Peripheral Blood Mononuclear Cells Culture Medium Research Articles

The Optimum Dose of Beta-Glucan for Stimulating Peripheral Blood Mononuclear Cells (PBMCs) to Produce Cytokines: In Vitro Study

Background: Beta-glucan has been frequently used in laboratory trials as an immunomodulator in both in vivo and in vitro studies, but the ef­fective dose for measuring its performance has not been established. Like other immunomodula­tors, researchers must establish the right dose of beta-glucan in their laboratory experiments for the purpose of testing substances as immuno­modulators without achieving false-positive or –negative results. This study aimed to determine the optimum dose of beta-glucan to induce cyto­kine production by peripheral blood mononu­clear cells (PBMCs) in vitro. Subjects and Method: This was a laboratory experimental study. This study measur­ed the production of two cytokines, including in­terferon gamma (IFN-γ) and interleukin 12 (IL-12), from the isolated PBMCs of healthy subjects. The doses of beta-glucan used as immunomodu­lator in­clu­ded 1, 5, 10, 20, and 50 μg/ml. Beta-glucan was add­ed to the PBMC culture medium, and the PBMCs were cultured for 6 days. On the sixth day, the supernatant was harvested and the cytokine production was analyzed using sandwich enzyme-linked immunosorbent assay (ELISA). Cytokines were also analyzed using the human IFN-γ ELISA kit and the human IL-12 ELISA kit, and data analysis was performed by one-way ANOVA. Results: IFN-γ levels were found to be increased in the group treated with 5 μg/ml beta-glucan. The highest IFN-γ levels (70.0 pg/ml) were observed in the group treated with 10 μg/ml beta-glucan. The production of IL-12 increased sharply in the group treated with5 μg/ml beta-glucan but decreased in the group treated with 10 μg/ml beta-glucan. The mean cytokine levels of the beta-glucan group were found to be significantly different from those of the control group (p=0.001). One-way ANOVA revealed that the highest IL-12 production (77.2 pg/ml) occurred at a dose of 5 μg/ml beta-glucan. This average value was significantly different from the average production of IL-12 in the control group (p=0.001). Conclusion: The optimum dose of beta-glucan for stimulating PBMCs to produce IFN-γ in vitro was 10 μg/ml, while for the production of IL-12, the dose was 5 μg/ml. Both cytokines can be mea­sured within 6 days of cell culture. Keywords: beta-glucan, immunomodulator, cy­to­kines Correspondence: Meira Erawati. Faculty of Medicine, Diponegoro University. Jl. Prof. Soedarto, S.H, Tembalang, Semarang, Indonesia. Email: meira­e­rawati­@­gmail.com. Mobile: +62-81915339685 Indonesian Journal of Medicine (2020), 5(2): 170-177 https://doi.org/10.26911/theijmed.2020.05.02.12

Subclinical Epstein-Barr Virus Primary Infection and Lytic Reactivation Induce Thyrotropin Receptor Autoantibodies.

Epstein-Barr virus (EBV) is a herpes virus that mainly infects in B lymphocytes and occasionally reactivates lytically. Most individuals have been infected with EBV primarily in their childhood with no symptoms, and the virus persists latently for life. We have previously reported that EBV-infected B cells with thyrotropin receptor autoantibodies (TRAbs) on their surface [TRAb(+) EBV(+) cells] were present in the peripheral blood mononuclear cells (PBMCs) of healthy adult controls and patients with Graves' disease, and that TRAbs released in the culture medium of PBMCs containing TRAb(+) EBV(+) cells by EBV reactivation. EBV lytic reactivation induced the differentiation of host B cells into plasma cells and antibody production. Various autoantibodies have been detected during the acute phase of infectious mononucleosis (IM) that is the symptomatic primary infection of EBV. Therefore, the autoantibody production may be induced by the asymptomatic primary infection. In this study, we examined the presence of TRAb(+) cells, EBV(+) cells, and TRAb(+) EBV(+) cells in PBMCs from 29 healthy or subclinical children without Graves' disease and one cord blood that were divided into six age groups, and also measured plasma TRAb levels. The results obtained demonstrated that low levels of TRAb production occurred with EBV primary infection and lytic reactivation in children without symptoms of IM. Furthermore, the populations of TRAb(+) cells, EBV(+) cells, and TRAb(+) EBV(+) cells were small in the period of primary infection, but they potentially expand with repeated EBV lytic reactivation. This may partly explain why the onset of Graves' disease often occurs in young adults, but rarely in infancy.

Cultures and co-cultures of human blood mononuclear cells and endothelial cells for the biocompatibility assessment of surface modified AISI 316L austenitic stainless steel

Samples of AISI 316L austenitic stainless steel were subjected either to grinding and polishing procedure, or to grinding and then low temperature glow-discharge nitriding treatment, or to grinding, nitriding and subsequently coating with collagen-I. Nitrided samples, even if only ground, show a higher corrosion resistance in PBS solution, in comparison with ground and polished AISI 316L. Biocompatibility was evaluated in vitro by incubating the samples with either peripheral blood mononuclear cells (PBMC) or human umbilical vein endothelial cells (HUVEC), tested separately or in co-culture. HUVEC-PBMC co-culture and co-incubation of HUVEC with PBMC culture medium, after the previous incubation of PBMC with metallic samples, allowed to determine whether the incubation of PBMC with the different samples might affect HUVEC behaviour. Many biological parameters were considered: cell proliferation, release of cytokines, matrix metalloproteinases (MMPs) and sICAM-1, gelatinolytic activity of MMPs, and ICAM-1 protein expression. Nitriding treatment, with or without collagen coating of the samples, is able to ameliorate some of the biological parameters taken into account. The obtained results point out that biocompatibility may be successfully tested in vitro, using cultures of normal human cells, as blood and endothelial cells, but more than one cell line should be used, separately or in co-culture, and different parameters should be determined, in particular those correlated with inflammatory phenomena.

Immunomodulatory effects of the pentapeptide YGSRS on human peripheral-blood mononuclear cells

Context: The pentapeptide YGSRS is originated from coffee bean, while its pharmacological features have little been examined.Objectives: We investigated the effects of YGSRS on proliferation, cytokine production and CD4+ CD25+ Foxp3+ regulatory T (Treg) cell frequency of human peripheral blood mononuclear cells (PBMCs) activated by T-cell mitogen.Materials and methods: The effects of YGSRS on T-cell mitogen-activated PBMCs were assessed by WST assay procedures. Concentrations of Th1/Th2/Th17 cytokines in the PBMCs culture medium were analyzed with beads-array procedures followed by analysis with flow cytometry. The CD4+ CD25+ Foxp3+ Treg cells in mitogen-activated PBMCs were stained with fluorescence-labeled specific antibodies followed by flow cytometry.Results: YGSRS at 1–10 000 ng/ml (1.56–15 600 nM) has a tendency to promote the mitogen-activated proliferation of PBMCs, but the effects were not statistically significant. YGSRS affect the production of tumor necrosis factor (TNF) α, interleukin (IL)-4, IL-6 and IL-10 from the activated PBMCs, and statistically significant increase in the concentrations of IL-6 and IL-10 in the medium were observed at 1–1000 ng/ml (1.56–1560 nM) (p < 0.05). YGSRS has a tendency to decrease the frequency of Treg cells in the activated PBMCs, but the difference was not statistically significant.Discussion and conclusions: The data suggest that the pentapeptide YGSRS affects the production of several types of cytokines from activated human peripheral T cells, which may modulate Th2 type immunity.

Effects of n-3 FA supplementation on the release of proresolving lipid mediators by blood mononuclear cells: the OmegAD study

Specialized proresolving mediators (SPMs) induce resolution of inflammation. SPMs are derivatives of n-3 and n-6 PUFAs and may mediate their beneficial effects. It is unknown whether supplementation with PUFAs influences the production of SPMs. Alzheimer's disease (AD) is associated with brain inflammation and reduced levels of SPMs. The OmegAD study is a randomized, double-blind, and placebo-controlled clinical trial on AD patients, in which placebo or a supplement of 1.7 g DHA and 0.6 g EPA was taken daily for 6 months. Plasma levels of arachidonic acid decreased, and DHA and EPA levels increased after 6 months of n-3 FA treatment. Peripheral blood mononuclear cells (PBMCs) were obtained before and after the trial. Analysis of the culture medium of PBMCs incubated with amyloid-β 1-40 showed unchanged levels of the SPMs lipoxin A4 and resolvin D1 in the group supplemented with n-3 FAs, whereas a decrease was seen in the placebo group. The changes in SPMs showed correspondence to cognitive changes. Changes in the levels of SPMs were positively correlated to changes in transthyretin. We conclude that supplementation with n-3 PUFAs for 6 months prevented a reduction in SPMs released from PBMCs of AD patients, which was associated with changes in cognitive function.

Effects of berberine and red yeast on proinflammatory cytokines IL-6 and TNF-α in peripheral blood mononuclear cells (PBMCs) of human subjects.

Background and Aims: Obesity is a condition associated with chronic or acute inflammatory response characterized by an increase of proinflammatory cytokine levels. Peripheral blood mononuclear cells (PBMCs) migrate in adipose tissue inducing synthesis and secretion of adipocytokines as IL-6 and TNF-α. The aim of this study was to investigate the effect of berberine (a natural alkaloid) and red yeast (a natural antioxidant) on IL-6 and TNF-α cytokines release and gene expression, in circulating lipopolisaccarides (LPS) stimulated PBMCs.Methods and Results: PBMCs isolated from whole blood of healthy donors were stimulated with LPS to induce cytokines production; simultaneously cells were treated with increasing doses of berberine and red yeast. The substances were administered alone or in association. IL-6 and TNF-α protein levels in the culture medium and their mRNA levels were assessed by ELISA and real time PCR, respectively. Berberine and red yeast treatment prevented the LPS induction of IL-6 release in the culture medium of PBMCs. In addition, berberine plus red yeast treatment showed a synergic inhibitory effect on IL-6 release at low concentration. Berberine and red yeast showed an inhibitory effect also on LPS induction of TNF-α release exerting a synergic effect mainly at high concentrations. On the contrary, berberine and red yeast did not significantly affect IL-6 and TNF-α mRNA levels induced by LPS. In this case, only concomitant treatment of PBMCs with high doses of berberine and red yeast inhibits LPS induced IL-6 or TNF-α mRNA levels.Conclusions: The results of our study show that both berberine and red yeast were able to carry out anti-inflammatory action through an inhibition of proinflammatory IL-6 and TNF-α protein release. Moreover, when given in combination these substances were able to inhibit IL-6 and TNF-α gene expression in PBMCs activated by LPS. Therefore, these substances could represent a useful pharmacological treatment to reduce the proinflammatory status accompanied with obesity.

Tax Posttranslational Modifications and Interaction with Calreticulin in MT-2 Cells and Human Peripheral Blood Mononuclear Cells of Human T Cell Lymphotropic Virus Type-I-Associated Myelopathy/Tropical Spastic Paraparesis Patients

The human retrovirus human T cell lymphotropic virus type-I (HTLV-1) is the etiologic agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Axonal degeneration in HAM/TSP patients occurs without neuron infection, with the secreted viral Tax protein proposed to be involved. We previously found that Tax secreted into the culture medium of MT-2 cells (HTLV-1-infected cell line) produced neurite retraction in neuroblastoma cells differentiated to neuronal type. To assess the relevance of Tax posttranslational modifications on this effect, we addressed the question of whether Tax secreted by MT-2 cells and peripheral blood mononuclear cells (PBMCs) of HTLV-1-infected subjects is modified. The interaction of Tax with calreticulin (CRT) that modulates intracellular Tax localization and secretion has been described. We studied Tax localization and modifications in MT-2 cells and its interaction with CRT. Intracellular Tax in MT-2 cells was assessed by flow cytometry, corresponding mainly to a 71-kDa protein followed by western blot. This protein reported as a chimera with gp21 viral protein-confirmed by mass spectrometry-showed no ubiquitination or SUMOylation. The Tax-CRT interaction was determined by confocal microscopy and coimmunoprecipitation. Extracellular Tax from HAM/TSP PBMCs is ubiquitinated according to western blot, and its interaction with CRT was shown by coimmunoprecipitation. A positive correlation between Tax and CRT secretion was observed in HAM/TSP PBMCs and asymptomatic carriers. For both proteins inhibitors and activators of secretion showed secretion through the endoplasmic reticulum-Golgi complex. Tax, present in PBMC culture medium, produced neurite retraction in differentiated neuroblastoma cells. These results suggest that Tax, whether ubiquitinated or not, is active for neurite retraction.

Biocompatibility evaluation of surface‐treated AISI 316L austenitic stainless steel in human cell cultures

The effects of AISI 316L austenitic stainless steel, tested in untreated state or subjected to glow-discharge nitriding (at 10 or 20 hPa) and nitriding + post-oxidizing treatments, on human umbilical vein endothelial cells (HUVEC) and on peripheral blood mononuclear cells (PBMC) were evaluated. All the treated samples showed a better corrosion resistance in PBS and higher surface hardness in comparison with the untreated alloy. In HUVEC put in contact for 72 h with the sample types, proliferation and apoptosis decreased and increased, respectively, in the presence of the nitrided + post-oxidized samples, while only slight differences in cytokine (TNF-alpha, IL-6, and TGF-beta1) release were registered. Intercellular adhesion molecule-1 (ICAM-1) increased in HUVEC incubated with all the treated samples, while vascular cell adhesion molecule-1 (VCAM-1) and E-selectin increased in the presence of all the sample types. PBMC incubated for 48 h with the samples showed a decrease in proliferation and an increase in apoptosis in the presence of the untreated samples and the nitrided + post-oxidized ones. All the sample types induced a remarkable increase in TNF-alpha and IL-6 release in PBMC culture medium, while only the untreated sample and the nitrided at 10 hPa induced an increase in ICAM-1 expression. In HUVEC cocultured with PBMC, previously put in contact with the treated AISI 316L samples, increased levels of ICAM-1 were detected. In HUVEC coincubated with the culture medium of PBMC, previously put in contact with the samples under study, a noteworthy increase in ICAM-1, VCAM-1, and E-selectin levels was always registered, with the exception of VCAM-1, which was not affected by the untreated sample. In conclusion, even if the treated samples do not show a marked increase in biocompatibility in comparison with the untreated alloy, their higher corrosion resistance may suggest a better performance as the contact with physiological environment becomes longer.

Serum hepatocyte growth factor levels are increased in patients with congestive heart failure

Background: Hepatocyte growth factor (HGF) is a potent endothelial cell–specific mitogen. We investigated the clinical importance of HGF in congestive heart failure (CHF). Methods and Results: Thirty-five patients with acute exacerbation of CHF and 7 control subjects were examined. Serum and peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood on days 1, 7, and 14 after admission. PBMCs were cultured at a density of 1 × 107 cells/mL for 24 hours. HGF levels in serum and the PBMC culture medium and serum interleukin 6 (IL-6) levels were measured by enzyme-linked immunosorbent assay. Serum HGF levels in patients with CHF were markedly increased at admission compared with those in control subjects and gradually returned to control levels during hospitalization. HGF levels in the PBMC culture medium were also significantly increased in CHF patients compared with control subjects. There was a positive correlation between HGF levels in serum or those in the PBMC culture medium and serum IL-6 levels. HGF levels in serum and the culture medium were not notably different between CHF patients regularly treated with and without angiotensin-converting enzyme inhibitors. Conclusions: HGF levels in serum are increased in patients with acute exacerbation of CHF.

Expression of matrix metalloproteinases in patients with acute myocardial infarction.

This study investigated the clinical significance of matrix metalloproteinases (MMPs) in acute myocardial infarction (AMI) and the involvement of peripheral blood mononuclear cells (PBMCs), which are a possible source of MMPs in AMI. Forty patients with AMI were recruited. Plasma and PBMCs were isolated from peripheral blood on days 1, 7, 14 and 21 after the onset of AMI. Levels of MMP-1 and MMP-2 were measured by enzyme-linked immunosorbent assay. The MMP-1 level in the culture medium of PBMCs after incubation for 24h was designated as 'PBMC-MMP-1 level.' Plasma MMP-1 did not significantly change during the course of AMI, but the plasma MMP-2 levels increased gradually after the onset of AMI with maximum elevation on day 21 after onset. Plasma MMP-2 activity also became significantly elevated during the course of AMI. PBMC-MMP-1 levels in the patients were significantly higher than those in control subjects over the course of AMI. Significant positive correlations were observed between maximum PBMC-MMP-1 levels and maximum plasma C-reactive protein levels (r=+0.55, p<0.01) and left ventricular end-diastolic volume index (r=+0.63, p<0.001). In conclusion, plasma MMP-2 levels and activity and MMP-1 production by PBMCs are increased in patients with AMI. Inflammation after AMI may enhance production of MMP-1 by PBMCs. These changes may play an important role in the ventricular remodeling that occurs after AMI by promoting the degradation of the extracellular matrix.

Expression of vascular endothelial growth factor in patients with acute myocardial infarction

OBJECTIVEThe purpose of this study was to investigate the clinical significance of vascular endothelial growth factor (VEGF) in acute myocardial infarction (AMI). We also examined the involvement of peripheral blood mononuclear cells (PBMCs), which are a possible source of VEGF in AMI.BACKGROUNDVEGF is a potent endothelial cell–specific mitogen and could affect the outcome of AMI.METHODSThirty patients with AMI were used for this study. Serum and PBMCs were isolated from peripheral blood on days 1, 7, 14 and 21 after the onset of AMI. PBMCs were cultured at a density of 5 × 106cells/ml for 24 h. VEGF levels in serum and the culture media were measured by enzyme-linked immunosorbent assay using a specific anti-human VEGF antibody.RESULTSSerum VEGF levels elevated gradually after the onset of AMI and reached a peak on day 14. VEGF levels in the culture medium of PBMCs after incubation for 24 h (PBMC-VEGF) were maximally elevated 7 days after the onset. Maximum serum VEGF levels showed significant positive correlations with maximum creatine phosphokinase (CPK) levels (r = +0.70, p < 0.001), but maximum PBMC-VEGF levels did not correlate with maximum CPK levels. Patients showing improvement in left ventricular systolic function during the course of AMI showed significantly higher PBMC-VEGF levels than patients without improvement.CONCLUSIONSThe extent of myocardial damage contributes to the elevation of serum VEGF levels in AMI. VEGF produced by PBMCs may play an important role in the improvement of left ventricular function by promoting angiogenesis and reendothelialization after AMI.