/ΔsCr/ΔsCr%) was clearly less favorable for L-TLR4ex. Tacrolimus expressed a stabilizing effect. Both the positive vasculitis score and chronic graft nephropathy were more frequent in the L-TLR4ex group. On TLR-day an association of renal function and Tac concentration with TLR4ex was clear only in the tacrolimus population. The TLR4ex was lower in patients with a future deterioration of the graft function. ConclusionIn kidney transplant recipients the occurrence of DGF results in a long-term reduction of the averaged TLR4ex in PBMC. Tacrolimus exerts a clear, stabilizing, positive and dose-dependent effect on TLR4ex. An improvement in renal transplant function may be expected in KT patients with high TLR4ex. Evaluation of the averaged TLR4ex can be used to assess the efficacy of immunosuppression in the treatment with tacrolimus and to estimate the likelihood of deterioration in renal function.

Glucocorticoid effects on sheep peripheral blood mononuclear cell proliferation and cytokine production under in vitro hyperthermia

The present experiment aimed at understanding the effects of cortisol levels on sheep peripheral blood mononuclear cell (PBMC) proliferation and cytokine production during hyperthermia. To mimic stress related to the exposition of high ambient temperatures, PBMC were cultured at 43°C for 12 h, and subsequently at 39°C for additional 12 h. Cells in normothermia were cultured at 39°C for 24 h. Phytohemagglutinin-stimulated PBMC were cultured with different cortisol levels: 0 ng/mL; 100 ng/mL, representing the physiological cortisol concentration simulating stress condition (Cort100); and 1,000 ng/mL, representing the hyperactivated hypothalamic-pituitary-adrenal axis (Cort1000). Phytohemagglutinin-stimulated PBMC with 0 ng/mL of cortisol concentration represented the positive control, whereas nonstimulated PBMC without cortisol represented the negative control (NC). The free cell supernatants were collected for the determination of IL-6, IL-1β, and IL-10 by ELISA. Bromodeoxyuridine assay was performed on cells to determine cell proliferation. Exposition to hyperthermia negatively affected cell proliferation, IL-6, IL-1β, and IL-10 concentrations in cell supernatants. The interaction of hyperthermia and cortisol level affected both cell proliferation and IL-10 production. Both PBMC proliferation and IL-10 production in positive control, Cort100, and Cort1000 decreased at 43°C as compared with 39°C NC. On average, the Cort100 treatment displayed higher concentrations of IL-6 than NC. The present experiment demonstrated that the action of cortisol concentration simulating stress condition on cell proliferation and cytokine production was a permissive/stimulatory action during normothermia, whereas it was a suppressive action during hyperthermia. These data confirmed that cortisol concentration simulating stress condition could have a role in the immune system of sheep via mediating cellular homeostasis in the condition of hyperthermia. The negative effects of hyperthermia on sheep immune responses were apparent when performing an immunological challenge.

Cytokine Production by Mononuclear Cells from Patients with Familial Infantile Bilateral Striatal Necrosis

Prompted by findings suggesting immune instability in infantile bilateral striatal necrosis (IBSN), we evaluated levels of proinflammatory (tumor necrosis factor α, interleukin [IL]-1β, IL-6, interferon [IFN]γ) and anti-inflammatory (IL-10 and IL-1ra) cytokines produced by peripheral blood mononuclear cells (PBMC) from 6 children with IBSN and 11 age-matched controls. Compared to controls, non-stimulated PBMC from the IBSN group produced a significantly lower level of IL-1ra (by 38%; p < 0.001) and significantly lower levels of TNFα, IL-1β, and IFNγ (by 36% [p < 0.001], 25% [p = 0.06], and 32% [p < 0.02]) under PBMC stimulation. The severe cachexia manifesting shortly after IBSN onset may impair the immunological state, placing patients at risk of death from hyperpyrexia and sepsis.

Does Iron Modulate Cytokine Production by Desferrioxamine Treated Mononuclear Cells?

Iron is an essential element for maintaining the normal function of cellular and multicellular organisms and it is closely associated with inflammation and immunity. Macrophages and lymphocytes play an important role in its homeostasis. Hereby we have evaluated cytokine production by iron depleted and iron supplemented human peripheral blood mononuclear cells (PBMC) from healthy adult donors. Non-stimulated or stimulated PBMC (lipopolysaccharide-LPS or phorbol-myristate acetate-PMA and ionomycin) were incubated for 24 h without or with desferrioxamine (DFO) and the quantity of TNFα, IL-1β, IL-6, IFNγ, IL-2, IL-10 and IL-1ra in the supernatants was evaluated using ELISA kits. In addition, cytokine levels were determined in supernatants of cultures to which DFO was added for 2 h followed by 24 h of incubation with iron. The secretion of IL-1β, IL-6 and IL-1ra by non-stimulated PBMC and that of IL-1β by LPS-stimulated cells was dose-dependently enhanced following incubation with increasing concentrations of DFO between 25 and 100 µM. On the other hand LPS-induced production of IL-10 and PMA/ionomycin stimulated IL-2 and IFNγ secretion was dose dependently reduced. The inhibited IFNγ, IL-2 and IL-10 secretion by stimulated PBMC caused by DFO was reversed after incubation with iron at concentrations similar to those in human serum. However, the inhibition of LPS-induced IL-1ra or IL-1β production induced by DFO was not affected by iron supplementation. The results of the study underline the importance of intra and/or extracellular iron concentration and DFO for the release of inflammatory cytokines by human peripheral blood mononuclear cells.

Hyporesponsiveness of natural killer cells and impaired inflammatory responses in critically ill patients

BackgroundTo investigate natural killer (NK) cell activity, circulating cytokine level and peripheral blood mononuclear cell (PBMC) cytokine production status in critically ill patients.MethodsBlood samples were collected <24 h after admission from 24 intensive care unit (ICU) patients and 24 age-, sex-, and body mass index (BMI)-matched healthy controls. Serum cytokine concentrations and cytokine production by PBMCs and lipopolysaccharide (LPS)-stimulated PBMCs were measured.ResultsThe ICU group showed lower NK cell activity than the controls under all conditions and an absence of interferon (IFN)-γ. After adjusting for triglycerides, LDL- and HDL-cholesterol, and glucose, the ICU group exhibited lower serum levels of albumin and interleukin (IL)-12 and higher leukocyte counts and hs-CRP and IL-6 levels than the controls. Non-stimulated PBMCs from ICU patients secreted significantly greater amounts of IL-6 and IL-1β than the controls; however, the production of IL-6, TNF-α and IL-1β in response to LPS stimulation was significantly lower in the ICU group.ConclusionsSignificant reductions in NK cell activity and serum IL-12 level, an absence of serum IFN-γ, and decreased cytokine production from LPS-stimulated PBMCs indicate the hyporesponsiveness of NK cells and an impaired early phase inflammatory response in critically ill patients (ClinicalTrials.gov NCT02565589:). Retrospectively registered; October 1, 2015.

Activity of the Calcineurin Pathway in Patients on the Liver Transplantation Waiting List: Factors of Variability and Response to Tacrolimus Inhibition.

We sought to evaluate, in patients on a liver transplantation waiting list, potential biomarkers of the base calcineurin pathway activity with use of a new model of nonstimulated peripheral blood mononuclear cells (PBMC) and ex vivo response to tacrolimus (TAC). The calcineurin pathway activity was explored ex vivo in stimulated and nonstimulated PBMC from 19 patients. The inhibition of NFAT1 translocation to PBMC nuclei, expression of intracellular IL-2, and membrane CD25 in different T-cell subsets were measured by multiparametric flow cytometry before and after exposure to TAC. We also studied the influence on the individual response of polymorphisms in 3 key genes of the calcineurin pathway: PPIA, PPP3CA, and IL2RA. All pharmacodynamics profiles closely fitted an I/Imax sigmoid model. Interindividual variability was higher in nonstimulated than in stimulated conditions, as well as in the presence of TAC. IL-2+CD8+ cells at TAC Imax showed the highest interindividual variability, suggesting its usefulness as a biomarker of individual TAC effects integrating many different sources of regulation and variability. Moreover, in the absence of TAC, patients with end-stage liver disease exhibited lower NFAT1 translocation and T-cell activation than healthy volunteers from a previous study under similar conditions. Multivariate statistical analysis showed strong and significant associations between TAC pharmacodynamic parameters and 2 polymorphisms in the gene-coding cyclophilin A (rs8177826 and rs6850). We show the feasibility of using nonstimulated PBMCs to explore the calcineurin pathway under more physiologic conditions and point toward potential biomarkers for TAC pharmacodynamic monitoring. ClinicalTrials.gov Identifier: NCT01760356.

Comparison of Flow-cytometric Antibody Secreting Cell Assay and Mabtech Immunoglobulin ELISpot Assay

BackgroundThe increase of donor-specific antibodies (DSA) after transplantation could be a more important marker than the level of DSA in pre-transplantation sera. The assessment of sensitized cells that can secrete DSA is needed. We developed an assay for antibody-secreting cells (ASCs) measured with the use of flow cytometry and compared it with the Mabtech immunoglobulin (IgG) ELISpot assay. MethodsThirteen patients who were awaiting or received organ transplantation and 15 healthy control subjects were included. All subjects were positive for anti-cytomegalovirus (CMV) IgG. Peripheral blood mononuclear cells (PBMCs) were seeded with CpG 2006 (5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′), 500 ng/mL human CD40 ligand, and 50 ng/mL interleukin-21 in complete RPMI media. Eight micrograms of CMV pp28 antigen were added to test wells and compared with nonstimulated PBMCs. After 72 hours, analysis with the use of the human IgG ELISpot kit (Mabtech) and flow cytometry with anti-CD19-PE, CD27-PE-Cy7, CD38-APC, IgG-FITC antibodies was performed. ResultsThe flow-cytometric ASC assay was moderately correlated with Mabtech IgG ELISpot assay (r = 0.554; P < .001). The ASCs measured by means of flow cytometry were significantly higher in healthy control subjects compared with patients (P < .001). ASCs measured by means of flow cytometry in CMV antigen–stimulated PBMCs were significantly higher compared with nonstimulated PBMCs (P < .001). The IgG-secreting cells measured by means of Mabtech ELISpot assay was not different between healthy control subjects and patients nor between CMV antigen–stimulated and nonstimulated PBMCs. ConclusionsFlow-cytometric ASC assay can differentiate ASCs for CMV antigen better than Mabtech IgG ELISpot assay. Flow-cytometric ASC assay might be useful for assessing sensitization status in patients awaiting organ transplantation.

Retinoic acid-mediated anti-inflammatory responses in equine immune cells stimulated by LPS and allogeneic mesenchymal stem cells

Retinoic acid (RA), an active metabolite of vitamin A, has shown potential therapeutic immunomodulatory properties. Allogeneic mesenchymal stem cells (MSCs)-based therapy is an effective approach to induce tissue healing and regeneration in many equine orthopedic conditions. However, MSCs-based therapies induced inflammatory responses in vivo. This study aimed to: 1. Determine the effect of RA cell culture treatment on inflammatory responses of lipopolysaccharides (LPS)- and allogeneic MSCs-stimulated peripheral blood mononuclear cells (PBMCs). 2. Determine the effect of RA on stimulated MSCs viability and morphology. Allogeneic MSCs-stimulated PBMCs had significant decreases in the anti-inflammatory cytokines (IL-10, IL-1ra, TGF-β1), increases in the pro-inflammatory mediators (IL-1β, IL-6, TNF-α, SAA), and increases of CD14 and MHC II percent positive cells compared to LPS- and non-stimulated PBMCs. Retinoic acid treatment of LPS- and allogeneic MSCs-stimulated PBMCs counterbalanced the induced inflammatory responses. Moreover, RA significantly improved the viability and morphology of stimulated MSCs. These findings highlighted the potential complications of equine allogeneic MSCs-based therapies and the immuno-modulatory effect of RA on equine stimulated cells. In conclusion, the use of RA to ameliorate allogeneic MSCs therapy associated inflammation may offer advantages that would require further investigations.