Peripheral Blood Mononuclear Cells Cytokine Secretion Research Articles

Manipulating host secreted protein gene expression: an indirect approach by HPV11/16 E6/E7 to suppress PBMC cytokine secretion

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.

In vitro immunomodulatory effects of novel strontium and zinc-containing GPCs.

Glass polyalkenoate cements (GPCs) are bio-adhesives which consist of ionomeric glass particles embedded in a poly-salt matrix. These materials have been used in dentistry and orthodontics extensively but are presently being optimized as bone putties for orthopedic applications. This study utilized a patented ionomeric glass (mole fraction: SiO2:0.48, ZnO:0.36, CaO:0.12, SrO:0.04) to formulate two GPCs: GPC A (<45μm particle size glass) and GPC B (45μm-63μm). These formulations were previously assessed for their effect on osteoblast viability and osteogenic function. However, the immunomodulatory effects of GPC A and B have not previously been investigated. Non-toxic concentrations of (a) GPC dissolution products and (b) fragmented GPC particles were tested for their ability to affect the secretion of cytokines (TNF-α, IL-1β, IL-6 and IL-10) by rat peripheral blood mononuclear cells (PBMCs), in the presence or absence of the stimulant liposaccharide (LPS). Additionally, the ionic concentrations of Sr, Zn, Ca, and Si were measured in GPC ionic extracts, and the size, shape and concentration of fragmented GPC particles in deionized water were characterized using an optical microscope-based particle analyzer. The results showed that GPC A ionic products reduced the concentration of TNF-α secreted by stimulated cells compared with cells stimulated in the absence of GPC products. Interestingly, the particles released from GPC A significantly increased the secretion of both TNF-α and IL-6 from unstimulated cells, compared to control cells. Neither GPC B ionic products nor released particles were found to be biologically active with respect to PBMC cytokine secretion.

EFFECT OF HCG ON HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) SECRETION OF PRO- AND ANTI-INFLAMMATORY CYTOKINES

Successful embryo implantation and appropriate trophoblast invasion is associated with an adequate communication and interaction between peripheral blood mononuclear cells (PBMC) and the human embryo. Human chorionic gonadotropin (hCG) is one of the earliest embryonic signals that affect PBMC cytokine secretion. Nowadays, the intrauterine administration of hCG-activated autologous PBMC is a widely used treatment for improvement of pregnancy outcome. However, the effect of hCG on PBMC cytokine secretion is scarcely studied.

Prognostic tools for hypertrophic scar formation based on fundamental differences in systemic immunity.

Unpredictable hypertrophic scarring (HS) occurs after approximately 35% of all surgical procedures and causes significant physical and psychological complaints. Parallel to the need to understanding the mechanisms underlying HS formation, a prognostic tool is needed. The objective was to determine whether (systemic) immunological differences exist between patients who develop HS and those who develop normotrophic scars (NS) and to assess whether those differences can be used to identify patients prone to developing HS. A prospective cohort study with NS and HS groups in which (a) cytokine release by peripheral blood mononuclear cells (PBMC) and (b) the irritation threshold (IT) after an irritant (sodium lauryl sulphate) patch test was evaluated. Univariate regression analysis of PBMC cytokine secretion showed that low MCP‐1, IL‐8, IL‐18 and IL‐23 levels have a strong correlation with HS (P < .010‐0.004; AUC = 0.790‐0.883). Notably, combinations of two or three cytokines (TNF‐a, MCP‐1 and IL‐23; AUC: 0.942, Nagelkerke R2: 0.727) showed an improved AUC indicating a better correlation with HS than single cytokine analysis. These combination models produce good prognostic results over a broad probability range (sensitivity: 93.8%, specificity 86.7%, accuracy 90,25% between probability 0.3 and 0.7). Furthermore, the HS group had a lower IT than the NS group and an accuracy of 68%. In conclusion, very fundamental immunological differences exist between individuals who develop HS and those who do not, whereas the cytokine assay forms the basis of a predictive prognostic test for HS formation, the less invasive, easily performed irritant skin patch test is more accessible for daily practice.

Alcohol abuse and smoking alter inflammatory mediator production by pulmonary and systemic immune cells.

Alcohol use disorders (AUDs) and tobacco smoking are associated with an increased predisposition for community-acquired pneumonia and the acute respiratory distress syndrome. Mechanisms are incompletely established but may include alterations in response to pathogens by immune cells, including alveolar macrophages (AMs) and peripheral blood mononuclear cells (PBMCs). We sought to determine the relationship of AUDs and smoking to expression of IFNγ, IL-1β, IL-6, and TNFα by AMs and PBMCs from human subjects after stimulation with lipopolysaccharide (LPS) or lipoteichoic acid (LTA). AMs and PBMCs from healthy subjects with AUDs and controls, matched on smoking, were cultured with LPS (1 μg/ml) or LTA (5 μg/ml) in the presence and absence of the antioxidant precursor N-acetylcysteine (10 mM). Cytokines were measured in cell culture supernatants. Expression of IFNγ, IL-1β, IL-6, and TNFα in AMs and PBMCs was significantly increased in response to stimulation with LPS and LTA. AUDs were associated with augmented production of proinflammatory cytokines, particularly IFNγ and IL-1β, by AMs and PBMCs in response to LPS. Smoking diminished the impact of AUDs on AM cytokine expression. Expression of basal AM and PBMC Toll-like receptors-2 and -4 was not clearly related to differences in cytokine expression; however, addition of N-acetylcysteine with LPS or LTA led to diminished AM and PBMC cytokine secretion, especially among current smokers. Our findings suggest that AM and PBMC immune cell responses to LPS and LTA are influenced by AUDs and smoking through mechanisms that may include alterations in cellular oxidative stress.

The effects of carboxymethyl chitosan on the regulation of the proliferation, differentiation and cytokine expression of peripheral blood mononuclear cells

To elucidate the effects of carboxymethyl chitosan (CMCTS) on the proliferation, differentiation and cytokine secretion of peripheral blood mononuclear cells (PBMCs), leukomonocytes were separated and cultured. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, acridine orange/ethidium bromide staining, flow cytometry and enzyme-linked immuno sorbent assay was used to evaluate different concentrations of CMCTS A (molecular weight (MW)=50 000) and CMCTS B (MW=500 000) in the proliferation, apoptosis, differentiation and cytokine secretion of PBMCs. The results demonstrated that CMCTS A and CMCTS B at concentrations of 500 and 100 μg ml−1, respectively, significantly enhanced the proliferation of PBMCs. Both CMCTS A and CMCTS B inhibited the apoptosis of PBMCs, and the optimal inhibitory concentration was 500 μg ml−1. Furthermore, the inhibitory efficacy of CMCTS A was higher than that of CMCTS B. CMCTS A induced the differentiation of PBMCs with CD4 antibody at concentrations of 500 and 100 μg ml−1. Furthermore, the differentiation of PBMCs with CD3–CD56 antibodies was induced by both CMCTS A and CMCTS B. Both CMCTS A and CMCTS B at concentrations of 500 μg ml−1 induced interleukin (IL)-2 secretion, with CMCTS A having a more marked effect than that of CMCTS B. CMCTS A at a concentration of 500 μg ml−1 induced interferon (IFN)-γ secretion. In conclusion, CMCTS with a low MW (50 000) and at a concentration of 500 μg ml−1 positively regulated the proliferation, differentiation and IFN-γ and IL-2 secretion of the PBMCs in vitro.

In vitro models for the assessment of inflammatory and immuno-modulatory effects of the volatile organic compound chlorobenzene

An in vitro cell culture system based on an air/liquid culture technique was developed which allows a direct exposure of cells to volatile chemicals without medium coverage. For the establishment of the experimental system, chlorobenzene was used as a model compound. Chlorobenzene is a volatile organic compound which is mainly used as a solvent. Beside other adverse health effects, chlorobenzene exposure has been shown to be associated with respiratory tract irritations, Th2 differentiation, and allergic sensitizations. Human peripheral blood mononuclear cells (PBMC) and lung epithelial cells (A549) were exposed to chlorobenzene via gas phase for 20 h. Additionally, PBMC were incubated with culture supernatants from exposed lung epithelial cells. High chlorobenzene concentrations (100 g/m 3) induced IL-8 production in A549 cells, whereby lower concentrations (10 μg/m 3–1 g/m 3) stimulated the secretion of the monocyte chemoattractant protein-1 (MCP-1). A direct effect of chlorobenzene on the cytokine secretion of PBMC was not found. However, if PBMC were incubated with culture supernatants of exposed lung cells, an enhanced production of the Th2 cytokine IL-13 was observed. This induction was prevented in the presence of an anti-MCP-1 antibody. Our data suggest that chlorobenzene induces the production of inflammatory mediators in lung cells. The primary chlorobenzene caused release of MCP-1 in lung epithelial cells may secondarily result in a Th2 differentiation in T lymphocytes. These findings may contribute to the understanding of how chlorobenzene mediates the development of inflammatory reactions in the airways and contributes to the development of an allergic reactivity.

Effects of melatonin on the cytokines production of neonatal cord blood mononuclear cell

Objective To investigate the effects of melatonin(MLT)on the cytokines production of neonatal cord blood mononuclear cells(CBMC)in vitro. Methods Ten blood specimens from placenta vena umbilicalis of 10 healthy term delivery newborns were obtained,and 10 healthy adult volunteers'peripheral blood mononuclear cells(PBMC)specimens were taken as controls.CBMC and PBMC suspensions were cultivated in the system without serum and hormone,and then 200 μl PBMC/CBMC suspensions(2 105 cells)were co-cultured with MLT with different concentrations(50 ng/ml,5 ng/ml,500 pg/ml,50 pg/ml)for 72 hours.The concentrations of cytokines including IL-1β,IL-2,IL-4,IL-6,IL-8,IL-12,IFN-γ and TNF-α in the cell supernatant were detected.Results The 500 pg/ml MLT induced secretions of IL-2[(12.18 7.31)pg/ml],IL-6[(2091.08±968.58)pg/ml],IL-8[(2779.04±1168.49)pg/ml],IL-12[(15.29±6.88)Pg/ml]and IFN-γ[(52.22 17.77)pg/ml]of CBMC were more than those of the control group,which were(6.99±4.94)pg/ml,(1017.06±452.60)pg/ml,(1293.33±791.34)pg/ml,(6.84±2.26)pg/ml and(22.24±10.38)pg/ml(P<0.05).Compared with control group[(175.63±62.10)Pg/ml],all concentrations of MLT could inhibit the TNF-α secretion[(24.87 11.21)pg/ml,(18.14±9.44)pg/ml,(31.38±15.28)pg/ml and(28.18±17.35)pg/ml]of CBMC significantly.The 50 ng/ml MLT could induce the PBMC's secretion of IL-2, IL-12, IFN-γ,and inhibit the secretion of TNF-α. The best effect of MLT on CBMC was at the concentration of 500 pg/ml, while that on PBMC was 5 ng/ml. Comparing the best effects of MLT, the levels of IL-12 and IFN-γ of CBMC were higher than those of PBMC, and IL-2 level of CBMC was lower than that of PBMC (P<0.01). Conclusions MLT could promote the cytokine secretion of PBMC and CBMC, and the effects of MLT on IL-12 and IFN-γ secretion of CBMC were stronger than those of PBMC. The results suggested that MLT may contribute to promote the immune function in newborn infants, and provide experimental evidence for clinical application of MLT. Key words: Melatonin; Fetal blood; Leukocytes, mononuclear; Cytokines

T cell reactivity to P0, P2, PMP-22, and myelin basic protein in patients with Guillain-Barré syndrome and chronic inflammatory demyelinating polyradiculoneuropathy

Objectives: It has been suggested that autoimmunity to peripheral myelin proteins is involved in the pathogenesis of Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). We aimed to compare...

Effects of Clozapine on In Vitro Immune Parameters: A Longitudinal Study in Clozapine-Treated Schizophrenic Patients

Clozapine is an atypical antipsychotic agent with immunomodulatory properties. We hypothesized that in vitro immune parameters of peripheral blood mononuclear cells (PBMC) are affected in the course of clozapine treatment and that clozapine per se, added in vitro to PBMC cultures of clozapine-treated patients, exerts differential effects in the timecourse of treatment in vivo. We measured proliferation and cytokine secretion of PBMC, serum autoantibodies, and immunoglobulin levels in 17 patients before and during the first 6 weeks of clozapine treatment. Independent of clozapine dosage and rectal temperature, clozapine treatment in vivo suppressed proliferation and shedding of sIL-2r by PBMC, and the addition of clozapine in vitro induced, relative to unstimulated conditions, PBMC proliferation and secretion of IL-6 and sIL-2r. Serum IgG levels were increased; whereas, autoantibody pattern was unaffected. Thus, clozapine treatment and the addition of clozapine in vitro exert differential effects on various in vitro immune parameters independent of clozapine dosage and rectal temperature in the course of treatment.

Cloning bovine cytokine cDNA fragments and measuring bovine cytokine mRNA using the reverse transcription-polymerase chain reaction

Bovine cytokine-specific primers and the reverse transcription-polymerase chain reaction (RT-PCR) were used to clone cDNA fragments that were specific for bovine IL-1α, IL-1β, IL-2, and IFN-γ. Specificity of the cDNA fragments was verified by sequence analysis based on known bovine IL-1α, IL-1β, IL-2, and IFN-γ gene sequences. In addition, RT-PCR was used to monitor cytokine mRNA expression in concanavalin A (Con A) and lipopolysaccharide (LPS)-stimulated bovine peripheral blood mononuclear cells (PBMC), and the results were compared with those obtained by measuring PBMC cytokine secretion using biologic assays. IL-1 activity in LPS-stimulated PBMC cultures was similar at 12 h and 24 h, although the activity decreased by approximately 40% at 48 h. IL-2 and IFN-γ activity in supernatants of Con A-stimulated PBMC cultures was low at 12 h and reached maximum levels at 48 h. RT-PCR transcript analysis detected an increase in IL-1α, IL-1β, IL-2, and IFN-γ mRNA expression that was usually correlated with the detection of these soluble cytokines by the bioassays. These results indicate that RT-PCR is a sensitive and effective method of obtaining cDNA probes and that this technique can be used to monitor bovine cytokine mRNA expression.